Why Study Microbiology?
- Balance of Nature – food source, play a role in decomposition, help other animals digest grass (cattle, sheep, termites).
- Environmental– provide safe drinking water; development of biodegradable products; use bacteria to clean up oil spills, etc. – called bioremediation.
- Industrial – foodstuffs (beer, wine, cheese, bread), antibiotics, insulin, genetic engineering
- Agricultural – research has led to healthier livestock and disease-free crops.
- Microbiology defined – The study of microbiology is the study of microorganisms, which are organisms that are invisible to the naked eye.
- Classification of Microorganisms
The 5 major groups of microorganisms: bacteria, algae, fungi, protozoa, and viruses. We will also study some other smaller groups such as prions and viroids. The one property that links these groups together is their very small size! 2 types of cells (viruses, prions and viroids are acellular – “without a cell”):
- Prokaryotic(“before nucleus”) – these guys are cells, but they have no internal membrane bound structures (no membrane-bound nucleus or membrane-bound organelles); includes only the bacteria.
- Eukaryotic(“true nucleus”) – do have internal membrane bound structures (membrane bound nucleus and membrane-bound organelles); includes organisms such as protozoans, fungi, algae, animals, plants.
- Bacteria (singular – bacterium) (study of bacteria – bacteriology)
- size: 1/1000 the volume of a typical eukaryotic cell
- 2 groups (discovered in 1970’s) – we’ll discuss more later
- Archaeobacteria – ancient bacteria
- Eubacteria – true bacteria
- some shapes: bacillus(rod), coccus (spherical), spirillum (spiral), vibrio (curved rod)
- motile or nonmotile
- how do they obtain their energy?
- photosynthetic autotrophs– use energy from the sun to produce their own carbohydrates for energy.
- chemosynthetic autotrophs– process inorganic molecules for energy (ex. sulfur or iron).
- heterotrophs– depend on outside sources of organic molecules (ex. carbohydrates or sugars) for energy
- temperature extremes: -20oC to 110oC (that’s really cold & really hot! freezing is OoC and boiling is 100oC)
- examples of diseases?
- Algae (singular – alga) – not a focus in this course.
- unicellular or multicellular
- size: some microscopic, some macroscopic (ex. kelp)
- motile or nonmotile
- how do they obtain their energy? photosynthetic autotrophs
- disease causing? no
- Fungi(singular – fungus) (study of fungi – mycology)
- unicellular or multicellular (yeasts are unicellular, molds are multicellular)
- how do they obtain their energy?
- Why are they ecologically important? Scavengers; they live off dead matter and thus, decompose it.
- examples of diseases (called mycoses)?
examples of nonpathogenic fungi?
- Protozoa (“first animals”)
- motile or nonmotile
- how do they obtain their energy? Heterotrophs
- disease causing – 2 examples: malaria & giardiasis (one of the “don’t drink the water diseases”)
- Viruses – (study of viruses – virology)
- acellular, so not considered prokaryotic or eukaryotic; obligate intracellular parasites; when they are outside of a host cell, there is no evidence that these guys are alive.
- basic structure of a virus – a piece of nucleic acid (RNA or DNA) enclosed by a protein coat (capsid); possess no nucleus, organelles, cell membrane, or cytoplasm.
- size – 1/10 to 1/1000 the size of an ordinary bacterial cell.
- examples of diseases?
Important Note: We will consider a sixth group, the helminths (worms), in our study of microbiology. While most of the adult stages of these worms are macroscopic, many of them go through a microscopic stage in their life cycles (egg & larval stages). Some examples of helminths are tapeworms, hookworms, pinworms, heartworms, and Chinese liver flukes. More to come later!! III. A Brief History of Microbiology
- Leeuwenhoeck (lived 1632-1723)
- What discovery is he credited with? First person to use microscopes to observe microbes; as a hobby he made small handheld microscopes; he called microorganisms “animalcules.”
- What discovery is he credited with? He first described “cellulae” (small rooms) in cork in 1665. His discovery led to the formulation of the cell theory, which states that cells are the basic organizational unit of all living things.
- Redi and Spontaneous Generation
- What is this theory?Living organisms arise from nonliving things (ex. maggots come from rotting meat)
- Who disproved this theory and how? In the late 1600’s Francisco Redishowed that maggots developed only in meat that flies could reach to lay eggs on.
- Many insisted that he only disproved spontaneous generation for macroorganisms; maybe microbes were an exception.
- Needham vs. Spallanzani – still trying to prove or disprove the theory of spontaneous generation.
- What was Needham’s hypothesis, experiment, & conclusions? Everyone knew boiling killed microbes; so, he would boil chicken broth, put it in a flask, & seal it; if microbes grew, then it could only be because of spontaneous generation; they did grow. [We now know that microbes grew because the flask was not sterilized before he poured in the broth!]
- What was Spallanzani’s hypothesis, experiment, & conclusions? He was not convinced by Needham’s experiment. He put broth in a flask, sealed it (creating a vacuum), & then boiled it. There were no microbes in the cooled broth! Critics said he didn’t disprove spontaneous generation – they said he just proved that spontaneous generation required air.
- Pasteur’s Epic Experiments (1859)
- What was his experimental method? To offset the argument that air was necessary for spontaneous generation, Pasteur allowed the free passage of air, but prevented the entry of microbes. He boiled meat broth in a flask & then drew out & curved the neck of the flask in a flame. No microbes developed in the flask. When he tilted the flask so some broth flowed into the curved neck & then tilted it back so the broth was returned to the base of the flask, microbes grew. Gravity had caused the microbes that had entered the flask in air & dust to settle at the low point of the neck, never reaching the broth in the base until the broth washed them in.
- Pasteur’s success was partly due to good luck. He used meat, which contains few bacterial endospores (endospores are resistant to heat; many experiments done prior to Pasteur’s used vegetable broths – plants contains many endospore-forming bacteria.)
- What 3 things did Pasteur’s experiments prove?
- No living things arise by spontaneous generation.
- Microbes are everywhere – even in the air and dust
- The growth of microbes causes dead plant & animal tissue to decompose & food to spoil (this led him to develop the technique of pasteurization– he developed it to keep wine from spoiling).
- Pasteur also contributed to the development of vaccines.
- The Germ Theory of Disease
- What is the germ theory of disease? Microbes (germs) cause disease and specific microbes cause specific diseases.
- Who proved this theory? Robert Koch in the late 1870’s.
- What disease was he studying? anthrax – disease of cattle/sheep; also in humans
- What was his experimental method? He observed that the same microbes were present in all blood samples of infected animals. He isolated and cultivated these microbes (now known to be Bacillus anthracis). He then injected a healthy animal with the cultured bacteria & that animal became infected with anthrax & its blood sample showed the same microbes as the originally infected animals.
- What did his experiments prove? Particular microbes cause particular diseases.
- What are Koch’s 4 Postulates?
1.) The causative agent must be present in every individual with the disease. 2.) The causative agent must be isolated & grown in pure culture (how did he invent pure cultures?; with Frau Hesse’s help, he developed the agar plate method (see p. 13). 3.) The pure culture must cause the disease when inoculated into an experimental animal. 4.) The causative agent must be reisolated from the experimental animal & reidentified in pure culture.
- What are Some Ways that We Can Control Infectious Diseases?
- Immunity– stimulating the body’s own ability to combat infection; from ancient times it was a recognized fact that people who suffered from certain diseases never got them again; infection could produce immunity.
- Immunizationdefined: produce immunity by providing exposure to altered organisms that do not cause disease.
- Jenner & Smallpox– observed that dairymaids that contracted a mild infection of cowpox seemed to be immune to smallpox. He inoculated a boy with fluid from a cow pox blister and he contracted cowpox; he then inoculated him with fluid from a smallpox blister; the boy did not contract smallpox; the term vaccination came from vacca for cow.
- The first vaccines:
1.) Pasteur’s discovery? attenuated bacteria can produce immunity 2.) Attenuated defined – weakened virus or bacteria that is unable to cause the disease (it was later discovered that killed microbes can also produce immunity) 3.) What vaccines did Pasteur develop? anthrax, rabies
- Public Hygiene
- Improving sewage disposal.
- Assuring a clean public water supply.
- Food preservation & inspection.
- Pasteurization – kills most microbes by exposing to heat.
- Improving personal hygiene.
Semmelweiss & childbed fever
- Developing antiseptic techniques.
Lister & carbolic acid – he developed the first aseptic techniques.
- Who is the father of chemotherapy? Paul Ehrlich – he discovered a drug treatment for syphilis; he developed the guiding principle of chemotherapy, which is selective toxicity (the drug must be toxic to the infecting microbe, but relatively harmless to the host’s cells).
- What was the first major class of drugs to come into widespread clinical use? sulfa drugs
- Who discovered the first antibiotic? Flemming discovered (penicillin); antibiotics are antibacterial compounds produced by fungi and bacteria.
CHAPTER 2 – INTRODUCTORY CHEMISTRY & BIOCHEMISTRY INTRODUCTORY CHEMISTRY Why is it necessary to study chemistry? Living things are made of matter (anything that occupies space & has mass) & matter follows the laws of chemistry. Even the characteristics we consider to be unique to living things are the result of chemical reactions (ex. movement as a result of muscle contractions).
- THE ATOM
- Atom– smallest unit of matter unique to a particular element.
- Element– A substance made up of only one kind of atom – ex. carbon contains only carbon atoms. Elements can’t be broken down into other substances by ordinary chemical means. Each element displays unique properties (ex. some are gases, some are solids, some are metals, etc.). About 92 elements occur naturally (there are also some man-made elements). Some of the elements important to our study of living systems are carbon, oxygen, hydrogen, nitrogen, sodium, chlorine, phosphorus, and potassium. You may also be familiar with the elements lead, iron, iodine, gold, silver, and copper, nickel, and platinum.
- Some Important Things to Know about Atoms & Elements:
- An atom consists of 2 basic parts:
- nucleus – the nucleus contains protons & neutrons:
1.) protons – positively charged; all atoms have protons; protons give the nucleus a positive charge. 2.) neutrons – neutral in charge; fxn.: “stabilizers”; all atoms except hydrogen have one or more neutrons.
- electrons– negatively charged; occupy orbit energy levels or shells around the nucleus; attracted to the positive charge of the nucleus; in an atom, the number of electrons always equals the number of protons, so the atom, as a whole, has no charge; electrons determine the chemical properties of elements (ex. whether they are a liquid, solid, or gas, etc.).
- atomic number= number of protons (or number of electrons); in an atom, the number of protons always equals the number of electrons; this number differs for each element.
- atomic mass number= number of protons + number of neutrons; the number of electrons is not included in the mass number due to their insignificant mass.
- The 6 elements important for building organic molecules like carbohydrates, lipids, proteins, and nucleotides are: carbon, hydrogen, oxygen, sulfur, nitrogen, & phosphorus.
Note: Be able to determine the number of electrons, protons, & neutrons in an atom, given the atomic number & atomic mass. Ex. Sodium (Na) has an atomic number of 11 & an atomic mass of 23. A Sodium atom has 11 protons, 11 electrons, & 12 neutrons.
- CHEMICAL REACTIONS
- More about Electrons
- Electrons orbit around the nucleus at different energy levels or shells the 1st shell (k shell) can hold no more than 2 electrons; the next shell (l shell) can hold 8 electrons, 2 electrons in each of 4 orbitals; the next shell (m shell) also holds 8 electrons. These will be the only shells that we will deal with in this class.
- An atom is the most stable when all of its shells are completely filled (the k shell fills first, then the l shell, then the m shell, & so on).
- The atoms of many elements have partially filled outer shells, therefore they are not very stable; these atoms tend to react with other atoms to completely fill their outer shells &, in doing so, they form chemical bonds; it is important to remember that it’s the electronsof an atom that participate in the chemical bonds that form between atoms. Moleculesare formed when 2 or more atoms are joined together by interactions between the electrons of their outer electron shells.
- Chemical Bonds
- Ionic bonds
- definition– in ionic bonds electrons are transferred to other atoms to completely fill outer shells; atoms are electrically neutral, but when they gain or lose electrons in combining with other atoms, they are called ions (charged atoms) & they take on a positive or negative charge; in other words, the transfer of electrons upsets the balance of protons & electrons in an atom; atoms that lose electrons are positively charged, atoms that gain electrons are negatively charged; ionic bonds involve the attractions between these oppositely charged ions. So, before you can have an ionic bond, you have got to have oppositely charged ions, & to create ions you have got to transfer electrons.
- example:NaCl (sodium chloride- table salt); Na (at. # 11) has one electron in its outer m shell – it needs 7 electrons to fill this shell – it is easier for Na just to give this electron away, & eliminate the m shell entirely. Cl (at. # 17) has 7 electrons in its outer m shell – it only needs 1 electron to fill its outer shell. Therefore, when Na & Cl atoms react, Na gives up its outer electron to Cl. Because Na gives up an electron, it now has 11 protons & 10 electrons, resulting in a positively charged atom. Cl now has 17 protons & 18 electrons, resulting in a negatively charged atom. Na+ & Cl– ions are attracted to each other because of their opposite charges & an ionic bond is formed. The attraction is the ionic bond! Only the electron # changes when ions are formed!
- Covalent bonds – more common in the human body & are more stable.
- definition – Electrons are not transferred, but are shared. The shared electrons spend part of their time around the nucleus of one atom & part of their time around the other. Each pair of electrons shared equals one covalent bond (if 2 pair of electrons are shared between 2 atoms, a double covalent bond is formed, a triple covalent bond occurs when 3 pr. of electrons are shared). We only discuss single covalent bonds.
- example: methane (CH4); a carbon atom can form four covalent bonds – it has 4 electrons in its outer shell, therefore it needs 4 more electrons to fill its outer shell; hydrogen has one electron in its outer shell, therefore it needs one electron to fill its outer shell. Rather than give 4 electrons away or accept 4 electrons, carbon shares its 4 electrons in its outer shell with 4 Hydrogen atoms.
- polar vs. nonpolar – covalent bonds can be polar or nonpolar; if both atoms exert the same pull on the shared electrons (equal sharing), the bond is nonpolar (example: methane); if there is unequal sharing of electrons, the bond is polar; in molecules with polar covalent bonds, there is an atom that has a much larger nucleus (more protons) than the other atoms in the molecule; the atom with the most protons is more attractive to the shared electrons, so the electrons spend mostof their time around this atom’s nucleus; all of these electrons spending most of their time around a particular nucleus gives this part of the molecule a partial negative charge; the other atom(s) in the molecule acquire a partial positive charge, because the shared electrons are not spending much time around them. Shared electrons in polar covalent bonds are not spending all of their time around a particular nucleus – if this were the case then we would be talking about electrons being transferred (as in ionic bonds).
- Hydrogen bonds – These bonds result from polar covalent bonds; they form betweenmolecules & occur between the slightly negative atom of one molecule & the slightly positive atom of another molecule. These bonds can occur between hydrogen & oxygen & between hydrogen & nitrogen. Hydrogen bonds are weaker than ionic & covalent bonds, because the charges on the molecules are “partial” or weak charges.
- Chemical Reactions– A chemical reaction when atoms or molecules (called reactants) collide and are transformed into different combinations of the same atoms or molecules (called products). In this process, chemical bonds break and new ones form. In living systems special proteins called enzymes catalyze these chemical reactions (they make them “go”). We’ll talk more about enzymes later.
- A single water molecule is made up of one oxygen atom & two hydrogen atoms (H2O).
- Oxygen has 8 protons, while each hydrogen has only one proton. Oxygen forms a covalent bond with each hydrogen so that the outer shells of each atom are complete.
- Because the oxygen atom has more protons (positives) than the hydrogen nuclei, the shared electrons have a greater attraction to the oxygen nucleus & spend more time around it than they do around the hydrogen nuclei (unequal sharing of electrons). The oxygen atom therefore has 10 electrons (8 of its own & 1 from each hydrogen) spending most of their time around its nucleus of 8 protons – this gives the oxygen end of the water molecule a partially negative charge. Since the hydrogen electrons are spending most of their time around the oxygen atom, the hydrogen atoms, which have one proton each, take on a partially positive charge. This results in a polar molecule (a molecule that has partially positive & negative regions). Their polarity allows water molecules to interact with one another, forming hydrogen bonds. The same type of interaction is possible between water & many other polar substances. Polar substances are hydrophilic (water-loving) & nonpolar substances are hydrophobic (water-fearing).
- PROPERTIES OF WATER – Hydrophilic & hydrophobic interactions underlie several properties of water that are biologically important.
- Temperature-Stabilizing Effects:
Note: The temperature of a substance is a measure of how fast its molecules are moving; the higher the temperature of a substance, the faster its molecules are moving. a.) heating water – It takes considerable heat to raise the temperature of water because the hydrogen bonds between the water molecules restrict the movement of the molecules; in order for the temperature of water to rise, a number of H bonds must be broken – this takes a lot of energy. This resistance to temperature change helps living cells to maintain a relatively constant temperature; this is important because biochemical reactions take place within a narrow temperature range (this has to do with the action of enzymes). This resistance to temperature change also helps organisms that live in aquatic or marine environments.
- Water As a Solvent – the polarity of water is also responsible for water’s capacity as a solvent(something that dissolves something else); water is an excellent solvent for ions & other polar molecules (solutes) in cells.
- ACIDS AND BASES
When molecules of inorganic acids, bases, or salts dissolve in water of body cells, they undergo ionization or dissociation(they break apart into their individual ions).
- Acids & Bases Generally Defined
Acid – Defined as a solute that releases H+ ions in a solution [ex. HCl – hydrochloric acid dissociates into H+ ions & Cl– ions] Base – Defined as a solute that removes H+ ions from a solution; many release OH– ions in this process. [ex. Mg(OH)2 – magnesium hydroxide dissociates into OH– ions & Mg++ ions].
- pH Scale– Fluids are assigned a pH value (0 -14), which refers to the hydrogen ion concentration present in the fluid. The hydrogen ion concentration is abbreviated as [H+].
- acid– pH below 7.0; base – pH above 7.0; neutral – pH = 7.0
- pH = – log [ H+] (formula for calculating pH)
- It is a common misconception to think that as the [H+] increases, the pH also increases! The rule is: As [H+] increases, pH decreases!This can be seen from the following example:
solution A: [H +] = 1 x 10–2 or 0.01 pH = -log[1 x 10-3] = 2 solution B: [H+ ] = 1 x 10–8 or 0.00000001 pH = -log[1 x 10-4] = 8 (A quick way to find the pH of these solutions is to look at the exponent or count the number of decimal places in the [H+]) Solution A is more acidic than Solution B – Solution has a higher [H+] than Solution B (0.001 > 0.0001); therefore, Solution A has a lower pH than B. When you think about a pH value, think that this number is really the number of decimal places in the hydrogen ion concentration. The larger the number, the more decimal places there are, indicating a smaller hydrogen ion concentration.
- Buffers– help maintain a constant pH by removing or adding H+ ions; the pH inside living systems is generally between 7.35-7.45 (exception: the hydrochloric acid in the digestive system makes the pH here 2-3); this pH range is important, as many biochemical reactions take place only within this range; buffers can combine with hydrogen ions &/or release them, & so help stabilize the pH.
4 Groups of Organic Compounds Important in Living Organisms: Carbohydrates Lipids Proteins Nucleotides Organic compound defined – A compound containing carbon (with exception of carbon dioxide); found in all living things. THE CENTRAL ROLE OF CARBON
- The Carbon Backbone – The processes of life are primarily the result of the chemistry compounds of carbon. Because of carbon’s tendency to form four covalent bonds in four different directions, carbon can form an unbelievably large number of different compounds of high complexity; organic compounds derive their basic shapes from the carbon atoms; this shape helps determine the compound’s function in living systems.
- Functional groups– The structure & behavior of organic compounds also depends on the properties of their functional groups; functional groups are groups of atoms (ex. hydrogen, oxygen, nitrogen, phosphorus, sulfur) attached to the carbon backbone.
- Structure: generally made up of only three elements: carbon, hydrogen, & oxygen
- Three Principle Classes of Carbohydrates:
- Structure – composed of single sugar molecule; the atoms in a sugar molecule can form a straight chain or a ring (rings are more common in the body).
- Examples – Glucose, Ribose, Fructose, Galactose
- Functions – monosaccharides are important energy molecules in living things; glucose is the primary energy source for humans & many other animals; also important as building blocks of larger sugars.
- Oligosaccharides – composed of short chains of monosaccharides; Examples:
- Sucrose(table sugar) is a disaccharide composed of glucose & fructose; sucrose is the form in which sugars are transported in plants.
- Lactose(milk sugar) is a disaccharide composed of glucose & galactose.
Sucrose = Glucose + Fructose
- Polysaccharides– straight or branched chains of many monosaccharide units
- Storage Polysaccharides
1.) Starch – sugar storage in plants. 2.) Glycogen – “animal starch;” principle storage form for glucose in higher animals; this energy storage is short term; lipids are used for long term energy storage.
- Structural Polysaccharides
1.) Cellulose – Principal component of the plant cell wall; also found in the cell walls of algae and fungi. Monosaccharides are bonded together in such a way that the molecule resists breakdown by multicellular organisms. We don’t have the digestive enzymes to break the bonds; however, some microorganisms do have these enzymes; this is why microbes are so important in the gut of a termite, cow, etc.) 2.) Chitin – contains nitrogen; forms the cell wall of some fungi (it’s the same stuff insect exoskeleton’s are made of!)
- Protein Structure:
- Proteins are composed of subunits called amino acids(there are 20 different amino acids that make up proteins). Amino acids contain carbon, hydrogen, oxygen, and nitrogen. Some also contain sulfur. Amino acids have a structure similar to the one below. The R stands for some other atom or group of atoms bonded to the central carbon atom in the molecule. The sequence of amino acids in a chain helps determine the structure & shape of the protein & therefore the function of the protein; there are many possible combinations of amino acids that produce the many different kinds of proteins.
H H O N—-C—-C H R OH
- Peptide bonds– linkage formed between one amino acid & another amino acid; the name of these bonds is why chains of amino acids are called polypeptides.
- Producing the three dimensional structure of a protein: We have been discussing proteins as “chains of amino acids.” However, the final structure of proteins is not a straight chain of amino acids. Proteins are very complex, three-dimensional molecules, with numerous twists & folds. The amino acid chain of every kind of protein is folded in a very specific way [the chain will twist & fold itself based on the linkage of amino acids in a specific sequence & the environmental conditions (i.e., temperature & pH)]. There are several bonds & forces which give a protein its specific 3-D structure (i.e., hydrogen bonds, ionic bonds, etc.); these bonds link distant parts of the molecule, forming loops, twists, etc. Destruction of a protein’s 3-D structure by extreme heat or pH is called denaturation(see “enzymes” on the next page for how this occurs). Analogy for protein structure: Think of a phone cord. Pull it straight (like a straight chain of a.a.), then let it twist, then roll the twisted cord into a ball. (Every type of protein folds and twists in a very specific way.)
- Some Specific Functions of Proteins:
- Structural Proteins: collagen in connective tissue, keratin in the skin, cytoskeleton in cells
- Functional Proteins:
- membrane transport proteins – transport substances across the cell membrane
- cell movement – ex. flagellum
- enzymes as catalysts (enzymes speed up the rate of chemical reactions); all life processes are primarily the result of chemical reactions; molecules in living things require enzymes in order to react; without enzymes, chemical reactions in living things can’t occur; see below for more information on enzymes.
- antibodies in the immune response
- Enzymes– a large, globular protein molecule that accelerates a specific chemical reaction. Virtually all chemical reactions that take place in cells involve enzymes!!! Most of a cell’s proteins are enzymes.
- Why are enzymes needed? In order for particular molecules to react with one another, they must be in close proximity & must collide with sufficient force to overcome the mutual repulsion of their negatively charged electron clouds & to break existing chemical bonds within the molecules. The force with which they collide depends on their kinetic energy (energy of motion). Most chemical reactions require an initial input of energy to get started, which increases the kinetic energy of the molecules, enabling a greater number of them to collide with sufficient force. In the chemistry lab, we can supply this energy with heat. In a cell, many different reactions are going on at the same time, therefore heat cannot be used as it would be nondiscriminatory (it would affect many reactions at the same time). Cells get around this problem by using enzymes, which serve as catalysts(they get the chemical reactions going). The enzymes form a temporary association with the molecules that are to react, bringing them close to one another & weakening the existing chemical bonds, making it easier for new ones to form.
- Enzyme Structure & Function– Enzymes are large, complex, globular proteins consisting of one or more polypeptide chains. The molecules that enzymes acts on are known as the substrates. Enzymes are folded to form a groove or pocket (called an active site) on their surface into which the substrate fits & where the chemical reactions take place. See diagram below:
- Effects of Temperature & pH on Enzyme Function
- Temperature:As the temperature increases, so does the rate of enzyme catalyzed reactions, but only up to a certain point. At high temperatures, the enzymes are denatured (due to the vibration of molecules at high temperatures, the bonds that maintain the enzyme’s structure are broken & the protein unfolds). If denaturation is severe, the damage to the enzyme is irreversible.
- pH:The shape of the enzyme depends partly on attraction between positively & negatively charged amino acids. As the pH changes (acidic – more H+, basic – fewer H+), these charges change, changing the shape of the enzyme & its function. Remember: the optimum pH for most enzymes is 6-8. (exception: the stomach which has a pH of 2)
Note: All proteins can be denatured by heat and extreme pH.
This molecule has both protein and polysaccharide components and it forms the cell wall of eubacteria. It may be one or several layers thick. It is an extremely strong protective covering. Glycan strands in all eubacteria are made of alternating units of 2 modified sugars, N-acetylglucosamine (NAGA) and N-acetylmuramic acid (NAMA). (It’s structure is similar to a chain link fence!) More later!
- General Structure– all lipids are mostly nonpolar (hydrophobic) & are insoluble in polar solvents such as water; lipid structure varies greatly & is discussed below for each type.
- Some General Functions:
- long term energy storage (example: glycerides); energy is stored in the chemical bonds; excess carbohydrates, proteins or fats are converted to triglycerides & are stored in adipose (fat) tissue.
- structural (example: phospholipids make up the cell membrane of cells)
- Types of Lipids:
- Lipids with Fatty Acids – Glycerides & Phospholipids:
1.) Structure: classified as mono-, di-, & triglycerides, depending on the number of fatty acids attached a single glycerol molecule; glycerol has 3 carbon atoms & 3 hydroxyl (OH) groups; fatty acids are long, nonpolar chains composed of hydrogen & 4 to 24 carbon atoms, with a carboxyl (COOH) group at one end. a.) Saturated fatty acids – all carbons in the fatty acid tails are joined together by single carbon to carbon bonds & as many hydrogen atoms as possible are linked to the carbons (the carbons are said to be “saturated” with hydrogens); triglycerides with many saturated fatty acids are solid at room temperature; occur mostly in animal tissues, but also in a few plant products; examples: butter, lard, cocoa butter, palm oil, coconut oil; the liver produces cholesterol from some breakdown products of saturated fats. b.) Unsaturated fatty acids – one or more double bonds occur between carbon atoms in the fatty acid tails; this cuts down on the number of hydrogen atoms that can bond to the carbons; liquids at room temperature; the double bonds create a kink in the shape of the molecule prevent the fatty acids from packing close together & becoming solidified; unsaturated fatty acids are more common in plants; monounsaturated fatty acids are better for you that the polyunsaturated ones; the poly’s can produce compounds called trans fatty acids, which increase the risk of heart disease. A triglyceride molecule: H H H H H H H H H H H H–C—O—C–C—C–C–C–C—C—C—C—C–C—H saturated f.a. O H H H H H H H H H H H H H H H H H H H H H–C—O—C–C—C–C–C–C–C—C—C—C—C–H saturated f.a. O H H H H H H H H H H H H H H H H H H H H H—C—O—C—C—C—C==C—C—C—C—C—C—C—H unsaturated f.a. H O H H H H H H H H 2.) Functions of Glycerides a.) Energy – For most organisms and cellular microorganisms, sugars in excess of what can be stored as glycogen are converted into fats for more permanent storage; this is not the case in bacteria!
1.) Structure – 2 fatty acids & 1 phosphate group are linked to a glycerol molecule; a small polar group is linked to the phosphate group; this results in a molecule with a dual nature – the molecule has a nonpolar, hydrophobic end & a polar, hydrophilic end. 2.) Function: Structural. The phosphate end of the molecule & its polar group are called the “head” of the molecule; the two fatty acids are called the “tails” of the molecule; the head is hydrophilic (“water-loving”), while the 2 fatty acid “tails” are hydrophobic (“water-fearing”). This arrangement forms the structural basis of cellular membranes & is called the phospholipid bilayer.
2 Fatty AcidTails (nonpolar) V
- Lipids without Fatty Acids: Steroids
- Structure– different from other lipids; they consist of 4 interlocking carbon rings with numerous hydrogens attached; while they have no fatty acids, they are still nonpolar & hydrophobic, so they are classified as lipids.
- Some Examples:
1.) cholesterol – important component in eukaryotic cell membranes & serves as the starting material for the synthesis of other steroids. Not found in the cell membranes of bacteria with the exception of the Mycoplasms.
- Structure: nucleotide = phosphate(s) + monosaccharide + a nitrogen-containing compound (called a base); it’s the bases that spell out the genetic message in DNA & RNA).
- Nucleotides are the basic subunits of nucleic acids such as
- DNA (deoxyribonucleic nucleic acid)– carrier of the genetic message – makes up chromosomes in the nucleus of the cell.
- RNA(ribonucleic acid) – transcribes genetic message present in DNA & produces proteins from it.
- Nucleotides also make up the adenosine phosphates(ex. ATP – adenosine triphosphate – used for energy molecule in the cell).
- Nucleotides make up some coenzymes(ex. NAD & FAD); these molecules function as electron carriers in some biochemical reactions; they are called the cell’s “reducing power.” We’ll talk about this more in the metabolism chapter.
Chp. 3 Microscopy and Staining
Microscopy is the technology of making very small things visible to the human eye. Most microbes are so small that they are measured in micrometers or nanometers. A typical bacterial cell is about 1 um while a virus is more in the range of 10-100 nm. Remember, Leeuwenhoek was probably the 1st to see microorganisms in the 1600’s with his invention of his simple microscopes. Resolution is the ability to see two objects as separate, discreet entities….kind of like the ability to see railroad tracks as being separate tracks….GOOD resolution is being able to distinguish the two tracks as separate…..once the two tracks merge into one, the resolution is poor!!! Refraction is the bending of light as it passes from one medium to another of different density. Immersion oil, which has the same index of refraction as glass, is used to replace air and to prevent refraction at a glass-air interface. An example would be when one looks at objects just below the surface of water in a pond or other body of water…..the objects become refracted or “distorted” from the true image. The total magnification of a light microscope is calculated by multiplying the magnifying power of the objective lens by the magnifying power of the ocular lens. Increased magnification is of no value unless good resolution can also be maintained. Scanning (3X) x (10X) = 30X total Low power (10X) x (10X) = 100X total High “dry” (40X) x (10X) = 400X total Oil immersion (100X) x (10X) = 1000X total Most microscopes are designed so that when the microscopist increases or decreases the magnification by changing from one objective lens to another, the specimen will remain very nearly in focus. Such microscopes are said to be parfocal (par means equal). Types of microscopes: Compound Light*** (this is what we will use…know the parts and functions….we will spend more time on this in the lab. Dark-Field- Phase-Contrast- Fluorescence- Transmission- Scanning Electron-
- Techniques of Light Microscopy
- Preparation of specimens
Wet mounts are used to view living organisms. The hanging drop technique is a special type of wet mount, often used to determine whether organisms are motile. Smears of appropriate thickness are allowed to air-dry completely and are then passed through an open flame. This process, called heat fixation, kills the organisms, causing them to adhere to the slide and more readily accept stains.
- Principles of Staining
A stain, or dye, is a molecule that can bind to a structure and give it color. Most microbial stains are cationic (positively charged), or basic dyes, such as methylene blue, crystal violet, or safrannin. Some are anionic dyes (negatively charged), or acidic dyes, such as nigrosin or India ink. MOST bacterial surfaces are negatively charged so they will attract the basic dyes. Simple stains use one dye and reveal basic cell shapes and arrangements. Differential stains use two or more dyes and distinguish various properties or organisms. The Gram stain, spore stain, and acid-fast stain are examples. Negative stains color the background around cells and their parts, which resist taking up the stain. (acidic dyes will “stick” to the glass slide since glass has a + charge). Imagine a magnet when thinking of basic and acidic dyes…..basic dyes (+) will attract to bacteria due to their (-) parts but will be repelled by the glass because of its (+) charge!! Acidic dyes, on the other hand, will attract to the (+) glass but be repelled by the (-) bacterial parts!! We will also be covering these in great detail in the laboratory. Go to Chp. Reference Chapter 4 – Characteristics of Prokaryotic & Eukaryotic Cells All cells have:
- Cell or plasma membrane (separates the cell from the outer environment)
- Genetic material (DNA)
- TWO GENERAL TYPES OF CELLS:
- Prokaryotic (“before nucleus”) – a cell lacking a membrane-bound nucleus & membrane-bound organelles (ex. bacteria); these cells do have some organelles, but they are not membrane-bound; all prokaryotic cells have a cell wall, its primary component being peptidoglycan; prokaryotic cells are much smaller than eukaryotic cells (about 10 times smaller); their small size allows them to grow faster & multiply more rapidly than eukaryotic cells (they have a higher surface area to volume ratio than larger cells; thus, because they are small, they can easily meet their modest nutritional needs and grow rapidly). This group includes all bacteria.
- Eukaryotic (“true nucleus”) – a cell having a membrane-bound nucleus & membrane-bound organelles (“little organs” – specialized structures that perform specific functions within the cell); evolved about 2 million years after the prokaryotes; cell walls are sometimes present, but they are composed of cellulose or chitin; organisms with eukaryotic cells include fungi, algae, protozoa, plants, & animals.
It is important to know the differences between prokaryotic and eukaryotic cells; allows us to control disease-causing bacteria without harming our own cells.
- PROKARYOTIC CELL STRUCTURE
- Pili– straight hairlike appendages; they are usually short; all gram negative bacteria have pili; function is to attach bacteria to other bacteria, other cells, or other surfaces (not for locomotion):
- sex piliallow one bacterial cell to adhere to another (cells can actually exchange genetic material through the pili – this is the closest bacteria get to sexual reproduction!); called conjugation.
- other types of pili attach bacteria to plant or animal cells to maintain themselves in a favorable environment; if pili have been lost (maybe due to a mutation) in disease-causing bacteria, the bacteria will not be able to establish an infection.
- Flagella(singular – flagellum) – long, thin structures that extend outward from the surface of the envelope; function is locomotion – bacteria with flagella are motile; flagella rotate to propel the bacterium. Bacteria can have 1, 2, or many flagella (ex. of a bacteria with many flagella – Salmonella).
- Axial Filaments – bundles of flagella which wrap around the cell body between the cell wall and the outer membrane; together they form a helical bulge that moves like a corkscrew as the entrapped flagella turn & propel the cell; found only in one type of bacteria called the spirochetes; this unique form of movement is well suited to the viscous environment (mud & mucous) where the bacteria is generally found. Ex. of bacteria with a.f. – Treponema (causes syphilis) and Borrelia(causes Lyme disease).
- Cell Envelope (layers from outside to inside) (BE ABLE TO DIAGRAM!)
- Glycocalyx– found in most bacteria; slimy or gummy substance that becomes the outermost layer of the cell envelope; a thick glycocalyx is often called a capsule; a thin glycocalyx is often called a slime layer; functions:
- protection from drying out
- helps a cell adhere to a surface where conditions are favorable for growth
- provide protection against phagocytosis(engulfment & destruction by cells such as white blood cells) – a slippery glycocalyx makes it difficult for the phagocyte to grab hold of the bacterium.
- Outer Membrane – primarily found in gram negative bacteria(ex. E. coli, Salmonella, Shigella, Pseudomonas, Proteus, Neisseria gonorrhoeae); composed of a bilayer membrane; the inner layer is composed of phospholipids; the outer layer is composed of lipopolysaccharides (LPS’s), a compound that’s not found in any other living organism!; part of the LPS is hydrophobic, part is hydrophilic; most molecules are transported across the outer membrane and into the cell through special proteins called porins; these porins create small pores or channels in the outer membrane that allow molecules to diffuse in; function of the outer membrane is mainly protection – because of the outer membrane, gram negative bacteria are generally more resistant than gram positive bacteria to many toxic compounds, including antibiotics (antibiotics are too large to diffuse through the porins).
More about LPS’s – These compounds are endotoxins and are only released when the bacteria die and their cell walls are broken down. Endotoxins cause fever and dilate blood vessels (drop in blood pressure results). Killing the bacteria may increase the concentrations of this toxin!
- The Cell Wall – The structure described below is found in all eubacteria except the mycoplasmas (these bacteria lack a cell wall); in archaeobacteria, the cell walls are composed of a different type of peptidoglycan or protein & some do not have cell walls. In gram negative bacteria, the cell wall lies just inside the periplasm; in gram positive bacteria, it lies just inside the glycocalyx, if one exists.
- Structure & Composition of Cell Wall in Eubacteria
1.) The chief component is peptidoglycan. 2.) Peptidoglycan is composed of long chains of polysaccharides (glycan) cross-linked by short proteins (peptides). 3.) When linked together these chains create the single rigid mesh-like molecule that forms the bacterial cell wall (resembles a chain link fence!) 4.) A major difference between G(+) & G(-) bacterial cell walls: a.) G(-): peptidoglycan mesh is only one layer thick. b.) G(+): peptidoglycan wall is many layers thick.
- Cell Wall Function – In many cases, the cell wall is very porous and does not regulate the transport of substances into the cell. Two major functions of the cell wall are maintaining shape and withstanding turgor pressure. Both are discussed below.
1.) Cell Shape – one fxn. of the cell wall is to confer shape on the bacterium; most bacteria fall into one of these general groups. However, some bacteria have irregular shapes. Even bacteria of the same kind or within the same culture sometimes vary in size and shape (especially in aging cultures). a.) cocci (singular – coccus) – spherical b.) bacilli (singular – bacillus) – rod-shaped c.) spirilli (singular – spirillum) – spiral-shaped d.) vibrio – comma-shaped In addition to these characteristic cell shapes, cells can also be found in distinctive groups of cells: pairs, chains, tetrads (cubes), grape-like clusters, etc. 2.) Withstanding Turgor pressure – A cell’s turgor pressure is the internal pressure from its contents. Ordinarily, a bacterium is in a hypotonic solution (a more dilute solution that has less solute and more water than the inside of the bacterium) and water tries to move from a high water concentration to a low water concentration; that is, water tries to move inside the bacterium (see tonicity under osmosis later in the handout). Without the cell wall, the water would continue to more inside the cell, and the cell would lyse or burst; the cell wall withstands turgor pressure, so that the cell does not lyse. Action of some antibiotics (ex. penicillin) – Bacteria produce enzymes that reseal breaks in the peptidoglycan cell wall that occur during normal growth and division; penicillin binds to these enzymes, inactivating the enzymes so that the breaks cannot be resealed. The bacteria then lyse. Lysozyme, an enzyme found in tears, digests (breaks down) peptidoglycan.
- Mycoplasmas – group of bacteria that lack a cell wall; they avoid lysis from turgor pressure by maintaining a nearly equal pressure between their cytoplasm and their external environment by actively pumping sodium ions out of the cell; additionally, their cell membranes are strengthened because they contain cholesterol, a lipid found in eukaryotic cell membranes.
- Periplasm – used to be called a space, because of the way it looked in electron micrographs; found between the cell membrane and the peptidoglycan cell wall; therefore, only found in gram negative cells; composed of a gelatinous material containing proteins; one function of these proteins is that break down certain nutrients into smaller molecules that can pass through the cell membrane.
- Plasma or Cell Membrane – membrane that encloses the cytoplasm of any cell; major function is to contain the cytoplasm and to transport and regulate what comes in and what goes out of the cell. Many prokaryotic cell membranes are similar to eukaryotic cell membranes. Its structure is referred to as the Fluid Mosaic Model, because the structure behaves more like a fluid than a solid. Contains:
Membrane Lipids: (composed primarily of phospholipid molecules) a.) phospholipid bilayer (hydrophobic fatty acid tails & hydrophilic phosphate heads review chemistry handout on phospholipids) Membrane Proteins: (proteins float in the fluid lipid bilayer) a.) Integral proteins – inserted in the bilayer; mainly involved in transport. 1.) carrier proteins – bind to specific substances & transport them across the cell membrane. 2.) channel proteins – proteins with a channel through which small, water soluble substances move across the cell membrane. b.) Peripheral proteins – usually attached to membrane surface; some are enzymes; some are involved in the electron transport chain and/or photosynthesis (we’ll talk about these processes in the metabolism chapter); others are involved in the changes in cell shape that occur during cell division. Note: Archaeobacteria Cell Membranes – there are different kinds of bonds in the phospholipid molecules that link the lipids (tails) to the glycerol molecule (head); these bonds are stronger and may help these bacteria survive extreme temperature and pH. Cell Membrane Invaginations – the cell membrane sometimes invaginates or folds back on itself, forming structures that extend into the cytoplasm; since prokaryotic cells lack organelles, these invaginations provide increased surface area for peripheral proteins (enzymes) to catalyze chemical reactions.
- Cytoplasm– matrix composed primarily of water (90%) & proteins. Contains the following:
- Nucleoid– or nuclear region is a mass of DNA; well defined, although it is not surrounded by a membrane; most of a bacterium’s DNA is arranged in a single circular molecule called a chromosome; some bacteria also contains smaller circular DNA molecules called plasmids (to be discussed later).
- Ribosomes– site of protein synthesis; prokaryotic ribosomes are smaller than eukaryotic ribosomes. Antibiotics such as tetracycline, erythromycin, and streptomycin can specifically target bacterial ribosomes & not harm the host’s eukaryotic ribosomes.
- Endospores– extremely hardy, resting (non-growing) structures that some bacteria, principally G(+), produce through the process of sporulation when nutrients are exhausted; when favorable conditions return, endospores germinate to produce new vegetative cells, which grow & reproduce; they are able to withstand harsh environmental conditions because they contain so little water and high concentrations of calcium and dipicolinic acid; when favorable conditions return, the spore germinates into a new vegetative cell.
Some of endospore-producing bacteria are pathogenic to humans. Ex. Clostridium tetani causes tetanus (other species of this genus cause botulism and gas gangrene). Bacillus is another genus of bacteria that forms spores. We will learn how to stain bacteria so you may observe these spores. III. EUKARYOTIC CELL STRUCTURE
- Cilia – short, hairlike, motile cellular extensions that occur on the surfaces of certain cells; ex. some protozoa (called Ciliates) use cilia for motility & feeding.
- Flagella– in humans, the single, long, hairlike cellular extension that occurs in sperm cells; beat in waves (prokaryotic flagella rotate!); some protozoans use flagella for motility.
- Cell Wall
- Animal cells – no cell wall!
- Plant cells – made of cellulose
- Fungi – in most made of cellulose; some made of chitin (polysaccharide containing nitrogen – similar to exoskeletons of insects) and cellulose.
- Algae – made of cellulose
- Protozoans – no cell wall!
- Glycocalyx – A glycocalyxmay exist outside the plasma membrane; composed of carbohydrate chains from glycoproteins in cell membrane.
- Plasma Membrane– already described; differences are between prokaryotes & eukaryotes:
- proteins involved in electron transport chain and photosynthesis are not found in cell membrane, but are found in cytoplasmic organelles (mitochondria and chloroplast respectively), and
- cell membrane contains cholesterol (in prokaryotes, only mycoplasmas have cholesterol in their cell membrane).
- Cytoskeleton (not found in prokaryotes)
- structure– network of filamentous protein structures.
- functions– give the cell shape (support & rigidity); anchor the organelles; transport substances through the cell (cytoplasmic streaming), cytoplasmic streaming also enables some eukaryotes to move (formation of pseudopods); involved in cell division; involved in cell motility (flagella).
- Structure in eukaryotic cells:
- nuclear envelope– double membrane with nuclear pores that surrounds the nucleus.
- chromosomes– genetic material composed of DNA & associated; chromosomes are linear.
- carrier of the hereditary information, which exerts a continuing influence over the ongoing activities of the cell through protein synthesis; “control center of the cell.”
- isolates the DNA in eukaryotic cells.
- Ribosomes (may be free in the cytoplasm or attached to rough endoplasmic reticulum & the nucleus)
- Structure– not membrane-bound; made up of RNA & protein.
- Function– sites of protein synthesis (where amino acids are assembled into polypeptides).
- Membrane-bound Organelles – Eukaryotic cells have specializes membrane-bound organelles that carry out specific functions such as photosynthesis (chloroplasts), ATP production (mitochondria), lipid & protein synthesis (endoplasmic reticulum, golgi complex), cellular digestion (lysosomes), & transport (vesicles). We will not discuss these organelles in detail, since the focus of this class will be on prokaryotes. You will discuss these organelles in detail in Anatomy & Physiology I.
- ENDOPLASMIC RETICULUM
1.) Structure: interconnecting flattened sacs, tubes, & channels. 2.) Types & Functions: (both types support the cytoplasm & provide more surface area inside the cell for chemical reactions to take place) a.) rough E. R. – (ribosomes are attached to it) – function: initial modification of proteins; process: polypeptide chains are formed at the ribosome & some of them are transported into the r. e. r. for modification; the polypeptides are then packaged in transport vesicles or sacs (a piece of the e. r. pinches off around the polypeptide); these vesicles transport the polypeptides to the golgi complex for further modification into proteins. b.) smooth E. R. – (no ribosomes attached) – function: main site of lipid synthesis; lipids are then sent to the golgi body in transport vesicles for further modification & distribution.
- GOLGI COMPLEX
1.) Structure – 4 to 8 flattened, membrane-bound sacs loosely stacked on top of one another surrounded by vesicles; looks like a stack of pancakes. 2.) Function – final modification of proteins & lipids. 3.) Process: transport vesicles from the r.e.r. or s.e.r. fuse with the golgi complex; proteins & lipids are processed in the golgi complex; the finished product is pinched off in a piece of golgi membrane (another vesicle) & is transported to the part of the cell where it is needed; the golgi complex processes, packages, & distributes the material the cell manufactures (“the Wal-Mart distribution center”).
1.) Structure – membrane-bound sacs that could be pinched off pieces of golgi complex, E.R., or cell membrane 2.) Function – transport material within the cell & into & out of the cell. 3.) Some specialized vesicles: a.) Lysosomes – contain enzymes for breaking down proteins, lipids, etc. (digestion within the cell); they fuse with other vesicles (such as phagocytic vesicles) to degrade or digest their contents. b.) Peroxisomes – contain enzymes (peroxisomes) that break down toxic hydrogen peroxide into water and oxygen (you see the oxygen bubbles when you apply hydrogen peroxide to tissue).
1.) Structure – usually shown oval shaped; double membrane: smooth outer membrane & a folded inner membrane (folds provide more surface area for chemical reactions to take place). 2.) Function – break down energy containing organic molecules (ex. carbohydrates) & repackage the energy into smaller units (ATP) that can be used by the cells; called the “powerhouse” of the cell.
1.) Structure – network of filamentous protein structures called microtubules & microfilaments. 2.) Functions – give the cell shape (support), anchor the organelles, transport substances through the cell, involved in cell division.
1.) Structure – paired cylindrical structures composed of protein filaments 2.) Function – during cell division, organize a microtubule network, called spindle fibers; spindle fibers are responsible for moving the chromosomes around in the cell during division.
- CELL MEMBRANE TRANSPORT
- PASSIVE TYPES OF TRANSPORT ACROSS THE CELL MEMBRANE
- Most passive transport processes depend on the process of DIFFUSION
- Definition– the net movement of particles from a greater concentration to a lower concentration (down a concentration gradient) to distribute the particles uniformly; it’s a passive process – molecules move by their own kinetic energy – requires no energy expenditure by the cell (no ATP); molecules will diffuse freely until an equilibrium is reached (equal distribution on both sides)
- Simple Diffusion through the Cell Membrane– The lipid interior of the cell membrane is a barrier to simple diffusion; most polar molecules (polar molecules get “stuck” in the nonpolar fatty acid tails). Small, nonpolar, lipid soluble molecules like fats, carbon dioxide, oxygen, & alcohol move easily through the cell membrane by simple diffusion. Polar & charged molecules can diffuse through the membrane if they are small enough to pass through pores in channel proteins.
- Osmosis– a special case of diffusion; the movement of water across a semipermeable membrane – water moves from a high water concentration to a low water concentration (or from a low solute concentration to a high solute concentration); water moves across cellular membranes through pores in channel proteins or through momentary openings in the membrane.
Tonicity: (describes the relative concentrations of solute in two fluids, such as the fluid inside & outside a cell); 3 cases: 1.) isotonic solutions (“iso” = same) – two or more solutions that have equal concentrations of solute. 2.) hypotonic solution (“hypo” = less) – one solution has less solute (more water) than the other; a cell that is in a hypotonic environment will lyse (burst); ex. placing a cell in distilled water would cause the cell to lyse – water would move into the cell to where the water concentration is lower. 3.) hypertonic solution (“hyper” = more) – one solution has more solute (less water) than the other; a cell that is in a hypertonic environment will crenate (shrink), because the water in the cell moves out of the cell to an area of lower water concentration; ex. placing a cell in water with a high salt or sugar concentration would cause the cell to crenate – water would move out of the cell to where the water concentration is lower. Note: The above examples describe the environment that the cell is in (i. e., the solution is hypotonic or hypertonic to the cell). You can also talk about the cell in relation to its environment (i. e., the cell is hypertonic or hypotonic to its environment). You have to make this distinction!! The cells in our bodies try to maintain the isotonic condition so that they are not in danger of lysing or crenating.
- Facilitated Diffusion– Again, only small, nonpolar molecules readily diffuse across the cell membrane. Polar & charged molecules get “stuck” in the fatty acid part of the lipid bilayer. Small, polar molecules, like water, and some ions can diffuse through channel proteins. Most biologically important molecules, however, are polar & are much larger than water (ex. glucose) and cannot fit through channel proteins. Special selective carrier proteins are located in the membrane to transport molecules like glucose. In facilitated diffusion, carrier proteins move molecules from a high concentration to a low concentration like in simple diffusion; it is believed that changes in the shape of the carrier protein allow it to envelop and then release the transported substance.
Note: Few prokaryotes transport in this way; but may compounds, including most sugars, enter most eukaryotic cells in this way.
- ACTIVE TYPES OF TRANSPORT ACROSS THE CELL MEMBRANE
These processes use energy (ATP)!!!
- Active Transport– Carrier proteins move molecules move from low concentration to high concentration (against the concentration gradient). Example:
- In prokaryotes – most nutrients are transported in this way because many prokaryotes live in low nutrient environments;group translocation is a form of active transport that occurs in some prokaryotes with certain molecules; in group translocation, a molecule is transported into the cell and at the same time chemically changed in to a slightly different molecule; this occur so that the molecule cannot leave the cell.
- Vesicle Mediated Transport by Eukaryotes– We will concentrate on the type of vesicle mediated transport called endocytosis, since this is how white blood cells eat bacteria, etc.
- Endocytosis– substances are imported into the cell; vesicles (sacs) are formed from the cell membrane, sometimes in response to the triggering of a receptor membrane protein (called receptor-mediated endocytosis); the cell membrane envelopes the substance to be imported & pinches off to form a vesicle that moves into the cytoplasm; endocytic vesicles can then fuse with enzyme-containing vesicles called lysosomes to digest their contents.
When solid material is imported into the cell, this type of endocytosis is specifically called phagocytosis (“cell eating”); ex. a white blood cell engulfing a bacteria. Return to Chp. index
Chapter 5….associated with the below chapters with respect to diseases
Chapter 18-23 – Eukaryotic Organisms [Fungi, Protozoans, Helminths, & Arthropod Vectors]
Features that distinguish protozoal & helminthic infections : 1.) More important in tropical countries than in countries like the U. S. However, parasitic diseases are becoming more prevalent in the U. S. as more infected people move here; also because people with immune deficiencies such as AIDS are more susceptible to certain parasites. 2.) How the immune system responds to these parasites is a mystery. An immune response is activated, but the immune system is seldom able to rid the body of them. 3.) They have more complex life cycles, with multiple hosts involved.
- Some General Characteristics:
- eukaryotic cells
- heterotrophic (use organic compounds a carbon source; they can’t make their own sugars; no photosynthesis)
- prefer more acidic conditions than bacteria
- can tolerate higher osmotic pressure and lower moisture than bacteria
- larger than bacteria and have more cellular and morphologic detail
- cannot tolerate the high temps. that bacteria can (fungal spores aren’t as resistant as bacterial spores)
- most are aerobic; some are facultative anaerobes (ex. yeasts) & some are anaerobes
- important in ecosystems as decomposers (called saprophytes– they obtain nutrients by decomposing dead & decaying matter); some are parasites, causing disease (mycosis; mycoses is plural); some produce toxins that cause disease (mycotoxicosis; mycotoxicoses is plural).
- major cause of plant diseases
- the study of fungi is mycology
- General Morphology:
- most, with exception of unicellular species, have a vegetative structure called a mycelium(a multinucleate mass of cytoplasm enclosed within a system of rigid, branched, tube-like filaments called hyphae).
- hyphaecan be coenocytic (undivided network of branching tubes) or have septa (cross walls).
- cells walls are composed of cellulose, chitin (contains nitrogen – also found in the exoskeletons of insects, crayfish, etc.), or a combination of the two.
- specific morphology will be discussed later for each group of fungi
- Reproduction – Fungi are classified by how they reproduce (sexually or asexually).
[functions of spores include dissemination and reproduction]
- Asexual Reproduction– Occurs by elongation of hyphae, budding, or asexual spore production.
Asexual spores are specialized cells that are dispersed & germinate in a favorable environment to produce a new fungus; they are products of a type of cell division called mitosis (one cell divides to form 2 daughter cells that are identical to one another and to the original parent cell). Types: sporangiospores , conidiospores.
- Sexual Reproduction– Occurs by producing sexual spores, which form following sexual fusion of gametes (similar to sperm & eggs). Types: zygospores, ascospores, & basidiospores.
D. 2 General Groups of Fungi – Yeasts vs. Molds
[These are descriptive terms, not taxonomic! These organisms belong to many groups of fungi.]
- Yeasts – characteristics:
- nonfilamentous, unicellular
- reproduce asexually by budding
- reproduce sexually by producing various kinds of spores
- aerobic or facultative anaerobes
- used to prepare bread, wine, beer, etc. (fermentation of carbohydrates produces ethanol & carbon dioxide) ex. Saccharomyces cerevisiae(cervesa means beer in Spanish)
- some are pathogenic; ex. Candida albicans(causes yeast infections, thrush; see below)
- Molds – characteristics:
- filamentous, multicellular
- have a vegetative structure called a mycelium(a multinucleate mass of cytoplasm enclosed within a system of rigid, branched, tube-like filaments called hyphae).
- hyphaecan be coenocytic (undivided network of branching tubes) or have septa (cross walls).
- also possess reproductive hyphae which produce different kinds of spores (discussed above and below)
- see below for examples.
- Classification of Some of the Lower Fungi:
- Characteristics: coenocytic hyphae, produce sporangiospores(asexual spores) & zygospores (sexual spores).
- Ex. Rhizopus nigricans– black mold that develops on stale bread; the tiny black dots on the mold are the sporangia, which hold the sporangiospores; sporangia look like tiny mushroom caps.
- can be opportunistic; some are pathogenic in the immnocompromised
- Classification of Some of the Higher Fungi:
- Ascomycetes (Sac Fungi)
- Characteristics: includes molds with septate hyphae and some yeasts; ascospores(sexual spores) develop within sacs called asci (sing. ascus); also produce conidiospores (asexual spores).
1.) Saccharomyces cerevisiae – yeast is used to make beer, bread, wine; cervesa means beer in Spanish. 2.) Trichophyton – causes athlete’s foot (tinea pedis); ringworm of the feet; other species infect different parts of the body (dandruff, nail fungus, jock itch) 3.) Penicillium spp. – conidiospores form long chains on branching conidiophores, creating a brush-like structure that looks like a broom (penicillus means “brush”); some species produce the antibiotic penicillin. 4.) Aspergillus spp. – form long chains on a globelike conidiophore; cause aspergillosis, a pulmonary disease of animals & humans; infection is often secondary to tuberculosis, immunodeficiency, & steroid therapy. 5.) Histoplasma capsulatum – causes Mississippi Valley fever (histoplasmosis); can get from bird droppings and bat guano; endemic disease in this area; pulmonary disease. 6.) Candida albicans – part of our natural flora; opportunistic; becomes a problem when defenses are weakened or balance of microbes is upset (ex. from antibiotic treatment); cause of vaginal & intestinal yeast infections & thrush in the mouth (“cottage cheese patches”) – called candidiasis.
- Basidiomycetes (Club Fungi)
- Characteristics: many form basidiocarps(mushrooms, puffballs, or shelflike bodies on trees); some are molds, a few are yeasts; produce conidiospores; also produce basidiospores (sexual spores); basidiospores form on the “gills” of mushroom basidiocarps.
1.) Amanita – poisonous mushroom; toxin causes a mycotoxicosis 2.) Cryptococcus – yeast cells surrounded by a capsule; causes fatal meningitis (cryptococcosis); transmission – inhalation of contaminated dust; found in 8% of AIDS patients.
- Deuteromycetes (Imperfect Fungi)
- Characteristics: called the imperfect fungi because no sexual stage has beenobserved; we put them in this group until a sexual stage is observed; these fungi grow as yeasts or molds; identify on basis of shape & arrangement of their conidiospores (asexual spores); some species are pathogenic; many of these fungi have recently been placed in other phyla.
- Dimorphic Fungi – Some fungi switch between a single-celled yeast phase of growth & a mycelial phase (called dimorphism); discovered by Pasteur; some species will switch if oxygen supply decreases. Pathogenic dimorphic fungi are mycelial outside of the host & single-celled inside the host. With pathogenic species, it is usually high body temperature that causes the switch. Candidachanges in response to the higher nutrient concentrations found in the body. The problem with dimorphism is that single cells are more readily spread in bloodstream, leading to systemic infections.
- Mycoses (Fungal Diseases)
- Humans usually acquire fungal disease from nature; they are not highly contagious.
- mycotoxicosisvs. opportunistic mycoses:
- See above for diseases
- Some produce toxins that are hallucinogenic; ex. muscarin– produced by a mushroom
- Some produce toxins that are highly poisonous; ex.
1.) Claviceps (rye mold) – produces ergot; causes death to anyone eating bread made from contaminated rye; LSD is made form fruiting structures (causes hallucinations) 2.) Aspergillus – produces aflatoxin; which grows in many plant materials; low levels of toxin can be carcinogenic. 3.) Amanita – poisonous mushroom
- Antibiotics: Penicillins produced by Penicillium; Cephalosporins produced by Cephalosporium.
- General Characteristics:
- Unicellular eukaryotes.
- The protistan lineages continue into the kingdoms of plants, fungi, and animals.
- Limited to a moist environment because they lack a cell wall
- Most reproduce asexually by fission (one cell divides to form 2 identical daughter cells & budding; some (ex. Plasmodiumthat causes malaria) under go schizogony (multiple fission). Sexual reproduction occurs by conjugation, the fusion of vegetative cells, or by the fusion of specialized gametes called gametocytes.
- Some have complex life cycles, requiring multiple hosts and changing their morphology (ex. Plasmodium uses the mosquito as an intermediate host)
- Trophozoite– active, motile, feeding stage of protozoans; parasitic stage that causes the disease in the host.
- Cyst– resistant, inactive stage; how diseases are usually transmitted by the fecal-oral route; usually more useful than trophozoites for lab identification.
- Classification: [based on mode of locomotion or motility]
- Mastigophora or Zoomastigophora (move by means of flagella)
- Trypanosoma gambiense– infects the blood and tissue fluids; causes African sleeping sickness (it leads to the loss of consciousness and death when it invades the CNS); can also infect cattle; vector is the tsetse fly.
- Giardia lamblia– body has the appearance of a human face (4 “eyes” are nuclei); have 2-6 flagella; form cysts; causes a waterborne dysentery (traveler’s diarrhea); one of the “don’t drink the water” diseases; firs sigh is usually an explosive, foul-smelling watery diarrhea followed by copious amounts of campers are a high-risk group because of a sylvatic cycle (parasite is found in mountain streams contaminated with human feces or animal feces, especially beavers).
- Trichomonas vaginalis – causes vulvovaginitis; numerous flagella
- Sarcodina (move by means of pseudopodiaor “false feet” – temporary extensions of the cell body caused by protein filaments of the cytoskeleton pushing on the cell membrane); feed on algae, bacteria, and other protozoans by phagocytosis.
- Amoeba proteus– freshwater; not pathogenic
- Entamoeba histolytica– causes amoebic dysentery; usually acquired by consuming fecally contaminated water or food; flies and cockroaches can also be mechanical vectors; produce cysts; first protozoan to be shown to be a pathogen (1875); one of the “don’t drink the water” diseases; trophozoites may invade the intestinal mucosa where they can cause ulceration and escape into the blood vessels; they may allow bacteria in fecal material to enter the body cavity and cause peritonitis.
- Naegleria fowleri – causes amoebic meningioencephalitis; usually seen in swimmers.
- Acanthamoeba polyphaga– accumulates on the water surface of contaminated hot tubs when tubs are covered; cause ulceration of the eyes and skin; can invade the central nervous system and cause meningioencephalitis.
- Ciliophora (move by means of cilia)
- Paramecium caudatum– freshwater; not pathogenic
- Balantidium coli– only ciliophoran that causes disease; produces cysts; causes diarrhea of large intestine; rare except in the Philippines; symptoms are similar to those of amoebic dysentery.
- Apicomplexa or Sporozoa or Haemosporina – Basically nonmotile. All have an infectious, sporelike stage (sporozoite) that is often transmitted to new hosts by an insect vector. All are parasitic (obligate parasites – cannot live apart from the host). Some have elaborate life cycles, changing body form (trophozoite, sporozoite, merozoite); life cycle includes schizogony(multiple fission). Examples
- Plasmodium vivax – causes malaria; vector is the mosquito; kills 1-3.5 million people each year; malaria = bad air; used to infect people with malaria to stop the progression of syphilis (fevers would kill the bacteria).
- Toxoplasma gondii– causes toxoplasmosis; humans acquire the disease by consuming cysts in the meat of infected animals or ingesting material contaminated by cat feces containing the parasite (can get it from cleaning the litter box – doctors warn pregnant women not to do this).
- Cryptosporidium– form cysts; cause enteritis & diarrhea; can occur in water supplies; can also be transmitted by fecal-oral transmission from kittens/puppies; resistant to chlorine (it can survive full-strength Chlorox!); threat only AIDS patients and those immunocompromised; no effective treatment found.
- Pneumocystis carinii– may be a fungus!!; causes pneumocystis pneumonia; spread in respiratory droplets; common in AIDS patients.
III. HELMINTHS – Flatworms & Roundworms General Characteristics: ¨ Animals ¨ Cephalization – concentration of sensory receptors toward the anterior end. ¨ Organ/system level or organization/ ¨ Sexual reproduction. Most flatworms are monoecious (male & female reproductive organs in same animal). Roundworms are dioceious(separate sexes).
- Platyhelminthes(Flatworms = Trematodes + Cestodes) – most are free-living; marine and freshwater; predators, scavengers, or parasitic; some have regenerative capabilities.
- Trematoda (Flukes) – all parasitic of vertebrates; have complex life cycles that include sexual and asexual phases; they require at least 2 kinds of organisms to complete the cycle – they reach sexual maturity in a primary or definitive host (always a vertebrate), their larval stages develop or become encysted in an intermediate host (usually an invertebrate).
- Clonorchis sinensis (Chinese or Human Liver Fluke)
¨ Adults live in bile ducts (in the liver) of humans (definitive host) ¨ Intermediate hosts: snail (first) and fish (second) ¨ Life cycle: a snail ingests the eggs; the eggs hatch & release a larval stage which goes through several transformation before finally forming a tadpole-like cercariae; the cercariae bore through the flesh of the snail, & escape into the water; they swim until they find the appropriate species of fish; they encyst in the muscle tissues of the fish (forming metacercariae); the adult flukes develop in livers of humans who eat raw, infected fish; eggs of the parasite are excreted in the feces; when human feces end up in ponds, etc., snails ingest the eggs & the cycle repeats itself.]
- Schistosomamansoni (Schistosomes or Blood Flukes) – adults live in circulatory system; spiny eggs break through the blood vessel wall and through the gut wall to be expelled in feces; eggs hatch into cercaria in water; cercaria then penetrate skin when a person is bathing or swimming; cause spleen and liver enlargement, dysentery, and cirrhosis of the liver
- Cestoda (Tapeworms) – intestinal parasites of vertebrates; no digestive system like in trematodes & nematodes; they absorb nutrients through their tegument!
Morphology: scolex (head) with suckers and/or hooks (for attachment), proglottids (body units – each one has male and female reproductive organs): immature proglottids (closest to the scolex), mature proglottids (next closest to the scolex), and gravid proglottids (furthest from the scolex – in these proglottids, the uterus is filled with eggs). General life cycle of tapeworms: the gravid proglottids break off and are passed in the definitive host’s feces; larval forms hatch when the eggs are ingested by the intermediate host; larvae then encyst in the intermediate host (called a cysticercus or bladder worm); adult worms usually develop in the definitive host when raw or poorly cooked infected meat is eaten. Examples:
- Taenia solium(pork tapeworm) – reaches a length of 2-7 meters; primary host: humans, etc.; intermediate host: swine
¨ Humans can be infected with the adults by consuming rare pork containing cysticerci larvae; larvae then develop into adults in digestive tract of the human. ¨ Humans can also be infected with larval forms when they accidentally ingest eggs (they get them from other infected humans who contaminate food, etc. with the eggs when they don’t use proper hygiene after going to the bathroom). In this case every organ in the body may harbor cysticerci. When a cysticercus dies, it releases toxins and usually causes a severe allergic reaction, which is sometimes fatal.
- Taenia saginata (beef tapeworm) – reaches a length of 5-25 meters; primary host: humans, etc.; intermediate host: cattle, sheep, etc.; life cycle similar to that of T. solium above; beef riddles with encysted larvae is called “measly beef.”
- Echinococcus granulosus (dog tapeworm); small – only 3 proglottids long; typical life cycle:
¨ dogs are infected by adults when they eat raw butchered livestock containing larvae (ex. raw bones, etc.) ¨ eggs are passed in feces of dog; livestock eat vegetation with eggs when grazing ¨ larvae hatch and encyst in the muscle tissue of livestock Humans can get hydatid cysts (larvae) from ingesting the eggs (the eggs are passed in feces of dog, dog licks himself, then dog licks your face). These cysts develop in the liver, lungs, and brain. Each fluid-filledcyst, containing many larvae, can reach the size of a grapefruit.
- Dipylidium caninum(dog & cat tapeworm) – often seen in children; flea is the intermediate host – it eats the eggs on an animal; larvae develop in flea; if a dog, cat, or human ingests the flea, the adult will develop. Note: Larvae are not transmitted through the bite of the flea!!
- Hymenolepis nana(dwarf tapeworm) – most common tapeworm of humans in the world; intermediate host is a grain beetle; humans can ingest the eggs in cereals and other foods that contain parts of the insects; intermediate host is optional (meaning that if you ingest the eggs of this worm, you get an adult infection).
- Nematoda (Nematodes)
General characteristics: ¨ Nematodes are everywhere!!!! They are freeliving in soil, fresh & salt water, & are parasitic in plants and animals. ¨ Dioecious (separate sexes). ¨ Possess a nonliving cuticle, which is secreted by the epidermis and is resistant to the digestive enzymes of the hosts. ¨ More highly developed than flatworms.
Chap. 6 – The Viruses
General Characteristics: ¨ virus means poison; someone once called them “a piece of bad news wrapped in a protein;” ¨ obligate intracellular parasites (can reproduce/replicate only inside a host cell) ¨ not cells; debate over whether or not they are considered “alive” (see below) ¨ consist of nucleic acids (DNA or RNA) in a protein coat, called a capsid (no cell membrane) ¨ they insert themselves into a host cell & direct the host cell’s metabolic machinery to make more virus; the virus supplies information (the plan) in the form of its nucleic acid – raw materials and driving force (ATP & reducing power) are supplied by the host cell. ¨ all cellular organisms can be attacked by viruses; however, viruses are very specific for the organisms & cells they infect. Are Viruses Alive? ¨ Characteristics of living things: reproduction, metabolism, organized as cells, contain all organic molecules (lipids, enzymes, nucleic acids, carbs), evolution & adaptation to changing environments. ¨ Viruses have some of these char’s: they can evolve, they contain some macromolecules, they direct their own reproduction; However, they are not cells – they do not have cytoplasm, a cell membrane, organelles, ribosomes, or a nucleus. They have DNA or RNA, unlike prokaryotic and eukaryotic cells, which have both. In addition, they lack a metabolism of their own (they cannot produce ATP, etc.) – raw materials and driving force (ATP & reducing power) are supplied by the host cell.
- HOW ARE THEY CLASSIFIED?(4 ways: size, structure, host range, life cycles)
- Size– range from about 1/10th to 1/3rd the size of a small bacterial cell.
- Structure– basic structure of a virus is a nucleic acid surrounded by a protein capsid; a membrane envelope may also be present outside of the capsid, but this is acquired from host cell. A complete viral particle (= capsid + nucleic acid + envelope if it is present) is called a virion.
- Nucleic Acid– Viruses can store their genetic info. in different types of nucleic acid (each virus has only type). Viruses can have DNA or RNA. Their nucleic acid can be double stranded (ds) or single stranded (ss); they can even have double stranded RNA! RNA viruses can have a (-) sense strand or a (+) sense strand of RNA.
(+) sense RNA acts like mRNA and can be translated into proteins by the host cell’s ribosomes. (-) sense RNA does not make sense to the host cell’s ribosomes. After the virus containing this type of RNA enters the host cell, a complementary (+) sense strand is made from its (-) sense strand. Only (+) sense strand RNA can be read by the host cell’s ribosomes!
- Capsids– protein coat that surrounds the nucleic acid; the constituent protein molecules making up the capsids are called capsomeres; there are 3 basic shapes based on how the capsomeres are arranged. See diagrams of these shapes!!
- helical– proteins fit together as a spiral to form a rod-shaped structure.
- polyhedral– proteins are arranged in equilateral triangles that fit together to form a geodesic dome-shaped structure; some appear almost spherical; you may have seen architectural structures that have this shape.
- complex– combination viruses with a helical portion (tail) attached to a polyhedral portion (head); ex. many bacteriophages; may also have a tail sheath (participates in injecting the viral nucleic acid into the host cell), plate, pins, & tail fibers (help virus attach to host cell).
- Viral Envelopes – pieces of the host cell’s cell membrane that the virus acquires as it emerges from its host cell; the virus pushes out of the cell membrane, forming a bud that encloses the virus – then the bud pinches off behind, resealing the cell – as a result the host cell is not lysed. Glycoprotein spikes from the host cell’s glycocalyx may stick out of the envelope. Viruses that lack envelopes are called naked viruses. Because envelopes are acquired from host’s cell membranes, viruses may be hidden from attach by the host’s immune system. Envelopes also help viruses infect new cells by fusion of the envelope with the host’s cell membrane. On the other hand, enveloped viruses are damaged easily by physical and chemical antimicrobial agents.
- Host Range – defined as the spectrum of organisms a virus attacks; viruses exhibit considerable specificity for hosts and even cells within that host; viral specificity is determined by whether or not a virus can attach to a cell. Attachment depends on the presence specific receptor siteson the surface of host cell and on specific attachment structures on the viral capsid or envelope. Examples of receptor sites are proteins, LPS’s, glycolipids, or glycoproteins.
- Life Cycles of Bacteriophages(viruses that infect bacteria – means “bacteria eating”)
- Replication [= Lytic Cycle] See diagram Events:
- Adsorption– the virion attaches itself to a specific receptor site on the surface of the host cell.
- Penetration – the viral nucleic acid penetrates the host cell
- Uncoating – removing the capsid & envelope; basically 2 ways it can happen:
1.) during penetration, the virion disassembles so that only the viral nucleic acid enters host cell 2.) the entire virion enters the host cell & uncoating occurs later
- Viral Synthesis(Latent Period) (also called biosynthesis) – more viral components
(nucleic acids & proteins for capsids) are synthesized by the host cell.
- Maturation(Assembly) – components are assembled into new viruses
- Release (Burst Period) – hundreds of intact virions exit host cell; 2 ways:
1.) If the virus is of the naked type, an encoded protein, lysozyme, dissolves the cell membrane &/or cell wall of the host cell, causing the cell to lyse & releasing the hundreds of viruses inside it. 2.) If the virus is to be an enveloped virus, it pushes out the cell membrane, forming a bud that encloses the virus – then the bud pinches off behind, resealing the host cell; as a result the host cell is not lysed.
- Lysogenic Cycle (Lysogeny or Temperance) – Temperence involves the capacity of certain viruses to set up long-term relationships with their host cells – the virus remains latent for many cellular generations by becoming integrated into a host cell’s chromosome (the integrated viral DNA is called a prophage). In this case no new viral components are synthesized & the host cell is not harmed. The virus may remain latent for long periods of time before initiating a lytic cycle. The problem with this type of cycle is that the viral nucleic acid that becomes integrated into the host cell’s chromosome gets replicated along with the host cell’s chromosome and is passed to daughter cells during cell division. In the prophage state, some viral genes are expressed, which may slightly change the host cell’s phenotype (ex. only lysogenic strains of Corynebacterium diphtheriaecause the disease diphtheria because the disease-causing toxin is encoded in the prophage of the infecting virus). Something (ex. temperature change) may trigger prophages to go into the lytic cycle. Released virions cannot infect cells that are carrying the same prophage – it makes the cell immune to attack by a virion of the same phage.
¨ Family names all end in viridae ; family names are often converted into English (ex. Retroviridae are called retroviruses). Genus names end in virus – species names are English words. Ex. Retroviridae, Lentivirus, Human Immunodeficiency Virus (HIV) ¨ Groupings reflect only common characteristics and are not intended to represent evolutionary relationships.
- Cultivating Animal Viruses & Diagnosing Viral Illnesses
¨ At one time animal viruses had to be cultivated & counted by infecting animals. ¨ In the 1930’s it was discovered that embryonated chicken eggs could be used to culture animal viruses; embryonated eggs are inoculated with dilutions of a virus sample to determine the highest dilution that kills the embryo; this procedure was more economical & efficient than using adult animals. ¨ In the 1950’s cell culture & tissue culture methods were developed. This solved the problem of viral specificity. Ex. Before cell cultures it was impossible to culture viruses in mice or chicken eggs that only infected humans (ex. HIV); continuous cell lines are usually derived from cancerous tissue & grow indefinitely in culture; regular cell lines grow increasingly slowly after 20-30 subcultures & eventually lose their ability to support viral replication; the most famous c.c.l. is the HeLa cell line (named after Helen Lack, the donor – from cervical cancer). ¨ Important Note: Physicians rely on symptoms to diagnose most viral illnesses. Culturing viruses takes too long & antibodies in the blood can usually be detected only after patient has recovered. ¨ Viral infections sometimes affect human cells in ways that can be seen under the microscope. For ex. the measles virus causes the membranes of neighboring cells to fuse, creating giant, multinucleated cells. Some virus-infected cells can be id. because they contain inclusion bodies, collections of viral components such as capsids and nucleic acid, waiting to be assembled into new viral particles. For example, the rabies virus produces inclusion bodies called negribodies in infected nerve cells (this is what we look for in suspected cases of rabid animals – have to look for negribodies in brain – animals have to be euthanized).
- Replication of Animal Viruses(Lytic cycle) – proceeds through similar stages as bacteriophage replication.
- Adsorption– Proteins in cell membrane act as receptor sites for a virus; remember, no cell walls in animal cells; adsorption is largely responsible for tissue specificity of animal viruses – only cells with a complementary receptor are attacked by a particular virus.
- Penetrationcan occur in 3 ways:
- viral envelope fuses with cell membrane, emptying the rest of the virion inside the cell.
- other enveloped viruses enter by being phagocytized by a host cell
- most naked animal viruses enter as most bacteriophages do – the capsid adsorbs to cell surface & only the viral nucleic acid enters cell.
- Uncoating– Envelopes/capsids are often removed in the penetration process; viruses that enter the cell partially or completely intact are uncoated inside the cell by the host cell’s own hydrolytic enzymes, sometimes those in its lysosomes.
- Viral Synthesis– The specifics of this process depend on which of the 5 types of nucleic acids is present in the virus.
- Maturation– Assembly not really understood
- Release– Enzymes cause lysis of the host cell or viruses “bud.” Viruses that kill the host cell by causing lysis are called cytocidal. Viruses that damage the host cell but do not kill it are called cytopathic. Persistent viral infections can last for years, producing new virus particles by budding without killing the infected cell.
- Latency(similar to temperance or lysogeny) – Sometimes the viral nucleic acid is integrated in the host cell’s DNA (called a provirus), allowing the infected animal cells to function normally for years (just as a lysogenic bacteriophage or prophage does).
Ex. Typical of DNA viruses belonging to Herpesvirus family – herpes simplex 1 (causes fever blisters) causes a symptomless latent infection of nerve cells of mouth & lips – infection can be reactivated by a fever, a cold, too much sun, or stress. Ex. Varicella Zoster (another Herpsevirus) causes chickenpox as the primary infection & shingles as the reactivation. Ex. HIV (Human Immunodefiency Virus) – belongs to the Retrovirus family; causes AIDS (Acquired Immune Deficiency Syndrome).
- Some Animal RNA Viruses
Retroviruses (Retroviridae) ¨ large group of RNA viruses; includes HIV (Human Immunodeficiency Virus) which causes AIDS (acquired immune deficiency syndrome); infects T cells (type of white blood cell). ¨ capsid contains 2 copies of the same (+) sense RNA molecule (called a diploid virus); capsid also contains the enzyme reverse transcriptase. ¨ Retro means “backward.” This virus uses the enzyme reverse transcriptase to make DNA from its RNA. This DNA can be integrated into the host cell’s chromosome. The proviral DNA can now be transcribed into mRNA and translated into viral proteins to assemble new viruses for release; As with prophages, the provirus can stay in a latent stage in which it is replicated along with host cell DNA, causing the host cell no damage. ¨ AZT (azidothymidine), which is used against HIV, helps stop reverse transcription by targeting the enzyme reverse transcriptase. Flaviviridae ¨ enveloped; polyhedral capsid; (+) sense RNA ¨ includes Yellow Fever (hemorrhagic fever)
¨ enveloped; polyhedral capsid; (+) sense RNA ¨ includes Rubella virus (Rubella or German measles) Picornaviridae ¨ naked; polyhedral capsid ¨ includes Enterovirus (causes polio); Rhinovirus (common cold); Hepatovirus (Hepatitis A) Orthomyxoviridae – Influenza Viruses ¨ Flu viruses; 3 types (A, B, C); A is the most common, infecting many species of animals, including humans; A is responsible for many pandemics (worldwide epidemics); B & C only infect humans & do not cause pandemics; Outbreaks of B occur every 2-3 years; C causes mild cold-like illnesses. ¨ enveloped RNA viruses; protein spikes in envelope; its (-) sense RNA is divided into 8 separate pieces, each one packaged in a helical capsid ¨ This virus exhibits antigenic shift– sudden changes in properties that id. the virus as a foreign invader to the defenses of the human immune system; occurs from genetic changes that can occur when 2 different flu viruses infect the same cell; when this happens it is likely that the RNA molecules of the 2 infecting virions recombine in various ways among the new virions, producing a virus that is significantly different from either of the original infecting strains. This is why you can get the flu over and over again! Rhabdoviridae ¨ enveloped; helical capsid; (-) sense RNA ¨ includes Rabies virus Paramyxoviridae ¨ enveloped; helical capsid; (-) sense RNA ¨ includes viruses that cause Mumps, Measles, Viral pneumonia, Bronchitis Bunyaviridae ¨ enveloped; segmented RNA; (-) sense RNA ¨ includes Hantavirus (“4 corners disease”)
¨ enveloped; filamentous capsid; (-) sense RNA ¨ includes Ebola virus Reoviridae ¨ naked; polyhedral capsid; ds RNA ¨ includes Rotavirus (most common cause of diarrhea in infants and young children under the age of 2)
- Some Animal DNA Viruses
Adenoviridae ¨ naked; polyhedral capsid; ds DNA ¨ mainly responsible for human respiratory diseases; also causes diarrhea in babies and young children Herpesviridae – enveloped; polyhedral capsid; dsDNA ¨ Simplex virus – Herpes simplex 1 (oral) and 2 (genital & neonatal) ¨ Varicellovirus – Varicella zoster – chicken pox and shingles ¨ Roseolovirus – Roseola infantum – roseola in infants (rash and fever) ¨ Lymphocryptovirus – Epstein Barr virus – causes infectious mononucleosis and Burkitt’s lymphoma; also linked to Hodgkin’s disease. Poxviridae ¨ enveloped; brick shaped capsid; ds DNA; largest of all viruses ¨ includes Orthopoxvirus – small pox & cow pox Papovaviridae ¨ naked; polyhedral capsid; ds DNA; replicate in nuclei of host’s cells. ¨ Includes Papillomavirus – warts (some associated with cervical cancer)
Chap. 7 – Microbial Growth
The term microbial growth refers to the growth of a population (or an increase in the number of cells), not to an increase in the size of the individual cell. Cell division leads to the growth of cells in the population. Two Types of Asexual Reproduction in Microbes: 1.) Binary Fission – Bacterial reproduction occurs through fission, a primitive form of cell division that does not employ a spindle fiber apparatus. [A spindle fiber apparatus made of protein filaments is responsible for moving the chromosomes around during cell division (mitosis & meiosis) in most eukaryotic cells. Bacteria do not have these structures.] The bacterial cell doubles in size and replicates its chromosome. Following DNA replication, the two chromosomes attach to separate sites on the plasma membrane, and the cell wall is laid down between them, producing two daughter cells. 2.) Budding – A few bacteria and some eukaryotes (including yeasts) may also replicate by budding, forming a bubble-like growth that enlarges and separates from the parent cell.
- Microbial Growth
- Phases of Growth – A microbial lab culture typically passes through 4 distinct, sequential phases of growth that form the standard bacterial growth curve: (Not all growth phases occur in all cultures). See graph; be able to draw & label.
- Lag Phase– In the lag phase, the number of cells doesn’t increase. However, considerable metabolic activity is occurring as the cells prepare to grow. (This phase may not occur, if the cells used to inoculate a new culture are in the log phase & provided conditions are the same).
- Log Phase(logarithmic or exponential phase) – cell numbers increase exponentially; during each generation time, the number of cells in the population increases by a factor of two). The number of microbes in an exponentially increasing population increases slowly at first, then extremely rapidly. Organisms in a tube of culture medium can maintain log growth for only a limited time, as nutrients are used up, metabolic wastes accumulate, microobes suffer from oxygen depletion.
- Stationary Phase– The number of cells doesn’t increase, but changes in cells occur: cell become smaller and synthesize components to help them survive longer periods without growing (some may even produce endospores); the signal to enter this phase may have to do with overcrowding (accumulation of metabolic byproducts, depletion of nutrients, etc.).
- Death Phase– In this phase, cells begin to die out. Death occurs exponentially, but at a low rate. Death occurs because cell have depleted intracellular ATP reserves. Not all cells necessarily die during this phase!
B. Continuous Culture of Microbes
In the lab, cultures usually pass through all growth phases – not in nature. In nature, nutrients continuously enter the cell’s environment at low concentrations, and populations grow continually at a low but steady rate. The growth rate is set by the concentration of the scarcest or limiting nutrient, not by the accumulation of metabolic byproducts – in nature there is always some other microbe that can use these metabolic byproducts for their own metabolism. In the lab, we have to continually replace the media.
II. Measuring Numbers of Microbes
- Indirect Measurements(measure a property of the mass of cells and then ESTIMATE the number of microbes)
- Turbidity– Can hold tube up to the light and look for cloudiness as evidence of growth (difficult to detect slight growth). A spectrophotometer can measure how much light a solution of microbial cell transmits; the greater the mass of cells in the culture, the greater its turbidity (cloudiness) and the less light that will be transmitted. Disadvantages: Not sensitive in terms of numbers of bacterial cells & not useful for detecting minor contamination.
- Metabolic Activity – 3 ways:
- The rate of formation of metabolic products, such as gases or acids, that a culture produces.
- The rate of utilization of a substrate, such as oxygen or glucose.
- The rate of reduction of certain dyes. Ex. methylene blue becomes colorless when reduced.
- Direct Measurements – Give more accurate measurements of numbers of microbes.
- Direct Counts– Coulter Counter – electronic counter; rapid & accurate only if bacterial cells are the only particles present in the solution. [gives a total count – live & dead cells].
- Plate Count– Bacterial colonies are viewed through the magnifying glass against a colony-counting grid; called a Quebec colony counter (we have this in the lab). [gives a viable count]
- Filtration– A known volume of liquid or air is drawn through a membrane filter by vacuum. The pores in the filter are too small for microbial cells to pass through. Then the filter is placed on an appropriate solid medium and incubated. The number of colonies that develop is the number of viable microbial cell in the volume of liquid that was filtered. This technique is great for concentrating a sample, ex. a swimming pool, where small populations may go undetected using some other methods. [gives a viable count]
III. Growth Factors – Microbes can exist in a great many environments because they are small, easily dispersed, need only small quantities of nutrients, are diverse in their nutritional requirements.
- Physical Factors
- pH – bacteria can classified as:
- acidophiles(acid-loving) – grow best at a pH of 1 to 5.4; Ex. Lactobacilllus (ferments milk)
- neutrophiles – exist from pH to 5.4 to 8.5; most bacteria that cause human disease are in this category.
- alkaliphiles(base loving) – exist from pH to 7.0 to 11.5; ex. Vibrio cholerae (causes cholera)
- Temperature– bacteria can be classified as:
- psychrophiles (cold-loving) 15oC to 20oC; some can grow at 0oC.
- mesophiles – grow best between 25oC and 40 C; human body temp is 37oC.
- thermophiles (heat-loving) – 50oC to 60oC; found in compost heaps and in boiling hot springs.
- Moisture– only the spores of sport-forming bacteria can exist in a dormant state in a dry environment.
- Hydrostatic pressure – pressure exerted by standing water (ex. lakes, oceans, etc.); some bacteria can only survive in high hydrostatic pressure environments (ex. ocean valleys in excess of 7000 meters); the high pressure is necessary to keep their enzymes in the proper 3-D shape; without it, the enzymes lose their shape and denature and the cell dies.
- Tonicity (hypotonic, hypertonic, isotonic) – The use of salt as a preservative in curing meats and the use of sugar in making jellies is based on the fact that a hypertonic environment kills or inhibits microbial growth. Halophiles (salt lovers) inhabit the oceans.
- Radiation– UV rays and gamma rays can cause mutations in DNA and even kill microorganisms. Some bacteria have enzyme systems that can repair some mutations.
Chapter 8 – Metabolism of Microbes
I. A Model for Metabolism (using E. coli as an example)
What is metabolism? All the biochemical reactions that take place in a cell. The Model: Metabolism leading to the synthesis of a new microbial cell has 3 requirements:
- Raw Materials – nutrients composed of carbon (carbohydrates, proteins, etc.)
- Driving Force
- energy – (ATP– adenosine triphosphate) – Some chemical produce ATP; some chemical reactions use ATP.
- reducing power– Many biochemical reactions involve oxidation (removal of electrons from a compound) & reduction (addition of electrons to a compound; E. coli stores electrons in coenzymes called NAD (nicotinamide adenine dinucleotide) & NADP(nicotinamide adenine dinucleotide phosphate). These compounds capture electrons in the form of hydrogen atoms from compounds that are being oxidized, thus forming NADH & NADPH). NAD & NADP stores the cell’s reducing power. Bacteria will then use this reducing power to build its cellular components (it will reduce other compounds in this process).
Hint: O.I.L. = oxidation is loss; R.I.G. = reduction is gain
- A Plan – contained in the cell’s DNA; this is “the genetic code.”
- Flow of Materials– Sequential Steps:
- Entry Mechanisms– Raw materials from the environment are transported into the cells by various mechanisms.
- Catabolic Reactions– reactions in which raw materials are broken down into smaller molecules (precursor molecules); there are 12 precursor molecules that are required to synthesize building blocks (amino acids, monosaccharides, nucleotides, fatty acids) that will build a new cell.
- Anabolic Reactions– reactions that build up larger molecules from smaller ones:
a.) biosynthesis – 12 precursor molecules are put together to produce building blocks. b.) polymerization – building blocks are joined together to form macromolecules (proteins, nucleic acids, lipids, polysaccharides, peptidoglycan); ex. amino acids are put together to form a protein, nucleotides are put together to form DNA. c.) assembly – macromolecules are assembled into biological structures; ex. peptidoglycan forms a cell wall. raw materials à precursor molecules à buliding blocks àmacromolecules
- Driving Force– Where is ATP & Reducing Power Produced & Used in the Above Reactions?
- Entry Mechanisms– Many materials that move into the cell are moving from low to high concentration; this requires ATP; remember the bacterial is usually hypertonic to its environment.
- Catabolic Reactions– In general, the catabolic reactions transform raw materials into precursor molecules, reducing power, & ATP.
- Anabolic Reactions– In general, these reactions use precursor molecules, ATP, & reducing power.
III. Aerobic Metabolism (We’ll use E. coli as the example) (Aerobic means that this type of metabolism requires oxygen)
- Catabolic Reactions– Supply the cell with the 12 precursor molecules, reducing power (NADH & NADPH), & ATP.
- Precursor Molecules – A minimum of 3 different pathways are required to produce all 12 precursor molecules: (We’ll look at these later in this handout)
- glycolysis – produces 6
- tricarboxylic acid (TCA) cycle – produces 4
- pentose phosphate pathway – produces 2 (we won’t discuss this one)
- Reducing Power– Many steps in a catabolic pathway involve oxidation (loss of electrons)/reduction (gain of electrons) reactions. The electrons (along with hydrogens) are usually transferred from the molecule being oxidized to the coenzymes NAD or NADP, to form NADH & NADPH. NADH & NADPH are used to reduce other molecules or are used to generate new ATP molecules.
- Stored Energy– The principal compound that stores chemical energy in all cells is ATP (adenosine triphosphate). The energy-storage capabilities of ATP depend on the 2 bonds that join the 3 phosphate groups in the ATP molecule. These bonds are among the most highly reactive bonds found in biochemicals. They are called high-energy bonds. The phosphate groups that are joined in ATP by high-energy bonds are readily donated to other compounds. These compounds that receive a phosphate group from ATP are termed phosphorylated compounds. After receiving a high-energy phosphate, phosphorylated compounds can then participate in chemical reactions that would not occur if the compounds were unphosphorylated. This is why we say that the energy “stored” in the bonds of ATP is used to drive other metabolic reactions.
- ATP Formation from ADP [most of the ATP is made by chemiosmosis]
1.) Chemiosmosis & the Electron Transport System– The electron transport system is made up of a series or chain of compounds. Some of these compounds are hydrogen-carriers and some are electron-carriers. NADPH/NADH transfers hydrogen atoms to a hydrogen-carrier in the chain. This hydrogen-carrier then passes hydrogen electrons to an electron-carrier in the chain; the hydrogen ions are pumped out of the cell (or out of the inner mitochondrial compartment, if you’re talking about eukaryotes). Each compound in the chain will then alternately accept & then release hydrogen electrons; as electrons are passed along the chain they drop to lower and lower energy levels. At the end of the chain, electrons are accepted by oxygen to form water. (The final electron acceptor is oxygen!! This is why this process is called aerobic respiration!). ATPase is an enzyme located in the cell membrane of prokaryotes; in eukaryotes it’s located in the inner mitochondrial membrane. In E. coli, the pumping of hydrogen ions (H+) out of the cell creates a H+ concentration gradient & an electrical gradient across the cell membrane (there are more H+ outside the cell than inside). H+ then flows back into the cell (H+ ions want to move from a high concentration to a low concentration). The ions move through the membrane-bound ATPase, converting ADP to ATP. This process is called chemiosmosis.
- Catabolic Pathways (breaking down) – Chemical reaction sequences that transform raw materials into the 12 precursor molecules, store energy in the form of ATP, & store reducing power in the form of NADPH/NADH. Central metabolism(described below) begins with the monosaccharide glucose. Most organisms have other catabolic pathways, which use substrates other than glucose (ex. humans can use protein & lipids, if glucose concentrations are low). These pathways all eventually feed into central metabolism at various points.
- Glycolysis(= “glucose splitting”) – This pathway begins with glucose (a 6 carbon sugar) and ends with 2 pyruvic acids or pyruvates (3 carbon molecules). The initial reactions in glycolysis require ATP. Later reactions in glycolysis produce a small amount of ATP. A small amount of NADH is also produced, which will result in the production of more ATP in the electron transport system. The main function of glycolysis is to split glucose.
- Kreb’s Cycle – Some of the pyruvate formed by glycolysis is used in biosynthesis, the rest is oxidized to another precursor molecule, acetyl CoA(coenzyme A). Acetyl-CoA then enters the Kreb’s Cycle by combining with a precursor molecule oxaloacetate to form citrate. In a series of 6 reactions, carbon dioxide is released & oxaloacetate is regenerated (this is where most of the carbon dioxide you exhale comes from!). In this cycle, only a small amount of ATP is formed (called substrate level phosphorylation). However, considerable reducing power is stored in the form of NADH, NADPH, & FADH2(flavine adenine dinucleotide), which are used to produce ATP in the electron transport system.
- Pentose Phosphate Pathway – we won’t discuss.
- Anabolic Pathways (building up) -use precursor molecules, ATP, & reducing power produced in above catabolic reactions.
- Biosynthesis– E. coli converts precursor molecules produced in catabolism into building blocks (amino acids, monosaccharides, nucleotides, fatty acids) of macromolecules (proteins, nucleic acids, lipids, polysaccharides, peptidoglycan). Organisms that can’t make a given building block grow only if that molecule is provided in the medium (or diet). Ex. E. coli can make all 20 amino acids. Humans are unable to make 9 of the 20, so these nutrients must come from our diets.
- Polymerization– In these reactions, building blocks produced in biosynthesis are joined to form macromolecules. The major cellular polymerization reactions are DNA replication, RNA synthesis, protein synthesis, & polysaccharide & peptidoglycan synthesis. For most macromolecules, building blocks must be joined in a specific order. For ex., amino acids must be arranged in the proper order to produce the right proteins. If you change the amino acid sequence, you change the protein’s structure and therefore change its function.
Some polymerization is determined directly by the organism’s DNA (ex. DNA replication, RNA synthesis, protein synthesis). Other reactions are indirectly determined by DNA (ex. polysaccharide & peptidoglycan synthesis). In the latter, building blocks are ordered by the enzymes that catalyze the polymerization reactions. Because enzymes are proteins & protein structure is determined by DNA, the reactions the enzymes catalyze are indirectly determined by DNA.
- Assembly– Assembly of macromolecules into cellular structures (ex. flagella, ribosomes, cell wall) may occur spontaneously (self-assembly) or as a result of reactions catalyzed by enzymes.
IV. Anaerobic Metabolism
Anaerobic metabolism allows cells to grow in the absence of oxygen. Strict anaerobes are capable of only anaerobic metabolism. Facultative anaerobes are capable of both aerobic & anaerobic metabolism. (ex. E. coli)
- Anaerobic Respiration– Involves an electron transport chain, but uses a compound other than oxygen as the final electron acceptor, allowing the cell to generate ATP by chemiosmosis. Compounds that can be used as final electron acceptors include sulfate & nitrate.
- Nitrate users: These organisms, including E. coli, play a role in the nitrogen cycle (removing nitrogen from terrestrial & aquatic environments & returning it to the atmosphere). Some microbes reduce nitrate to nitrite. Some microbes reduce nitrite further to nitrogen gas. We’ll see this in lab!
- Sulfate users: These organisms, called sulfur reducers, play a role in the sulfur cycle. Sulfate is reduced to hydrogen sulfide gas. Sulfate reducers typically grow in marine & river mud flats, giving these environments a rotten-egg odor & turning the mud black. We’ll see this in lab!
- Fermentation– This type of anaerobic metabolism uses no electron transport chain.
Some differences between fermentation & respiration (aerobic or anaerobic):
- Fermentation generates fewer ATP per molecule of substrate. (ex. E. colican produce 38 ATP/molecule of glucose by aerobic respiration, but only 3 ATP by fermentation.)
- Because many molecules of substrate must be metabolized to supply a cell’s ATP requirements, the substrate must be in abundance in order for the microbe to grow.
- Sugars are usually the only substrate that can be used in fermentation.
- Lactic Acid Fermentation– This type of fermentation is carried out by lactic acid bacteria, the microbes that cause milk to sour (used to produce yogurt & buttermilk). Muscle tissue of animals also carries out lactic acid fermentation, when deprived of oxygen (during strenuous exercise – this is what makes your muscles sore). Steps:
- Glycolysis – Glucose is metabolized to produce 2 molecules of pyruvic acid (a little ATP & NADH is also produced).
- Pyruvic Acid Oxidation – pyruvic acid is oxidized to lactic acid using NADH, thus using up the reducing power stored in glycolysis.
Parts of the Kreb’s cycle & Pentose Phosphate pathway are used to help generate the 12 precursor metabolites; they cannot all be formed in fermentation.
- Alcoholic Fermentation
This type of fermentation is typical of yeast, a type of fungi. In this pathway, pyruvic acid is converted to carbon dioxide and ethanol. glucose à pyruvic acid à carbon dioxide + ethanol
V. Nutritional Classes of Microorganisms
Microbes are classified according to nutritional class, which depends on 2 factors: 1.) How it generates ATP & reducing power: chemo- (from oxidation of inorganic compounds like sulfur nitrite, ammonia, iron) or photo-(sun) 2.) The source of carbon atoms it uses to make precursor metabolites: auto- (obtain carbon from carbon dioxide) or hetero- (obtain carbon from organic compounds like carbs, proteins, lipids, etc.) Can have combinations of all of these: chemoautotrophs, chemoheterotrophs, photoautotrophs, photoheterotrophs. Return to Chp. index Chap. 9 & 10 – Microbial Genetics (DNA Replication & Protein Synthesis), Recombinant DNA, Genetic Engineering
- THE BASIS OF HEREDITY
All information necessary for life is stored in an organism’s chromosomes, which are made up of DNA (exception: some viruses only have RNA). A chromosome is circular (prokaryotes) or linear (eukaryotes). Remember the organic molecules stuff we covered at the beginning of the semester? Nucleic acids (DNA & RNA) are made up of building blocks called nucleotides (more on structure below). In DNA, nucleotides are arranged in a twisted double chain called a helix. The particular nucleotide sequence spells out the “genetic code” that provides information for the synthesis of new DNA (DNA replication necessary for cell division) and for the synthesis of proteins. A typical prokaryotic cell contains a single circular chromosome. Bacteria may also have a small, circular piece of extrachromosomal DNA called a plasmid. Human body cells, examples of eukaryotic cells, have 46 linear chromosomes. A gene, the basic unit of heredity, is a liner sequence of DNA nucleotides that form a functional unit of the chromosome or plasmid. All information for the structure and function of an organism is coded in its genes. The information in specific gene is not always the same; different versions of the same gene are called alleles. Using humans as an example, the hair color gene is always found at the same location on a chromosome, but the different versions or alleles that can exist for hair color are blond, brunette, red, etc. Because prokaryotes only have one chromosome, so they generally only have one allele for a particular gene. Many eukaryotes have 2 sets of chromosomes and thus 2 alleles of each gene, which may be the same or different. For example in humans, we have 46 chromosomes or 23 pair. In each pair, you get one chromosome from your mom and one for your dad. You may have a blonde hair allele from your mom and a dark hair allele from your dad (so you get dark hair since the dark hair allele is dominant).
- DNA STRUCTURE – THE WATSON-CRICK MODEL [DNA = deoxyribonucleic acid]
In the Watson-Crick model, the DNA molecule is a double-stranded helix, shaped like a twisted “ladder.” Remember that nucleic acids (DNA & RNA) are made up of building blocks called nucleotides. Each nucleotide is made up of a sugar, a phosphate, and a nitrogenous base. When we put these nucleotides together to build a DNA ladder, the sides of the ladder are composed of alternating phosphate groups & sugar molecules. The rungs of the ladder are made up of paired nitrogenous bases joined in the middle by hydrogen bonds. The nitrogenous bases are adenine, thymine, guanine, & cytosine; adenine always pairs with thymine (A-T or T-A) & guanine always pairs with cytosine (G-C or C-G) [This is called complementary base pairing]. These 4 bases spell out the genetic message or code! DNA enters into 2 kinds of reactions: 1.) Replication – replicates the DNA before cell division, so that each new daughter cell will receive a copy. 2.) Protein Synthesis (Gene Expression); 2 steps: transcription & translation III. DNA REPLICATION IN PROKARYOTES [Remember bacteria have a circular chromosome.] Replication begins by an enzyme breaking the hydrogen bonds between the nitrogenous bases in the DNA molecule; the double stranded DNA molecule “unzips” down the middle, with the paired bases separating. As the 2 strands separate, they act as templates, each one directing the synthesis of a new complementary strand along its length. If a nucleotide with thymine is present on the old strand, only a nucleotide with adenine can fit into place in the new strand; if a nucleotide with guanine is present on the old strand, only a nucleotide with cytosine can fit into place in the new strand, & so on. This is called complementary base pairing. DNA replication is called semiconservative replication since half of the original DNA molecule is conserved in each new DNA molecule. Like other biochemical reactions, DNA replication requires a number of different enzymes, each catalyzing a particular step in the process.
- GENE EXPRESSION – PROTEIN SYNTHESIS
- FROM DNA TO PROTEIN: THE ROLE OF RNA
By the 1940’s biologists realized that all biochemical activities of the cell depend on specific enzymes; even the synthesis of enzymes depends on enzymes! Remember that the DNA molecule is a code that contains instructions for biological function & structure. Proteins (enzymes) carry out these instructions. The linear sequence of amino acids in a protein determines its 3-D structure & it is this 3-D structure that determines the protein’s function. The big question was: How does the sequence of bases in DNA specify the sequence of amino acids in proteins? The search for the answer to this question led to the discovery of RNA (ribonucleic acid), which is similar in structure to DNA (deoxyribonucleic acid). Three types of RNA:
- messenger RNA(mRNA) – single stranded; contains codons (3 base codes); mRNA is constructed to copy or transcribe DNA sequences.
- ribosomal RNA(ribosomes!) (rRNA) – ribosomes “read” the code on the mRNA molecule & send for the tRNA molecule carrying the appropriate amino acid.
- transfer RNA(tRNA) – clover leaf shaped; at least one kind for each of the 20 a. a. found in proteins; each tRNA molecule has 2 binding sites – one end, the anticodon (also a 3 base code), binds to the codon on the mRNA molecule; the other end of the tRNA molecule binds to a specific amino acid; each tRNA & its anticodon are specific for an a. a.!!
Differences between RNA & DNA:
- RNA nucleotides contain a different sugar than DNA nucleotides. (ribose vs. deoxyribose).
- RNA is single stranded – DNA is double stranded.
- In RNA, uracil replaces thymine. There is no thyamine in RNA!!! But, there is adenine.
- TWO MAJOR EVENTS IN PROTEIN SYNTHESIS:
- Transcription[mRNA copies or transcribes DNA sequences]
This process is similar to what occurs in DNA replication. A segment of DNA uncoils unzips. Free RNA nucleotides, are then added one at a time to one end of the growing RNA chain. Cytosine in DNA dictates guanine in mRNA, guanine in DNA dictates cytosine in mRNA, adenine in DNA dictates uracil in mRNA, thymine in DNA dictates adenine in RNA. This complementary base pairing is just like what occurs in DNA replication. An enzyme catalyzes this process. After transcription the mRNA goes out in search of a ribosome. This mRNA molecule will now dictate the sequence of a. a. in a protein in the next step called translation.
- Translation– actual synthesis of polypeptides or proteins; translate information from one language (nucleic acid base code) into another language (amino acids); remember, the sequence of amino acids (the protein’s primary structure) determines what the protein’s 3-D globular structure is going to be & structure determines function.
- Initiation– Begins when the ribosome attaches to the mRNA molecule, reading its first or START codon. The first tRNA comes into place to pair with the initiator codon of mRNA (it occupies the peptide site in the ribosome). The START codon is AUG, which specifies the amino acid methionine. All newly synthesized polypeptides have to start with methionine.
- Elongation– The second codon of the mRNA molecule is then read and a tRNA with an anticodon complementary to the second mRNA codon attaches to the mRNA molecule; with its a. a. this second tRNA molecule occupies the aminoacyl site of the ribosome. When both the P & A sites are occupied, an enzyme forges a peptide bond between the 2 a. a. & the first tRNA is released. The first tRNA cannot be released until this peptide bond is formed, as it will take its a. a. with it!! The second tRNA is then transferred from the A site to the P site & a third tRNA is brought into the A site. The ribosome continues to move down the mRNA molecule in this fashion, “reading” the codons on the mRNA molecule & adding amino acids to the growing polypeptide chain.
- Termination– Toward the end of the coding sequence on the mRNA molecule is a codon that serves as a termination signal. There are no tRNA anticodons to complementary base pair with this codon. Translation stops and the polypeptide chain is freed from the ribosome. Enzymes in the cell then degrade the mRNA strand.
[In eukaryotic cells, the polypeptide is taken up by the rough e.r. & is modified into a 3-D protein; the proteins are then packaged into transport vesicles (a piece of the e. r. pinches off around the protein); these vesicles transport the proteins to the golgi complex for further modification; the finished protein is pinched off in a piece of golgi membrane (another vesicle) and is transported to the part of the cell where it is needed. In the prokaryotic cell, none of these organelles exist, modification/processing of the polypeptide into a protein occurs in the cytoplasm.] The genetic code. The mRNA codons for the 20 universal amino acids. See the table in your text of mRNA codons for the 20 amino acids. The 3-base codons are written to the left and the abbreviations of the amino acids they correspond to are written to the right. The amino acid abbreviations in the table are: Ala – alanine; Arg – arginine; Asn – apararagine; Asp – aspartamine; Cys – cysteine; Glu – glutamic acid; Gln – glutamine; Gly – glycine; His – histidine; Ile – isoleucine; Leu – leucine; Lys – lysine; Met – methionine; Phe – phenylalanine; Pro – proline; Ser – serine; Thr – threonine; Trp – tryptophan; Tyr – tyrosine; Val – valine. The code has been proven to be the same for all organisms from humans to bacteria – it’s known as the universal genetic code. Notice that most of the amino acids have more than one code (ex. Arg has 6 codes!). However, each code is specific for an amino acid (ex. UUU only codes for the amino acid Phe). Three of the 64 codons do not specify amino acids. Instead they indicate STOP or termination of the translation process (they say “This is the end of the polypeptide.”) The START codon is AUG, which specifies the amino acid methionine. All newly synthesized polypeptides have to start with methionine. Since AUG is the only codon for methionine, when it occurs in the middle of a message, it is ignored as a START codon and is simply read as a methionine-specifying codon.
- A mutationis any chemical change in a cell’s genotype (genes) that may or may not lead to changes in a cell’s phenotype (specific characteristics displayed by the organism). Many different kinds of changes can occur (a single base pair can be changed, a segment of DNA can be removed, a segment can be moved to a different position, the order of a segment can be reversed, etc.). Mutations account for evolutionary changes in microorganisms and for alterations that produce different strains within species. Mutations often make an organism unable to synthesize one or more proteins. The absence of a protein often leads to changes in the organism’’ structure or in its ability to metabolize a particular substance.
- Spontaneous mutations– occur by chance, usually during DNA replication. Only about one cell in a hundred million (108) has a mutation in any particular gene. Since full-grown cultures contain about 109 cells per milliliter, each milliliter contains about 10 cells with mutations in any particular gene. Because the bacterial chromosome contains about 3,500 genes, each ml of culture contains about 35,000 mutations that weren’t present when the culture started growing. Wow, when you think about it that’s a lot of mutations in just one ml!
- Induced mutationsare caused by chemical, physical, or biological agents called mutagens.
- Chemical Mutagens– ex. Nitrates and nitrites are added to foods such as hot dogs, sausage, and lunch meats for antibacterial action. Unfortunately these same compounds have been proved to cause similar mutations and cancer in lab animals
- Physical Mutagens– Include UV light, X-rays, gamma radiation, & decay of radioactive elements; heat is slightly mutagenic.
- Consequences of Mutations– Most mutations do not change the cell’s phenotype. If the mutation changes the codon to another that encodes the same amino acid, the protein remains the same. For example if the DNA code is changed from AGA to AGG, the mRNA codon would change from UCU to UCC. Check your table! The amino acid would not change. The amino acid would stay serine. In this case the genotype is altered, but the phenotype stays the same. Having more than one codon for each amino acid allows for some mutations to occur, without affecting an organism’s phenotype. A mutation that changes a codon to one that encodes a different a. a. may alter the protein only slightly if the new a. a. is similar to the original one. However, if a mutation changes an a. a. to a very different one, there may be a drastic change in the structure of the protein, causing major complications for the cell. For example, if the structure of an enzyme called DNA polymerase was greatly altered, the cell would not be able to replicate its DNA and thus would not be able to multiply.
- Repair of DNA Damage – Bacteria & other organisms have enzymes that repair some mutations.
VI. GENETIC TRANSFER
Gene transfer refers to the movement of genetic information between organisms. In most eukaryotes, it is an essential part of the organism’s life cycle and usually occurs by sexual reproduction. Male and female parents produce sperm and egg which fuse to form a zygote, the first cell of a new individual. Of course, sexual reproduction does not occur in bacteria, but even they have mechanisms of genetic transfer. Gene transfer is significant because it greatly increases the genetic diversity of organisms. We’ve already discussed how mutation account for some genetic diversity, but gene transfer between organisms accounts for even more. In recombinant DNA technology, genes from one species of organism are introduced into the genetic material of another species of organism. For example, human genes can be inserted into the bacterial chromosome.
- BACTERIAL PLASMIDS & CONJUGATION
Most bacteria carry additional DNA molecules known as plasmids:
- Plasmids are circular DNA molecules, much smaller than the bacterial chromosome.
- Plasmids can move in and out of the bacterial chromosome.
- Two important plasmids are fertility (F) plasmids and drug resistant (R) plasmids.
- The F Plasmid – This plasmids contains about 25 genes, many of which control the production of F pili. F piliare long, rod-shaped protein structures that extend from the surface of cells containing the F plasmid. Cells that lack the F plasmid are known as female (recipient) or F(-) cells. Cells that possess the F plasmid are known as male (donor) or F(+) cells. F(+) cells attach themselves to F(-) cells by their pili and transfer a copy of an F plasmid to the F(-) cells through a pilus. The once F(-) cells are now F(+) and will now produce pili, because they now have the F plasmid that contains the plasmid genes that code for these pili. This transfer of DNA from one cell to another by cell-to-cell contact is known as conjugation and is a form of sexual recombination because new genetic material is introduced into the cell. This is as close to sex as bacteria get!
- The R Plasmid– In 1959 a group of Japanese scientists discovered that resistance to certain antibiotics and other antibacterial drugs can be transferred from one bacterial cell to another. It was subsequently found that genes conveying drug resistance are often carried on plasmids. Over the last few decades, R factors have proliferated to the point that some infections are difficult to cure with antibiotics.
Note: Plasmids are very important to scientists involved in recombinant DNA research. Genes of interest can be inserted into plasmids. The plasmids are introduced to bacteria and the bacteria take them up by endocytosis. As the bacteria reproduce themselves by mitosis, they replicate the plasmid during interphase and pass it to their daughter cells. The plasmids can then be isolated from all of these bacterial cells and the gene of interest can be excised. In this way a large quantity of a gene of interest can be produced. We’ll talk about this more later.
- TRANSFORMATION– A genetic change in which DNA leaves one cell, exists for a time in the aqueous extracellular environment, & then is taken into another cell where it may become incorporated into the genome. Ex. Extracts from killed, encapsulated, virulent (disease causing) bacteria, when added to living, harmless, unencapsulated bacteria, can convert the latter to the virulent type. By endocytosis, the living, nonvirulent bacteria pick up the DNA from the dead, virulent bacteria and incorporate the DNA into their own DNA. The nonvirulent bacteria now have the genes that code for proteins that transform them into virulent bacteria.
A LITTLE ABOUT VIRUSES Viruses consist essentially of a molecule of nucleic acid (RNA or DNA) enclosed in a protein coat called a capsid. No cytoplasm, ribosomes, or other organelles are present. Viruses move from cell to cell, utilizing the host cell’s chromosomes, enzyme systems, and organelles to replicate the viral nucleic acid and synthesize new capsid proteins. They are obligate parasites in that they can’t multiply outside the host cell. Viruses that infect bacteria are called bacteriophages. A virus will attach to a host cell and inject its nucleic acid into the host cell. The viral nucleic acid takes over the host cell’s genetic material and causes it to help the virus replicate the viral nucleic acid and to produce its capsids. The new viruses are assembled in the host cell (nucleic acids are inserted into the capsids) and the host cell is lysed to release the new viruses to go and attack other cells. This cycle is known as the lytic cycle. Viruses that infect bacterial cells are called bacteriophages.
- Temperance or Lysogeny– This mechanism gives viruses the capacity to set up long-term relationships with their host cell. Instead of going through the lytic cycle, the virus nucleic acid remains integrated in the host cell’s chromosome. The virus may remain in this latent stage for long periods of time before initiating a lytic cycle. The problem with this type of cycle is that the integrated viral nucleic acid gets replicated along with the host cell’s chromosome during cell division & is passed to daughter cells (& then they pass it to their daughter cells, & so on). Something (ex. temperature change, stress) may later trigger these latent viruses to go into the lytic cycle all at once, destroying all of the infected host cells.
- Transduction– Viruses can serve as vectors or carriers of genetic information from one bacterium to another. During the reproduction of the virus in the bacterium, fragments of bacterial DNA instead of viral nucleic acid may become accidentally incorporated into a viral capsid. Such “viruses” may be able to infect a new host cell, but they are not able to complete a lytic cycle. The genes they carry from a previous bacterial host may become incorporated into the chromosome of the new bacterial host, possibly giving the bacterium new characteristics (ex. drug resistance).
VII. GENETIC ENGINEERING
Genetic engineering refers to the purposeful manipulation of genetic material to alter the characteristics of an organism in a desired way. One of the most useful of all techniques of genetic engineering is the production of recombinant DNA – DNA that contains information from two different species of organisms.
For example a particular human gene can be removed from a human chromosome. Recombinant DNA is then constructed by inserting that gene into a bacterial plasmid, which serves as a carrier. The recombinant DNA is then introduced host bacterium, which takes up the plasmid. The host bacterial cell then divides and its daughter cells divide, producing millions of cells that all contain a copy of the human gene of interest. This process serves at least 2 purposes: 1.) Large quantities of the human gene of interest are produced. 2.) The bacteria can read the human gene of interest, producing the protein coded for on the gene by protein synthesis. The genetic code is universal! We can obtain large quantities of a particular protein using bacteria.
B. MEDICAL APPLICATIONS – (See Table 8.3 p. 211 for a more extensive list) – Products such as insulin to treat diabetes, human growth hormone to treat dwarfism, blood-clotting proteins to treat hemophilia, antibiotics, and vaccines have all been produced by bacteria using recombinant DNA technology.
- Vaccines – Immunizationmeans deliberately introducing an antigen into the body that can provoke an immune response & the production of memory cells. The first injection elicits a primary immune response, which provokes the production of antibodies & memory cells to provide long-lasting protection against disease. Many vaccines are made from killed pathogens (called inactivatedvaccines); pathogens can be killed/inactivated by heat or chemicals (such as formaldehyde). Too much heat denatures proteins (changes their shape). When the body comes into contact with the real thing it won’t have the right antibodies and memory cells & the person may get the disease. If the pathogens aren’t heated enough, some live pathogens may be injected in the vaccine! Attenuated (weakened) pathogens are also used in vaccines; these have been cultured in abnormal conditions so that they are no longer pathogenic. Sometimes these organisms cause adverse side effects & can sometimes revert to their pathogenic forms, causing the disease.
Using “genetically engineered viruses:” Recently, genetic engineering & recombinant DNA technology has allowed us to use bacteria to produce the protein antigens found in the protein capsids of certain viruses (remember, viruses don’t have phospholipid cell membranes – they have proteins coats or capsids). Scientists determine the genetic code for these proteins & insert the gene into the chromosome of bacterial cells. The bacteria produce the proteins coded for on the inserted genes when they go through their regular process of protein synthesis. These proteins can then be injected as a vaccine (your body doesn’t care if the proteins are in the real viral capsid or if they were made by a bacterium; they are the same proteins & your body’s immune system will respond to these antigens in the same way). “Genetically engineered viruses” (ex. hepatitis B, influenza, rabies) do not pose the same risks as inactivated and attenuated viruses! Return to Chp. index Chapter 11 – Controlling Microbes (Sterilization & Disinfection) Some Important Terms Defined: sterilization – treatment to destroy all microbial life (even destroys bacterial endospores and fungal spores); there are no degrees of sterility! disinfection (sanitation) – treatment to reduce the number of pathogens to a level at which they pose no danger of disease; disinfectants are used to kill microbes on inanimate objects (most disinfectants are too harsh for use on delicate tissue); most disinfectants do not kill spores. antisepsis – kill microbes or inhibit their growth on skin or other living tissue; antiseptics are applied to living tissue. sanitizer – typically used on food-handling equipment and eating utensils to reduce bacterial numbers so as to meet public health standards (may mean just washing with soap in some cases). “-static” – treatments that inhibit rather than kill; ex. refrigeration. (bacteriostatic, fungistatic, etc.) “-cidal” – treatments that kill. (bactericidal, fungicidal, viricidal, etc.) (germicidal is a more general) chemotherapeutic agents – chemicals, incl. antibiotics, used to treat disease (discussed in Chap. 12)
I. Physical Controls
- Advantages – simple, inexpensive, effective penetrates to kill microbes throughout the object; best method if material being treated is not damaged by heat.
- Mode of Action – denatures proteins.
- Dry Heat Sterilization– ex. flaming loops, tubes in lab & hot air ovens (171oC, 1hr., 160oC for 2 hr., 121oC for 16 hrs.); used to sterilize materials that can withstand high temps. & any materials damaged by moisture.
- Moist Heat Sterilization– ex. boiling or in autoclaves; effective at a lower temperature than dry heat & it penetrates more quickly; disadvantages of boiling – does not kill thermophiles, endospores; autoclave is more effective than boiling- it uses pressure to raise the temperature above that of boiling (121oC, 15psi, for 20 min.); used to sterilize liquids and material easily charred; used in food canning & the lab to sterilize glassware & media.
- Pasteurization– limits growth, but does not sterilize; used to slow spoilage of milk & dairy products, wine, beer; advantage: causes minimal damage to the product; developed by Louis Pasteur; standard treatment: heat to 63oC for 30 min. or 72oC for 15 sec.
- Effect – microbiostatic; does not sterilize; slows down enzymes.
- Refrigeration– preserves food because it stops the growth of most species of microbes (slows chemical reactions); most disease-causing microbes are mesophiles, not psychrophiles; an exception is Listeria spp., which causes listeriosis (food poisoning).
- Freezing– kills most bacteria, but survivors can remain alive for long periods in the frozen state; bacteria cultures can be preserved by rapid freezing, sometimes with the addition of a compound called DMSO, milk, or glycerol to protect proteins.
- Electromagnetic Spectrum– Radiation is classified by wavelength with ionizing and UV light radiation at the short-wavelength end, visible light in the middle, & radio waves at the long-wavelength end. The shorter the wavelength, the greater its energy, & the more lethal it is. Mode of Action: denatures DNA.
- Two types of radiation that kill bacteria directly are UV (ultraviolet) Light& Ionizing Radiation. The effect of both is sterilization.
- UV Light– bacteria actually have special enzymes that can correct some damage done by UV light!; in the lab mercury vapor lamps (germicidal lamps) are used; disadvantages: kills only on surfaces & these wavelengths can also be harmful to humans.
- Ionizing Radiation– 2 forms; both cause a chain of ionizations by stripping electrons from atoms, resulting in cell death; disadvantages: technically complex; is being used to sterilize some produce, much to the public’s dismay.
1.) X rays 2.) Gamma rays
- Membrane Filtration
- Effect – physically removes cellular organisms (not viruses – they are too small).
- Uses – in lab, used with media, antibiotics, & other heat sensitive materials; filtration is replacing pasteurization in some causes, because filtration causes even less damage; you may have heard of the new “cold filtered” beers.
- Defined – the removal of water.
- Two processes:
- evaporation involving heat– effect – kills many microbes; rarely used in lab because the high heat causes chemical changes (denaturation); is used in food industry.
- lyophilization[freeze drying] – removes water directly by converting water from a solid state (ice) to a gaseous state; materials are frozen & placed in a chamber to which a partial vacuum is applied; avoids the chemical changes caused by heat drying; effect – stops microbial growth by stopping most chemical reactions (just like regular freezing) frequently used in the microbiology lab to preserve perishable materials such as proteins, blood products, & reference cultures of microbes; used in food industry to make instant coffee, etc.; disadvantage – expensive.
- Osmotic Strength
- Method – high concentrations of salt or sugar.
- Mode of action – microbes cannot grow if they are deprived of water; also, crenation or shrinkage can occur (you’re placing the microbes in a hypertonic environment).
- Disadvantage – once added, solutes (such as salt or sugar) cannot be easily removed; not used in lab.
II. Chemical Control – The effectiveness of a chemical anitmicrobial agent is affected by time, temperature, pH, and concentration.
- Testing Germicides – 3 ways:
- Phenol coefficients:Germicides can be tested by comparing their effectiveness to phenol, a traditional germicide. It was phenol that Lister first used – he called it carbolic acid. The procedure involves preparing several dilutions of a chemical agent, inoculating them with the bacteria Salmonella typhi (a digestive tract pathogen) or Staphylococcus aureus (a wound pathogen), incubating the tubes, and then checking for cloudiness in the tubes, indicating growth. The ratio of the effective dilution of the chemical agent to the dilution of phenol that has the same effect is the phenol coefficient. A disinfectant with a phenol coefficient of 1.0 has the same effectiveness as phenol. Less than 1.0 means it’s less effective. Greater than 1.0 means it’s more effective.
- Paper disc method– paper discs are saturated with the chemical agent and placed on the surface of an agar plate inoculated with a test organism. Clear “zones of inhibition” appear around the discs if the chemical agent is effective.
- Use-dilution test– The test microbe is added to dilutions of the chemical agent. The highest dilution that remains clear after incubation indicates a germicide’s effectiveness.
B. Mechanisms of Action
- Affect Proteins – The alteration of protein structure is called denaturation.Denaturation can be permanent (bacteriocidal) or temporary (normal structure can be restored – bacteriostatic). Mechanisms of denaturation include:
- Hydrolysis– breakdown of a molecule by addition of water
- Oxidation– addition of oxygen or removal of hydrogen
- Attachment of atoms or chemical groups – ex. heavy metals (mercury), alkylating agents (ex. –CH )
- Affect Membranes
- membrane proteins – denaturation (see above)
- membrane lipids –can be dissolved.
- Affect Cell Wall Formation
- Affect Nucleic Acid Structure
- Affect Metabolism
- Types of Germicides
- Structure – compounds with hydrophilic & hydrophobic parts.
- Mode of action – Penetrate oily substances in water & break them apart into small droplets that become coated with surfactant molecules. The hydrophobic end of the surfactant stick into the droplets & the hydrophilic end is attracted to the water. The result is an emulsion, a fine suspension of oily droplets in water, which can now be rinsed away.
- Effect of soaps & detergents – wash away microbes, but do not kill them.
- Wetting agents are surfactants that are often used with other chemical agents to help the agent penetrate fatty substances. Surfactants are not germicidal by themselves!
- Quaternary ammonium salts – four organic groups attached to a nitrogen atom. Effect: kill all classes of cellular microbes & enveloped viruses by disrupting membranes. Uses: nontoxic & widely used in the home, industry, labs, & hospitals. Their effectiveness is decreased in the presence of soap. Actually support Pseudomonasgrowth! Now being mixed with other agents to overcome some of these problems.
- Phenol & Phenolics
- Structure – compounds with hydroxyl groups (-0H) attached to a benzene ring.
Mode of Action – denature cell proteins, disrupt cell membranes.
- Effect – kill most organisms; action is not impaired by organic materials (remain active even in the presence of blood, feces, etc.)
1.) Lysol 2.) Cresol – found in creosote; plant derivative used to prevent the rotting of wooden posts, fences, railroad ties. 3.) Hexachlorophene – chlorinated phenolic; effective as an antiseptic; once widely used as an ingredient in soaps & lotions; in 1970’s was found to increase risk of brain damage in babies; has now been replaced with chlorhexidine in hospitals – good agent for surgical scrubs.
- Structure – compounds with a hydroxyl group (-OH).
- Mode of Action – when mixed with water disrupt lipids in cell membranes & denature proteins.
- Ethanol & Isopropanol– widely used as skin antiseptics; a 50 to 70% solution in water is the most effective concentration (one of the few exceptions to the rule: increase effectiveness by increasing concentration); does not sterilize skin because it evaporates quickly and does not penetrate deeply enough into skin pores.
- Main disadvantage – do not kill endospores.
- Mode of Action – inactivates enzymes by oxidation.
1.) Iodine – antiseptic a.) Tincture – iodine in a dilute alcohol solution; one of first skin antiseptics. b.) Iodophor – mixture of iodine and surfactants; ex. Betadine and Isodine (used for surgical scrubs and to prepare skin for surgery) 2.) Chlorine – disinfectant; ingredient in household bleach; added to drinking water and swimming pools; inactivated by the presence of organic materials.
- Hydrogen peroxide
- Mode of Action – oxidizing agent (denatures proteins)
- Uses of H2O2: antiseptic for cleaning wounds, disinfect medical instruments & soft contact lenses. When H2O2comes into contact with tissue, it bubbles producing oxygen gas. This is because all aerobes (incl. eukaryotes) produce the enzymes catalase & peroxidase which decompose H2O2 into oxygen & H2O. H2O2 generally kills microbes before it is destroyed by catalase or peroxidase. (You can differentiate between Staphylococcus & Streptococcus using H2O2; Staph is relatively resistant to H2O2 because of the large amounts of catalase & peroxidase it produces.) May be used to clean deep puncture wounds, because the oxygen produced kills obligate anaerobes present in the wound (ex. Clostridium).
- Heavy Metals
- Mode of Action – heavy metals (mercury, copper, silver) react with the sulfhydryl groups of proteins (denaturation)
- Effect – kills many microbes.
1.) Mercuric chloride – once widely used as an antiseptic; highly toxic; now merthiolate & mercurochrome are used (less toxic); merthiolate is prepared as a tincture; use – basic first aid kit supplies for disinfecting skin & mucous membranes. 2.) Silver Nitrate – once applied to eyes of newborns to prevent gonorrhea; the trend for a while was toward using antibiotics instead, but the development of antibiotic-resistant strains has necessitated the use of silver nitrate again. 3.) Selenium sulfide – kills fungi, including spores; commonly used to treat fungal skin infections; included in dandruff shampoos (dandruff is often caused by a fungus).
- Alkylating Agents
- Mode of action – they alkylate(attach short chains of carbon atoms) to proteins and nucleic acids. Must not be used where they may effect human cells (these agents are carcinogenic).
- Formalin– 37% solution. of formaldehyde used to preserve tissues & to embalm; kills all microbes, including spores; lower concentrations are used to inactivate microbes for killed vaccines.
- Glutaraldehyde– used to sterilize surgical instruments if equipment for heat sterilization is not readily available.
- Ethylene oxide– gas; advantages: disappears from the object after treatment; disadvantage: extremely toxic to humans so must be used in a sealed chamber; kills all bacteria, including endospores; used to sterilize materials destroyed by heat (plastic, rubber gloves, animal feed, mattresses, telephones).
- Dyes – Ex. Crystal violet blocks cell wall synthesis. It effectively inhibits growth of G(+) bacteria in cultures and in skin infections. It can be used to treat yeast infections.
III. Food Preservation
- Temperature – Environmental factor most often used to preserve food. Canning is the oldest method. Two factors, time & temp., determine safe heat treatments for canning. Refrigeration is low enough to stop the growth of most microbes. Psychrophilic (ex. Listeria) microbes are the exception. See pg. 1 of this handout for more info. on temp.
- pH – Acidity (low pH) prevents the growth of most microbes, especially in an anaerobic environment. Ex. adding vinegar (acetic acid) to foods. Low pH also increases the effectiveness of heat treatments (ex. acidic foods like tomatoes can be canned merely by boiling).
- Drying – drying & salting do not sterilize but preserve food by making it unable to support microbial growth for lack of water, an essential nutrient. See pg. 1 & 2 of this handout for more info. on drying & freeze drying.
- Chemicals – Various chemical preservatives are added to commercially prepared foods. Ex.:
- calcium propionate – antifungal agent added to bread.
- sorbic acid – antifungal agent added to soft drinks, salad dressings, cheeses.
- sodium benzoate– antifungal agent added to soft drinks, salad dressings, cheeses.
- sodium nitrate(nitrite) – antibacterial agent that prevents germination of Clostridium botulinum spores when added to bacon, ham, hot dogs.
Return to Chp. Index Chap. 12 – Antimicrobial Therapy I. TERMS
- Chemotherapy – term coined by Paul Ehrlich(father of chemotherapy) – He discovered a drug treatment for syphilis; he also developed the guiding principle of chemotherapy, which is selective toxicity (the drug should be toxic to the infecting microbe, but relatively harmless to the host’s cells). Now the term chemotherapeutic agent describes any chemical substance used in medical practice.
- Antimicrobial agent – drug used to treat disease caused by microbes.
- Antibiotic –chemical substance produced by a microorganism that has the capacity to inhibit the growth of bacteria and even destroy them. One of the first antibiotics was penicillin, discovered by Alexander Fleming in while he was carrying out experiments on Staphylococcus (1929). Some of his plates became contaminated with mold spores. As he examined them, Fleming noticed that the Staphylococcus colonies were dissolving as they neared the area where the mold was growing. He reasoned that the mold was secreting something that killed the bacteria. The mold was found to be a member of the genus Penicillium, so he named the bacteria destroying substance “penicillin.” Ten years later Florey and Chain had purified enough penicillin to begin experiments involving the treatment humans. It was enormously useful in the latter part of World War II.
II. GENERAL PROPERTIES OF ANTIBACTERIAL AGENTS
(Antifungal and antiviral agents will be discussed later in the semester as treatment for specific diseases)
- Selective Toxicity – drug harms the microbe without causing significant damage to the host. When searching for ways to treat disease, scientists look for differences between the human (or animal) host and the pathogen. Ex. Penicillin interferes with cell wall synthesis. Animal cells have no cell walls, so penicillin is not toxic to animals.
- Spectrum of Activity –the range of different microbes against which an antimicrobial agent acts. Example: Broad spectrum: G(+) and G(-) bacteria vs. Narrow spectrum: G(-) only;
C. Modes of Action (How Do the Drugs Work?)
- Inhibition of Cell Wall Synthesis – attach to enzymes that cross-link peptidoglycans.
Penicillins – Bactericidal. Natural penicillins are extracted from cultures of the mold Penicillium notatum. Structure: contain beta lactam rings. The discovery of resistant bacterial strains led to the development of semisynthetic penicillins (resistant bacteria have beta lactamaseenzymes that can break down beta lactam rings). The first of the semisynthetic penicillins was methicillin (not broken down by beta lactamase enzymes). Other familiar semisynthetics are ampicillin and amoxicillin. Allergy is rare in children but occurs in 1-5% of adults. Also used prophylactically (to prevent infection). Penicillins are not as effective against G(-) due to the presence of the outer membrane. Cephalosporins – Derived from several species of the fungus Cephalosporium. Have limited antimicrobial action. Their discovery led to the development of a large number of bactericidal, semisynthetic derivatives of cephalosporin. Structure: contain beta lactam rings. Frequently used cephalosporins include cephalexin (Keflex) and ceftriaxone (these 2 are 3rd generation cephalosporins). Have a fairly wide spectrum of activity, rarely cause serious side effects, and can be used prophylactically. Many people that are allergic to penicillin may also be sensitive to the cephalosporins. Others – Carbapenems – new group of extremely broad spectrum antibiotics that have a 2-part structure (Ex. Primaxin – consists of a beta lactam antibiotic and cilastatin sodium, a compound that prevents degradation of the drug in the kidneys); Bacitracin – derived from the bacterium Bacillus (highly toxic, so only used topically – used on lesions and wounds of skin and mucous membranes); Vancomycin – produced by Streptomyces (used to treat infections caused by methicillin-resistant staphylococci and enterococci).
- Disruption of Cell Membrane Function
Antibiotics such as polymyxins act as detergents and distort cell membranes. Polymyxins are obtained from Bacillus polymyxa and are especially effective against G(-) bacteria such as Pseudomonas that have phospholipids in their outer membrane (along with the lipopolysaccharides).
- Inhibition of Protein Synthesis
Protein synthesis requires the DNA code, RNA (mRNA, tRNA, and rRNA). The difference between bacterial and animal ribosomes allows antimicrobial agents to attack bacterial cells without damaging animal cells. Ex. streptomycin, erythromycin, chloramphenicol (These antibiotics can affect mitochondria. They have their own ribosomes that are similar to bacterial ribosomes.) Aminoglycosides – obtained from various species of Streptomyces and Micromnospora. The first was streptomycin (1940’s); many bacteria are now resistant to it. This antibiotic can damage kidneys and the inner ear nerves, so is used only in special situations and usually in combination with other drugs. These drugs have a great ability to act synergistically with other drugs. Gentamycinis a mainstay for the treatment of Pseudomonas (if resistant, then use polymyxins). Tetracyclines – several are obtained from Streptomyces. Semisynthetic tetracyclines have been developed. All are bacteriostatic. They have the widest spectrum of activity of any antibiotic. Unfortunately, because of this they destroy both the pathogenic bacteria and the normal flora. Can cause a variety of mild to severe toxic effects (kidney and liver damage, light sensitivity, interferes with effectiveness of birth control pills, staining of teeth, abnormal bone development in fetus). Used to treat Lyme disease. Chloramphenicol – originally obtained from Streptomyces, but is now fully synthesized in the lab. It is bacteriostatic and has a broad spectrum of activity. Because of its toxic effects on bone marrow, it is the drug of last choice in the U.S. Be careful – it is sometimes sold without prescription outside the U.S. Macrolides – Erythromycin, a commonly used macrolide, is produced by Streptomyces. It is bacteriostatic and is valuable in treating infections caused by penicillin-resistant organisms or in patients allergic to penicillin. Considered one of the least toxic of commonly used antibiotics.
- Inhibition of Nucleic Acid Synthesis
Target enzymes involved in nucleic acid synthesis (ex. DNA replication, transcription). Rifamycin – specifically targets the enzyme involved in the transcription process (mRNA synthesis); produced by Streptomycesand only used in the U.S. for treating tuberculosis; has high drug interaction. Ex. Rifampicin. Quinolones –new group of broad spectrum antibiotics; targets enzyme that unwinds DNA prior to replication; especially effective against traveler’s diarrhea and UTI’s. Ex. Nalidixic acid used against G(-)’s.
- Interference of Metabolism
Antimicrobial compounds can function in 2 ways: 1.) by competitively inhibiting enzymes and 2.) by being erroneously incorporated into important molecules such as nucleic acids. The actions of these compounds are sometimes called molecular mimicry because they mimic the normal molecule, preventing a reaction from occurring or causing it to go awry.
- Competitive Inhibition– Remember our discussion on enzymes, their active sites, and their substrate? In competitiveinhibition an antimicrobial compound binds to an enzyme’s active site, so that the enzyme cannot react with its “true” substrate. To bind to the active site, the antimicrobial compound must be similar in structure to the true substrate.
Ex. The drug sulfanilamide is very similar to the compound para-aminobenzoic acid (PABA). Sulfanilamide competitively inhibits an enzyme that acts on PABA. Many bacteria require PABA in order to make folic acid, which they use in synthesizing nucleic acids and other compounds. When sulfanilamide is bound to the enzyme, a bacterium cannot make folic acid. Animals obtain folic acid from their diets (they don’t have the enzymes to make it themselves), so their metabolism is not disturbed by these competitive inhibitors.
- Nucleic Acid Incorporation– Antimicrobial compounds such as vidarabine and idoxuridine are erroneously incorporated into nucleic acids. These molecules are very similar in structure to the nitrogenous bases. When incorporated into a nucleic acid, they garble the information that it encodes because they cannot form the correct base pairs during replication and transcription. These compounds can harm the host cells as well as the microorganisms (animal cells use the same nitrogenous bases to make nucleotides). These agents are most useful in treating viral infections, because viruses incorporate these “fakes” more rapidly that do cells and are more severely damaged.
III. KINDS OF SIDE EFFECTS
- Toxicity – Some antimicrobials do exert toxic effects on the patients receiving them. These effects are discussed later in connection with specific drugs.
- Allergy – An allergyis a condition in which the body’s immune system responds to a foreign substance, usually a protein. For Ex., breakdown products of penicillins combine with proteins in body fluids to form a molecule that the body treats as a foreign substance.
- Disruption of Normal Microflora – Antimicrobials, especially broad-spectrum antibiotics, mat exert their adverse effects not only on pathogens but also on the normal or indigenous microflora(the microorganisms that normally inhabit the skin and the digestive, respiratory, and urogenital tracts and keep numbers of unwanted “nonnative” microorganisms in check). When these native populations are reduced, other organisms not susceptible to the antimicrobial agent invade and multiply rapidly (called superinfections) Ex. Oral ampicillin and long-term use of penicillin can often leads to destruction of normal microflora in the gut and in turn, growth of the yeast Candida. Cephalosporins, tetracyclines, and chloramphenicol often lead to oral and vaginal yeast infections. Live-culture yogurts or acidophilus in the form of a pill (both contain lactobacilli) can be given to counteract this effect (basically you are reestablishing the normal microflora).
IV. GENERAL PROPERTIES OF ANTIBACTERIAL AGENTS
A. How Resistance is Acquired
- Spontaneous Mutations – Most bacteria acquire antibiotic resistance by spontaneous mutationsin their genetic material. Bacteria reproduce so rapidly that billions of cells can be produced in a short time; among them there will always be a few mutants. If a mutant happens to be resistant to a drug, that mutant and its progeny will survive, whereas the nonresistant cells will die. After a few generations, most of the survivors will be resistant to the drug. Understand that antibiotics do not induce mutations, but they do create environments that that favor the survival of mutant resistant organisms (see section E below).
- R Plasmids – Resistant genes usually found on R plasmids can transferred from one bacterium to another by conjugation through pili, transduction (using a viral vector), or transformation (Remember Chapter 8?)
- L forms – Some species of bacteria can lose their cell wall and swell into irregularly shaped cells called L forms. L forms can arise spontaneously and can persist and divide repeatedly. They can spontaneously revert to normal-walled cells. In the L form, bacteria are resistant to antibiotics that effect cell wall formation (ex. penicillin).
B. Mechanisms of Resistance – include the following: development of an enzyme that destroys or inactivates the drug; alteration of an enzyme that allows a formerly inhibited reaction to occur (the drug no longer works on the target enzyme); alteration of a metabolic pathway – a new chemical reaction bypasses the chemical reaction effected by the drug; alteration of membrane permeability – drugs can no longer cross the membrane, so they have no effect.
C. First Line, Second-Line, and Third-Line Drugs – as a strain of bacteria acquires resistance to a drug, another drug must be found, and so on.
D. Cross-resistance – resistance to 2 or more similar antimicrobials via a common mechanism. Ex. Penicillin contains a structure called a beta lactam ring. Some bacteria have an enzyme called beta lactamase that will break the beta lactam ring in penicillins. Bacteria that have this enzyme are also resistant to some cephalosporins that also contain beta lactam rings.
E. Limiting Drug Resistance
1. High levels of antibiotic can be maintained in the bodies of patients long enough to kill all pathogens, including resistant mutants or to inhibit them so that body defenses can kill them.
- Two antibiotics may be administered simultaneously so they can exert an additive effect (synergism). Ex. penicillin is often added to another antibiotic (the penicillin damages the cell wall, allowing better penetration by the other antibiotic). In the following example, another antibiotic is added to penicillin to increase its effectiveness. Augmentin = penicillin (amoxicillin) and clavulanic acid. Clavulanic acid binds to beta lactamases and prevents them from inactivating the amoxicillin.
- Antibiotics can be restricted to essential uses only (ex. not for colds, etc.). In addition, the use of antibiotics in animal feeds could be banned.
F. Special Problems with Drug-Resistant Hospital Infections – Resistant organisms are found more often in hospitalized patients than among outpatients. Why?
- There are many different kinds of infectious agents in confined area.
2. Sick people live in close proximity.
3. Hospitalized patients tend to be more severely ill and many have lowered resistance to infections because of their illness or because they are taking immunosuppressant drugs.
4. Hospitals typically make intensive use of a variety of antibiotics (resistant strains readily emerge and spread among patients).
V. DETERMINING MICROBIAL SENSITIVITIES TO ANTIMICROBIAL AGENTS
- The Disk Diffusion Method(Kirby-Bauer method) – A bacteria is uniformly spread over an agar plate. Filter paper disks are saturated with the drug and placed on the agar surface. Clear areas called zones of inhibition appear on the agar as round disks where the drugs inhibit the bacteria. Important to realize the results obtained in vitro (in the lab) often differ from those obtained in vivo (in a living organism). Metabolic processes in a living organism may inactivate or inhibit a drug. We’ll do this procedure in lab.
- The Dilution Method – A constant quantity of microbial inoculum is introduced into a series of broth cultures containing decreasing concentrations of a drug. After incubation, the tubes are examined and the lowest concentration of the drug that prevents visible growth (indicated by turbidity) is noted. Advantage to using this method over the disk method: Samples from tubes that show no growth can be used to inoculate broth that contains no drug to see if the drug was bactericidal or bacteriostatic.
- Serum Killing Power – Obtain patient’s blood sample while the patient is receiving an antibiotic. Bacteria are added to the patient’s serum (blood plasma minus clotting proteins). Growth (turbidity) after incubation indicates that the antibiotic is ineffective.
- Automated Methods– Bacteria are added to wells in trays to which a variety of antimicrobial agents have been added. The trays are inserted into a machine that measures microbial growth. Advantages: efficient, fast, inexpensive, and allows physicians to prescribe an appropriate antibiotic early in an infection rather than prescribing a broad-spectrum antibiotic while awaiting lab results.
VI. ATTRIBUTES OF AN IDEAL ANTIMICROBIAL AGENT
- Solubility in body fluids
- Selective toxicity
- Toxicity not easily altered (no food or drug interactions)
- Stability (should be degraded and excreted by the body slowly)
- Resistance by microorganisms not easily acquired
- Long shelf life.
- Reasonable cost
Return to Chp. index
Chap. 13 – Epidemiology
- SOME IMPORTANT DEFINITIONS:
Epidemiology – study of when & where diseases occur & how they are transmitted in human populations (focuses on groups of people rather than individuals); the modern definition does not limit this study to that of epidemic diseases; knowing the source of the disease can help prevent transmission even while the causative microorganism (etiologic agent) is still unknown. Epidemics – a pattern of disease transmission that affects many members of a population within a short time (ex. cholera in South America, flu, etc.). Pandemic – an epidemic that spreads world wide (ex. AIDS, flu). Endemic – numbers stay too low to constitute a public health concern (ex. chicken pox). Sporadic – diseases occurring only occasionally in a population (ex. tetanus, trichinosis).
- Sources of Information
- vital statistics– birth, death, marriage, & divorce records
- census data– number of people living in an area & their distribution by age, race, sex, marital status.
- disease reports– doctors are required to report certain diseases to public health dept.; local public health stats are forwarded to state agencies (ex. Texas Dept. of Health) and the Centers for Disease Control & Prevention (CDC); the CDC prepares the Morbidity & Mortality Weekly Report (MMWR) for the U.S.; stats included in this report:
- morbidity rate– the number of individuals affected by a disease during a set period in relation to the total number in the population (expressed as number of cases per 100,000 people per year).
- mortality rate– the number of deaths due to a disease in a population during a specific period in relation to the total population (expressed as number of deaths per 100,000 people per year).
- other sources– surveys, questionnaires, interviews, hospital records.
B. Use of Stats
- incidence rate– number of new cases within a set population during a specified period of time divided by the total number of people in the population; incidence rates measure the growth or spread of a disease; ex. this stat tells us how many people develop AIDS in the U.S. per year.
- prevalence rate– number of people who have a certain disease at any particular time (old and new cases) divided by the total number of people in the population; ex. this stat tells us how many people currently have AIDS in the U.S.
III. HOSPITAL EPIDEMIOLOGY – NOSOCOMIAL INFECTIONS
Among patients admitted to hospitals each year about 10% (2 million) acquire a nosocomial infection; about 20,000 of those infected die from their infection.
A. Organisms Causing Nosocomial Infections
Escherichia. coli, Enterococcus, Staphylococcus aureus, and Pseudomonas are responsible for one half of all nosocomial infections.
- Factors Fostering Nosocomial Infections
- immunocompromised patients– people with AIDS, organ transplant recipients (they take immunosuppressants so that the organ will not be rejected by their body), the elderly, cancer patients, patients taking steroids (ex. those with asthma).
- invasive medical procedures– ex. blood drawing, i.v.’s, urinary catheters, endoscopes, implants, coronary bypass surgery, hemodialysis, gynecological equipment, tooth extractions, injections
- antibiotic resistance– many bacteria found in hospitals have developed antibiotic resistance.
- Types of Nosocomial Infection: (From most common to least common)
- UTI’s (urinary tract infections)– usually E. coli, Proteus, Klebsiella, Enterobacter; can be from catheterization; more commonly results from improper hygiene (wiping the wrong way).
- surgical wound infections– most commonly Staphylococcus aureus & enterics; at least 10% of surgery patients develop an infection despite scrubbing, etc.!
- respiratory tract (ex. pneumonia) – include Streptococcus, Staphylococcus, Pseudomonas aeruginosa,enterics.
- skin infections– particularly in newborns (usually Staphylococcus aureus ) & burn victims (usually
Pseudomonas aeruginosa ).
- Nosocomial Infection Control
- hospitals hire hospital epidemiologists.
- once an epidemic is recognized, take cultures from hospital workers.
- patient isolation; reverse isolationseparates infection-prone patients from sources of infection (ex. the boy in the plastic bubble).
- enforce CDC program.
- treat every patient as if they are infected with AIDS.
IV. PUBLIC HEALTH: PREVENTING DISEASE – PROPHYLAXIS
Public health deals with disease prophylaxis (prevention); 2 methods of prophylaxis: 1.) decrease or eliminate the reservoir or interrupt disease transmission. 2.) immunization – artificially augments the body’s natural immune defenses.
- Decrease or Eliminate the Reservoir or Interrupt Disease Transmission
- Clean Water – diseases such as cholera, typhoid fever, & diarrhea can be spread when human sewage contaminates the water supply.
- Clean Food – pasteurization, boiling, adequate cooking, refrigeration prevent food poisoning, trichinosis (roundworm), salmonellosis, tapeworm infection, etc.
- Personal Cleanliness – hand washing of #1 importance.
- Insect Control– to decrease mosquito populations early programs drained swamps, screened living areas, used mosquito netting, used insecticides such as DDT (until it was found to be carcinogenic to humans!); now efforts concentrate on educating the public to remove stagnant water; biological control is also used – ex. Gambusia, the mosquito fish, was introduced to the U.S. – this fish feeds on mosquito larvae
- Prevention of STD’s– public education, limit sexual exposure, use of condoms.
- Prevention of Respiratory Diseases– isolate infected individuals, wear face masks; most effective way is immunization.
- Active Immunization (= Immunization or Vaccination)
- Active Immunization Defined – a person’s own immune system is stimulated, memory cells are produced to protect against future natural infection.
- Vaccine Defined– an agent containing antigen capable of inducing active immunity without causing disease; vaccines must be safe & immunogenic (stimulate an immune response strong enough to confer protection against natural infection); vaccines can be given orally, subcutaneously (below skin), or intramuscularly; some stimulate both Ab & cell mediated immune responses, other stimulate primarily Ab mediated immunity.
- Types of Active Vaccines
1.) attenuated – Live, weakened viruses or bacteria; virus is cultivated in the lab until it loses its virulence; the organism is then injected into a human and allowed to multiply; may cause a limited infection, usually without serious illness; provides strong & long-lasting immunity. Ex. tuberculosis (b), oral Sabin polio (v), mumps (v), measles (v), rubella (German measles) (v). The latter 3 are referred to as MMR. The fairly new chicken pox vaccine is also attenuated. This type of vaccine is not recommended for those who are immunocompromised. 2.) inactivated (killed) – By heat or chemical agents such as formalin, phenol, or acetone; process can destroy the Ag’s that stimulate immunity (ex. heat denaturation of protein Ag’s); inactivated microorganisms can’t multiply in host so vaccine dose must contain enough Ag to produce a protective immunologic reaction; usually requires a booster; Ex. pertussis (b), typhoid fever (b), rabies (v), Haemophilus influenzae type B (b) (causes meningitis), injectable Salk polio (v) (sometime referred to as IPV – inactivated polio vaccine), cholera (b), viral influenza Haemophilus influenzae type B – combined polysaccharide Ag with a protein to make it more powerful (polysaccharides are weak stimulants of Ab production); called a protein conjugate vaccine. 3.) genetically engineered – Genetic engineering & recombinant DNA technology have allowed us to use bacteria to produce the protein antigens found in the capsids of certain viruses and the cell envelopes of bacteria. Scientists determine the genetic code for these antigens & insert the gene into the chromosome of bacterial cells. The bacteria produce the antigens coded for on the inserted genes when they go through their regular process of protein synthesis. These antigens can then be injected as a vaccine (your body doesn’t care if the protein antigens are in the real viral capsid or if they were made by a bacterium; they are the same proteins & your body’s immune system will respond to these antigens in the same way). These vaccines do not pose the same risks as inactivated and attenuated viruses! Examples: Pertussis (b) – the inactivated vaccine contains many Ag’s that contribute to the frequent undesirable side effects of this vaccine; an acellular, genetically engineered vaccine has recently been licensed; it has fewer side effects, but may not stimulate vigorous immunity. Hepatitis B (v) – originally produced from recovering viruses from the serum of infected patients, which could have other diseases like AIDS (made people nervous); now a genetically engineered vaccine. 4.) toxoids – for diseases caused by exotoxins rather than the microorganisms themselves, vaccines are made of toxoids(toxins that have been modified by heat or chemical agents to render them harmless); toxoids stimulate the production of Ab’s called antitoxins; ex. tetanus (b), diphtheria (b).
- Passive Immunization
- Defined– Ab’s from an immune person or animal are transferred to a patient; like an Ab transfusion!
- gamma globulin, a collection of Ab’s from the pooled serum of many different donors;
- special preparations contain high titers of specific Ab’s; ex. varicella zoster (v), (chickenpox & shingles), tetanus (b), mumps (v), measles (v), hepatitis A & B (v), rabies (v), pertussis (b)
- Advantages – even severely immunosuppressed patients can be protected & protection is immediate.
- Disadvantages– protection lasts only as long as the Ab molecules survive in the recipient – months if from a human, only weeks if from an animal; also a risk of serum sickness.
Serum sickness – Occurs when proteins from animal serum are used in medical therapy; ex. horse antiserum is used in the treatment of venomous snake bites. Small concentrations of venom are injected into the horse to get it to produce antibody against the toxin. Patients then receive an infusion of these horse antibodies to bind to the snake venom antigen in their blood. The patients may produce antibodies against the horse antibodies, forming large complexes that are deposited in the tissues.
- Boosters– Immunity is not always life-long. Booster shots boost immunity by greatly increasing the numbers of antibody.
V. NOTIFIABLE DISEASES
These are infectious diseases that are potentially harmful to the public’s health and must be reported by physicians to the CDC. Make sure you can list some of these! Return to Chp. Index Chap. 13 – Host-Microbe Relationships and Disease Processes
- HOST-MICROBE RELATIONSHIPS
Pathogen – parasite capable of causing disease Host – an organism that harbors another organism
- Symbiosis– two different organisms living together.
- commensalism – one organism benefits, the other is neither harmed or benefited. Many bacterial species fall into this category. Many individual bacterial species by themselves don’t provide direct benefit to the host.
- mutualism – both partners benefit.
Ex. ruminants (cud-chewing animals) and termites have microbial species that break down cellulose from plant cell walls so that it can be used for energy by the animal; this relationship is essential for the ruminant. Ex. Large numbers of E. coli live in the large intestine of humans. These bacteria release vitamin K, which we use to make certain blood-clotting factors. Ex. Collectively an organism’s natural flora protects the host by competing with and edging out many pathogens. This phenomenon is called microbial antagonism.
- parasitism – host is harmed, the parasite benefits; microbial parasites = pathogens. A narrow definition of parasites would include only eukaryotic pathogens such as protozoa, helminths, and arthropods (ticks, lice, fleas). A broader definition of this term includes viruses, bacteria, and fungi.
- Contamination , Infection, and Disease – A Sequence of Events
- contamination – the microbes are present.
- infection –multiplication of any parasitic organism within or upon the host’s body; growth of normal flora is usually not considered an infection; infection does not always cause disease
- disease – disturbance in the state of health (state of relative equilibrium in which the body’s organ systems are functioning adequately); disease is characterized by changes in the host that interfere with normal function.
- Types of Flora: (define and give specific examples for each)
- Resident flora(= normal) – microbial species present in/on human body throughout life; permanent species; coexist with humans in a stable relationship.
- What does washing do to these guys? Reduces, but does not eliminate.
- What parts of the body inhabited by normal flora?(external vs. internal surfaces)
External – skin, conjunctiva Internal – nose, mouth, intestinal tract, vagina, urethra, ear
- Transient flora – microbial species that can be cultured from body surfaces under certain circumstances, but are not permanent residents.
- What does washing do to these guys? Usually eliminates.
- Why aren’t they part of the body’s normal flora? Not well enough adapted to life on human body.
- Noscomial infections(hospital-acquired infections) – Hospital workers have a large transient flora population because of large number of pathogens they are exposed to every day (ex. pathogenic Staphylococcus aureus ); therefore, hospital workers must be extremely careful about hand washing.
- Opportunists – microbial species that cause disease when the proper opportunity arises, but are usually harmless; infections usually occur when bacteria get into a place where they don’t belong (ex. nonpathogenic bacteria that are part of the normal flora of the colon go crazy when they get into the urinary tract.)
- Name three opportunities for infection – breakdown in immune system, antibiotic treatment, bioimplantation of artificial devices (catheters, pacemakers, artificial joints);
- What’s one reason for a vaginal yeast superinfection? Antibiotic treatments reduce numbers of normal vaginal bacterial species; these bacteria usually keep the yeast Candida albicans in check.
- Changing Flora – Examples:
What is one good reason why mothers should breastfeed? It’s not important just for the nutrients and the antibodies the baby receives in breast milk. While on breast milk, a baby’s intestinal flora is composed mostly of Bifidobacterium, which metabolizes milk sugars into acetic and lactic acid. These acids reduce the pH of the intestine, making it inhospitable to many disease-causing microbes, most importantly those causing diarrhea. The intestinal flora changes when the baby is on formula and the same protection is not provided. What is the effect of estrogen on the vaginal pH? What effect does this have on the normal flora? Estrogen increases the growth of lactobacilli which produce an acidic vaginal environment, making it inhospitable to disease-causing microbes (ex. E. coli from feces). Newborns have high estrogen levels from estrogen that crosses the placenta. In a few weeks, this estrogen level falls off. It doesn’t increase again until puberty. This is important for when sexual activity could begin.
II. ESTABLISHING DISEASE
Pathogenicity is the capacity to produce disease. An organism’s pathogenicity depends on its ability to invade a host, multiply in the host, and avoid being damaged by the host’s defenses. Virulence refers to the intensity of the disease produced by pathogens, and it varies among different microbial species. A pathogen must overcome the following seven challenges if it is to survive on or in a human host & cause disease. Pathogenesis, a microbe’s ability to cause disease, depends upon its meeting all of these challenges. The seven challenges are:
- Maintain a reservoir(a place in which a pathogenic microorganism is maintained between infections).
- Human reservoirs – ex. pertussis, measles, gonorrhea, common cold.
A person who is ill from an infection is a reservoir. Healthy people can also be reservoirs – called carriers. incubatory carrier – in early symptomless stages of illness (most diseases have specific incubation periods associated with them; you may not realize you have an infection, but you can still be contagious). chronic carrier – person who harbors a pathogen for an extended period of time without becoming ill (ex. people who are HIV+ but have not developed AIDS); this group also includes people who recover from an illness, but harbor the pathogen (ex. Hepatitis B); people with herpes are also chronic carriers.
- Animal reservoirs– ex. for rabies the animal reservoirs are skunks, possums, bats, raccoons, etc.; in this case, the pathogen is spread through the bite of the rabid animal reservoir; insect vectors can also be involved in spreading pathogens from animal reservoirs to humans – ex. lyme disease (Borrelia uses deer and mice as a reservoir; a tick is the vector). Zoonosis – a human disease caused by a pathogen that maintains an animal reservoir. Mutations & genetic variation can occur in the reservoir, causing new strains to emerge.
- Environmental reservoirs– ex. soil, water, house dust; Clostridium tetani uses soil – it’s able to survive in this environment because of its ability to produce endospores; Vibrio cholerae uses water.
- Leave its reservoir & enter the body of a human host.
Disease transmission takes place when a pathogen leaves a reservoir and enters the body of a host. Most have a preferred portal of entry. The number of pathogens that reach the portal of entry influences the likelihood of successful disease transmission. The number of microbes that must enter the body to establish infection in 50% of test animals is expressed as the ID50 (infection dose). The LD50 (lethal dose) measures fatal infections – the number of microbes that must enter the body to cause death in 50% of test animals. The most common portals of entry for disease-causing microbes are external & internal body surfaces: skin, conjunctivae (around eyes), nasal cavity & nasopharynx, mouth, intestinal tract, vagina, urinary tract, etc. Others include tissues below the skin in the case of an open wound or the placenta. Transmission can occur in several ways:
- Human-to-human (communicable diseases– transmitted from one person to another)
- Respiratory droplets expelled by coughing, sneezing, talking; ex. Bordetella pertussis(whooping cough); more human diseases are transmitted by respiratory transmission than by any other method.
- Direct body contact or person-to-person or horizontal transmission – transmission by touching, kissing, sexual intercourse; includes STD’s (sexually transmitted diseases); ex. gonorrhea; herpes is most commonly spread by kissing or exchange of saliva (common in young children).
- Vertical transmission – transmission from mother to infant; prenatal transmission – occurs across the placenta; perinatal transmission– occurs during passage through the birth canal; ex. STD’s such as syphilis, gonorrhea, HIV, Herpes.
- Fecal-oral route – can involve direct contact (ex. a person does not wash his hands after defecating and then shakes hands with someone); this transmission can also be by vehicles such as water, food, fomites, or vectors (flies, etc.); (crops and water supplies may be contaminated with fecal matter).
- Airborne Transmission – these pathogens are hardy enough to withstand prolonged drying; can be transmitted across great distances (greater than a meter); can remain viable in dust & reenter the air; ex. Mycobacterium tuberculosis.
- Vehicle Transmission (objects such as food, water, fomites) Fomites– inanimate objects such as cups, towels, bedding, eating utensils, bedding, & handkerchiefs; ex. common cold viruses.
- Parenteral Transmission– occurs when a biological arthropod vector introduces pathogens during a skin-penetrating bite or when breaks in the skin or mucous membranes provide microbes with access to deeper tissues; can also occur from penetration with a hypodermic needle; ex. HIV, hepatitis B virus, Plasmodium (a protozoan that causes malaria; mosquito vector), Clostridium tetani (causes tetanus when anaerobic conditions are created in deep wounds)
- Vectors – usually insects or other arthropods(mosquito, tsetse fly, tick, flea, lice, etc.)
- mechanical vector – ex. a fly lays its eggs in dog feces and then lands on your sandwich.
- biological vector– ex. the protozoan parasite that causes malaria goes through a stage in its life cycle in the mosquito. Could bacteria use biological vectors?
- Adhere firmly to the surface of the host’s body and thereby colonize it.
Pathogens, like normal flora, attach to specific types of target cells by means of adhesins (protein molecules that are very specific for the receptors that they bind to on target cell surfaces); many adhesins are molecular components of capsules or pili, thus these structures are are responsible for the virulence of many strain of bacteria; some bacteria have a repertoire of adhesins – this versatility makes them extremely virulent.
- Invade the body in order to enter cells or deeper tissues.
Only a few pathogens cause disease by colonizing surfaces; most have additional virulence factor that enable the pathogen to invade tissues (in other words, most pathogens are invasive – they penetrate the body’s surface to enter cells or deeper tissues). This ability allows them to escape certain host defenses and to gain access to a nutrient-rich environment that is free of competing microbes. Streptococcusproduces the enzyme hyaluronidase that digests hyaluronic acid, a glue like substance that helps hold the cells of certain tissues together. Some pathogens actually enter cells to live and multiply inside them; they are called intracellular pathogens. Ex. of intracellular pathogenic bacteria – Rickettsias (ex. Rocky Mt. Spotted Fever) & Chlamydias. Some bacteria gain entry into cells by adhering to surface receptors that fold into the cell during endocytosis. Most eukaryotic pathogens do not invade cells. An example of one that does is Plasmodium (a sproozoan protozoan that causes malaria); this parasite enter red blood cells.
- Evade the body’s elaborate defenses against microbial invaders. Here are just a few of the ways:
- Protection Against Phagocytosis by White Blood Cells.
- capsules– make bacteria slippery and hard for wbc’s to phagocytize; some bacteria are virulent only if they produce a capsule; ex. Streptococcus pneumoniae, Haemophilus influenzae.
- surface proteins– interference with phagocytosis; ex. Streptococcus pyogenes – produces M proteins – hairlike projections on the surface of its cell wall (kind of makes them prickly); because of these projections, these guys can be phagocytized only if antibodies bind to the bacteria, masking the M proteins.
- living inside the phagocyte (white blood cell)– only pathogens that possess special adaptations can survive the enzymes produced by the phagocyte; ex. Mycobacterium tuberculosis – survives because the phagocyte’s enzymes cannot break through its waxy outer layer.
- Antigenic Variation – some microbes mutate & change their surface antigens; a person may be immune to one strain, but not to another. Ex. Neisseria gonorrhoeae, HIV, cold virus, influenza virus
- Production of Exoenzymes (enzymes produced and then released by bacteria) – Ex. Coagulase – triggers blood clotting mechanism, allowing bacteria protection from immune
defenses. Ex. Staphylococcus IgA Proteases – enzymes produced by bacteria that destroy the IgA class of antibody. Ex. Neisseria. Streptokinase – dissolves blood clots so bacteria can spread to other tissues.
- Multiply within the body, perhaps producing toxic products or stimulating host reactions that cause disease.
The 2 most common forms of bacterial pathogenesis are
- the production of toxins (poisonous products that harm human cells and tissues ) & exoenzymes.
- stimulation of the body’s defenses.
- Produced by G(+) or G(-) bacteria.
- Bind to receptors on the surfaces of different types of cells.
- Specific for the cells they infect (ex. neurotoxins, such as tetanus & botulinum toxins effect only nervous tissue. Enterotoxins, such as those produced by Vibrio cholerae& Shigella effect only epithelial cells lining the intestinal tract).
- After binding, the enzymes enter the cell & disrupt cellular function, usually by inhibiting one specific metabolic reaction.
- Some exotoxins are unbelievably potent; ex. tetanus toxin – an amount about equal to the size of the period at the end of this sentence can kill an adult!
- Some exotoxins enter the bloodstream, causing systemic disease or toxemia(tissues throughout the body are affected).
- Diseases that result from the ingestion of a toxin are termed intoxicationsrather than infections. Ex. botulism food poisoning is the result of injesting toxins made by pathogens.
- Exotoxins can be neutralized with special antibodies called antitoxins. Certain exotoxins can be modified in the lab by treatment with heat or chemicals such as formaldehyde to produce toxoids; these molecules that have lost their disease-causing properties, but still stimulate the immune system to produce antitoxins (antibodies against toxin); vaccines can be produced from toxoids (ex. tetanus).
- In most exotoxin-caused diseases, such as cholera, tetanus, E. colidiarrhea, shigellosis, & pertussis, occur only if the bacteria multiply in the body. In other diseases, such as botulism (food poisoning caused by Clostridium botulinum ), the toxin is produced outside the body & is ingested with contaminated food – called a food intoxication.
- Some are enzymes. Ex. Hemolysinswhich lyse red blood cells. Alpha-hemolysin partially breaks down hemoglobin (the oxygen-carrying protein in rbc’s), leaving a greenish halo around colonies grown on blood plates. Beta-hemolysin completely breaks down hemoglobin, leaving a clear ring around colonies. Hemolysins are produced by streptococci and staphylococci.
- Leukocidins produced by stretptococci and staphylococci damage or destroy certain kinds of white blood cells. While most diseases are characterized by an elevated white cell count, some may result in a decrease in numbers of wbc’s.
- May be named after the part of the body they affect. Ex. neurotoxin, enterotoxin
- Released only by G(-) bacteria; all G(-) bacteria produce endotoxins.
- Endotoxins are lipopolysaccharides molecules (LPS’s) in the outer membrane.
- Its effects include: fever, increased or decreased #’s of wbc’s, shock, death, diarrhea
- Endotoxins are not secreted by bacteria, but are released into the environment when the bacterial cell dies.
- Unlike exotoxins, endotoxins are not proteins, so they are relatively heat stable.
- Unlike exotoxins, endotoxins don’t stimulate the immune system to produce antibody; therefore, toxoid vaccines would be useless.
- Unlike exotoxins, endotoxins are not specific for the cells they effect.
- Normally clinically significant only when large numbers of dying bacteria are circulating in the bloodstream; paradoxically, agents that kill G(-) bacteria (antibiotics, etc.) may actually increase endotoxin-mediated damage.
- Stimulation of the Body’s Defenses
Ex. Streptococcus pneumoniae – when it multiplies in the lungs, phagocytes (white blood cells) come to combat the infection; however, this bacteria is protected by a capsule so more and more phagocytes arrive to help; dead bacteria & phagocytes accumulate in the lungs, impairing normal gas exchange & making breathing difficult.
- Leave the body and return to a reservoir &/or enter a new host.
The anatomic route through which a pathogen usually leaves the body of its host is called its portal of exit. For most respiratory pathogens, the portal of exit is the same as the portal of entry. For most gastrointestinal pathogens, the portal of entry is the mouth & the portal of exit is the anus. STD’s exit the same way they enter – across the mucous membrane surfaces of the genital tract. Parenterally transmitted pathogens exit the same way they enter – in the blood.
III. TYPES OF INFECTIOUS DISEASE
Acute disease – develops rapidly and runs its course quickly Chronic disease – develops more slowly, is usually less severe, and persists for a long period. Latent disease – characterized by period of inactivity (ex. Herpes) Local infection – confined to a specific area Systemic infection – generalized infection; affects most of the body. Septicemia – pathogens are present in and multiply in the blood Primary infection – initial infection in a previously healthy person. Secondary infection – follows a primary infection (ex. a bacterial infection following a cold). Superinfection – secondary infection that results from the destruction of normal microflora and often follows the use of broad-spectrum antibiotics. Mixed infection – caused by several species of organisms present at the same time.
- STAGES OF INFECTIOUS DISEASE
A. INCUBATION PERIOD – Time between infection and the appearance of signs and symptoms. Although the infected person is not aware of the presence of an infectious agent, she can spread the disease to others. Each infectious disease has a typical incubation period.
- PRODROMAL PHASE(prodromos = forerunner) – Short period during which nonspecific, often mild, symptoms such as malaise and headache sometimes appear. You feel like you’re coming down with something.
- INVASIVE PHASE– Period during which the individual experiences the typical signs and symptoms of the disease (fever, nausea, rash, cough, etc.). During the acme part of this phase, signs and symptoms reach their greatest intensity. In some diseases this phase may be fulminating (sudden and severe), in others it may be persistent or chronic. A period of chills followed by fever marks the acme of many diseases. The battle between pathogens and host defenses is at its height during this stage.
- DECLINE PHASE – Symptoms begin to subside as the host defenses and the effects of treatment if being administered finally overcome the pathogen. Secondary infections may occur during this phase.
- CONVALESCENCE PERIOD – Tissues are repaired, healing takes place, and the body regains strength and recovers. Individuals no longer have disease symptoms, but they may still be able to transmit pathogens to others.
Return to Chp. Index Chapter 14 – Nonspecific Defense Responses NONSPECIFIC DEFENSE RESPONSES – 1st & 2nd LINES OF DEFENSE [Nonspecific defenses are general attack responses; the response is the same, no matter who the “invader” is.]
- The Body’s First Line of Defense: Structural, Mechanical, & Chemical Defense Responses on Internal & External Body Surfaces:
- Skin & mucous membranes (epithelial surface tissues)
- Cells are tightly joined together, preventing bacteria from invading deeper tissues.
- Sloughing of dead cells prevents microbial population from continually increasing.
- The protein, keratin, fills the cells in the outer layers of the epidermis. These cells then contain little water, making the skin dry & inhospitable to many microbes.
- Ciliated, mucous membranes (ex. in the respiratory tract) trap microbes, dust etc. in mucous & cilia move mucous toward mouth, where it is coughed up and swallowed.
- Normal flora– Normal bacterial inhabitants of the skin, gut, & vagina – the “natives” outcompete the “foreigners” for resources. Also, some normal bacteria produce acid from sugar fermentation, creating an acidic environment that keeps other populations in check (ex. lactic acid produced by bacteria in the vagina keep the yeast Candida albicans under control).
In the vagina, low estrogen concentrations in prepubertal and postmenopausal women result in a decrease in bacterial numbers in the vagina; this can lead to vaginal yeast infections. Yeast infections can also result from antibiotic treatments (broad spectrum antibiotics kill the pathogen and the normal flora) & douching.
- Movement of body fluids dislodges microbes. Ex. urine, tears, saliva. Peristalsis in digestive tract causes food & digestive juices to sweep microbes away. (Urine itself is not microbiocidal!)
- Tears, perspiration, & saliva contain lysozyme, an enzyme that destroy the bacterial cell wall. Lysozyme is especially destructive to G(+) bacteria because they lack an outer membrane.
- Perspiration also contains high concentrations of salt, creating a hypertonic environment.
- Bile, produced by the liver, also disrupts the bacterial cell wall. Bile is secreted into the small intestine to aid in the digestion of lipids. It passes from the small intestine into the colon in feces; the bacterium E. coli, which is part of the normal flora of the colon, is resistant bile. Remember that bile salts are an important ingredient in some selective media that select for G(-) bacteria and against G(+) bacteria.
- Hydrochloric acid produced in the stomach (pH of the stomach is 2!).
- Fatty acids are contained in the oil secreted from oil glands in the skin. It makes the skin slightly acidic.
- The Body’s Second Line of Defense – What Happens Once the Microbes Get Past the Surface Defenses:
First a little about the types of white blood cells (called leukocytes):
- macrophages– phagocytic
- eosinophils – phagocytic
- neutrophils– phagocytic
- basophils – release histamine; involved in the inflammatory response.
- lymphocytes – 3 types: B cells, T cells, Natural Killer cells.
(Be careful not to get the terms leukocyte and lymphocyte confused!)
- Natural Killer Cells– Type of lymphocyte (type of wbc); most lymphocytes are involved in specific defense responses (ex. B & T lymphocytes). NK cells are unlike other lymphocytes in that they lack antibodies & antigen receptors (we’ll talk about these under the specific defenses); they are like a specific type of T lymphocyte called a killer T cell in that they release perforins [chemicals that cause lysis of the bacterium – they perforate or punch holes in the cell envelope of bacterium].
- Phagocytic White Blood Cells (Phagocytes) – Phagocytosisoccurs in 3 phases:
- Chemotaxis– the chemical attraction of phagocytes to a particular location; chemotactic chemicals that attract phagocytes include bacterial toxins components of damaged tissue cells, complement proteins, & antibodies.
- Adherenceor Attachment – Because of certain microbial defenses, adherence of the phagocyte cell membrane to the surface of the microbe may be difficult (for example some bacteria produce a slimy outer capsule that makes them slippery). Opsonization of microbes by complement proteins and antibody facilitates phagocytosis.
- Ingestion– The phagocyte engulfs the microbe with its cell membrane. The engulfed microbe moves into the cytoplasm of the phagocyte inside a vesicle (sac); these vesicles fuse with lysosomes containing digestive enzymes; phagocytes include the wbc’s such as neutrophils, eosinophils, & macrophages; phagocytes circulate within blood vessels, & are also located in the lymph nodes, spleen, liver, kidneys, lungs, joints, skin, red bone marrow, & brain.
Fever – When phagocytes ingest certain bacteria, the phagocytes secrete a type of interleukin, which circulates to the hypothalamus & causes it to secrete prostaglandins; these chemicals “reset” the hypothalamic thermostat at a higher temperature; temperature-regulating mechanisms (vasoconstriction, increased metabolism, shivering) act to bring the core body temperature to this new setting. [Aspirin, ibuprofen, & acetaminophen inhibit the synthesis of prostaglandins.] A low grade fever has a beneficial effect on the body: 1.) It inhibits the growth of some microbes. 2.) It increases the heart rate so that white blood cells, etc. are delivered to infection sites more rapidly. 3.) B cell & T cell proliferation (division) increases. 4.) Heat speeds up chemical reaction rates. High grade fevers are dangerous – they can denature the body’s own enzymes & other proteins. What’s low grade? What is considered low grade in infants is much lower in adults. This is due to a baby’s higher surface area to volume ratio. Basically, a baby has more surface area compared to her volume than an adult does. So, it’s easier for heat to reach the skin and dissipate into the air. Heat does not dissipate as easily from an adult’s body (too much volume for it to move through) and so it does more damage to internal organs.
- Interferon (IFN) – Interferons are proteins that are produced by certain viral-infectedcells (particularly macrophages). Once interferons are released from viral-infected cells, they diffuse to neighboring uninfected cells & bind to their surface protein receptors. This binding induces the uninfected cells to synthesize antiviral proteins that interfere with or inhibit viral replication. In other words, interferons serve as a red flag to warn uninfected cells that there’s a “stranger among us” & the uninfected cells take action to protect themselves.
Certain interferons also enhance the activity of phagocytes & natural killer cells. Certain interferons also inhibit cell growth & suppress tumor formation. Ex. Alpha-IFN is approved in the U.S. for treating Kaposi’s sarcoma, a cancer that often occurs in patients infected with HIV; it is also used for treating genital herpes & hepatitis B & C.
- Complement System– When certain microorganisms invade the body, about 20 complement proteins in blood plasma & on cell membranes interact as a system. When activated, these proteins “complement” or enhance certain immune, allergic, & inflammatory reactions; therefore, the complement system enhances the effectiveness of both nonspecific & specific defense responses. Complement proteins respond to the binding of antibodies to the cell membrane of the invading microbe; the complement proteins are activated one after another in a “cascade” of reactions [one reaction catalyzes the next.] These reactions have the following results: (use the acronym COLA to remember them!)
- Chemotaxis– They act as chemotactic chemicals to attract phagocytes to the scene.
- Opsonization– Complement proteins bind to the surface of the microbe & then interact with receptors on phagocytes to promote phagocytosis. In this way complement proteins give macrophages a “foot hold.”
- Lysis– Other complement proteins kill the microbe by causing lysis.
- Activation of Inflammatory response(See below).
- Inflammatory Response – Many cells, the complement system, & other substances take part in this response. This response is a series of events that destroys invaders & restores damaged tissues to normal. The 4 major symptoms of inflammation are redness, heat, swelling, & pain(think about what happens when a bee stings you or a cut gets infected). Inflammation is a nonspecific defense – the response of a tissue to a cut is similar to the response that results from a burn, radiation, or microbial invasion. The inflammatory response involves the following events:
- Vasodilation & Increased Permeability of Blood Vessels –
¨ Vasodilation is an increase in diameter of the arterioles. Arteriole dilation enables white blood cells & other substances to more easily penetrate the tissues. Increased permeability means that an increased amount of material is allowed to pass out of the blood vessels. ¨ Blood vessels dilate & become more “leaky” due to the release of histamine by basophils, which are activated by the complement system. Prostaglandins, released by damaged cells, intensify the effects of histamine. ¨ Within minutes after injury, dilation & increased permeability of blood vessels produces heat, redness, & swelling. ¨ Warmth & redness occurs from the large amount of warm blood flowing through the area. Temperatures will continue to rise due to the release of heat energy from chemical reactions (increased metabolic activity). ¨ Fluid seeping from “leaky” capillaries causes swelling. ¨ Pain can result from injury of nerve fibers, from irritation by toxins produced by microbes, from increased pressure due to swelling, or from prostaglandin release.
¨ Fluid seeping from “leaky” arterioles causes local swelling & delivers more complement proteins to the tissues (remember, proteins are large molecules – they would normally stay in the blood vessels & couldn’t get into the interstitial spaces between cells). ¨ Phagocytes, following increased concentrations of complement proteins to affected tissues, engulf foreign invaders & damaged cells. ¨ Eventually phagocytes die. Within a few days a pocket of dead phagocytes & damaged tissue forms (called pus).
- Tissue Repair
¨ Platelets initiate clotting mechanisms, help wall off the pathogen, & help repair tissues. Return to Chp. Index Chapter 15 – Specific Defenses
THIRD LINE OF DEFENSE – SPECIFIC DEFENSE RESPONSES:
THE IMMUNE SYSTEM
When general attack responses are not enough to stop the spread of an invader & illness follows, three types of white blood cells (macrophages, T cells, & B cells) will counterattack. Their interactions are the basis of the immune system. Two important characteristics of the immune system are its specificity & its memory. Summary Table: White Blood Cells Involved in Specific Defense Responses:
- Macrophages – phagocytic; involved in inflammatory, antibody-mediated, & cell-mediated responses (nonspecific responses); important not only for phagocytosis, but also for antigen presentation.
- Lymphocytes (these are not all of the lymphocytes, but some of the important ones)
- B cells– produce antibodies (Ab) [Y-shaped protein molecules which bind to specific targets (antigens) & tag them for destruction by phagocytes or the complement system].
- Cytotoxic T cells – involved in the cell-mediated response; directly destroy body cells already infected by certain viruses or parasitic fungi.
- Helper T cells– involved in the antibody-mediated & cell-mediated responses; they stimulate the rapid division of B cells & cytotoxic T cells by producing compounds called interleukins.
- Memory cells– certain B cell & T cells, which are produced during a first encounter with a specific invader (primary immune response), but are not directly involved in this first attack; they circulate freely & respond rapidly to any subsequent attacks (secondary immune response) by the same type of invader.
Recognition of Self & Nonself Among the surface proteins on your own body cells are MHC markers (“self” markers), which are normally ignored by your own white blood cells; MHC markers are unique to each individual (no one has the same kinds, except in the case of identical twins); microbes, etc. also have markers [called antigens (Ag) because they are foreign to your cells] on their surfaces which are not ignored by white blood cells; antigens are usually surface proteins with distinct configurations that trigger immune responses.
- PRIMARY IMMUNE RESPONSE:
A first-time encounter with an antigen elicits a primary immune response from the lymphocytes & their products. We will consider an antibody-mediated immune response & a cell-mediated immune response to such an encounter:
- Antibody-Mediated (Humoral) Immune Response – The main targets of this type of response are extracellular organisms: bacteria, extracellular phases of viruses, some fungal parasites, & some protozoans. Antibodies can’t bind to antigen if the invader has already entered the cytoplasm of a host cell!! The following events involve a bacterial infection:
- Macrophages– When bacteria enter the body, their invasion triggers a general inflammatory response, & macrophages engulf some of the bacterial cells by phagocytosis. The engulfed bacteria move into the cytoplasm of the macrophages inside vesicles. These vesicles fuse with lysosomes (vesicles containing digestive enzymes) & enzymes digest the bacterial cells, but do not destroy their surface antigens. At the same, the cell synthesizes MHC markers & is packaging them into vesicles in its golgi apparatus. The vesicles containing the antigen & the vesicles containing the MHC markers fuse. Inside the vesicle, the antigen binds to the MHC markers (now called antigen-MHC marker complexes). The vesicle containing the complexes undergoes exocytosis & the complexes are inserted in the cell membrane of the macrophage. Macrophages can now present the antigen to other white blood cells (ex. helper T cells).
- Helper T cells – When the appropriate helper T cells make contact with the macrophages, some of their membrane-bound antigen-receptorsbind to the macrophage antigen-MHC complexes (these receptors are specific for these particular complexes – they won’t bind to any other type!). This binding causes macrophages to secrete a compound called interleukin that stimulates the helper T cells to secrete their own interleukins. The helper T cell interleukins will cause activated B cells to start dividing (see below).
- B cells
- B cells “mature” in bone marrow. While each B cell is maturing, it makes many copies of just one kind of antibody (each B cell is unique in that it will only make one kind of antibody that no other B cell makes – each kind of antibody will only react to one antigen). While the B cell is maturing, some of the antibodies it is producing become positioned at the cell’s surface, where they will later bind to a specific antigen. The “tail” of each antibody is embedded in the cell membrane, & the “arms” stick out above the cell membrane’s surface. From the bone marrow, B cells migrate to the lymph nodes, the spleen, or lymphatic tissue in the g.i. tract.
- When a B cell is released from the bone marrow into circulation, it is known as a “virgin” B cellbecause its antibodies have not yet made contact with antigen.
- A virgin B cell with the right antibodies binds to a specific antigen. Some of the antigen is then taken into the B cell, combined with MHC markers, & moved to the B cell surface (see how macrophages do this above). The B cell is now said to be activated; it’s no longer virgin – it’s come into contact with a specific antigen.
- If an activated B cell interacts with the appropriate interleukin-producing helper T cell (see above under helper T cell), the B cell will start dividing quickly, giving rise to a clonal populationof identical B cells.
- Part of the B cell clonal population differentiates into plasma cells, which secrete thousands of copies of the particular antibody that had been produced by the virgin B cell (the antibody actually leaves the plasma cells!). Antibodies have different effects on antigen:
1.) Neutralizing – the binding of Ab with AG blocks or neutralizes the damaging effect of some bacterial toxins and prevents attachment of some viruses to body cells. 2.) Immobilization – If Ab forms against cilia or flagella of motile bacteria, the Ab-Ag complex may cause the bacteria to lose their motility, limiting their spread into nearby tissues. 3.) Agglutination – Because antibodies have tow or more sites for binding to Ag, the Ab-Ag reaction may cross-link pathogens to one another, causing agglutination (clumping together); this enhances phagocytosis. 4.) Activation of complement system– complement proteins cause lysis of microbe; complement also cause opsinization, which enhances phagocytosis. 5.) Opsinization – Antibodies enhance phagocytosis by coating the microbe (remember that complement protein can also be opsonins).
- Some of the B cell clones differentiate into memory B cells, which are involved in a secondary response (will be discussed later).
How do B cells produce the millions of different antibodies required to detect all of the millions of possible antigens? Part of each arm of an antibody is a polypeptide chain made up of amino acids, folded into a groove or cavity, which “fits” with the antigen (there are poor fits & better fits – the better the fit the better the immune response). All B cells have the same genes for coding the amino acids in the chain, but each maturing B cell shuffles the genetic code into one of millions of possible combinations, so that the sequence of amino acids then gets shuffled (this changes the shape of the protein, thus changing the shape of the antibody). So B cells can give rise to virtually unlimited chain configurations. Therefore, when an antibody comes into contact with an antigen for the very first time, the right antibody just happened to be there! Your immune system did not produce the virgin B cell antibodies in response to a particular antigen! It is our genes that determine what specific foreign substances our immune system will be able to recognize & resist! (The same rule applies to T cell Ag receptors.) The 5 Classes of Antibodies (Ab): (Ab’s are part of the immunoglobulin (Ig) family of proteins) 1.) IgG – largest class, activates the complement system; effective opsonin (enhances phagocytosis); only Ab that can cross the placenta (protects the newborn for several months after birth); predominates in secondary immune responses; all antitoxins belong to this class (remember antitoxins are antibodies made against exotoxins made by G(+) & G(-) bacteria); found in blood & extracellular fluids. 2.) IgA – second largest class; found in blood and body secretions (saliva, milk, mucus, tears); protects mucosal surfaces, especially preventing attachment of viruses; its presence in colostrum defends the g.i. tract of newborn humans against infection. 3.) IgM – extremely effecting in fixing complement; sometimes called early Ab, because it is the first Ab to form during a primary immune response; its structure allows it to build complex Ag-Ab lattices that clump, forming a visible precipitate; it’s found in blood & extracellular fluids. 4.) IgD – main type of Ab displayed on the surface of B cells. 5.) IgE – fixed to the surface of basophils; stimulates the microbe to release histamine when the basophil binds to Ag; basophils then releases histamine; this can contribute to allergies
- Cell-Mediated Immune Response (Cellular Immunity) – This type of response deals with viruses & other pathogens that have already penetrated host cells (they are intracellular!), where they remain hidden from antibodies. In the cell-mediated immune response, the host cells are killed by cytotoxic T cells (killer T cells) before the pathogens can replicate & spread to other cells. The following events involve a viral infection:
- Cytotoxic T cellsor Killer T cells – Cells in the bone marrow give rise to forerunners of killer T cells, which travel to the thymus gland, where they mature into killer T cells. Each T cell produces antigen receptors that become positioned at its surface (these receptors are not antibodies, but are similar!!!). Ag receptors recognize specific Ag-MHC marker complexes. When an Ag enters the body, only a few T cells have receptors that can recognize & bind to the Ag.
- Killer T cells are released into circulation by the thymus gland. When a virus infects a cell, viral proteins become associated with MHC markers on the host cell’s surface.
- The antigen receptors of killer T cells, bind to the antigen-MHC complexes of macrophages, infected cells, etc.
- Cytotoxic T cells secrete perforins(proteins that punch holes in the infected cell’s cell membrane).
- This kills the infected cell, but prevents the virus from replicating & spreading to other cells.
Note: As in antibody-mediated immune responses, macrophage-stimulated helper T cells stimulate killer T cells to divide by secreting interleukins. This creates a clonal population of killer T cells, all with the same antigen receptor as the original killer T cell (these cells are not called plasma cells!!!!!). As with B cells, some of the clones become memory T cells & will be involved in a secondary immune response. (When the body rejects a tissue graft or an organ transplant, cytotoxic T cells are one of the reasons why. They recognize MHC markers on the grafted cells as being foreign. Organ recipients take drugs to destroy cytotoxic T cells, but this compromises their ability to mount immune responses against pathogens.)
II. TYPES OF IMMUNITY
- Active– a product of a person’s own immune system.
- naturally acquired– comes from infections encountered in daily life.
- artificially acquired– stimulated by vaccines.
- Passive– Ab’s produced elsewhere are given to a person.
- naturally acquired– refers to Ab’s transferred from mother to fetus across the placenta & to the newborn in colostrum & breast milk.
- artificially acquired– consists of Ab’s formed by an animal or a human & administered to an individual to prevent or treat infection; ex. hepatitis A, diphtheria.
III. SECONDARY IMMUNE RESPONSES: A secondary immune response to a previously encountered antigen can occur in 2 or 3 days. It is greater in magnitude than the primary response & of longer duration. This is because some of the B & T cells of the clonal populations do not get involved in the primary response attack. They circulate for years as memory cells. When a memory cell encounters the same type of antigen that initiated the primary response, it divides at once (no helper T cells are needed to stimulate cell division!). A large clonal population of active B or T cells can then be produced in just a matter of days.
Defined: Immunization means deliberately introducing an antigen into the body that can provoke an immune response & the production of memory cells. The first injection elicits a primary immune response. A second injection (the “booster shot”) elicits a secondary response, which provokes the production of even more antibodies and memory cells to provide long-lasting protection against the disease. We will discuss the types of vaccines in chapter 15. Return to Chp. Index Chapter 16 & 17 – Pracatical applications of immunology & Immune Disorders There are many practical applications that one can use in the diagnostic immunology laboratory with respect to disease detection and disease monitoring. Many of the same processes that occurr in the human body, such as Ag-Ab interactions, can be utilized in a laboratory setting to augment disease conditions. The following 3 laboratory based detection methods are examples of very common diagnostic tools used in clinical settings, public health labs, and in research settings. Be aware that there are MANY more of these types of detection methods. I have chosen to describe the following 3 because of there “basic” principles behind each technique and due to their popularity with respect to high sensitivity and specificity. 1) Agglutination reactions occurr when an Ab and Ag are specific for each other and a “lock and key” lattice of Ab-Ag forms to allow for an observable agglutination reaction. An example is the Rapid Plasma Reagin (RPR) kit used for the detection/screening of syphillis caused by the bacterium, Treponema pallidum. The IgM antibody is often referred to as early Ab or the 1st Ab to appear in an infection. It is also known as the Ab of agglutination because of it’s molecular make-up. IgM is typically arranged as a pentamer (5 Ab’s bound together which allows for 10 sites of Ag attachment). An IgM pentamer (see figure in text) allows for many Ag’s to bind and form a complex lattice that will become insoluble in solution and be visible to the naked eye. Thus, one can use specific Ab for syphillis to bind to specific Treponema pallidum Ag’s and form a complex (this is typically called a “reactive”). The test is easy and low cost….because of these features, it makes an RPR test a useful and cheap screening test for this disease. 2) Fluorescent Antibody (FA) assays are one of the most common kits available to diagnostic labs for the detection of a variety of microbes. A kit will simply have an Ab that has been “labeled” with a fluorochrome such at FITC. The FITC will fluoresce when exposed to a uv light source on a microscope and “light up” as apple green fluorescence. If the reaction on a slide is not specific (ie. the Ag doesn’t match the labeled Ab) then there is no fluorescence. See text for illustrations of this test. 3) Enzyme Linked Immunoabsorbent Assays (ELISA) are also a very common kit available for diagnostic labs. The HIV screening test is an ElISA based test. The kit will have a specific Ab for HIV in a plastic coated well. A technician will then add serum from a patient. If the serum has HIV Ag, it will bind to the Ab in the plastic coated well. This step is followed by another Ab that is labeled with an enzyme. Then a substrate is added to the “sandwich” ELISA. If the labeled enzyme has attached in the previous step, the substrate will fit into the enzymes active site and a colorimetric reaction will take place. So, a typical color like yellow will be produced with a positive test. See the text for illustrations of this test. Immunological disorders are caused when the immune system malfunctions, producing either an inappropriate or inadequate immune response. Hypersensitivity – a misdirected response in which either Ab’s or T cells cause damage. While there are 4 types of hypersensitivities, I have chosen to discuss only those that are related to responses to microbes. You will probably learn about the others in an Anatomy & Physiology course.
- Types of Hypersensitivity:
- Type III (Immune-complex ) hypersensitivity – antibody mediated; IgM & IgG antibodies react with a person’s own antigens – bind to antigens that are free in circulation (in Type II reactions, Ab binds to Ag on tissues & cells); Ab-Ag binding creates complexes that remain soluble in body fluids; complexes can lodge in capillaries, activating complement & provoking an inflammatory response & attracting phagocytes; Ex:
- viral hepatitis – chronic infection from long-term exposure to microbial antigens
- farmer’s lung– contracted by people who continually inhale certain antigens from molds, plants, or animals
- serum sickness– occurs when proteins from animal serum are used in medical therapy; ex. horse antiserum is used in the treatment of venomous snake bites; patients receive an infusion of horse antibodies to bind to the snake venom antigen; the patients may produce antibodies against the horse antibodies, forming large complexes.
- Type IV (Cell Mediated or Delayed) hypersensitivity– does not involve Ab’s; a T cell encounters an Ag that matches its Ag receptors; this stimulates the T cell to divide; when sensitized or activated T cells encounter the Ag again they release interleukins that stimulate macrophages and initiate inflammation, causing tissue damage. Ex.:
- organ transplants(tissue grafts); immunosuppressive drugs are used; cyclosporine is an improvement – a product of a fungus, it interferes with T cell function, but not with B cell function.
- tuberculosis, leprosy– damage is caused by a granulomatous reaction (when a macrophage cannot completely destroy microbial Ag, it persists within the cell; these persistent Ag’s cause T cells to release interleukins that stimulate the production of a granuloma, a nodule of activated macrophages; the continuing inflammation that goes on within & around the granulomas displaces & destroys normal cells).
- Immunodeficiency Disorders
Immunodeficiency – failure to mount an adequate immune response
- Congenital – inherited or develop before birth & usually appear early in life; Ex.:
- severe combined immunodeficiencies (SCID)– B & T cell immunity are disabled.
- Acquired– occur later in life; caused by infection, cancer, or the side effects of immunosuppressive medication (ex. steroids); Ex.:
- AIDS – acquired immunodeficiency syndrome
- Cancer & the Immune System
Cells become cancerous when they under transformation, a process leading to uncontrolled division that produces large numbers of undifferentiated cells. These primitive, rapidly dividing cells form large growths called tumors that crowd and eventually kill normal neighbor cells. Transformed cells often express new surface antigens that are not found on normal cells & mark the cancer cells for destruction by T cells, NK cells, & macrophages. Cell transformation occurs frequently, but the immune system eliminates most malignant cells before they cause cancer. AIDS patients are at an increase risk for cancer (ex. Karposi’s sarcoma) because they lack the white blood cells to kill off the malignant cells. As immune defenses weaken with age, the elderly are also at an increased risk for cancer. Many anticancer drugs are highly toxic because they are nonspecific (nonspecific tumor destruction) – they kill normal rapidly dividing cells as well as cancer cells (ex. hair cells, intestinal cells, bone marrow stem cells). Cancer immunotherapy would be more effective. If we can determine unique antigens on the surface of cancer cells, then we can target only those cells for destruction. Return to Chp. Index