BACTERIAL PHYSIOLOGY SMALL GROUP. Monday, August 25, :00pm. Faculty: Adam Driks, Ph.D. Alan Wolfe, Ph.D.

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1 BACTERIAL PHYSIOLOGY SMALL GROUP Monday, August 25, :00pm Faculty: Adam Driks, Ph.D. Alan Wolfe, Ph.D.

2 Learning Goal To understand how bacterial physiology applies to the diagnosis and treatment of infections. Enabling Objectives To understand how the fundamentals of bacterial physiology apply to clinical problems, you will be able to: Identify the components of the bacterial cell Describe how these components are useful in diagnosis and contribute to virulence Describe how drugs that impair these components are used to treat disease Reading Assignment Lecture handouts Developed by Adam Driks, Ph.D.

3 Introduction and instructions This Patient-Oriented Problem-Solving (POPS) activity is composed of an outline of bacterial physiology and a pre-test. Before the POPS session: In the POPS session that precedes this one, each group will assign each of the problems to a different group member, who will then be responsible for leading a discussion on that problem during this POPS session. The discussion leader will not be responsible for providing an answer, but for coming prepared to direct the group effort to solve the problem. It is the responsibility of the discussion leaders to insure that each discussion is focused and does not take up too much time. Also, before the POPS session, answer the pre-test questions (on your own) and come prepared to discuss your answers. Use the outline of bacterial physiology to help you organize the information in the reading assignment. During the POPS session: 1) The class will go over the pre-test answers with the TA. 2) Discuss and answer the problems. The discussion leaders should record the answers. These problems are designed to illustrate how your knowledge of bacterial physiology will influence your approach to treatment. How you treat an infection is largely dependent on what you know about the infecting organism, and for many cases you will not have much detailed information about the weak points of the infecting bacterium. In such instances, your plan of treatment may have to rely on the fact that broad groups of bacteria share common architecture, and therefore may share common weak points. A challenge in your future practice will be to use the clinical presentations of diseases to determine which of these common features any given infecting organism possesses. 3) When the session is finished, each group will discuss their answers to the problems in sessions led by the TAs. Do not leave before you have discussed the answers with the TA or a faculty member. The group discussion with the TA is as important as any other part of the POPS.

4 OUTLINE OF BACTERIAL PHYSIOLOGY-From nutrition to reproduction I. Cells sense nutrients (small molecules) in the environment A. Cells sense and move towards nutrient by chemotaxis. Chemoreceptors bind nutrient molecules. As a result, a signal is transduced to the flagellar apparatus that directs the cell to swim up the nutrient gradient. II. Cells move to nutrient A. Cells swim towards food by means of a helical propeller (the flagellar filament) mounted on a rotary motor (the basal body). The motor, for many bacteria, is powered by a proton gradient across the plasma membrane, which generates a proton motive force. III. Cells take up (transport) molecules from the environment to their interiors A. Active transport. Many types of molecules are transported by this system. An important example is iron transport, in which extracellular iron is trapped by molecules called siderophores which are secreted by the cell and which bind the extracellular iron. The siderophore-iron complexes are then transported back into the cell. B. Passive transport. This occurs by diffusion. IV. Cells use nutrient to produce energy and synthesize macromolecules A. Production of molecules 1. Production of ATP a. Fermentation: A relatively inefficient process that produces alcohols and other organic compounds. b. Respiration: Used by aerobes. Electrons are passed step-wise from carrier protein to carrier protein, resulting in more efficient production of ATP than is possible during fermentation. It also generates a proton gradient (a difference in concentration of H + across the cell envelope; i.e. between the inside and the outside of the cell) and a proton motive force. 2. Synthesis of proteins a. Ribosomes: These are large complex structures built of RNA and protein that serve as protein-building factories. Ribosomes from all organisms are composed of a large and a small subunit. Bacterial ribosomal subunits are somewhat different than eukaryotic subunits, and this allows many antibiotics to target them uniquely. Chloramphenicol, lincomycin and erythromycin all inhibit the function of the large subunit. Tetracyclines, streptomycin and spectinomycin inhibit the function of the small subunit. b. Translation: mrna, encoding the primary sequence of the protein, feeds through the ribosome. The sequence of the mrna is read by the ribosome in groups of three nucleotides, each of which specifies a particular amino acid to be incorporated into the protein. As each successive triplet of mrna feeds through the ribosome and is decoded, an additional amino acid is fused onto the end of the growing protein chain. In prokaryotes, translation occurs in the same cellular compartment as

5 does transcription. Ribosomes and can even bind to an mrna before that mrna s synthesis is complete, and it is still attached to a polymerase molecule. This is in contrast to eukaryotes, in which transcription occurs in the nucleus, but translation only occurs after the mrna has been exported out of the nucleus. c. Folding: The nascent protein folds into a specific structure as it exits the ribosome. This structure may not be correct, and additional folding may be required for correct protein function. 3. Synthesis of DNA and RNA a. Complex macromolecular machines called polymerases synthesize nucleic acids by copying a nucleic acid template. These polymerases are fundamentally different between bacteria and other organisms in terms of both the numbers and types of subunits. b. DNA (replication): The chromosome is used as a template to produce a DNA copy of the genome, so that cell division may occur. The (usually) circular bacterial chromosome is replicated bidirectionally, meaning that replication starts at a specific point and proceeds in two directions, away from the origin of replication. c. RNA (transcription): specific regions of the chromosome containing individual genes are used as templates to produce mrna molecules that bear these gene sequences, so that proteins may be synthesized according to the gene sequences. B. Assembly of macromolecules 1. Assembly of peptidoglycan a. Precursors are synthesized in the cytoplasm b. The precursors are transported across the inner membrane to the region of the peptidoglycan. During transport, the precursor undergoes further modifications. c. The modified precursor is attached to a growing peptidoglycan-chain and crosslinked to other preexisting chains. This crosslinking is inhibited by penicillin. 2. Assembly of macromolecules containing protein: Including macromolecules that contain protein subunits exclusively (polymerases, flagella, exotoxins) and macromolecules that contain both RNA and protein (ribosomes, certain enzymes). a. Self assembly: Proteins contain all the information needed to bind to the correct partner within their own sequences. b. Guided assembly: Other cellular systems are needed to insure that molecules find their correct partners. Ex.s: Two molecules may bind only when they dock at a preexisting structure; a molecule can be synthesized adjacent to its binding partner; binding of two molecules may be facilitated by ensuring that they are both synthesized only at a discrete time in the organism s life (and other competitor molecules are not synthesized at this time); the molecules might be restricted to a particular region of the cell (i.e. the cell membranes) where they are more likely to find their binding partners.

6 V. Cell division A. Cells determine that it is the correct time to divide: cells monitor the DNA replication process and other cell parameters and permit division when conditions are adequate. B. The dividing cell separates into two distinct envelope-bounded cells. One cell can divide into two by either generating a ring of constriction in the cell envelope that eventually pinches the cell in two, or by forming a new region of cell envelope (the septum) at a mid-point within the cell that partitions it into two cells. When the division process is disrupted with low concentrations of drugs that inhibit peptidoglycan formation, long or oddly shaped cells often result.

7 Pretest. Answer these questions on your own before coming to the POPS session. Begin the session by comparing your answers with those of the other members of your group. When you have discussed the answers within your group, alert your TA. 1) a. Cells need specific enzymes to survive in an oxygen-containing environment. What are the cells protecting themselves against? b. What are these enzymes and what do they do? 2) a. What is the major environmental requirement for fermentation? b. What are the environmental requirements for respiration? c. Is one a more efficient way to generate energy than the other? 3. Describe a mechanism for sequestering iron when it is limiting in the host. 4. What is a multidrug efflux system, and why is it especially problematical for treatment of bacterial disease? 4. a. What is the target of lysozyme? b. Name two ways in which the peptidoglycan is important to bacteria. 5. a. What are the major components of the Gram-positive envelope? b. Structurally, the Gram-negative envelope appears very different than the Gram-positive envelope. What are the major reasons for this? c. What component of the outer membrane is toxic? d. What component of the Gram-negative envelope is most useful diagnostically? Problems. The person already assigned to each question will begin the discussion for that question, and direct it so that it makes efficient use of the available time. Problem I. Drug X causes rod-shaped bacteria to round up and become sensitive to osmotic shock, but has no effect on eukaryotic cells. When the treated bacteria are analyzed for the levels of total proteins, lipids, and peptidoglycan-related peptides and sugars, they are all roughly normal. The drug has this effect on both Gram-positive and Gram-negative bacteria, although it is more effective against Gram-positive than Gram-negative bacteria. 1) What structure is the most likely target for this drug? 2) Why would the drug have these effects on cell morphology and sensitivity to osmotic shock?

8 3) How does this drug cause its effect on bacteria without altering the levels of these compounds? 4) What is the most likely cause for the difference in action between Gram-positive and Gram negative organisms? Problem II. A company wishes to develop a system to deliver drugs to cancer cells using a bacterium that will secrete the appropriate drug in the patient s body. In outline, they plan to construct a DNA plasmid that has a gene whose expression results in the production, and secretion by bacterial cells, of the drug. They will then introduce the plasmid into a bacterial host, and inject bacterial cells bearing these plasmids into tumor bearing regions within patients. (Note: Pugsley, PNAS 93: , 1996, has a very interesting spin on an issue raised by this question. The paper is worth looking at.) 1) Design several (between two and five) drugs that can kill the target cancer cells but not inhibit the growth of the bacterial delivery system. In answering the problem, you may assume that for any drug you choose, the appropriate gene is available and that it is able to result in the production of the drug. There are several ways that you could tackle this problem. For example, you could design a system that would be toxic only to cancer cells. In this case, you could use the fact that cancer cells divide more rapidly then normal cells as a way to target them specifically. One way to approach this would be to design an analogue of some eukaryotic protein as a competitive inhibitor. Another approach would be to develop a system in which the bacteria would tend to migrate to specifically cancer cells or flourish only in direct contact with tumor cells. In this case, you would be focusing on specificity of targeting as opposed to specificity of toxicity. Would only one or the other strategy be sufficient for the therapy to be effective? Try to ensure that the therapy is not harmful to the patient. It is appropriate to be especially creative in answering this particular problem-there are a variety of good approaches to this problem. Problem III. A major determinant of bacterial infectivity is the ability of the pathogen to survive entry into the host. In the case of food-born bacterial pathogens, significant hurdles to overcome are the highly acidic environment of the stomach and the weakly acidic environment of the intestines. Pathogenic bacteria have a remarkable ability to survive the acidic conditions of the gut. In fact, a large part of the differences between infectious doses of organisms like Shigella (inf. dose bacteria), Salmonella typhi (inf. dose 10 5 bacteria) and Vibrio cholerae (inf. dose 10 8 bacteria) are due to different levels of stomach acid tolerance. Recent studies raise the possibility that there is a common mechanism used by related pathogens to survive exposure to acid. This mechanism is normally not activated, but becomes active in the presence of acid. In this view, the differences in survival in the stomach of various bacterial pathogens is largely due to differences in their ability to induce this mechanism under acidic conditions. If there was a

9 general way to defeat this protective mechanism, then the danger of contaminated food could be greatly reduced. In an effort to identify these common mechanisms, researchers set up an experimental animal model in which they can simulate the acidic conditions of the gut and study how pathogens sense and respond to the environment. They determined that some one hundred proteins are synthesized by the pathogen within minutes of contact with acid. 1) Clearly, these one hundred proteins will have a variety of very different functions, most or all of which will be involved in some way in promoting virulence. What would you speculate are some of the roles, in general terms, of the one hundred proteins? Of the various functions that you would predict, which of these should the researchers study in detail in order to design the most effective and powerful antibacterial therapies? Why? 2) The researchers succeed in identifying a single gene, that when mutated, results in cells which cannot produce any of the one hundred proteins. Of course, the cells have lost all acid tolerance. Using what you have learned in lecture, what is a likely function of the protein encoded by the gene? 3) The researchers succeed in characterizing the genes encoding several of the one hundred proteins. It turns out that four of them appear to have a special role, in that when any one of the four is deleted from the genome, twenty of the other proteins are no longer made. A different set of twenty proteins is absent for a mutation in each of the four genes. Therefore, the proteins encoded by these four genes are each involved in controlling, in some way, a separate set of twenty other proteins. What are likely roles for these four proteins? 4) After a long period of time, the genes for each of the one hundred proteins are identified and, one by one, they are deleted from the genome and their effects on infection in the animal model are assessed. The researchers discover that, for a group of seventy of the proteins, when any of the genes are deleted, the only protein that is subsequently missing is the one encoded by the gene. Furthermore, there is only a 5 % reduction in acid tolerance. Given that these genes do not seem to have an important role in regulating the appearance of any of the other one hundred proteins, and that they make a significant but small contribution to virulence, what is the most likely role of these proteins? When you are done: Alert a TA that you have completed the problems. They will lead your group in a discussion during which you will go over your answers to the problems. The POPS is not finished until you have had the TA led discussion.

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