Examination Candidate Number: Desk Number: BA, BSc, and MSc Degree Examinations 2017-8 Department : BIOLOGY Title of Exam: Bacterial pathogenesis Time Allowed: 2 hours Marking Scheme: Total marks available for this paper: 100 Sec on A: Short Answer / Problem / Experimental Design ques ons (50 marks) Sec on B: Essay ques on (marked out of 100, weighted 50 marks) The marks available for each ques on are indicated on the paper Instructions: Sec on A: Answer all ques ons in the spaces provided on the examina on paper Sec on B: Answer either ques on A or ques on B. Write your answer on the separate paper provided and a ach it to the back of the ques on paper using the treasury tag provided. For marker use only: For office use only: 1 2 3 4 5 6 Module total as % DO NOT WRITE ON THIS BOOKLET BEFORE THE EXAM BEGINS DO NOT TURN OVER THIS PAGE UNTIL INSTRUCTED TO DO SO BY AN INVIGILATOR page 1 of 9
SECTION A: Short Answer / Problem / Experimental Design questions Answer all questions in the spaces provided Mark total for this section: 50 1. You wish to determine a competitive index (CI) experiment to assess the role of a specific gene in causing bacterial diarrhea. The bacterial species is poorly characterized but the gene s sequence resembles an enterotoxin in a different pathogenic species. Your supervisor asks you to address the 3Rs first and also to convince him of the value of your proposed CI experiment. Explain the supervisor s request and outline how you would address both points. (10 marks) The answer should identify what the 3Rs are (reduced refine replace, animal welfare issue), and outline an approach to test toxicity in vitro (tissue culture cells, intestinal cells) with the mutant or purified protein first, and mention that if absence of toxicity can be shown to relevant tissue culture cells an animal experiment may be justified. The answer should clarify what the CI means and how this value is interpreted. The answer needs to address what it will mean if the CI is >1, or <1 for the role of the gene, or if it =1. The value of the CI should be clear, why competition with a WT has value. Thus, briefly if CI <1, it indicates that the gene is important in causing the symptoms, and that its role for the mutant can not be contributed by the wild type cells. If CI=1 then it indicates the gene does not contribute to gut colonization in the animal model system; if >1 is suggests the mutant is more virulence. Marks released if this is identified and discussed briefly further, for example identifying that if toxin is secreted CI may be close to 1, as function complemented by wt strain even though it is a virulence factor; if not secreted if colonies form CI around 1, or otherwise,1). Credit also awarded if the in vitro experimental data is tied back to a CI outcome. Marks will be released for conveying understanding of CI and 3Rs (4), for the approaches outline above or other sensible approaches (6). Higher marks will reflect clear and considered answers with full marks for original insight or comments as well. LO Define key terminology and describe important experimental approaches page 2 of 9
in the field of bacterial pathogenesis. Interpret and critically discuss experimental data and approaches in the field of bacterial pathogenesis. Compare virulence factors and strategies that contribute to the virulence strategies of different bacterial pathogens. 2. Briefly discuss the benefits and challenges of phase variation of an adhesin for a bacterial pathogen. A good answer should first clarify that phase variation generates randomly, phenotypic hetereogeneity among clonal cells that is reversible but heritable. (1) Some points that can be mentioned: Benefit: Phenotypic heterogeneity in general allows for this allows bet-hedging (no need to respond to environment) or division of labor. An off phase can be an advantage if the structure is immunogenic and thus will allow the bacterium to hide from the immune system (bet hedging). This could also facilitate release and thus spread to other tissues. (2) Challenge: off phase does mean that a part of the population is not expressing this factor, and thus can not benefit from it. The fact that it is random should be put into context, for example comparing with environmental regulation- evading the immune system is more generic and not tied into too many environmental cues. (2) A possible inclusion of regulation superimposed on the phase variation is correct, if put into context, but not required. A benefit of reversible (as opposed to mutations) is that the pathogen retains the full phenotypic capacity at the population level, providing the opportunity to deal with a new host etc. Other relevant points may also release marks. LOs Define key terminology and describe important experimental approaches in the field of bacterial pathogenesis. Describe critical stages of infection and identify different virulence strategies in this context. Compare virulence factors and strategies that contribute to the virulence strategies of different bacterial pathogens. page 3 of 9
3. In what ways is anaerobic respiration important for the success of E. coli in colonising host tissue? (10 marks) An ability to grow anaerobically is important, since t he environment in its natural habitat (the colon) is anaerobic. It is anaerobic due to t he abundance of carbon and a bsence of perfusion with oxygen / high population density of respiring bacteria. Respiration is significantly energetically more favourable than the alternative of fermentation. E. coli uses a variety of respiratory pathways to support generation of ATP anaerobically, such as nitrate respiration, nitrite respiration, DMSO respiration, TMAO respiration ( up to 2 marks for naming respiratory pathways ). These processes potentially allow E. coli to out-compete other bacteria lacking these capabilities. Biofilms have a tendency to be anaerobic, and so respiration may be important here. Host generates nitrate as part of the inflammatory response, and E. coli is able to take advantage of this. LO: Evaluate the role of the microbiome and non-pathogenic traits of bacteria in virulence. 4. a) What determines the size of a bacterial pathogen genome? Depends on selection. Selection for versatility (large genome) to cope in multiple environmental conditions inside and outside the host, or for persistence under tightly controlled and defined conditions such as in an intracellular pathogen (small genome). Gene gain processes include phages and other methods for horizontal acquisition. Gene duplication and diversification. Gene loss events include pseudogenization (not really affecting genome size), but this is eventually followed by loss of pseudogenes over a long timescale. Organisms in restricted environments may get limited access to new genes, and so will be dominated by gene loss events. LO: Compare virulence factors and strategies that contribute to the virulence strategies of different bacterial pathogens. b) You suspect a particular gene from an enteric pathogen is important in gut colonisation. Describe how you could identify the function of the gene, and determine whether it is important for colonisation of a mammalian host. Make a mutant and test its ability to grow in laboratory culture in vitro. Deciding on experimental conditions is really dependent on having good * predictions* for gene function, typically based on bioinformatic analysis. Compare growth (or other behavioural trait) under relevant conditions in the lab, compared to a wild type. Inoculate the bacterial strain for the mutant (and the wild-type) into an experimental model -e.g. Germ-free (or pathogen free) mouse (answers should give suitable consideration to whether or not the animal has page 4 of 9
been made free of the pathogen in question before the experiment!). Compare the relative ability to grow by absolute numbers of bacteria or relative survival (competition index). LO: Define key terminology and describe important experimental approaches in the field of bacterial pathogenesis. 5. The electron micrograph (below) shows part of a eukaryotic cell infected with a mutant bacterium that has a gene of unknown function ( unfun1 ) deleted. a) Suggest what membranes (indicated by arrows) could be surrounding the bacterium? (2 marks) Without further information: phagosomal membrane (1 mark) or an autophagosomal membrane (1 mark) b) The membrane surrounding the bacterium was subsequently shown to contain the lipidated protein LC3/ATG8. What process is likely to clear the bacterium from the cell? (2 marks) LC3 means the membrane is an autophagosomal membrane (1 mark) and the bacterium is likely to be degraded by lysosomes via autophagy (1 mark). c) In wild type bacteria containing unfun 1, intracellular bacteria are not seen associated with eukaryotic membranes. What can you conclude about the function of UNFUN1? (1 mark) UNFUN1 prevents bacteria being destroyed by autophagy page 5 of 9
d) The predicted structure of UNFUN1 is shown (above), where the numbers refer to the positioning of amino acids. You perform a GST pulldown experiment with GST fused to different parts of UNFUN1. You subsequently undertake a western blot (above) for binding of LC3 to the GST-fusion proteins. Explaining your answer, where does LC3 bind to UNFUN1? There are two binding sites (1 mark). Fragment 400-810 does not bind therefore binding is before aa 400 (1 mark). 1-350 binds, but not 20-350, therefore there is one binding site at the N-terminus 1-20 (1 mark). However 20-400 binds indicating there must be a second site (1 mark). Both 20-400 binding and 350-760 or 350-810 indicate the second site must be between the WD40 and beta-barrel domains (2 marks). LOs: Interpret and critically discuss experimental data and approaches in the field of bacterial pathogenesis. Describe critical stages of infection and identify different virulence strategies in this context. Define key terminology and describe important experimental approaches in the field of bacterial pathogenesis. page 6 of 9
6. A signature tagged mutagenesis experiment is undertaken, which identifies a gene that is essential for bacterial intracellular survival. Describe two experiments and/or analyses that you could perform to determine the function of the protein. A lot of experimental methods could be used here. Typical answers: Genomic analyses to see whether the gene is within a known region e.g. pathogenicity island encoding T3SS system (1), or is it encoded next to a chaperone protein which is often next to a T3SS effector protein (1), bioinformatic modelling of the primary amino acid sequence/ structure for homology to proteins of known function (1 mark). Could add adenylate cyclase to see if the protein is secreted from the bacterium (1 mark). Could add a tag such as GST or His and then use affinity chromatography to see what mammalian protein it interacts with (1 mark) suggesting a function. LOs: Define key terminology and describe important experimental approaches in the field of bacterial pathogenesis. Interpret and critically discuss experimental data and approaches in the field of bacterial pathogenesis. page 7 of 9
SECTION B: Essay question Answer one question on the separate paper provided Remember to write your candidate number at the top of the page and indicate whether you have answered question A or B Mark total for this section: 50 EITHER A) Discuss strategies employed by bacterial pathogens to optimise their success in colonising host tissues. Answers will typically focus on how enteric bacterial pathogens are able to exploit the availability of carbon sources (sugars, such as sialic acid) and respiratory electron acceptors (such as tetrathionate) as ways to continue to grow in the host environment. Success is relative to the other microbes in the tissue in question, and essays should address this. Some of the strategies discussed in lectures can explain how pathogens succeed relative to other organisms in the gut. Additionally, the impact of the microbiome, and host side processes are relevant -host products and activities of other members of the microbiome both contribute to success by the pathogens. Other examples could be drawn in from lectures discussing biofilm production, control strategies, phase variation, etc. All of which could be made relevant to the answer. Very good answers will be well structured and built around themes, rather than simply long descriptions of mechanisms employed in particular pathogens. Addresses LOs: Evaluate the role of the microbiome and non-pathogenic traits of bacteria in virulence. Compare virulence factors and strategies that contribute to the virulence strategies of different bacterial pathogens. OR B) What makes the perfect intracellular bacterial pathogen? Needs to be able to gain entry into the host - by inference it is then not seen by the immune system and therefore a strong immune response is not generated. Then needs to be able to survive inside the host: Either lives within a vacuole/membrane and therefore needs to acquire nutrients with vesicular delivery but at the same time needs to either avoid delivery to the lysosome or page 8 of 9
manipulate the lysosome itself. If in the lysosome it needs to be able to survive at low ph, and inside vesicles it needs to be able to survive an oxidising environment. It needs to be able to protect itself from host responses such as ROS. If it can escape into the cytoplasm (needs a mechanism to do this) then it needs to be able to have its own metabolic pathways for a reduced environment low in aromatic amino acids. Would also need a mechanism to avoid autophagy. The ability to replicate and spread is also important. So must be able to divide in its niche and transfer from cell to cell either using a system such as Listeria that uses actin or activating the immune system to a certain extent to bring other cells in close proximity that allows dissemination throughout the host. Inside vacuolar membranes means drugs have limited access to the pathogen. The archetypical perfect pathogen is TB: intracellular, slow metabolism, avoids the lysosome, can live in an anaerobic environment, drug resistant, only kills 10% of hosts. A good answer evaluates the whole of the intracellular pathway. LO: Describe critical stages of infection and identify different virulence strategies in this context. page 9 of 9