University of York Department of Biology B. Sc Stage 3 Degree Examinations Human Molecular Parasitology. Submission deadline: 12 noon

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1 Examination Candidate Number: University of York Department of Biology B. Sc Stage 3 Degree Examinations Human Molecular Parasitology Submission deadline: 12 noon Work should be submitted via the Yorkshare VLE Total marks available for this paper: 100 Your assignment will be put through turnitin on submission. We recommend you also put your work through turnitin prior to submission. All questions should be answered on this question paper. All questions should be answered using Arial font size 11 or larger Question 1: It has been postulated that exosomes may be produced by some parasites. Cite references where appropriate. LO1, LO2 a) How has the discovery of exosomes impacted the field of parasitology? (300 word limit) (6 marks) It has provided an alternative pathway of parasite factor transfer to host cellular/tissue compartments beyond what the parasite itself occupies (1) and independent of the necessary markers and receptors from traditional characterized pathways (1). Thus expanding current models of host manipulation and opening the field to additional functions and renewed interest in parasite-derived proteins and ncrnas (1). Virulence factors involved in host:parasite interactions; host manipulation by parasites. Multitude of proposed roles offered up for specific molecules along these lines; host mimicry to compete with endogenous factors and block signaling, translational arrest, protease degradation of host cell response pathways. b) Outline an experimental design strategy to isolate, identify and functionally characterise exosomes from a specific parasite culture. (300 word limit) (10 marks) Any workable strategy accurately described and outlined will suffice. Grow human-infective stage Plasmodium, Leishmania, helminths or Trypanosomes at high density in culture. Stimulate cultures as appropriate to parasites. Precipitate vesicles. Stack a light gradient. Ultracentrifuge and fractionate gradient with a spectrophotometer running concurrently or spec each fraction individually to isolate page 1 of 7

2 fractions with high nucleotide and protein content. Isolate exosomes and expose host cells to them independent of parasites and look for gene/protein expression. DNA, ncrnas, mrnas, mrbps, proteins, enzymes - factors which can alter host cell behaviour. Many correct answers possible. Ex. By isolating exosomes from parasite-derived culture and exposing host immune cells to exosomes independent of parasites. c) Evaluate potential strengths and weaknesses of using culture-derived parasites for your exosomal analyses. (200 word limit) (5 marks) +s Culture-derived can increase concentrations of factors for better isolation and identification efficiency. No animals necessarily sacrificed. Higher yields and replicates possible. Lower costs. -s Exosomes released from parasites grown in culture may not be representative of those produced in vivo (in a patient)(1). Growing parasites in culture may miss important parasite:host interaction dynamics which may alter appropriate stimulation, formation, composition or function of parasite-derived exosomes (2). This answer relates to the use of parasites grown in culture (students were told to do this) not the answer provided by the student (precipitation, centrifugation etc). d) Compare obstacles to exosomal function for an extracellular versus an intracellular parasite. Provide specific examples. (200 word limit) (4 marks) Exosomes in helminths or T.brucei (extracellular) vs. L.mexicana, T.cruzi or Plasmodium (intracellular). Discussion of distinctions in compartmental obstacles, immune cellular/genetic system targeted and molecules involved in parasite:host communication and manipulation. Multiple combinations possible, all correct answers accepted. Question 2: Paper analysis (25 marks). The following questions refer to Valentim et al. Science, 2013 (doi: /science ); the pdf is supplied on the VLE. LO1, LO3 page 2 of 7

3 a) Summarise the significance of this paper to the research field. (200 word limit) (4 marks) In the field of human helminth parasitology this paper is significant in that, by using a genetic mapping approach and a variety of biochemical techniques, the authors determine the molecular basis by which Schistosoma mansoni displays OXA resistance. However, it could be argued that it is less important to know the molecular basis of OXA resistance than, say, praziquantel (PZQ) resistance. This is because OXA was only previously used in Brazil (as not active against S. haematobium or japonicum) and has now been replaced as the drug of choice by PZQ in worldwide efforts to eliminate schistosomiasis (PZQ active against all human-infective schistosomes). Nevertheless, this paper showcases a successful approach that can be used in the future to understand PZQ resistance (which may become widespread, especially as mass drug administration efforts intensify). It is an open question as to whether this approach will work for PZQ resistance if it is caused by mutations in multiple genes. Also significantly, this study provides a framework to re-design OXA in a rational manner such that it can be improved to target all species of human infective schistosomes (e.g. Taylor J et al Biol Chem 2017; /jbc.M ). This study also allowed Chevalier et al (2016 Int J Parasitol; /j.ijpara ), to infer that OXA resistance has emerged independently several times (i.e. distinct mutations in same gene). b) Why are all F1 progeny of the LE x HR cross sensitive to OXA? Were the frequencies of resistant and susceptible F2 parasites expected and why? (100 word limit) (3 marks) - Schistosomes have diploid genomes page 3 of 7

4 - OXA resistance is recessive so SS x RR cross (susceptible x resistant) generates susceptible F1 = SR. - F1 x F1 cross generates SS, SR, RS and RR = ¼ resistant c) Compare and contrast the implications of the data from Fig. 2A and Fig. 2C. (200 word limit) (4 marks) Experiment presented in Fig. 2A involves expressing 6 candidate genes (from 16 present in QTL) as bacterially-expressed recombinant proteins. These proteins are then tested for their ability to activate OXA in a biochemical assay (i.e. to promote OXA binding to soluble extracts obtained from OXA-resistant adult worms). This experiment shows that the recombinant sulfotransferase Smp_89320 is capable of activating OXA binding in this acellular assay. Extracts from susceptible and resistant worms are included as positive and negative controls, respectively. The susceptible extract is included as it is likely to contain native (correctly folded) parasite proteins that activate OXA. This experiment establishes that Smp_89320 is a good candidate for the OXA-activating protein and so should be tested in live worms. Experiment presented in Fig. 2C shows that knockdown of Smp_89320 in live worms converts an OXA-susceptible parasite into a resistant parasite. As such, this experiment reveals that Smp_89320 is the OXA-activating protein in intact worms. So, whilst 2A establishes that Smp_89320 can activate OXA in a biochemical assay, 2C shows that this is the only (or at least predominant) molecule that does so in live worms. The authors do not demonstrate that Smp_89320 is the OXA-activating protein in vivo (i.e. in the mammalian host). d) Design an experiment to test why oxamniquine shows variable activity against different species of schistosome. Explain your rationale. (200 word limit) (4 marks) - Mutate specific sulfotransferase residues in S. mansoni Smp_ to that those present in S. haematobium Sha_ Prediction is mutant Sm protein will not activate OXA. page 4 of 7

5 - Mutate specific sulfotransferase residues in S. haematobium Sha_ to those in S. mansoni Smp_ Prediction mutant Sh protein will activate OXA e) Parasite drug resistance is prevalent in veterinary helminth infections whilst OXO and PZQ resistance is observed in laboratory and field schistosome isolates. Explain why widespread resistance has not developed despite mass drug administration programs. (200 word limit) (4 marks) - Veterinary resistance likely consequence of high level sustained use of anthelminthics (i.e. treat all animals for sustained period; all surviving worms are drug resistant). - OXA and PZQ resistance in schistosomes are thought to be associated with a fitness cost i.e. they are only selected for/expanded in a population following drug selection. - Schistosome MDA programmes target w school aged children. This leaves large untreatead populations i.e. adults and pre-school children. As such, larger reservoirs of non-selected parasites remain and resistance does not spread. Increased drug treatment may lead to increased field resistance. f) Describe a molecular mechanism through which OXA might kill susceptible schistosomes. (100 word limit) (3 marks) The authors suggest the sulfotransferase sulfonates OXA to generate an unstable intermediate. This decays to generate an electrophilic alkylating agent that binds and damages parasite DNA, ultimately leading to parasite death. g) What is the evidence that OXA resistance has evolved more than once? (100 word limit) (3 marks) This paper reveals two independent SULT mutations (E142 and C35R) that cause loss of function. Whilst E142 originally characterized in this paper using a laboratory isolate, a more recent study has shown that this also occurs in field isolates (Chevalier et al, Int J Parasitol 2016). Chevalier 2016 also show two further page 5 of 7

6 inactivating mutations = 4 distinct mutations in total. This is suggestive of multiple independent origins. Question 3: Essay. Answer either question A or B. Word limit 1,000 (50 marks) A. What factors have been instrumental in the success of a phenotypic screening approach to malaria drug discovery?. LO1, LO3 Factors Search for new drug candidates facilitated by high throughput screening of chemical libraries. Factors: high quality compound libraries available, many in pharmaceutical companies. Open innovation developments allow shared IP. Access to funding through MMV, availability of high quality in vitro and in vivo assays to test compounds. Engagement of both academic and pharmaceutical (industry) partners. Greater understanding of plasmodium biology allows mode of action and mechanism of resistance studies. Examples of phenotypic screens o Asexual blood stages of plasmodium o Spiroindolone target deconvolution identified ATPase4 as the target o MMV08138 targets IspD in the apicoplast o Hexahydroquinalones o Proteasome inhibitors o Protein synthesis inhibitors o PI(4)K inhibitors o Malaria box of compounds MMV have extensive portfolio most come from phenotypic screens First class students will realise that new target based drug discovery can arise from phenotypic following successful target deconvolution (but not the other way round). Very good students will discuss PK-PD and the quality of compound libraries. page 6 of 7

7 B. Compare and contrast the gene expression and function of surface proteins of mammalian stage Trypanosoma brucei vs Trypanosoma cruzi parasites. LO1, LO2 T.brucei - Pol I drives VSG gene expression with only 1 VSG expression site driven at a time. Antigenic variation of VSG permits survival of extracellular T.brucei in mammalian bloodstream. Variable Surface Glycoprotein host immune cell evasion due to VSG remodeling, recycling and surface coat switching. VSG protein switches with each wave of parasitemia (SL-ST differentiation). 4 mechanisms of recombination enable switching, known transcriptional repressors of quiescent Expression Sites, VSG switching triggers; role of camp pathway. Expression Site Associated Genes (ESAGs) role in survival. ESAG6/7 Fe++ receptor transport, susceptibility to TLF of humans. T.cruzi - All surface proteins are Pol II-driven and upregulated via gene duplication; trans-sialidases (TS), mucins, MASP genes represent 50% of total genome content. Surface proteins are necessary for host complement-mediated triggering of endocytic update, enables infectivity of all mammalian cell types. Contribute to immune evasion, host cell invasion and infection. page 7 of 7