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dc.contributor.advisorRussell, Bruce
dc.contributor.authorWard, Kurt Edward
dc.date.available2021-02-22T01:00:35Z
dc.date.copyright2021
dc.identifier.citationWard, K. E. (2021). Genetic manipulation of Plasmodium cynomolgi as a model for investigating Plasmodium vivax drug resistance (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/10705en
dc.identifier.urihttp://hdl.handle.net/10523/10705
dc.description.abstractPlasmodium vivax is the most widely distributed cause of human malaria, and thought to be the most difficult to diagnose and treat. Until recently this species has largely been neglected compared to the most significant cause of fatal human malaria, Plasmodium falciparum. One of the reasons behind this comparative neglect is that, unlike P. falciparum, P. vivax is not amenable to continuous in vitro culture. The absence of a tractable continuous culture method has hampered mechanistic biological studies of P. vivax, and in particular has frustrated the development of methods for the genetic manipulation of this species. As reverse genetics is the gold standard for the investigation of putative drug resistance markers, our current inability to easily manipulate the genome of P. vivax has impeded our understanding of drug resistance in vivax malaria. Efforts to verify putative markers of drug resistance in vivax malaria are of particular importance given the recent emergence and spread of multiple drug resistance throughout Southeast Asia. To date, the identification of molecular markers of P. vivax drug resistance has wholly relied on ex vivo and clinical drug susceptibility studies. While a range of molecular markers have been proposed, including mutations in mdr1, crt, and dhfr-ts, these phenotypic sensitivity studies are subject to a range of confounding factors (i.e., patient immunity, geographic gene fixation). Until we can verify these putative markers using reverse genetics, our understanding of vivax drug resistance and the mechanistic action of antimalarials against P. vivax will remain obscured. We have been fortunate to be at the forefront of a revolution in the in vitro continuous culture of P. cynomolgi, a sister species to P. vivax which shares many of its distinctive features (including dormant hypnozoite stages), and a high degree of genetic similarity. Considering the lack of a method for the continuous culture of P. vivax, the use of the P. cynomolgi Berok model provides for an exciting opportunity investigate P. vivax drug resistance markers through reverse genetics. While P. cynomolgi has been transfected with episomal plasmids using ex vivo and in vivo methods, the future of such work, which relies heavily on the use of nonhuman primates, faces increasing ethical and practical obstacles. Although the tractable continuous culture method for P. cynomolgi has resulted in a range of important phenotypic studies, conditions for the genetic manipulation of this species have not been optimised. Indeed, no in vitro episomal or integrative genetic manipulation has been reported in this species. As no framework existed for the integrative genetic manipulation of P. cynomolgi, we undertook training in a well-established method for Plasmodium genetic manipulation in the Fidock Lab (Columbia University, NY) using CRISPR/Cas9. We used this system to investigate the role of putative drug markers for artemisinin sensitivity, and trained in the construction, selection, and phenotypic assessment of recombinant P. falciparum parasites. This experience allowed us to design a CRISPR/Cas9 system for the genetic manipulation of P. cynomolgi. Our experience working with the well-established P. falciparum system highlighted the requisite attributes for the successful genetic manipulation of Plasmodium species. These include culture scalability, long term, contamination free culture, and the ability to enrich mature asexual forms for transfection. Therefore, the next focus of this study was to optimise the P. cynomolgi Berok continuous culture for reverse genetics. As many of the conditions used for P. falciparum were not compatible with P. cynomolgi culture, we identified viable alternatives for our model. These included a cost-effective substitution for nonhuman primate serum in culture media (horse serum and Albumax), a combination of antibiotics amenable for contamination prevention in P. cynomolgi cultures (penicillin and cefquinome), and a practical method for schizont enrichment (Nycodenz gradient centrifugation). These basic culture condition improvements facilitated the first in vitro selection of an episomal expression plasmid in P. cynomolgi. Although initial transfections of episomal plasmids were successfully undertaken using the Bio-Rad Gene Pulser, we found that larger plasmids were more efficiently delivered using the Amaxa Nucleofector 4D. The later system was used to deliver CRISPR Cas9 plasmids for the insertion of P. vivax putative resistance markers in mdr1, dhfr-ts, and k13. Of these, we were only able to successfully insert the mdr1 Y976F mutation, which has been associated with chloroquine resistance in ex vivo and epidemiological drug resistance studies of vivax malaria. This marks the first recorded integrative genetic manipulation of P. cynomolgi. Preliminary phenotypic assays indicate that the Y976F mutation does not alter the sensitivity of P. cynomolgi to chloroquine, mefloquine, or lumefantrine. A small decrease in amodiaquine sensitivity was observed, which requires further investigation. While this study has demonstrated that P. cynomolgi is amenable to genetic manipulation in vitro, there are some important challenges and concerns relating to this model which still need to be addressed. Nonetheless, the undeniable similarity between the genome and phenotype of P. cynomolgi and P. vivax warrants further investment into the development of a more tractable system for the genetic manipulation of this species.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectPlasmodium
dc.subjectcynomolgi
dc.subjectvivax
dc.subjectdrug resistance
dc.subjectmdr1
dc.titleGenetic manipulation of Plasmodium cynomolgi as a model for investigating Plasmodium vivax drug resistance
dc.typeThesis
dc.date.updated2021-02-18T01:21:58Z
dc.language.rfc3066en
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanno
otago.openaccessAbstract Only
otago.evidence.presentYes
otago.abstractonly.term335d*
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