Abstract
Malaria is a life-threatening disease caused by Plasmodium spp. parasites. Although Plasmodium falciparum is the deadliest form of malaria, Plasmodium vivax is the most globally widespread, with 40 % of the world’s population at risk of infection. P. vivax is particularly difficult to diagnose and treat, an issue that is confounded by a lack of a continuous in vitro culture method. Without the ability to culture P. vivax, our knowledge of this important parasite has lagged behind P. falciparum (which has had a well-established in vitro culture method available since the 1970s).
The recent re-establishment and optimisation of a continuous in vitro culture method for P. vivax’s sister taxon, Plasmodium cynomolgi (a vivax-like parasite found in old world monkeys) however, has for the first time allowed the mechanistic investigation of key biological processes, such as P. vivax biology (as it shares many of its distinct phenotypic characteristics), and the molecular basis for drug-resistant P. vivax (as it has a high degree of genetic similarity). Without a continuous culture method, previous studies in these areas have had to rely on ex vivo and clinical drug susceptibility studies to investigate P. vivax drug resistance. Reverse genetic studies in P. cynomolgi will for the first time provide the ability to validate putative drug resistance markers in P. vivax, a crucial early step in mitigating the further emergence and spread of drug resistance through South-East Asia.
To date the only published report on P. cynomolgi transfection involved episomal plasmids in an in vivo model (monkey infection). Our laboratory has focused on the establishment and optimisation of an in vitro integrative transfection protocol for P. cynomolgi; using a ‘one plasmid’ CRISPR-Cas9 system adapted from the Fiddock Lab (University of Columbia, USA). This system was used to investigate validated molecular markers of P. falciparum drug resistance (the Y268S mutation in pfcytb, and the K76T mutation in pfcrt) and whether they would confer drug resistance in P. cynomolgi, as a model for P. vivax. We have been able to develop CRISPR plasmids for the Y268S mutation in cytochrome b (cytb) which is suspected to confer atovaquone resistance to parasites. The isolation of the P. cynomolgi crt gene was difficult however, and this, combined with the challenge of understanding transfection efficiency, led to a decision to try a second approach to the transfection of the crt gene. A plasmid containing cas9 and the gRNA was produced and was transfected into P. cynomolgi parasites alongside a single stranded oligodeoxynucleotide repair template.
For the transfection of P. cynomolgi to be successful, there needs to be optimal culture conditions for parasites, to encourage growth and prevent contamination. Throughout our study, P. cynomolgi has proven to be an excellent model for P. vivax, however there are still obstacles associated with P. cynomolgi in vitro culture that need to be overcome in order to allow more efficient, ethical, and cheaper culturing of these parasites. To reduce costs and increase culture volumes, we investigated several serum combinations as alternatives to non-human primate serum. We also investigated antibiotic combinations to prevent bacterial contamination of cultures and sought to optimise the synchronisation of parasites (a key component of the transfection procedure).
Finally, we sought to investigate the roles of the dbp1 and dbp2 genes in P. vivax invasion of RBCs by tagging these proteins with GFP to visualise them under electron microscopy. We were able to successfully construct a plasmid to tag the dbp2 gene in P. cynomolgi.
Our laboratory has been able to show that P. cynomolgi is a tractable P. vivax model, receptive to genetic manipulation by CRISPR-Cas9. This work is the first of its kind for P. cynomolgi, but still requires further optimisation before it is available as a toolbox for further research into the neglected area of vivax malaria molecular biology.