Plasmodium vivax malaria is the most prevalent of all the human malaria species and can account for up to 391 million cases annually. P. vivax malaria is endemic in many parts of Asia, Central and South America, Oceania and certain Caribbean Islands. P. vivax malaria causes a debilitating disease that is responsible for considerable morbidity and economic loss in these endemic countries. Over the last 20 years, there has been a significant increase in resistance of P. vivax malaria to chloroquine and the antifolate drug combination sulfadoxine/pyrimethamine.
While the mechanism of chloroquine resistance in P. vivax remains unknown the mechanism of antifolate resistance has been determined to be mutations within the parasite's dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. The mechanism of action of antifolate and sulfa drugs is to target the parasite's DHFR and DHPS enzymes, respectively. Previous studies with P. falciparum have shown that it is the accumulation of mutations within these genes that are responsible for the reduced susceptibility to antifols. Amino acid mutations in P. vivax dhfr (pvdhfr) has been reported at positions 51, 58, and 117, which correspond to amino acids 50, 59 and 108 in P. falciparum dhfr (pfdhfr) and the existence of such mutants correlates geographically with antifolate use against P. falciparum. However, due to the inability to culture P. vivax parasites long term in the laboratory, surrogate biological systems, such as Saccharomyces cerevisiae (yeast) and Escherichia coli have been used to determine the correlation of the presence of mutations in the pvdhfr to the reduction in susceptibility to antifolates. While these systems are informative, they are however, quite biologically distinct from Plasmodium parasites, particularly in membrane structures and transporters that can potentially affect the susceptibility to drugs.
This thesis is aimed at developing a more homologous system that will be able to study gene(s) responsible for antimalarial resistance, in particular the effect of the mutations within pvdhfr in conferring resistance to antifolates. The thesis has three main sections: firstly to determine the pvdhfr and pvdhps allelic types and their origins in P. vivax isolates collected from the Asia-Pacific region; secondly, to define the role of mutations in the pvdhfr using an episomal P. falciparum transfection system and; thirdly to stably express the wild type and various mutant pvdhfr alleles using the piggyBac P. falciparum transposon mutagenesis system and then test and select for new drugs that are effective in killing these parasites.
The hypothesis for the first part of this thesis was the Western Pacific region, much like other P. vivax endemic areas, will have a higher number and range of mutations within pvdhfr and/or pvdhps in areas that have had a higher exposure to antifolates. This may have been the result of the spread of the resistance conferring pvdhfr and pvdhps alleles into the region. To test this hypothesis, pvdhfr and pvdhps from 70 P. vivax isolates from several Asian-Pacific countries (China, East Timor, Papua New Guinea, the Philippines, Vanuatu and Vietnam) were PCR amplified and sequenced to investigate the prevalence, extent and origins of P. vivax dhfr and dhps allelic types. The key outcomes were: the majority of the isolates had a double mutation of 58R/117N; the greatest range of mutations were observed in parasites from the South West Pacific countries of PNG and Vanuatu, which correlates with the higher antifolates use in these regions; the pvdhfr in the P. vivax isolates from Asian countries were found, at most, to have double mutations; only a small number of mutations were detected in pvdhps and these mutations only coincided with mutations within the pvdhfr and; the triple and quadruple pvdhfr mutant alleles appear to have at least independent origins in Thailand, Indonesia and Papua New Guinea/Vanuatu.
The hypothesis for the second part of the thesis was that the development of an episomal expression system would provide a more homologous system to investigate the level of susceptibility that specific point mutations within the pvdhfr confer against antifolates. To test this hypothesis, I used a previously developed episomal P. falciparum transfection system to express both wild type and various mutant pvdhfr alleles in a P. falciparum antifolate-sensitive line (D6) and assessed their influence on the susceptibility of the recipient P. falciparum line to commonly used and new drugs. The key outcomes of this study were: the recipient P. falciparum line (D6) transiently expressing the wild type pvdhfr was at least as susceptible to antifolates as the parent strain D6; in most instances, the susceptibility profile of the transfected parasites decreased as the number of pvdhfr mutations increased; the quadruple pvdhfr mutant (F57L/S58R/T61M/S117T) had the greatest level of resistance; in contrast, the pvdhfr triple mutant allele (S58R/T61M/S117T), in some instances, was as susceptible to antifolates as the pvdhfr wild type allele, which may indicate the T61M mutation to be a compensatory mutation; the S117N mutation is responsible for pyrimethamine resistance; whereas the S117T and F57L mutations may be responsible for the increase in resistance to the triazine compounds (cycloguanil and WR99210).
The hypothesis for the last part of the thesis was that integration of the pvdhfr into the P. falciparum genome would provide a more stable expression system to enable the investigation into the effect of new drugs or combination of drugs or new treatment regimens required to combat P. vivax infections. To test this hypothesis, I used a previously developed P. falciparum transposon mutagenesis method, known as piggyBac, to integrate the wild type and various mutant pvdhfr alleles into the P. falciparum genome. This was the first time this system had been used to transfect P. vivax genes into the P. falciparum genome. I also used this system to assess the response of the P. vivax dhfr alleles to conventional as well as newer generation antifolate drugs, such as JPC-2067. The major outcomes of this research were: integration of the P. vivax dhfr alleles occurred rapidly, within 2-3 weeks; integration of the pvdhfr alleles occurred only once within the P. falciparum genome, with the majority (81%) of the integrations occurring in non-coding regions of the genome; the levels of the pvdhfr transcription driven by the pfdhfr promoter were not different between the integrants of non-coding and coding regions; the pfdhfr promoter also had no effect on the expression and transcription of the transfected parasite's endogenous pfdhfr; the integrated wild type and single mutant pvdhfr alleles had the same susceptibility profile as the episomally expressed pvdhfr alleles, while the quadruple mutant pvdhfr allele conferred less resistance to pyrimethamine than the same episomally expressed allele; the parasite with the integrated quadruple pvdhfr mutant allele was much less susceptible to antifolates than the wild type and single mutant pvdhfr alleles; the resistance phenotype of integrated parasites were stable without drug pressure, while the level of resistance in parasites episomally expressing the pvdhfr quadruple mutant allele reduced with the decreasing copy number of episomes; the novel antifolate, JPC-2067, was determined to be as effective against the mutant pvdhfr as it was to the wild type pvdhfr and native parasites.
This thesis has contributed to a better understanding of the roles of vivax-specific genetic mutations in the resistance to antifolate drugs; has provided a platform to test newer generation antifolates against P. vivax parasites with mutant dhfr and; have provided a novel biological system that will have the potential to investigate, systematically, functions of other P. vivax gene(s), which are largely unknown, and currently cannot be done due to the inability to culture P. vivax long term in the laboratory. The importance of this system is to help guide the production of better antimalarials that can be used, particularly in regions where P. vivax and P. falciparum parasites coexist.