Exploring the contribution of new genetic markers of drug resistance in human malaria parasites

Antimalarial drugs remain as one of the most powerful tools in the fight against malaria with artemisinin derivatives now standing as the cornerstone of anti-malarial drug therapy. Unfortunately, evidence of delayed in vivo parasite clearance after artemisinin treatment is accumulating on the Thai-Cambodian border and in nearby countries. A better understanding of the mechanisms of artemisinin (ART) resistance may contribute to the development and validation of new tools for the surveillance of resistance. One promising approach to identifying candidate genetic markers of ART resistance is genetic analysis of drug-pressured mutants of the rodent malaria parasite Plasmodium chabaudi. This experimental system has identified a number of genetic mutations in parasites artificially selected for resistance to ART derivatives. These mutations encode alterations in a de-ubiquitinating enzyme (UBP-1) and in a 26S proteasome subunit (26SPS), both involved in the ubiquitin-proteasome pathway, responsible for protein turnover through selective degradation. An additional mutation was found to have occurred in a gene encoding the “mu” chain of the AP2 adaptor protein complex, a component of the endocytic machinery. The importance of the above mentioned markers in modulating susceptibility to different drugs in the human malaria parasite remains unclear. In that context, the hypothesis to be tested in this thesis is that the three loci implicated in ART resistance in experimentally selected in P. chabaudi will similarly modify ART response in natural parasite populations of P. falciparum. Increased artemisinin resistance in a P. chabaudi parasite derived from a chloroquine resistant parasite after prolonged and progressive artemisinin selection was phenotypic and genetically characterized. The whole genome sequencing identified a mutation in a gene encoding the mu chain of the AP2 adaptor protein complex. To explore the genetic variability of the ap2-mu gene in P. falciparum and its associations with artemisinin in vitro responses we sequenced field isolates from Brazil, São Tomé and Rwanda. Analysis of P. falciparum field isolates showed a weak association between a Ser160Asn mutation and in vitro dihydroartemisinin responses. To investigate the correlation between polymorphisms in pfubp-1 and pfap2-mu and in vivo parasite susceptibility to ART we genetically characterized samples from an ACT clinical trial carried out in Kenya. Previously work done on the same ACT clinical trial samples described sub-microscopic persistent parasites on day 3 post-treatment samples. These parasites were only detected by qPCR but the children carrying these parasites had a higher transmission potential and were far more likely to go on to classical treatment failure at day 28 or day 42 post-treatment. The molecular work carried out here demonstrates that a Ser160Asn/Thr mutation in the pfap2-mu gene and an E1528D mutation in the pfubp-1 gene might be associated with in vivo responses to artemisinin derivatives. Polymorphisms on the pfubp-1 gene and pfap2-mu genes were further studied using field isolates from an ACT clinical trial in Burkina-Faso which were also tested in vitro for their response to dihydroartemisinin and several other antimalarial drugs. Using these samples, we also investigate the genetic polymorphisms of the pf26S-protSU, another drug resistant candidate gene identified in the studies of P. chabaudi. Data revealed that polymorphisms in pfubp1 and pf26S-protSU, can modulate in vitro responses to lumefantrine. However, this work did not reveal any significant association between polymorphisms in pfubp-1 and pfap2-mu genes and in vitro artemisinin susceptibilities or treatment outcomes. In order to validate the pfap2-mu candidate marker as an important modulator of parasite sensitivity to artemisinins and to improve understanding of the biological mechanisms of resistance to this class of drugs we further performed gene functional characterization using transfection techniques. Transgenic parasites carrying the 160Asn allele of pfap2-mu were significantly less sensitive to dihydroartemisinin using a standard in vitro test. Sensitivity to chloroquine and quinine were also reduced. Localization studies of pfap2-mu were performed by transfection of fluorescent-tagged gene construct into P. falciparum and expression of fluorescent fusion protein in parasites was observed using a confocal microscope. The findings from this study provide the first in vivo evidence that polymorphisms in the pfap2-mu and pfubp-1 genes modulate P. falciparum responses to artemisinins. Additionally, transgenic laboratory lines of P. falciparum carrying the 160Asn mutation in pfap2-mu gene have altered in vitro responses to dihydroartemisinin, quinine and chloroquine. We therefore propose these genes should be evaluated further as potential molecular markers of artemisinin resistance