| Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been shown to be central to the molecular mechanism of quinoline antimalarial drug resistance. However, additional facets to resistance biochemistry are emerging, and it is now clear that multiple quinoline drug resistance phenotypes exist in different regions of the globe. Different public health policies and drug use histories in different regions, along with natural genetic drift, have created this diversity, such that there are now at least 53 distinct isoforms of PfCRT protein expressed in various strains or isolates of Plasmodium falciparum malarial parasites around the globe. Some of these can be described in detail, but information is incomplete. This leads to some degree of continued uncertainty on how best to proceed in controlling malaria in some regions.;Resistance-conferring PfCRT isoforms (which harbor mutation K76T and are distinguished by 3-8 additional mutations) are believed to confer resistance to the 4-amino quinoline drug chloroquine (CQ) by transporting CQ away from the drug's digestive vacuole (DV) - localized heme target. One theory then is that variable CQ transport catalyzed by different mutant PfCRTs are responsible for the range of CQ sensitivities now found for P. falciparum around the globe. Alternatively, additional mutations in drug- selected parasites may complement PfCRT in conferring a range of resistance phenotypes. In this thesis, I further optimize a convenient method for screening for CQ transport mediated by PfCRT, involving heterologous expression in Saccharomyces cerevisiae. I use this method and other techniques to quantify activity for all currently known naturally occurring PfCRT isoforms. My results show that chlorquine resistance (CQR) in isolates expressing certain PfCRT isoforms likely depends upon additional genetic events. The data also suggest that some mutant PfCRTs do not confer typical CQR phenotypes and that in and of itself the common K76T PfCRT mutation may not be a fully reliable indicator of CQR status. I also analyze targeted artificial mutations in PfCRT to better understand the function of specific isoforms.;Finally, I also carry out a large-scale chemogenetic screen of the Saccharomyces cerevisiae deletion mutant library to identify yeast genes relevant for resistance and sensitivity to CQ. This approach reveals overlapping gene pathways relevant to CQ toxicity, some of which have also been previously identified in related chemogenomic screens vs other quinoline drugs (e.g. quinine). Significantly enriched gene ontology (GO) terms persisted across increasingly stringent subsets of phenotypic grouping down to <1% of the entire library. GO terms enriched in our datasets are associated with vesicular traffic, mitochondrial function, and protein synthesis. A number of genes we identify by this approach encode orthologues of proteins expressed in genetic loci that our group has recently associated with increased resistance to the cytocidal activity of CQ in P. falciparum.. |
