Theses Doctoral

Investigating the determinants of resistance to quinine and chloroquine using a novel Plasmodium falciparum genetic cross

Kanai, Mariko

The repeated emergence of Plasmodium falciparum resistance to first-line antimalarial drugs necessitates understanding the underlying resistance mechanisms to detect and monitor resistance in the field and to inform drug discovery efforts. With the advent of the FRG NOD human liver-chimeric (huHep) mouse model for P. falciparum genetic crosses, interest has renewed in harnessing this forward genetics tool to study traits including drug resistance. The antimalarial quinine (QN) is of particular interest as it has retained efficacy over 400 years as parasite resistance has been slow to develop against the drug, likely due to a multifactorial mechanism of which only several genes have been partially implicated. Chloroquine (CQ) is a former first-line drug for P. falciparum (that is still in use for P. vivax), and it’s phasing out has been associated with the recent emergence of CQ-sensitive P. falciparum parasites. While the CQ resistance transporter (PfCRT) is known to be the primary driver of resistance, studies have provided evidence for secondary modulators of CQ, of which only the multidrug resistance protein 1 (PfMDR1) transporter has been identified. This thesis addresses the hypotheses that additional mediators are involved in the parasite resistance mechanism to QN and that genes other than pfmdr1 modulate parasite resistance to CQ.

In chapter 3, we present the P. falciparum genetic cross that we conducted between the QN- and CQ-sensitive African NF54 and QN- and CQ-resistant Cambodian Cam3.II parasites in huHep mice, in collaboration with Dr. Photini Sinnis’s laboratory at Johns Hopkins University. By applying different selective conditions to cross progeny bulk pools prior to cloning these bulks, we were able to recover 120 unique recombinant progeny from this cross. We observed minimal overlap in the progeny genotypes obtained from CQ and QN pressure, suggesting distinct mechanisms for parasite resistance to these drugs. Bulk progeny selection and progeny clone-based QN linkage mapping approaches identified quantitative trait loci (QTLs) on chromosomes 7 and 12, as well as minor QTLs on other chromosomes, consistent with a multifactorial resistance mechanism. We applied the latter approach to investigate parasite response to CQ and its active metabolite monodesethyl-CQ (md-CQ) and identified a novel chromosome 12 QTL in addition to pfcrt. Interestingly, while the chromosome 12 QTLs overlapped, the chromosome 7 QTL for high-grade QN resistance did not contain pfcrt.

In chapter 4, we used bioinformatic approaches, whole-genome sequence data from our cross and field isolates, and literature review to identify the drug/metabolite transporter 1 (DMT1) as the top candidate of the chromosome 7 QTL, and S-adenosylmethionine mitochondrial carrier protein (SAMC), hydroxyethylthiazole kinase (ThzK), and ATP-dependent zinc metalloprotease (FtsH1) as the top candidates for the chromosome 12 QTLs. By harnessing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing methodologies (SNP-editing, knockout, and tagging), we obtained evidence favoring DMT1 as a marker of QN resistance and localized this transporter to structures associated with vesicular trafficking, PVM, lipid bodies, and the lysosome-like digestive vacuole. We also harnessed SNP-editing and identified FtsH1 as a potential mediator of QN resistance and a modulator of CQ and md-CQ resistance. QN, mefloquine, and lumefantrine belong to the same aryl-amino alcohol class, and we found that QN is structurally more similar to mefloquine than lumefantrine. We also showed that QN can partially inhibit heme detoxification.

While conducting the work outlined in chapters 3 and 4, we identified an unmet need for quickly identifying clonal recombinant progeny and validating parasite identity, which inspired the study presented in chapter 5. We developed a genotyping method that can assess drug resistance-conferring SNPs directly from P. falciparum culture or infected blood as well as a multiplexed microsatellite genotyping method with five broadly informative markers. Both methods were applied in chapter 3 to identify clonal recombinant progeny, and the SNP genotyping method was used in chapter 4 to validate gene editing and progeny identity. We also tested the resolution, sensitivity, time, and cost of each method as well as whole-genome sequencing and recommended the ideal application for each genotyping method.

Our data demonstrate that DMT1 is a novel marker for QN resistance, and a new chromosome 12 locus associates with CQ response, of which ftsh1 is a potential candidate. In chapter 6, we discuss the potential mechanisms by which DMT1 is involved in QN resistance, the potential impact of our findings, and future experiments that can further characterize the QN and CQ resistance mechanisms and the functional role of these candidate genes.

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More About This Work

Academic Units
Cellular, Molecular and Biomedical Studies
Thesis Advisors
Fidock, David Armand
Degree
Ph.D., Columbia University
Published Here
July 19, 2023