The emergence and spread of drug resistance in Plasmodium falciparum has consistently been a major barrier to the control and eradication of malaria. Resistance to the affordable and fast-acting former first-line drug chloroquine (CQ) was first observed in the 1950s near the Thai-Cambodian border and in South America. Resistance later spread from Asia to highly endemic regions in Africa, with reports of up to 6-fold increases in regional malaria mortality rates. The replacement drug, sulfadoxine-pyrimethamine (SP), encountered resistance within one year of clinical use. Artemisinin-(ART) based combination therapies (ACTs), which consist of a fast-acting ART derivative and a slower-acting partner drug, became the global first-line standard in 2000 and, along with mosquito vector control measures, helped decrease mortality rates by 60%. Unfortunately, parasites resistant to ART derivatives arose in Southeast Asia. This compromised the effectiveness of the ACT partner drug piperaquine (PPQ) and resistance to this drug was first reported in 2015, around a decade after the introduction of dihydroartemisinin-PPQ. By 2019, PPQ resistance, driven primarily by a series of mutations in the P. falciparum chloroquine resistance transporter (PfCRT), was widespread in Southeast Asia, resulting in >50% failure upon treatment with dihydroartemisinin-PPQ.
Malaria mortality rates have surged recently, causing an estimated 619,000 deaths in 2021. Sub-Saharan Africa is most heavily affected by this disease where 78.9% of deaths are of young children. To this date, PPQ remains effective in Africa. It is a major concern that PPQ resistance will arise on this continent, however, given the importance of PPQ in current efforts to expand the range of antimalarial interventions and reverse the current rise of malaria cases in Africa. Understanding PPQ resistance mechanisms and their effect on parasite biology is critical to creating effective treatments and minimizing the impact of drug-resistant P. falciparum malaria. This thesis aims to investigate the earliest reports of PPQ resistance, to define the PPQ susceptibility and parasite fitness of contemporary SE Asian parasite strains, and to predict future dominant strains in the field to further our understanding of parasite resistance mechanisms and combat the spread of drug-resistant malaria.
In Chapter 3, we show that earlier reports of PPQ resistance in Yunnan Province, China could be explained by the unique China C PfCRT variant. Using gene editing, we reveal that this variant confers a loss of fitness and parasite re-sensitization to the chemically related former first-line antimalarial CQ, while acquiring PPQ resistance via drug efflux. We employ biochemical assays to measure mutant PfCRT-mediated drug transport and molecular dynamics simulations with the recently solved PfCRT structure to assess changes in the central drug-binding cavity. This study provides impetus for adding CQ into an antimalarial treatment regimen where PPQ has lost efficacy.
In response to widespread treatment failures, PPQ was removed as a first-line partner drug. Recently, additional mutations have been observed on the highly-resistant Dd2+F145I PfCRT isoform. These mutations developed in parasites in long-term in vitro culture or in Southeast Asian field isolates. In Chapter 4, I characterized the impact of these mutations on parasite fitness and antimalarial susceptibility by editing asexual blood stage parasites to express these mutant PfCRT haplotypes. Competitive growth assays with a GFP-expressing reporter line revealed that these additional mutations reduce the fitness defect imposed by F145I, likely the primary driver of their emergence. I found that these mutations differentially impact parasite susceptibility to PPQ and CQ in in vitro dose-response assays. I used proteoliposome-based drug uptake studies, molecular dynamic simulations, and peptidomics to detail the molecular features of drug resistance and parasite physiology of these lines. These experiments provide insight into parasite responses to the changing drug selective pressures in SE Asia to inform treatment strategies in this region moving forward.
In Chapter 5, I sought to determine whether Asian PPQ-resistant PfCRT mutations could also mediate PPQ resistance on African PfCRT haplotypes. Using zinc-finger nuclease-based gene editing, I introduce the most common African mutant pfcrt alleles with a SE Asian PfCRT mutation into Dd2 parasites. In PPQ survival assays, these mutations only confer high-grade PPQ resistance (defined as ≥10% survival at 200 nM) on the FCB PfCRT background. I assessed the susceptibility of these gene-edited isogenic lines to other clinical antimalarials and the relative fitness of these engineered lines with in vitro assays. These experiments clearly show that there is a genetic path to PPQ resistance in African parasites; however, they also suggest that fitness costs associated with these mutations may hinder the spread of resistance.
Our data provide important insights into PPQ resistance. In chapter 6, these findings are summarized along with future studies to strengthen and expand on the findings presented herein.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/tpc2-qq39 |
| Date | January 2024 |
| Creators | Hagenah, Laura Marie |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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