The spread of resistance to artemisinin (ART) derivatives is an emerging public health crisis, but also presents a valuable opportunity to understand the dynamics and evolution of resistance in real-time, so we can better manage resistance in the future. My laboratory will focus on three different, but complimentary research paths during the Merit extension period, with the central aim of understanding the evolutionary dynamics, genetic architecture and mechanism of ARTresistance. First, in collaboration with Franois Nosten, we will continue our long term monitoring of parasite populations on the Thai-Burma border, by sequencing genomes from ~100 parasites per year. We will document the change in frequency of kelch mutations over space and time, and critically examine evidence that additional genes are selected by ART treatment. These analyses will explore different statistical approaches for inferring adaptive evolution using longitudinal data and will generate testable hypotheses about resistance evolution. Second, to compliment our descriptive and statistical work and to test hypotheses generated, we will exploit our new-found gene-editing skills using CRISPR/Cas9 methods. We will examine functional differences between kelch alleles and experimentally investigate the role of additional genes selected by ART or incriminated in genome-wide association or linkage analysis studies. These studies will involve (i) editing of kelch alleles in parasite clones isolated from the Thailand-Burma border, (ii) phenotypic analyses of fitness, resistance and genome-wide transcription in edited parasites to determine the costs and benefits of different kelch alleles, (iii) functional analyses of additional candidate genes implicated in ART-resistance evolution, and (iv) development of high-throughput CRISPR/Cas9 methods using pools of repair template sequences or guide sequences together with deep sequence readout for gene and genome-wide analyses of loci underlying resistance. Lastly, we will examine how single SNP changes in the kelch locus impact protein structure. This work will be conducted with John Hart (UTHSCSA), with whom we have previously collaborated in studies of schistosome drug resistance. We will determine crystal structures of wild-type and ART-resistant kelch proteins encoded by different kelch alleles at high resolution. The molecules and biochemical pathways involved in resistance are currently being identified. Co-crystallization of kelch and interacting partner molecules will help to determine the mechanisms by which structural changes impact molecular interactions and generate ART-resistance phenotypes. The genome sequences, edited parasite clones, and crystal structures generated during this work will be made available to the research community. The outcome of this research will be a detailed understanding of a drug resistance selective sweep at population, genomic, functional and structural levels.
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