Graduation Year

2015

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Medicine

Major Professor

Dennis E. Kyle, Ph.D.

Committee Member

John H. Adams, Ph.D.

Committee Member

Burt Anderson, Ph.D.

Committee Member

Yu Chen, Ph.D.

Keywords

Artemisinin, Cell Cycle Phenotypes, Drug Resistance, Fitness, Malaria

Abstract

Resistance to artemisinin combination therapies (ACTs) has emerged in southeast Asia threatening the most widely used treatment against antimalarial-resistant Plasmodium falciparum worldwide. Artemisinin resistance has been associated with a reduced rate of parasite clearance following treatment with an ACT and is attributed to increased survival of ring-stage parasites. Single nucleotide polymorphisms (SNPs) in kelch gene (K13) has been associated with delayed in vivo clearance half-life of artemisinin-resistant P. falciparum and is the only known molecular marker of resistance. The absence of reliable in vitro phenotypes for artemisinin resistance has limited our understanding of the resistance mechanism(s) and fitness costs, therefore we have culture adapted and cloned patient isolates from Thailand and Cambodia that had clinical resistant phenotypes. Stable reduced susceptibility to artemisinin derivatives and mefloquine was observed using a modified [3H]hypoxanthine drug susceptibility assay. In addition we devised an in vitro phenotype assay of artemisinin resistance, known as the delayed clearance assay (DCA), that was positively correlated with the in vivo delayed clearance and presence of K13 SNPs of artemisinin resistance. Remarkably we discovered for the first time altered patterns of intraerythrocytic development in artemisinin-resistant P. falciparum. In the absence of drug pressure most artemisinin-resistant clones have a prolonged ring phenotype with temporally compressed trophozoite stage, yet with the normal overall asexual life cycle period. Parasites remain in ring-stage up to 14 hours longer than wild-type, whereas time spent in trophozoite-stage is dramatically reduced. One parasite, PL08-09 (5C), exhibited an accelerated 36-hour life cycle in the absence of drug pressure and progressed through asexual development in equal time spent at each intraerythrocytic stage. These data support our hypothesis that parasite resistance to artemisinin results from reduced exposure to drug at the most susceptible stage of development (trophozoite). Interestingly, the most prevalent K13 mutation C580Y is associated with both cell cycle phenotypes. Another cell cycle phenotype, ring-stage dormancy, has been associated with artemisinin resistance in vitro reducing the dormant period of artemisinin-resistant parasites following dihydroartemisinin exposure allowing resistant parasite cultures recrudesce before wild-type. However, sensitive parasites have the ability to enter ring-stage dormancy causing recrudescence in vitro. It is possible that multiple cell cycle phenotypes enhance the survival and fitness of the resistant parasite population as a whole in the face of antimalarial exposure. We have demonstrated that there is a fitness cost associated with artemisinin resistance and remains an important component in the spread of genetic mutations associated with artemisinin resistance. Resistant parasites outcompeted sensitive in vitro only when exposed to dihydroartemisinin. Two mutations associated with artemisinin resistance, including a mutation in K13, were lost in artemisinin resistant parasite by the end of the study. Conversely, parasite cultures maintained artemisinin resistance phenotypes in vitro only if exposed to artemisinin drug pressure every 21-42 days. The mechanism of artemisinin resistance remains elusive and how the parasites alter their intraerythrocytic development is unknown. Therefore. we transfected green fluorescent protein (GFP) or luciferase into artemisinin-resistant P. falciparum clones from Thailand and Cambodia to aid the study the cell cycle phenotypes associated with artemisinin resistance. Artemisinin resistance phenotypes were maintained in stably transfected clones. Increased growth of artemisinin-resistant clones was observed following exposure to ACT partner drug. Low concentrations of antimalarials synchronized the luciferase expression of artemisinin-resistant parasites having different cell cycle phenotypes in the absence of drug pressure. Ring-stage dormancy was observed with many antimalarial drugs and contributes to recrudescence observed by antimalarials other than artemisinin. Our results show evidence that current ACT treatments are selecting multidrug resistant parasites in the field that are better able to tolerate all antimalarials through regulatory cell cycle mechanisms. These cell cycle phenotypes associated with artemisinin resistance contribute to reducing the fitness cost associated with genetic mutations causing artemisinin resistance. This leads to the survival of the most fit population of parasites that survive combination drug treatments, thus demonstrating the importance of discovering novel drugs to target ACT-resistant P. falciparum.

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