Graduation Year

2016

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Dennis E. Kyle, Ph.D.

Committee Member

Burt Anderson, Ph.D.

Committee Member

Bill Baker, Ph.D.

Committee Member

Gloria Ferreira, Ph.D.

Committee Member

Robert Kennedy Keller, Ph.D.

Committee Member

John B. Dame, Ph.D.

Keywords

Malaria, dormancy, abscisic acid, gibberellic acid, isoprenoids

Abstract

Our ability to control malaria has been challenged by increasing antimalarial resistance. Plasmodium falciparum undergoes dormancy in the blood stages which is hypothesized to be a means by which they are able to survive under drug pressure. This helps select for resistant parasites which grow following removal of drug. The mechanisms behind dormancy and the subsequent recrudescence are not fully understood but translating knowledge from related organisms which undergo a similar phenomenon might shed some light. Higher plants utilize dormancy during the early development stages to survive under unfavorable conditions, increasing fitness of the seedling and ensuring viability when this is released and it develops into a mature plant. Abscisic acid (ABA) and gibberellic acid (GA) antagonistically regulate this in response to environmental cues. We have found that both can be supplemented to dihydroartemesinin-induced dormant parasites to stimulate early recovery. Fluridone, an ABA inhibitor that releases dormancy in plants, was found to prolong it and cause a delay in recrudescence. These effects were observed in artemisinin sensitive and resistant strains.

The apicoplast is required for recovery and supplementation of essential isoprenoid, isopentyl pyrophosphate (IPP), in apicoplast deficient parasites is sufficient enough to compensate for the lack of the organelle in antibiotic treated parasites. IPP plays an important role in development and metabolism of blood stage parasites as a key component of numerous secondary metabolites and protein activity by prenylation of isoprenoids. Its role in dormancy has not been explored prior to this study.

Carotenoids are long-chained ABA precursors consisting of two molecules of geranylgeranyl pyrophosphate (GGPP). Several carotenoids as well as enzymes in that pathway have been identified in the blood stages of P. falciparum. The Apicomplexan parasite, Toxoplasma gondii synthesizes ABA, where it plays a role in signaling and development. To date ABA has not been detected in P. falciparum due to limitations in methods previously utilized. We have found that parasites with fosmidomycin inhibition of isoprenoids can be rescued with GGPP supplementation which we planned to use to further elucidate the carotenoid biosynthetic pathway.

We hypothesized that Plasmodium has retained the ability to biosynthesize ABA and aimed to confirm this. We developed a novel method to label GGPP with 13C on three of its isoprene units. This would be used to metabolically label isoprenoid inhibited P. falciparum for incorporation through the carotenoid pathway for detection of 13C-ABA.

Available for download on Saturday, December 09, 2017

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