Selection at Work in Plasmodium Falciparum: Lessons From the Expanded Acyl CoA Synthetase Gene Family and in Vitro Artemisinin Resistance.

Abstract

Approximately one third of the world’s population is at risk of contracting malaria. The World Health Organization estimates there were over 200 million news cases of malaria in 2015, resulting in nearly 500,000 deaths from this preventable disease. The majority of fatalities occur in Sub-Saharan Africa, where Plasmodium falciparum malaria causes severe disease in children under the age of five and pregnant women. In the last decade, increased anti-malaria interventions have resulted in substantial decreases in cases and fatalities. However, the recent emergence of artemisinin drug resistance in Southeast Asia threatens these gains, and the loss of another first-line antimalarial therapy would be a devastating setback.

The first goal of this work was to identify genetic markers of artemisinin drug resistance. Identifying the genetic determinants and molecular mechanisms of artemisinin resistance is crucial for understanding the emergence of this phenomenon and tracking the spread of these drug resistant parasites. Over the course of four years, we used an in vitro drug resistance selection approach to generate three independent artemisinin-resistant lines. Here we characterize those lines, and present Pfcoronin, a kelch13-like protein, as a novel candidate marker for artemisinin resistance. This study identifies additional non-kelch13 molecular markers of artemisinin resistance, increases our understanding of how this resistance is acquired, and sheds light on the molecular mechanisms of artemisinin resistance in the parasite.

In contrast to in vitro selection, natural selection of parasites occurs during natural infection. Investigation of specific genes under selection in the parasite will increase our understanding of biological processes that provide a fitness advantage, and potentially identify novel pathways for therapeutic development.

Here, we focused on the acyl Co-A synthetase (ACS) gene family, previously shown to be under recent positive selection in P. falciparum. The signatures of recent positive selection identified in natural parasite populations suggest that particular ACS alleles may confer a selective advantage. Using molecular genetics approaches, we show distinct expression and localization patterns for individual ACS isoforms, and identify a growth defect in the ACS5 knockout line. Follow up studies characterize the fatty acid and metabolic profiles of individual ACS knockout lines, and point to a role for ACS5 in central carbon metabolism in P. falciparum.

Our investigation of the ACS gene family and their role in P. falciparum growth and metabolism led us to hypothesize a link between ACS activity and central carbon metabolism. In the final chapter, we explore the basic fatty acid and glucose requirements for P. falciparum growth in vitro, and present a metabolic profile for these starved parasites. Under starvation conditions, we were able to demonstrate fatty acid oxidation activity in the parasite. This is an unexpected finding, as this pathway was not previously annotated in the genome.

Taken together, these two projects tell a story of the selective pressures acting on P. falciparum parasites. Investigating in vitro selected artemisinin-resistant lines provides important insights into genetic markers and acquisition of resistance. Molecular and biochemical characterization of a gene family under natural selection in P. falciparum increases our understanding of important metabolic pathways that support parasite growth.