Person:

Demas, Allison

Background: Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin–piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. Methods: Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0–3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. Results: Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0–3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. Conclusions: This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1845-5) contains supplementary material, which is available to authorized users.

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.

Summary Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.