Cellular and Molecular Physiology of Bloodstream Malaria Parasites

National Institute Of Allergy And Infectious Diseases

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Abstract

In 2019, the Apicomplexan Molecular Physiology Section examined calcium transport and utilization by malaria parasites. We previously reported that malaria parasites require extracellular Ca++ for their development and that infected cells have increased Ca++ permeability. The sites and mechanisms of Ca++ utilization are, however, not well understood. We therefore used geographically divergent lines of the human pathogen, P. falciparum, to examine uptake and utilization. We used the Ca++ chelator EGTA and determined that six divergent parasite lines from four available genetic crosses exhibit stable difference in their Ca++ requirement for in vitro propagation. We identified a significant difference in Ca++ requirement between the parents of the HB3 x Dd2 genetic cross and used genetic mapping in 34 progeny clones to identify a single significant genomic locus on the parasite chromosome 7. Although encoded by a gene in the significant locus and a proposed Ca++ target, PfCRT (P. falciparum chloroquine resistance transporter), the primary determinant of clinical resistance to the antimalarial drug chloroquine, does not appear to contribute to this quantitative trait. Stage-specific application of extracellular EGTA also excluded determinants associated with merozoite egress and erythrocyte reinvasion. Ca++ utilization is therefore under genetic regulation and is inherited as a complex trait in bloodstream malaria parasites. This study will guide future research into the roles and molecular mechanisms of Ca++ uptake and utilization and may lead to the development of new antimalarial therapies. BMC Genomics 20:47 (2019). doi: 10.1186/s12864-018-5418-y. In another study, we examined and compared methods for genetic manipulation of the human malaria parasite. DNA transfection of malaria parasites is aggravated by a low transfection efficiency, a long incubation period prior to parasite outgrowth, a paucity of selectable markers and a biased A/T-rich genome that complicates gene cloning. While various enabling technologies have been introduced over the past two decades, facile and broad-range modification of essential genes remains challenging. Our laboratory recently designed and implemented a new application of the Bxb1 integrase strategy to meet this need through an intronic attB sequence within the gene of interest. The intronic attB element is silent and without effect on intron splicing or protein translation and function, but it allows efficient gene modification with minimal risk of unwanted changes at other genomic sites. Our study describes the range of applications for this new method as well as specific cases where it is preferred over CRISPR-Cas9 and other technologies. The advantages and limitations of various strategies for endogenous gene editing are also discussed and should benefit basic and translational studies of this pathogen. Parasites & Vectors 11:548 (2018). doi: 10.1186/s13071-018-3129-5.