In 2018, the Apicomplexan Molecular Physiology Section examined structure and function of a conserved high-molecular weight protein complex, termed the RhopH complex, in malaria parasites. We previously reported that the RhopH complex consists of three proteins (RhopH2, RhopH3, and one or more members of the CLAG multigene family). We also determined that this complex contributes to two essential activities in bloodstream parasites: erythrocyte invasion and nutrient uptake via an essential ion and nutrient channel at the host membrane. This channel, termed the plasmodial surface anion channel (PSAC), is an established antimalarial drug target. Here, we used DNA transfection and biochemical studies to examine how these proteins may contribute to PSAC formation. We used the Bxb1 integrase and a novel intronic attB sequence to produce a transfected parasite line expressing two isoforms of CLAG3 simultaneously. Biochemical studies using this parasite revealed an intermediate channel phenotype, consistent with trafficking of both CLAG3 isoforms to the host membrane. Co-immunoprecipitation and surface labeling studies revealed formation of CLAG3 oligomers in association with RhopH2 and RhopH3 at the host erythrocyte membrane. Along with in vitro selections applied to two distinct transfectant lines, these findings support direct contribution of the RhopH complex to PSAC formation and parasite nutrient acquisition. This dual-function protein complex is a validated target for development of new antimalarial therapies. MBio 9:e02293-17 (2018). We also examined the increased Ca++ permeability of P. falciparum-infected erythrocytes with a kinetic fluorescence assay developed by our group. This Ca++ permeability is important because Ca++ uptake is required for intracellular parasite development and it is mediated by a poorly characterized mechanism distinct from PSAC. We used cell surface labeling to determine that the uptake is mediated by new or altered proteins at the host membrane; conditional knockdown of parasite protein export significantly reduced infected cell Ca++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. We also used high-throughput screening to identify the first Ca++ transport inhibitors active against Plasmodium-infected cells. Ca++ uptake and utilization is essential for bloodstream parasites and represents an unexplored target for drug development. Cell Microbiol. doi: 10.1111/cmi.12853 (2018). In another study, we examined the optimal conditions for CRISPR-Cas9 gene editing in malaria parasites. We used a genome-wide computational approach to create a comprehensive database of single guide RNA sequences in the Plasmodium falciparum genome. This database is publicly available and includes information required for optimal design of CRISPR-Cas9 transfections for the full range of target gene modifications, including mutagenesis, gene knockout and gene knockdown. Our study also tabulated features of a large number of successful transfections in our laboratory, providing specific guidelines that should facilitate molecular studies over the broad range of basic and translational malaria research. Int J Parasitol. doi: 10.1016/j.ijpara.2018.03.009 (2018).
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