Cellular and Molecular Physiology of Bloodstream Malaria Parasites

National Institute Of Allergy And Infectious Diseases

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Abstract

In 2021, the Apicomplexan Molecular Physiology Section Malaria determined the cryo-EM structure of the RhopH complex, implicated in both erythrocyte invasion and formation of the essential plasmodial surface anion channel (PSAC) at the host membrane of malaria-parasite infected erythrocytes. Because the RhopH complex consists of three proteins unique to Plasmodium spp., how these subunits interact and traffic through subcellular sites to function in two unrelated but essential activities was unknown. We found that RhopH is synthesized as a soluble complex of CLAG3, RhopH2, and RhopH3 with 1:1:1 stoichiometry. After transfer to a new host cell, the complex crosses a vacuolar membrane surrounding the intracellular parasite and becomes integral to the erythrocyte membrane through a PTEX translocon-dependent process. Our 2.9 single-particle cryo-electron microscopy structure of the trafficking complex revealed that CLAG3 interacts with the other subunits over large surface areas. This soluble complex is tightly assembled with extensive disulfide bonding and predicted transmembrane helices shielded. Through this study, we proposed a large protein complex stabilized for trafficking but poised for host membrane insertion through large-scale rearrangements, paralleling smaller two-state pore-forming proteins in other organisms. eLife 10:e65282 (2021). PMID: 33393463. In another study, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Frster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell's parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation. mBio 11:e01354-20 (2020). PMID: 32900800 We also developed an improved method for limiting dilution cloning of Plasmodium falciparum cultures, as required for molecular and biochemical studies of clinical isolates and transfected lines. This process has been laborious, time-consuming, and subject to errors due to inaccurate dilutions at the onset. Historically, experiments frequently failed to cover clonal lines from microplate dilutions. Our improved method precisely controls the number of parasites dispensed into each microplate wellby measuring parasitaemia and total cell counts with flow cytometry. It also enables robust and facile detection of parasite growth in microplate wells using the c-SNARF fluorescent pH indicator. This combination of technologies should enable efficient cloning of individual parasite lines from large scale transfections such as those generated by chemical mutagenesis or directed saturation mutagenesis of genes of interest. Malaria J. 20:279 (2021). PMID: 34162381.