Gabriela, M. | Search bioRxiv

bioRxiv2022

The sulfonylpiperazine MMV020291 prevents red blood cell invasion by the malaria parasite Plasmodium falciparum through interference with actin-1/profilin dynamics

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell invasion, and have generated analogues with improved potency. Here, we identify actin and profilin as putative targets of the MMV020291 series through resistance selection and whole genome sequencing of three MMV020291 resistant populations. This revealed three non-synonymous single nucleotide polymorphisms in two genes; two in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wildtype parasites which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade red blood cells. Additionally, the series inhibits the actin-1 dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro co-sedimentation experiments using recombinant P. falciparum actin-1 and profilin proteins indicate that potent MMV020291 analogues amplify the actin-monomer sequestering effect of profilin, thereby reducing the formation of filamentous actin. Altogether, this study identifies the first compound series targeting the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

molecular biology↗

bioRxiv2021

A revised mechanism for how Plasmodium falciparum recruits and exports proteins into its erythrocytic host cell.

Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cells physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins are processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasites ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasites ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101s specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte. Author SummaryPlasmodium falciparum, the most lethal species of human malaria parasite, infects erythrocytes and develops within a parasitophorous vacuole. To support rapid parasite growth and immune evasion, the parasite remodels its erythrocyte by exporting a myriad of proteins into the erythrocyte compartment. Parasite proteins destined for export are first imported into the endoplasmic reticulum (ER) and then secreted into the parasitophorous vacuole, where they are translocated across the parasitophorous vacuole membrane into the erythrocyte via a protein-conducting channel called PTEX. A missing link in the story has been how proteins destined for export are specifically guided from the ER to PTEX at the parasitophorous vacuole membrane. In this study, we found that one of the core PTEX components, HSP101, resides within the parasites ER, in addition to its PTEX-related location at the parasitophorous vacuole. We also found that ER-located HSP101 can interact transiently with cargo proteins en route to the parasitophorous vacuole membrane. Our findings support a model in which HSP101 forms an initial interaction with exported proteins in the ER and then chaperones them to the rest of PTEX at the parasitophorous vacuole membrane for export into the erythrocyte.

microbiology↗