Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell’s 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 is 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 parasite’s 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 parasite’s 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 HSP101’s specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte.

Plasmodium parasites responsible for the disease malaria reside within erythrocytes. Inside this niche host cell, parasites internalize and digest host hemoglobin to source amino acids required for protein production. However, hemoglobin does not contain isoleucine, an amino acid essential for Plasmodium growth, and the parasite cannot synthesize it de novo. The parasite is also more metabolically active than its host cell, and the rate at which some nutrients are consumed exceeds the rate at which they can be taken up by erythrocyte transporters. To overcome these constraints, Plasmodium parasites increase the permeability of the erythrocyte membrane to isoleucine and other low-molecular-weight solutes it requires for growth by forming new permeation pathways (NPPs). In addition to the erythrocyte membrane, host nutrients also need to cross the encasing parasitophorous vacuole membrane (PVM) and the parasite plasma membrane to access the parasite. This review outlines recent advances that have been made in identifying the molecular constituents of the NPPs, the PVM nutrient channel, and the endocytic apparatus that transports host hemoglobin and identifies key knowledge gaps that remain. Importantly, blocking the ability of Plasmodium to source essential nutrients is lethal to the parasite, and thus, components of these key pathways represent potential antimalaria drug targets.

α-Carbonic anhydrase of Helicobacter pylori (HpαCA) plays an important role in the acclimation of this oncobacterium to the acidic pH of the stomach. Sulfonamide inhibitors of HpαCA possess anti-H. pylori activity. The crystal structures of complexes of HpαCA with a family of acetazolamide-related sulfonamides have been determined. Analysis of the structures revealed that the mode of sulfonamide binding correlates well with their inhibitory activities. In addition, comparisons with the corresponding inhibitor complexes of human carbonic anhydrase II (HCAII) indicated that HpαCA possesses an additional, alternative binding site for sulfonamides that is not present in HCAII. Furthermore, the hydrophobic pocket in HCAII that stabilizes the apolar moiety of sulfonamide inhibitors is replaced with a more open, hydrophilic pocket in HpαCA. Thus, our analysis identified major structural features can be exploited in the design of selective and more potent inhibitors of HpαCA that may lead to novel antimicrobials.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry-based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J-dot complex, which overall helps clarify protein-protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane.