Identification of a non-exported Plasmepsin V substrate that functions in the parasitophorous vacuole of malaria parasites

Abstract

Malaria parasites alter multiple properties of the host erythrocyte by exporting proteins into the host cell. Many exported proteins contain a five-amino acid motif called the Plasmodium export element (PEXEL) that is cleaved by the parasite protease Plasmepsin V (PM V). The presence of a PEXEL is considered a signature of protein export and has been used to identify a large number of exported proteins. The export of proteins becomes essential midway through the intraerythrocytic cycle—preventing protein export blocks parasite development 18–24 h after invasion. However, a genetic investigation revealed that the absence of the PEXEL protein PFA0210c (PF3D7_0104200) causes parasite development to arrest immediately after invasion. We now show that this protein is cleaved by PM V but not exported into the host erythrocyte and instead functions in the parasitophorous vacuole; hence, the protein was renamed PV6. We additionally show that the lysine residue that becomes the N-terminus of PV6 after processing by PM V prevents export. This is the first example of a native Plasmodium falciparum PM V substrate that remains in the parasitophorous vacuole. We also provide evidence suggesting that the parasite may produce at least one additional essential, non-exported PM V substrate. Therefore, the presence of a PEXEL and, hence, processing of a protein by PM V do not always target a protein for export, and PM V likely has a broader function in parasite growth beyond processing exported proteins. Furthermore, we utilized this finding to investigate possible requirements for protein export further. IMPORTANCE In the manuscript, the authors investigate the role of the protease Plasmepsin V in the parasite–host interaction. Whereas processing by Plasmepsin V was previously thought to target a protein for export into the host cell, the authors now show that there are proteins cleaved by this protease that are not exported but instead function at the host–parasite interface. This changes the view of this protease, which turns out to have a much broader role than anticipated. The result shows that the protease may have a function much more similar to that of related organisms. The authors also investigate the requirements for protein export by analyzing exported and non-exported proteins and find commonalities between the proteins of each set that further our understanding of the requirements for protein export.

Aryl amino acetamides prevent Plasmodium falciparum ring development via targeting the lipid-transfer protein PfSTART1

“…For Pf START1 proc2 , a similar conclusion is harder to draw considering the degradation products co-migrate in the same size. Altogether this data corroborates previous work 12 , 16 , 17 that indicates Pf START1 is most strongly expressed in schizonts, associated with membranes, and that more is located in the PV than in the parasite.…”

“…Pf START1 contains an unusual PEXEL motif and was recently described as not being exported to the RBC in trophozoites 37 . To investigate Pf START1 localisation in schizonts, 3D7 schizonts were Percoll-purified and sequentially lysed in equinatoxin II (EqtII), saponin (Sap) and Triton X100 (TX100) to collect the supernatant (SN) corresponding to the RBC cytosol soluble fraction, the parasitophorous vacuole (PV) soluble fraction, and the parasite fraction, respectively (Fig.…”

“…We found that Pf START1 inhibitors were active in a standard membrane-feeding assay. Whilst the functionality of Pf START1 has been previously explored in the asexual blood stage 16 , 17 , 37 , there is little known about Pf START1 in the mosquito stage of infection. Transcriptomic studies have shown that Pf START1 is expressed in salivary gland sporozoites 62 , 63 and ookinetes 64 .…”

“…One hit compound, MMV006833 (M-833), had a novel effect whereby the merozoite entered its target RBC but failed to differentiate into an amoeboid ring-stage parasite during the formation of the parasitophorous vacuole membrane (PVM) around the parasite 11 . Here, we generated M-833 resistant parasites and whole genome sequencing revealed mutations in the Plasmodium falciparum Steroidogenic Acute Regulatory protein-related lipid Transfer (START) domain-containing phospholipid transfer protein (PF3D7_0104200, PFA0210c, also called PV6 12 ).…”

PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins

“…Collectively, this suggests that PEXEL P1′ residues with charged or certain bulky side chains (phenylalanine but not tyrosine) are competent for processing but not export. Consistent with this, a recent report indicates lysine is also not tolerated for export at P1′ ( 42 ). For PV resident proteins that do not function in the host cell, these P1′ residues would provide a control mechanism to allow for proteolytic maturation by PMV without risking unproductive export, presumably preventing recognition by the PTEX unfoldase HSP101 ( 43 ).…”