Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in Plasmodium falciparum Protein Export

Abstract

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.

Author Summary

Malaria parasites multiply within erythrocytes, highly specialized cells that require profound alterations to support parasite survival. In order to understand how the parasite takes over the host cell it is necessary to know its molecular toolbox to carry out this process, which consists of the proteins exported from the parasite into the host cell. While many such proteins are known because they contain the clearly defined PEXEL/HT motif, an unknown number of further proteins lack such a motif. Here we present the identification of several of these PEXEL negative exported proteins (PNEPs). We analyzed the sequences mediating export in a subset of these proteins and find that in PNEPs lacking an N-terminal signal peptide, the N-terminal region of these proteins is sufficient to mediate export. Thus, despite the lack of a clear signature sequence, these regions share a property mediating export. In addition, we found evidence that suggests that soluble PNEPs get transported into the host cell by translocation across the parasitophorous membrane that surrounds the parasite. This adds a further group of proteins to share this property and highlights protein translocation as a general means of export in all types of malaria proteins tested so far.