Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is key to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here, we identify a Plasmodium Translocon of EXported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered and comprises HSP101, which is a ClpA/B-like AAA+ ATPase of a type commonly associated with protein translocons, a novel protein termed PTEX150 and a known parasite protein EXP2. EXP2 is the potential channel as it is the membrane-associated component of the core PTEX complex. Two other proteins, a novel protein PTEX88 and a thioredoxin known as TRX2, were also identified as PTEX components. As a common portal for numerous crucial processes, this novel translocon offers an exciting new avenue for therapeutic intervention.

Plasmodium falciparum causes the virulent form of malaria and disease manifestations are linked to growth inside infected erythrocytes. In order to survive and evade host responses the parasite remodels the erythrocyte by exporting several hundred effector proteins beyond the surrounding parasitophorous vacuole membrane. A feature of exported proteins is a pentameric motif (RxLxE/Q/D) that is a substrate for an unknown protease. Here, we show the protein responsible for cleavage of this motif is Plasmepsin V, an aspartic acid protease located in the endoplasmic reticulum. Plasmepsin V cleavage reveals the export signal (xE/Q/D) at the N-terminus of cargo proteins. Expression of an identical mature protein with xQ at the N-terminus generated by signal peptidase was not exported demonstrating Plasmepsin V activity is essential and linked with other key export events. Identification of the protease responsible for export into erythrocytes provides a novel target for therapeutic intervention against this devastating disease.

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

The mechanisms responsible for the immunosuppression associated with sepsis or some chronic blood infections remain poorly understood. Here we show that infection with a malaria parasite (Plasmodium berghei) or simple systemic exposure to bacterial or viral Toll-like receptor ligands inhibited cross-priming. Reduced cross-priming was a consequence of downregulation of cross-presentation by activated dendritic cells due to systemic activation that did not otherwise globally inhibit T cell proliferation. Although activated dendritic cells retained their capacity to present viral antigens via the endogenous major histocompatibility complex class I processing pathway, antiviral responses were greatly impaired in mice exposed to Toll-like receptor ligands. This is consistent with a key function for cross-presentation in antiviral immunity and helps explain the immunosuppressive effects of systemic infection. Moreover, inhibition of cross-presentation was overcome by injection of dendritic cells bearing antigen, which provides a new strategy for generating immunity during immunosuppressive blood infections.