Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease,ASP5, at the Host-Parasite Interface

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses.     

The Plasmodium falciparum exportome contains non‐canonical PEXEL/HT proteins

The pathogenicity of Plasmodium falciparum is partly due to parasite‐induced host cell modifications. These modifications are facilitated by exported P. falciparum proteins, collectively referred to as the exportome. Export of several hundred proteins is mediated by the PEXEL/HT, a protease cleavage site. The PEXEL/HT is usually comprised of five amino acids, of which R at position 1, L at position 3 and E, D or Q at position 5 are conserved and important for export. Non‐canonical PEXEL/HTs with K or H at position 1 and/or I at position 3 are presently considered non‐functional. Here, we show that non‐canonical PEXEL/HT proteins are overrepresented in P. falciparum and other Plasmodium species. Furthermore, we show that non‐canonical PEXEL/HTs can be cleaved and can promote export in both a REX3 and a GBP reporter, but not in a KAHRP reporter, indicating that non‐canonical PEXEL/HTs are functional in concert with a supportive sequence environment. We then selected P. falciparum proteins with a non‐canonical PEXEL/HT and show that some of these proteins are exported and that their export depends on non‐canonical PEXEL/HTs. We conclude that PEXEL/HT plasticity is higher than appreciated and that non‐canonical PEXEL/HT proteins cannot categorically be excluded from Plasmodium exportome predictions.     

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 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’s 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.     

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

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.     

The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells

Toxoplasma gondii and Plasmodium species are obligatory intracellular parasites that export proteins into the infected cells in order to interfere with host‐signalling pathways, acquire nutrients or evade host defense mechanisms. With regard to export mechanism, a wealth of information in Plasmodium spp. is available, while the mechanisms operating in T. gondii remain uncertain. The recent discovery of exported proteins in T. gondii, mainly represented by dense granule resident proteins, might explain this discrepancy and offers a unique opportunity to study the export mechanism in T. gondii. Here, we report that GRA16 export is mediated by two protein elements present in its N‐terminal region. Because the first element contains a putative Plasmodium export element linear motif (RRLAE), we hypothesized that GRA16 export depended on a maturation process involving protein cleavage. Using both N‐ and C‐terminal epitope tags, we provide evidence for protein proteolysis occurring in the N‐terminus of GRA16. We show that TgASP5, the T. gondii homolog of Plasmodium plasmepsin V, is essential for GRA16 export and is directly responsible for its maturation in a Plasmodium export element‐dependent manner. Interestingly, TgASP5 is also involved in GRA24 export, although the GRA24 maturation mechanism is TgASP5‐independent. Our data reveal different modus operandi for protein export, in which TgASP5 should play multiple functions.     

New insights into protein export in malaria parasites

In order to survive and promote its virulence the malaria parasite must export hundreds of its proteins beyond an encasing vacuole and membrane into the host red blood cell. In the last few years, several major advances have been made that have significantly contributed to our understanding of this export process. These include: (i) the identification of sequences that direct protein export (a signal sequence and a motif termed PEXEL), which have allowed predictions of the exportomes of Plasmodium species that are the cause of malaria, (ii) the recognition that the fate of proteins destined for export is already decided within the parasite's endoplasmic reticulum and involves the PEXEL motif being recognized and cleaved by the aspartic protease plasmepsin V and (iii) the discovery of the Plasmodium translocon of exported proteins (PTEX) that is responsible for the passage of proteins across the vacuolar membrane. We review protein export in Plasmodium and these latest developments in the field that have now provided a new platform from which trafficking of malaria proteins can be dissected.     

Role of Plasmepsin V in Export of Diverse Protein Families from the Plasmodium falciparum Exportome

Plasmodium falciparum exports several hundred effector proteins that remodel the host erythrocyte and enable parasites to acquire nutrients, sequester in the circulation and evade immune responses. The majority of exported proteins contain the Plasmodium export element (PEXEL; RxLxE/Q/D) in their N‐terminus, which is proteolytically cleaved in the parasite endoplasmic reticulum by Plasmepsin V, and is necessary for export. Several exported proteins lack a PEXEL or contain noncanonical motifs. Here, we assessed whether Plasmepsin V could process the N‐termini of diverse protein families in P. falciparum. We show that Plasmepsin V cleaves N‐terminal sequences from RIFIN, STEVOR and RESA multigene families, the latter of which contain a relaxed PEXEL (RxLxxE). However, Plasmepsin V does not cleave the N‐terminal sequence of the major exported virulence factor erythrocyte membrane protein 1 (PfEMP1) or the PEXEL‐negative exported proteins SBP‐1 or REX‐2. We probed the substrate specificity of Plasmepsin V and determined that lysine at the PEXEL P3 position, which is present in PfEMP1 and other putatively exported proteins, blocks Plasmepsin V activity. Furthermore, isoleucine at position P1 also blocked Plasmepsin V activity. The specificity of Plasmepsin V is therefore exquisitely confined and we have used this novel information to redefine the predicted P. falciparum PEXEL exportome .     

Shared elements of host‐targeting pathways among apicomplexan parasites of differing lifestyles

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal‐mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host‐targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal‐mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal‐mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.     

The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network

For membrane‐bound intracellular pathogens, the surrounding vacuole is the portal of communication with the host cell. The parasitophorous vacuole (PV) harboring intrahepatocytic Plasmodium parasites satisfies the parasites' needs of nutrition and protection from host defenses to allow the rapid parasite growth that occurs during the liver stage of infection. In this study, we visualized the PV membrane (PVM) and the associated tubovesicular network (TVN) through fluorescent tagging of two PVM‐resident Plasmodium berghei proteins, UIS4 and IBIS1. This strategy revealed previously unrecognized dynamics with which these membranes extend throughout the host cell. We observed dynamic vesicles, elongated clusters of membranes and long tubules that rapidly extend and contract from the PVM in a microtubule‐dependent manner. Live microscopy, correlative light‐electron microscopy and fluorescent recovery after photobleaching enabled a detailed characterization of these membranous features, including velocities, the distribution of UIS4 and IBIS1, and the connectivity of PVM and TVN. Labeling of host cell compartments revealed association of late endosomes and lysosomes with the elongated membrane clusters. Moreover, the signature host autophagosome protein LC3 was recruited to the PVM and TVN and colocalized with UIS4. Together, our data demonstrate that the membranes surrounding intrahepatic Plasmodium are involved in active remodeling of host cells.      

Spatial organization of protein export in malaria parasite blood stages

Plasmodium falciparum, which causes malaria, extensively remodels its human host cells, particularly erythrocytes. Remodelling is essential for parasite survival by helping to avoid host immunity and assisting in the uptake of plasma nutrients to fuel rapid growth. Host cell renovation is carried out by hundreds of parasite effector proteins that are exported into the erythrocyte across an enveloping parasitophorous vacuole membrane (PVM). The Plasmodium translocon for exported (PTEX) proteins is thought to span the PVM and provide a channel that unfolds and extrudes proteins across the PVM into the erythrocyte. We show that exported reporter proteins containing mouse dihydrofolate reductase domains that inducibly resist unfolding become trapped at the parasite surface partly colocalizing with PTEX. When cargo is trapped, loop‐like extensions appear at the PVM containing both trapped cargo and PTEX protein EXP2, but not additional components HSP101 and PTEX150. Following removal of the block‐inducing compound, export of reporter proteins only partly recovers possibly because much of the trapped cargo is spatially segregated in the loop regions away from PTEX. This suggests that parasites have the means to isolate unfoldable cargo proteins from PTEX‐containing export zones to avert disruption of protein export that would reduce parasite growth.      

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P‐mediated autophagy‐independent pathway

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.     

The Plasmodium translocon of exported proteins component EXP2 is critical for establishing a patent malaria infection in mice

Export of most malaria proteins into the erythrocyte cytosol requires the Plasmodium translocon of exported proteins (PTEX) and a cleavable Plasmodium export element (PEXEL). In contrast, the contribution of PTEX in the liver stages and export of liver stage proteins is unknown. Here, using the FLP/FRT conditional mutatagenesis system, we generate transgenic Plasmodium berghei parasites deficient in EXP2, the putative pore‐forming component of PTEX. Our data reveal that EXP2 is important for parasite growth in the liver and critical for parasite transition to the blood, with parasites impaired in their ability to generate a patent blood‐stage infection. Surprisingly, whilst parasites expressing a functional PTEX machinery can efficiently export a PEXEL‐bearing GFP reporter into the erythrocyte cytosol during a blood stage infection, this same reporter aggregates in large accumulations within the confines of the parasitophorous vacuole membrane during hepatocyte growth. Notably HSP101, the putative molecular motor of PTEX, could not be detected during the early liver stages of infection, which may explain why direct protein translocation of this soluble PEXEL‐bearing reporter or indeed native PEXEL proteins into the hepatocyte cytosol has not been observed. This suggests that PTEX function may not be conserved between the blood and liver stages of malaria infection.     

A crucial role for the C‐terminal domain of exported protein 1 during the mosquito and hepatic stages of the Plasmodium berghei life cycle

Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N‐terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C‐terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild‐type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention.     

The Exported Protein PbCP1 Localises to Cleft-Like Structures in the Rodent Malaria Parasite Plasmodium berghei

Protein export into the host red blood cell is one of the key processes in the pathobiology of the malaria parasite Plasmodiumtrl falciparum, which extensively remodels the red blood cell to ensure its virulence and survival. In this study, we aimed to shed further light on the protein export mechanisms in the rodent malaria parasite P. berghei and provide further proof of the conserved nature of host cell remodeling in Plasmodium spp. Based on the presence of an export motif (R/KxLxE/Q/D) termed PEXEL (Plasmodium export element), we have generated transgenic P. berghei parasite lines expressing GFP chimera of putatively exported proteins and analysed one of the newly identified exported proteins in detail. This essential protein, termed PbCP1 (P. berghei Cleft-like Protein 1), harbours an atypical PEXEL motif (RxLxY) and is further characterised by two predicted transmembrane domains (2TMD) in the C-terminal end of the protein. We have functionally validated the unusual PEXEL motif in PbCP1 and analysed the role of the 2TMD region, which is required to recruit PbCP1 to discrete membranous structures in the red blood cell cytosol that have a convoluted, vesico-tubular morphology by electron microscopy. Importantly, this study reveals that rodent malaria species also induce modifications to their host red blood cell.     

Role of the Plasmodium Export Element in Trafficking Parasite Proteins to the Infected Erythrocyte

The intracellular survival of Plasmodium falciparum within human erythrocytes is dependent on export of parasite proteins that remodel the host cell. Most exported proteins require a conserved motif (RxLxE/Q/D), termed the Plasmodium export element (PEXEL) or vacuolar targeting sequence (VTS), for targeting beyond the parasitophorous vacuole membrane and into the host cell; however, the precise role of this motif in export is poorly defined. We used transgenic P. falciparum expressing chimeric proteins to investigate the function of the PEXEL motif for export. The PEXEL constitutes a bifunctional export motif comprising a protease recognition sequence that is cleaved, in the endoplasmic reticulum, from proteins destined for export, in a PEXEL arginine- and leucine-dependent manner. Following processing, the remaining conserved PEXEL residue is required to direct the mature protein to the host cell. Furthermore, we demonstrate that N acetylation of proteins following N-terminal processing is a PEXEL-independent process that is insufficient for correct export to the host cell. This work defines the role of each residue in the PEXEL for export into the P. falciparum -infected erythrocyte.     

Trafficking of PfExp1 to the parasitophorous vacuolar membrane of Plasmodium falciparum is independent of protein folding and the PTEX translocon

Having entered the mature human erythrocyte, the malaria parasite survives and propagates within a parasitophorous vacuole, a membrane‐bound compartment separating the parasite from the host cell cytosol. The bounding membrane of this vacuole, referred to as the parasitophorous vacuolar membrane (PVM), contains parasite‐encoded proteins, but how these membrane proteins are trafficked to the PVM remains unknown. Here, we have studied the trafficking of PfExp1 to the PVM. We find that trafficking of PfExp1 to the PVM is independent of the folding state of the protein and also continues unabated upon inactivation of the PVM translocon Plasmodium Translocon of Exported proteins (PTEX). Our data strongly suggest that the trafficking of membrane proteins to the PVM occurs by as yet unknown mechanism, potentially unique to Plasmodium.     

Comparison of dinucleotide frequency and codon usage inToxoplasma andPlasmodium: Evolutionary implications

Summary The weight-averaged observed/expected dinucleotide frequencies for the sum total of the coding regions of fiveToxoplasma genes were compared with the same parameters previously determined for the coding regions of 21Plasmodium genes. In addition, codon usage in the fiveToxoplasma genes was compared with that in the 21Plasmodium genes, and the percent distribution of amino acids in theToxoplasma protein pool and thePlasmodium protein pool were compared with that in a general protein pool of 314 proteins. The results are consistent with the hypothesis that, contrary to currently held opinion, the generaToxoplasma andPlasmodium are not especially closely related.     

The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill‐defined due to the lack of functional studies. In this study, we show that the rhoptry‐associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post‐invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra‐erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite–host cell interface are not simply by‐products of the invasion process but contribute to the growth of Plasmodium in vivo.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.     

Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

The immunity‐related GTPases (IRGs) constitute an interferon‐induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion‐driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild‐type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A–D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.