The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol

The malaria parasite exports numerous proteins into its host red blood cell (RBC). The trafficking of these exported effectors is complex. Proteins are first routed through the secretory system, into the parasitophorous vacuole (PV), a membranous compartment enclosing the parasite. Proteins are then translocated across the PV membrane in a process requiring ATP and unfolding. Once in the RBC compartment the exported proteins are then refolded and further trafficked to their final localizations. Chaperones are important in the unfolding and refolding processes. Recently, it was suggested that the parasite TRiC chaperonin complex is exported, and that it is involved in trafficking of exported effectors. Using a parasite‐specific antibody and epitope‐tagged transgenic parasites we could observe no export of Plasmodium TRiC into the RBC. We tested the importance of the parasite TRiC by creating a regulatable knockdown line of the TRiC‐θ subunit. Loss of the parasite TRiC‐θ led to a severe growth defect in asexual development, but did not alter protein export into the RBC. These observations indicate that the TRiC proteins play a critical role in parasite biology, though their function, within the parasite, appears unrelated to protein trafficking in the RBC compartment.     

Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.     

Targeting malaria parasite proteins to the erythrocyte

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.     

Peculiarities of host cholesterol transport to the unique intracellular vacuole containing Toxoplasma

The intracellular protozoan Toxoplasma gondii is auxotrophic for low-density lipoprotein (LDL)-derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post-endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein-2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.     

Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.     

Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem)

We present a short insight into the problem of parasitophorous vacuole (PV) formation as a most peculiar kind of cell vacuolization occurring in the course of intracellular development of coccidian pathogens of the genera Eimeria, Isospora, Toxoplasma, Sarcocystis, Cryptosporidium, Epieimeria, and Karyolysus. The review focuses on the morpho-functional diversity of PVs in these parasites. By the present time, the PVs containing different parasite genera and species have been examined to different extent. The membrane of the PV (PVM) obviously derives from the host cell plasmalemma. But soon after parasite penetration, the morphofunctional organization and biochemical composition of the PVM drastically changes: its proteins are selectively excluded and those of the parasite are incorporated. As the result, the PV becomes not fusigenic for lysosomes or any other vacuoles or vesicles, because host cell surface markers necessary for membrane fusion are eliminated from the PVM during parasite invasion.The pattern of the PVs is parasite specific and demonstrates a broad diversity within the same genera and species and even at different stages of the endogenous development. The PV is far from being an indifferent membrane vesicle containing the parasite. Instead, it represents a dynamic system that reflects the innermost events of host-parasite relationships, thus promoting the accomplishing of the parasite life cycle, which, in its turn, is a necessary prerequisite of the parasite eventual survival as a species.     

LISP1 is important for the egress of Plasmodium berghei parasites from liver cells

Most Apicomplexa are obligatory intracellular parasites that multiply inside a so-called parasitophorous vacuole (PV) formed upon parasite entry into the host cell. Plasmodium , the agent of malaria and the Apicomplexa most deadly to humans, multiplies in both hepatocytes and erythrocytes in the mammalian host. Although much has been learned on how Apicomplexa parasites invade host cells inside a PV, little is known of how they rupture the PV membrane and egress host cells. Here, we characterize a Plasmodium protein, called LISP1 ( li ver- s pecific p rotein 1), which is specifically involved in parasite egress from hepatocytes. LISP1 is expressed late during parasite development inside hepatocytes and locates at the PV membrane. Intracellular parasites deficient in LISP1 develop into hepatic merozoites, which display normal infectivity to erythrocytes. However, LISP1-deficient liver-stage parasites do not rupture the membrane of the PV and remain trapped inside hepatocytes. LISP1 is the first Plasmodium protein shown by gene targeting to be involved in the lysis of the PV membrane.     

Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite''s growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.     

GRA12, a Toxoplasma dense granule protein associated with the intravacuolar membranous nanotubular network

The intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuole (PV), an intracellular niche in which it secretes proteins from secretory organelles named dense granules and rhoptries. Here, we describe a new dense granule protein that should now be referred to as GRA12, and that displays no homology with other proteins. Immunofluorescence and immuno-electron microscopy showed that GRA12 behaves similarly to both GRA2 and GRA6. It is secreted into the PV from the anterior pole of the parasite soon after the beginning of invasion, transits to the posterior invaginated pocket of the parasite where a membranous tubulovesicular network is first assembled, and finally resides throughout the vacuolar space, associated with the mature membranous nanotubular network. GRA12 fails to localise at the parasite posterior end in the absence of GRA2. Within the vacuolar space, like the other GRA proteins, GRA12 exists in both a soluble and a membrane-associated form. Using affinity chromatography experiments, we showed that in both the parasite and the PV soluble fractions, GRA12 is purified with the complex of GRA proteins associated with a tagged version of GRA2 and that this association is lost in the PV membranous fraction.     

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii is a gram‐negative intracellular bacterium that forms a large, lysosome‐like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol‐binding protein‐related protein 1 long (ORP1L) is a mammalian lipid‐binding protein that plays a dual role in cholesterol‐dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N‐terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co‐localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L‐depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.     

Toxoplasma gondii uses unusual sorting mechanisms to deliver transmembrane proteins into the host-cell vacuole

A critical step in infection by the apicomplexan parasite Toxoplasma gondii is the formation of a membrane-bound compartment within which the parasite proliferates. This process relies on a set of secretory organelles that discharge their contents into the host cell upon invasion. Among these organelles, the dense granules are specialized in the export of transmembrane (TM) GRA proteins, which are major components of the mature parasitophorous vacuole (PV) membrane. How eukaryotic pathogens export and sort membrane-bound proteins destined for the host cell is still poorly understood at the mechanistic level. In this study, we show that soluble trafficking of the PV-targeted GRA5 TM protein is parasite specific: when expressed in mammalian cells, GRA5 is targeted to the plasma membrane and behaves as an integral membrane protein with a type I toplogy. We also demonstrate the dual role of the GRA5 N-terminal ectodomain, which is sufficient to prevent membrane integration within the parasite and is essential for both sorting and post-secretory membrane insertion into the vacuolar membrane. These results contrast with the general rule that states that information contained within the cytoplasmic tail and/or the TM domain of integral membrane proteins dictates their cellular localization. They also highlight the diversity of sorting mechanisms that leads to the specialization of secretory processes uniquely adapted to intracellular parasitism.     

Toxoplasma gondii manipulates host cell signaling pathways via its secreted effector molecules

The obligate intracellular parasite Toxoplasma gondii secretes a vast variety of effector molecules from organelles known as rhoptries (ROPs) and dense granules (GRAs). ROP proteins are released into the cytosol of the host cell where they are directed to the cell nucleus or to the parasitophorous vacuole (PV) membrane. ROPs secrete proteins that enable host cell penetration and vacuole formation by the parasites, as well as hijacking host-immune responses. After invading host cells, T. gondii multiplies within a PV that is maintained by the parasite proteins secreted from GRAs. Most GRA proteins remain within the PV, but some are known to access the host cytosol across the PV membrane, and a few are able to traffic into the host-cell nucleus. These effectors bind to host cell proteins and affect host cell signaling pathways to favor the parasite. Studies on host–pathogen interactions have identified many infection-altered host signal transductions. Notably, the relationship between individual parasite effector molecules and the specific targeting of host-signaling pathways is being elucidated through the advent of forward and reverse genetic strategies. Understanding the complex nature of the host–pathogen interactions underlying how the host-signaling pathway is manipulated by parasite effectors may lead to new molecular biological knowledge and novel therapeutic methods for toxoplasmosis. In this review, we discuss how T. gondii modulates cell signaling pathways in the host to favor its survival.     

Proteases and chaperones are the most abundant proteins in the parasitophorous vacuole of Plasmodium falciparum-infected erythrocytes

After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole (PV) which forms an interface between the host cell cytosol and the parasite surface. This vacuole protects the parasite from potentially harmful substances, but allows access of essential nutrients to the parasite. Furthermore, the vacuole acts as a transit compartment for parasite proteins en route to the host cell cytoplasm. Recently we developed a strategy to biotin label soluble proteins of the PV. Here, we have paired this strategy with a high-throughput MALDI-TOF-MS analysis to identify 27 vacuolar proteins. These proteins fall into the following main classes: chaperones, proteases, and metabolic enzymes, consistent with the expected functions of the vacuole. These proteins are likely to be involved in several processes including nutrient acquisition from the host cytosol, protein sorting within the vacuole, and release of parasites at the end of the intraerythrocytic cycle.     

Formation and diversity of parasitophorous vacuoles in parasitic protozoa. The Coccidia (Sporozoa, Apicomplexa)

Data on parasitophorous vacuole (PV) formation in host cells (HC) harbouring different intracellular protozoan parasites have been reviewed and critically analysed, with special reference to the main representatives of the Coccidia. The vacuole membrane (PVM) is the interface between host and parasite, playing a role in nutrient acquisition by the parasite from the HC. The PV phenomenon is regarded as a generalized HC response to the introduction of alien bodies (microorganisms), which eventually reflects the evolutionary established host-parasite relationships at cellular, subcellular and molecular levels. Special attention has been paid to the existing morpho-functional diversity of the PVs within the same genera and species of parasites, and even at different stages of the parasite life cycle. The PVM is generally considered to derive from the HC plasmalemma, whose biochemical composition undergoes significant changes as the intravacuolar parasite grows. The original HC proteins are selectively excluded from the PVM, while those of the parasite are incorporated. As the result, the changed PVM becomes not fusigenic for HC lysosomes. For Toxoplasma gondii and other cyst-forming coccidia (Isospora, Sarcocystis), a definite correlation has been noticed between the extent of rhoptry and dense granule secrets released by a zoite during HC internalization, on the one hand, and the pattern of the PV that forms, on the other one. In T. gondii, tachyzoites, known to discharge abundant secrets, commonly force the development of PVs limited with a single unit membrane and equipped with a tubulovesicular network in the lumen. Unlike, bradyzoites known to be deficient in secretory materials trigger the formation of PVs with a three-membrane lining composed of the changed invaginated plasmalemma in addition to two membranes of endoplasmic reticulum. The two different types of PV harbour, respectively, exoenteric and enteric stages of T. gondii, the latter being confined to the cat intestine only. Unlike, all endogenous stages of the classic intestinal coccidia (Eimeria spp.) develop within PVs limited with a single membrane, with some invaginations extending into the PV lumen. Unusual PV patterns are characteristic of the extracytoplasmic eimerian coccidia (Cryptosporidium, Epieimeria) and adeleid haemogreagarines (Karyolysus). In cyst-forming coccidia, the PVM is actively involved in tissue cyst wall formation, thus protecting the encysted parasites from recognition by the host immune system. All this strongly suggests that the PV is far from being an indifferent membraneous vesicle containing a parasite, but represents a metabolically active compartment in infected cells. Since all the coccidia are obligate intracellular parasites, the mode of their intimate interaction with the HC, largely accomplished via the PV and its membrane, is vital for their survival as biological species.     

Export of proteins via a novel secretory pathway.

The intraerythrocytic location of the malaria parasite necessitates modification of the host cell. These alterations are mediated either directly or indirectly by parasite proteins exported to specific compartments within the host cell. However, little is known about how the parasite specifically targets proteins to locations beyond its plasma membrane. Mark Wiser, Norbert Lanners and Richard Bafford here propose an alternative secretory pathway for the export of parasite proteins into the host erythrocyte. The first step of this pathway is probably an endoplasmic reticulum (ER)-like organelle that is distinct from the normal ER. Possible mechanisms of protein trafficking in the infected erythrocyte are also discussed. The proposed ER-like organelle and alternative secretory pathway raise many questions about the cell biology of protein export and trafficking in Plasmodium.     

Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte.     

Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space

The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.     

Determination of the Plasmodium vivax schizont stage proteome

With the genome of the malaria parasite Plasmodium vivax sequenced, it is important to determine the proteomes of the parasite in order to assist efforts in antigen and drug target discovery. Since a method for continuous culture of P. vivax parasite is not available, we tried to study the proteome of the erythrocytic stages using fresh parasite isolates from patients. In schizont-enriched samples, 316 proteins were confidently identified by tandem mass spectrometry. Almost 50% of the identified proteins were hypothetical, while other major categories include proteins with binding function, protein fate, protein synthesis, metabolism and cellular transport. To identify proteins that are recognized by host humoral immunity, parasite proteins were separated by two-dimensional gel electrophoresis and screened by Western blot using an immune serum from a P. vivax patient. Mass spectrometry analysis of protein spots recognized by the serum identified four potential antigens including PV24. The recombinant protein PV24 was recognized by antibodies from vivax malaria patients even during the convalescent period, indicating that PV24 could elicit long-lasting antibody responses in P. vivax patients.     

Characterization of host cell-derived membrane proteins of the vacuole surrounding different intracellular forms of Trypanosoma cruzi in J774 cells. Evidence for phagocyte receptor sorting during the early stages of parasite entry.

Trypanosoma cruzi, an obligate intracellular protozoan parasite, exhibits developmental regulation of virulence. Although both noninfective epimastigote and infective trypomastigote stages of T. cruzi enter phagocytic cells via the formation of a parasitophorous vacuole (PV), only the latter developmental stages survive ingestion and perpetuate the infection. To determine whether the membrane composition of PV surrounding these different stages might contribute to differences in the outcome of infection, we identified selected membrane constituents by immunofluorescence and intracellular radioiodination, and studied their incorporation into PV. Complement receptors (CR3) are incorporated preferentially into the PV membrane surrounding serum-opsonized epimastigotes but not culture-derived metacyclic trypomastigotes. FcR are not preferentially incorporated into PV membranes unless epimastigotes or culture-derived metacyclic trypomastigotes are opsonized with anti-T. cruzi antibody. PV surrounding either parasite stage contain beta 1 integrins and lysosomal membrane glycoproteins (lgp). These results indicate that the plasma membrane glycoproteins incorporated into the surrounding PV membrane differ depending upon the stage of parasite being internalized, and that these differences reflect, at least in part, selective ligation of cell surface receptors mediating uptake. Furthermore, they imply that although virulent trypomastigote stages may avoid host cell uptake by conventional phagocytic receptors, i.e., CR3 or FcR, they do not escape fusion with an lgp-containing vacuole where they could still be exposed to lysosomal antimicrobial mechanisms.