Proximity-Labeling Reveals Novel Host and Parasite Proteins at the Toxoplasma Parasitophorous Vacuole Membrane.

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCEToxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

Contacting domains segregate a lipid transporter from a solute transporter in the malarial host-parasite interface.

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.

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.

High-Content Screening of the Medicines for Malaria Venture Pathogen Box for Plasmodium falciparum Digestive Vacuole-Disrupting Molecules Reveals Valuable Starting Points for Drug Discovery.

Plasmodium falciparum infections leading to malaria have severe clinical manifestations and high mortality rates. Chloroquine (CQ), a former mainstay of malaria chemotherapy, has been rendered ineffective due to the emergence of widespread resistance. Recent studies, however, have unveiled a novel mode of action in which low-micromolar levels of CQ permeabilized the parasite's digestive vacuole (DV) membrane, leading to calcium efflux, mitochondrial depolarization, and DNA degradation. These phenotypes implicate the DV as an alternative target of CQ and suggest that DV disruption is an attractive target for exploitation by DV-disruptive antimalarials. In the current study, high-content screening of the Medicines for Malaria Venture (MMV) Pathogen Box (2015) was performed to select compounds which disrupt the DV membrane, as measured by the leakage of intravacuolar Ca2+ using the calcium probe Fluo-4 AM. The hits were further characterized by hemozoin biocrystallization inhibition assays and dose-response half-maximal (50%) inhibitory concentration (IC50) assays across resistant and sensitive strains. Three hits, MMV676380, MMV085071, and MMV687812, were shown to demonstrate a lack of CQ cross-resistance in parasite strains and field isolates. Through systematic analyses, MMV085071 emerged as the top hit due to its rapid parasiticidal effect, low-nanomolar IC50, and good efficacy in triggering DV disruption, mitochondrial degradation, and DNA fragmentation in P. falciparum These programmed cell death (PCD)-like phenotypes following permeabilization of the DV suggests that these compounds kill the parasite by a PCD-like mechanism. From the drug development perspective, MMV085071, which was identified to be a potent DV disruptor, offers a promising starting point for subsequent hit-to-lead generation and optimization through structure-activity relationships.

Plasmodium falciparum exported protein PFE60 influences Maurer's clefts architecture and virulence complex composition.

Plasmodium falciparum, the most lethal malaria parasite species for humans, vastly remodels the mature erythrocyte host cell upon invasion for its own survival. Maurer's clefts (MC) are membraneous structures established by the parasite in the cytoplasm of infected cells. These organelles are deemed essential for trafficking of virulence complex proteins. The display of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell and the subsequent cytoadhesion of infected cells in the microvasculature of vital organs is the key mechanism that leads to the pathology associated with malaria infection. In a previous study we established that PFE60 (PIESP2) is one of the protein components of this complex. Here we demonstrate that PFE60 plays a role in MC lamella segmentation since in the absence of the protein, infected cells display a higher number of stacked MC compared with wild type infected red blood cells. Also, another exported parasite protein (Pf332) failed to localise correctly to the MC in cells lacking PFE60. Furthermore - unlike all other described resident MC membrane proteins - PFE60 does not require its transmembrane regions to be targeted to the organelle. We also provide further evidence that PFE60 is not a red blood cell surface antigen.

Three-dimensional analysis of morphological changes in the malaria parasite infected red blood cell by serial block-face scanning electron microscopy.

The human malaria parasite, Plasmodium falciparum, exhibits morphological changes during the blood stage cycle in vertebrate hosts. Here, we used serial block-face scanning electron microscopy (SBF-SEM) to visualize the entire structures of P. falciparum-infected red blood cells (iRBCs) and to examine their morphological and volumetric changes at different stages. During developmental stages, the parasite forms Maurer's clefts and vesicles in the iRBC cytoplasm and knobs on the iRBC surface, and extensively remodels the iRBC structure for proliferation of the parasite. In our observations, the Maurer's clefts and vesicles in the P. falciparum-iRBCs, resembling the so-called tubovesicular network (TVN), were not connected to each other, and continuous membrane networks were not observed between the parasitophorous vacuole membrane (PVM) and the iRBC cytoplasmic membrane. In the volumetric analysis, the iRBC volume initially increased and then decreased to the end of the blood stage cycle. This suggests that it is necessary to absorb a substantial amount of nutrients from outside the iRBC during the initial stage, but to release waste materials from inside the iRBC at the multinucleate stage. Transportation of the materials may be through the iRBC membrane, rather than a special structure formed by the parasite, because there is no direct connection between the iRBC membrane and the parasite. These results provide new insights as to how the malaria parasite grows in the iRBC and remodels iRBC structure during developmental stages; these observation can serve as a baseline for further experiments on the effects of therapeutic agents on malaria.

Genetic basis for phenotypic differences between different Toxoplasma gondii type I strains.

BACKGROUND: Toxoplasma gondii has a largely clonal population in North America and Europe, with types I, II and III clonal lineages accounting for the majority of strains isolated from patients. RH, a particular type I strain, is most frequently used to characterize Toxoplasma biology. However, compared to other type I strains, RH has unique characteristics such as faster growth, increased extracellular survival rate and inability to form orally infectious cysts. Thus, to identify candidate genes that could account for these parasite phenotypic differences, we determined genetic differences and differential parasite gene expression between RH and another type I strain, GT1. Moreover, as differences in host cell modulation could affect Toxoplasma replication in the host, we determined differentially modulated host processes among the type I strains through host transcriptional profiling. RESULTS: Through whole genome sequencing, we identified 1,394 single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) between RH and GT1. These SNPs/indels together with parasite gene expression differences between RH and GT1 were used to identify candidate genes that could account for type I phenotypic differences. A polymorphism in dense granule protein, GRA2, determined RH and GT1 differences in the evasion of the interferon gamma response. In addition, host transcriptional profiling identified that genes regulated by NF-ĸB, such as interleukin (IL)-12p40, were differentially modulated by the different type I strains. We subsequently showed that this difference in NF-ĸB activation was due to polymorphisms in GRA15. Furthermore, we observed that RH, but not other type I strains, recruited phosphorylated IĸBα (a component of the NF-ĸB complex) to the parasitophorous vacuole membrane and this recruitment of p- IĸBα was partially dependent on GRA2. CONCLUSIONS: We identified candidate parasite genes that could be responsible for phenotypic variation among the type I strains through comparative genomics and transcriptomics. We also identified differentially modulated host pathways among the type I strains, and these can serve as a guideline for future studies in examining the phenotypic differences among type I strains.

Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

Molecular make-up of the Plasmodium parasitophorous vacuolar membrane.

Plasmodium, the causative agent of malaria, is an obligate, intracellular, eukaryotic cell that invades, replicates, and differentiates within hepatocytes and erythrocytes. Inside a host cell, a second membrane delineates the developing pathogen in addition to the parasite plasma membrane, resulting in a distinct cellular compartment, termed parasitophorous vacuole (PV). The PV membrane (PVM) constitutes the parasite-host cell interface and is likely central to nutrient acquisition, host cell remodeling, waste disposal, environmental sensing, and protection from innate defense. Over the past two decades, a number of parasite-encoded PVM proteins have been identified. They include multigene families and protein complexes, such as early-transcribed membrane proteins (ETRAMPs) and the Plasmodium translocon for exported proteins (PTEX). Nearly all Plasmodium PVM proteins are restricted to this genus and display transient and stage-specific expression. Here, we provide an overview of the PVM proteins of Plasmodium blood and liver stages. Biochemical and experimental genetics data suggest that some PVM proteins are ideal targets for novel anti-malarial intervention strategies.

A helical membrane-binding domain targets the Toxoplasma ROP2 family to the parasitophorous vacuole.

During invasion, the obligate intracellular pathogen, Toxoplasma gondii, secretes into its host cell a variety of effector molecules, several of which have been implicated in strain-specific variation in disease. The largest family of these effectors, defined by the canonical member ROP2, quickly associates with the nascent parasitophorous vacuole membrane (PVM) after secretion. Here we demonstrate that the NH(2)-terminal domain of the ROP2 family contains a series of amphipathic helices that are necessary and sufficient for membrane association. While each of the amphipathic helices is individually competent to bind cellular membranes, together they act to bind the PVM preferentially, possibly through sensing its strong negative curvature. This previously uncharacterized helical domain is an evolutionarily robust and energetically efficient design for membrane association.

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.

An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface.

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are 'hypothetical' proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be 'knocked out'. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of 'hypothetical' P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host-microbial interactions for prophylaxis against this major human pathogen.

Characterization of events preceding the release of malaria parasite from the host red blood cell.

The process of merozoite release involves proteolysis of both the parasitophorous vacuole membrane (PVM) and red blood cell membrane (RBCM), but the precise temporal sequence remains controversial. Using immunofluorescence microscopy and Western blotting of parasite-infected RBCs, we observed that the intraerythrocytic parasite was enclosed in a continuous ring of PVM at early stages of parasite development while at the segmented schizont stage, the PVM appeared to be integrated in the cluster of newly formed merozoites. Subsequently, such clusters were detected extraerythrocytically together with single merozoites devoid of the PVM at low frequency, suggesting a primary rupture of RBCM, followed by PVM rupture and release of invasive merozoites. Secondly, since cysteine proteases are implicated in the process of parasite release, antimalarial effects of 4 cysteine protease inhibitors (leupeptin, E64, E64d, and MDL) were tested at the late schizont stage and correlated with the integrity of PVM and RBCM. We observed that leupeptin and E64 treatment produced extraerythrocytic clusters of merozoites associated with PVM suggesting inhibition of PVM lysis but not RBCM lysis. Merozoites in these clusters developed into rings upon removal of the inhibitors. In contrast, E64d and MDL caused an irreversible parasite death blocking further development. Future characterization of the mechanism(s) of inhibition may facilitate the design of novel antimalarial inhibitors.

GRA9, a new Toxoplasma gondii dense granule protein associated with the intravacuolar network of tubular membranes.

Important components of the parasitophorous vacuole in which the intracellular protozoan parasite Toxoplasma gondii develops, comprise proteins secreted from apicomplexan specific secretory organelles named the dense granules. Here, we confirm by immunofluorescence and by cryo-electron microscopy that the recently isolated B10 protein (318 amino acids, 41kDa) is a new dense granule protein that should now be referred to as GRA9. Within the vacuolar compartment, GRA9, like GRA2, GRA4 and GRA6, associates with the network of tubular membranes connected to the parasitophorous vacuole delimiting membrane. Like the other GRA proteins, GRA9 is secreted into the vacuole from the anterior end of the parasite. However, unlike GRA2 or GRA6, GRA9 does not transit by the posterior invaginated pocket of the parasite where the network first assembles. Within the dense granules, GRA9 exists in both a soluble and an insoluble state. Like the other GRA proteins, GRA9 is secreted as a soluble form only and like most of the GRA proteins, two forms of GRA9 of the similar molecular weight are detected within the vacuolar space: a soluble form and a membrane associated form. The dual properties of GRA9 are not only ascribed by the presence of amphipathic and hydrophobic alpha-helices but also by the fact that the protein is mainly hydrophilic.

Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion.

Malaria sporozoites have to cross the layer of sinusoidal liver cells to reach their initial site of multiplication in the mammalian host, the hepatocytes. To determine the sinusoidal cell type sporozoites use for extravasation, endothelia or Kupffer cells, we quantified sporozoite adhesion to and invasion of sinusoidal cells isolated from rat liver. In vitro invasion assays reveal that Plasmodium berghei and P. yoelii sporozoites attach to and enter Kupffer cells, but not sinusoidal endothelia. Unlike hepatocytes and other nonphagocytic cells, which are invaded in vitro only within the first hour of parasite exposure, the number of intracellular sporozoites in Kupffer cells increases for up to 12 hours. By confocal and electron microscopy, sporozoites are enclosed in a vacuole that does not colocalize with lysosomal markers. Inhibition of phagocytosis with gadolinium chloride has no effect on Kupffer cell invasion, but abolishes phagocytosis of inactivated sporozoites. Furthermore, sporozoites traverse in vitro from Kupffer cells to hepatocytes where they eventually develop into exoerythrocytic schizonts. Thus, malaria sporozoites selectively recognize and actively invade Kupffer cells, avoid phagosomal acidification, and safely passage through these phagocytes.

Intracellular calcium and pH conditions of cultured cells infected with Eimeria bovis or E. separata.

Loading of Eimeria bovis-infected Vero cells with membrane-permeant acetoxymethyl esters (AM-esters) of ion-sensitive dyes provided us with a noninvasive method for investigation of the permeability of the parasitophorous vacuole membrane (PVM) and simultaneous measurement of Ca2+ and H+ concentrations in different compartments of the infected cells. The distribution patterns of the cleaved membrane-impermeant dyes argue against the existence of nonselective pores in the PVM. There is also no indication of a parasitophorous duct connecting the vacuolar space with extracellular media. The pH inside the parasitophorous vacuole (PV) was lower than that in the cytoplasm of the host cell or the parasite, whereas the [Ca2+] in these compartments did not differ significantly. In HT29 cells infected with E. separata for 24 h the Ca2+ response to extracellular adenosine triphosphate (ATP) was significantly reduced, indicating influences on the host cell's intracellular signaling.

Actin-rich structures formed during the invasion of cultured cells by infective forms of Trypanosoma cruzi.

Previous work has shown that Trypanosoma cruzi extracellular amastigotes as well as metacyclic trypomastigotes infect cultured cells in a highly specific parasite form-cell type interaction. In this work we have investigated the mode of interaction of both forms with HeLa and Vero cells using scanning electron and confocal fluorescence microscopy. We examined the distribution of several host cell components as well as extracellular matrix elements during cell invasion by both T. cruzi infective forms. Scanning electron microscopy showed that membrane expansions formed during the invasion of cells by extracellular amastigotes. These expansions correspond to small cup-like structures in HeLa cells and are comparatively larger "crater"-like in Vero cells. We detected by confocal microscopy actin-rich structures associated with the internalisation of both infective forms of the parasite that correspond to the membrane expansions. Confocal fluorescence microscopy combining DIC images of cells labelled with monoclonal antibodies to phosphotyrosine, cytoskeletal elements, integrins, and extracellular matrix components revealed that some of the components like gelsolin and alpha-actinin accumulate in actin-rich structures formed in the invasion of amastigotes of both cell types. Others, like vinculin and alpha2 integrin may be present in these structures without evident accumulation. And finally, some actin-rich processes may be devoid of components like fibronectin or alphaV integrin. These studies provide evidence that the repertoire of host cell/extracellular matrix components that engage in the invasion process of T. cruzi forms is cell type- and parasite form-dependent.

The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve.

The obligate intracellular protozoan parasite Toxoplasma gondii creates and enters into a unique membrane-bounded cytoplasmic compartment, the parasitophorous vacuole, when invading mammalian host cells. By microinjecting polar fluorescent molecules into individual T. gondii-infected fibroblasts, we show here that the parasitophorous vacuole membrane (PVM) surrounding the parasite functions as a molecular sieve. Lucifer yellow (457 Da) displayed free bidirectional flux across the PVM and distinctly outlined the parasites, which did not take up the dye, within the vacuole. This dye movement was not appreciably delayed by pretreatment of cells with 5 mM probenecid or chilling the monolayer to 5 degrees C, suggesting that dye movement was due to passive permeation through a membrane pore rather than active transport. Calcein, fluo-3, and a series of fluorescein isothiocyanate-labeled peptides up to 1291 Da crossed the PVM in a size-restricted fashion. A labeled peptide of 1926 Da and labeled dextrans and proteins (> or = 3000 Da) failed to transit the PVM. This putative channel in the PVM therefore allows exchange of molecules up to 1300-1900 Da between the host cell cytoplasm and the parasitophorous vacuolar space.