Parasitophorous Vacuole Membrane Research Articles

Sequential Membrane Rupture and Vesiculation during Plasmodium berghei Gametocyte Egress from the Red Blood Cell

Malaria parasites alternate between intracellular and extracellular stages and successful egress from the host cell is crucial for continuation of the life cycle. We investigated egress of Plasmodium berghei gametocytes, an essential process taking place within a few minutes after uptake of a blood meal by the mosquito. Egress entails the rupture of two membranes surrounding the parasite: the parasitophorous vacuole membrane (PVM), and the red blood cell membrane (RBCM). High-speed video microscopy of 56 events revealed that egress in both genders comprises four well-defined phases, although each event is slightly different. The first phase is swelling of the host cell, followed by rupture and immediate vesiculation of the PVM. These vesicles are extruded through a single stabilized pore of the RBCM, and the latter is subsequently vesiculated releasing the free gametes. The time from PVM vesiculation to completion of egress varies between events. These observations were supported by immunofluorescence microscopy using antibodies against proteins of the RBCM and PVM. The combined results reveal dynamic re-organization of the membranes and the cortical cytoskeleton of the erythrocyte during egress.

Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually all nucleated cells in warm-blooded animals. The ability of Toxoplasma tachyzoites to infect and successfully manipulate its host is dependent on its ability to transport “GRA” proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside. GRAs have diverse roles in Toxoplasma’s intracellular lifecycle, including co-opting crucial host cell functions and proteins, such as the cell cycle, c-Myc and p38 MAP kinase. Some of these GRA proteins, such as GRA16 and GRA24, are secreted into the parasitophorous vacuole (PV) within which Toxoplasma replicates and are transported across the PV membrane (PVM) into the host cell, but the translocation process and its machinery are not well understood. We previously showed that TgMYR1, which is cleaved by TgASP5 into two fragments, localizes to the PVM and is essential for GRA transport into the host cell. To identify additional proteins necessary for effector transport, we screened Toxoplasma mutants defective in c-Myc up-regulation for their ability to export GRA16 and GRA24 to the host cell nucleus. Here we report that novel proteins MYR2 and MYR3 play a crucial role in translocation of a subset of GRAs into the host cell. MYR2 and MYR3 are secreted into the PV space and co-localize with PV membranes and MYR1. Consistent with their predicted transmembrane domains, all three proteins are membrane-associated, and MYR3, but not MYR2, stably associates with MYR1, whose N- and C-terminal fragments are disulfide-linked. We further show that fusing intrinsically disordered effectors to a structured DHFR domain blocks the transport of other effectors, consistent with a translocon-based model of effector transport. Overall, these results reveal a novel complex at the PVM that is essential for effector translocation into the host cell.

Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes.

The causative agent of malaria, Plasmodium, replicates inside a membrane-bound parasitophorous vacuole (PV) that shields this intracellular parasite from the cytosol of the host cell1. One common threat for intracellular pathogens is the homeostatic process of autophagy through which cells capture unwanted intracellular material for lysosomal degradation2. During the liver stage of a malaria infection, Plasmodium parasites are targeted by the autophagy machinery of the host cell and the PV membrane (PVM) becomes decorated with several autophagy markers, including LC3 (microtubule-associated protein 1 light chain 3)3,4. Here we show that Plasmodium berghei parasites infecting hepatic cells rely on the PVM transmembrane protein UIS3 to avoid elimination by host cell-mediated autophagy. We found that UIS3 binds host LC3 through a non-canonical interaction with a specialised surface on LC3 where host proteins with essential functions during autophagy also bind. UIS3 acts as a bona fide autophagy inhibitor by competing with host LC3-interacting proteins for LC3 binding. Our work identifies UIS3, one of the most promising candidates for a genetically-attenuated vaccine against malaria5, as a unique and potent mediator of autophagy evasion in Plasmodium. We propose that the protein-protein interaction between UIS3 and host LC3 represents a target for antimalarial drug development.

The parasite Toxoplasma sequesters diverse Rab host vesicles within an intravacuolar network.

Many intracellular pathogens subvert host membrane trafficking pathways to promote their replication. Toxoplasma multiplies in a membrane-bound parasitophorous vacuole (PV) that interacts with mammalian host organelles and intercepts Golgi Rab vesicles to acquire sphingolipids. The mechanisms of host vesicle internalization and processing within the PV remain undefined. We demonstrate that Toxoplasma sequesters a broad range of Rab vesicles into the PV. Correlative light and electron microscopy analysis of infected cells illustrates that intravacuolar Rab1A vesicles are surrounded by the PV membrane, suggesting a phagocytic-like process for vesicle engulfment. Rab11A vesicles concentrate to an intravacuolar network (IVN), but this is reduced in Δgra2 and Δgra2Δgra6 parasites, suggesting that tubules stabilized by the TgGRA2 and TgGRA6 proteins secreted by the parasite within the PV contribute to host vesicle sequestration. Overexpression of a phospholipase TgLCAT, which is localized to the IVN, results in a decrease in the number of intravacuolar GFP-Rab11A vesicles, suggesting that TgLCAT controls lipolytic degradation of Rab vesicles for cargo release.

Suppressors for Human Epidermal Growth Factor Receptor 2/4 (HER2/4): A New Family of Anti-Toxoplasmic Agents in ARPE-19 Cells.

The effects of tyrosine kinase inhibitors (TKIs) were evaluated on growth inhibition of intracellular Toxoplasma gondii in host ARPE-19 cells. The number of tachyzoites per parasitophorous vacuolar membrane (PVM) was counted after treatment with TKIs. T. gondii protein expression was assessed by western blot. Immunofluorescence assay was performed using Programmed Cell Death 4 (PDCD4) and T. gondii GRA3 antibodies. The TKIs were divided into 3 groups; non-epidermal growth factor receptor (non-EGFR), anti-human EGFR 2 (anti-HER2), and anti-HER2/4 TKIs, respectively. Group I TKIs (nintedanib, AZD9291, and sunitinib) were unable to inhibit proliferation without destroying host cells. Group II TKIs (lapatinib, gefitinib, erlotinib, and AG1478) inhibited proliferation up to 98% equivalent to control pyrimethamine (5 μM) at 20 μM and higher, without affecting host cells. Group III TKIs (neratinib, dacomitinib, afatinib, and pelitinib) inhibited proliferation up to 98% equivalent to pyrimethamine at 1–5 μM, but host cells were destroyed at 10–20 μM. In Group I, TgHSP90 and SAG1 inhibitions were weak, and GRA3 expression was moderately inhibited. In Group II, TgHSP90 and SAG1 expressions seemed to be slightly enhanced, while GRA3 showed none to mild inhibition; however, AG1478 inhibited all proteins moderately. Protein expression was blocked in Group III, comparable to pyrimethamine. PDCD4 and GRA3 were well localized inside the nuclei in Group I, mildly disrupted in Group II, and were completely disrupted in Group III. This study suggests the possibility of a vital T. gondii TK having potential HER2/4 properties, thus anti-HER2/4 TKIs may inhibit intracellular parasite proliferation with minimal adverse effects on host cells.

Inverted recruitment of autophagy proteins to the Plasmodium berghei parasitophorous vacuole membrane.

Selective autophagy and related mechanisms can act as variable defense mechanisms against pathogens and can therefore be considered as intracellular immune responses. When in hepatocytes, Plasmodium parasites reside in a parasitophorous vacuole (PV) and the PV membrane (PVM) is the main contact site between host cell and parasite. Early in infection, the PVM is directly labeled with host cell autophagy proteins LC3B and p62 (nucleoporin 62). We investigated the recruitment of different selective autophagy receptors and could show that mainly p62 and NBR1 (neighbour of BRCA1 gene 1) and to a lesser extent NDP52 (nuclear dot protein 52) associate with the PVM. To investigate the recruitment of these receptors to the PVM in Plasmodium-infected cells, we generated LC3B knock out HeLa cells. In these cell lines, autophagosome formation and autophagic flux are not different to those in WT cells. Unexpectedly, p62 and NBR1 recruitment to the PVM was strongly impaired in LC3B-negative host cells, suggesting that LC3B recruits both receptors to the PVM of Plasmodium parasites. We also noticed that LC3B recruited ubiquitin to the PVM. This indicates that, in comparison to classical selective autophagy, in P. berghei-infected cells the order of membrane labeling with autophagy proteins appears to be inverted from canonical ubiquitin-receptor-LC3B recruitment to LC3B-receptor and possibly ubiquitin.

The effect of lopinavir/ritonavir and lopinavir/ritonavir loaded PLGA nanoparticles on experimental toxoplasmosis

A marked reduction has been achieved in the incidence and clinical course of toxoplasmic encephalitis after the introduction of protease inhibitors within the treatment regimen of HIV (HIV-PIs). This work was undertaken to study for the first time, the efficacy of HIV-PIs, lopinavir/ritonavir (L/R), as a therapeutic agent in acute experimental toxoplasmosis. Lopinavir/ritonavir (L/R) were used in the same ratio present in aluvia, a known HIV-PIs drug used in the developing countries in the treatment regimens of AID's patient. Poly lactic-co-glycolic acid (PLGA) nanoparticles were used as a delivery system to L/R therapy. L/R alone or after its encapsulation on PLGA were given to Swiss strain albino mice that were infected with RH virulent toxoplasma strain. Both forms caused parasitological improvement in both mortality rate and parasite count. The higher efficacy was achieved by using L/R PLGA together with minimizing the effective dose. There was significant reduction in the parasite count in the peritoneal fluid and the liver. Parasite viability and infectivity were also significantly reduced. The anti-toxoplasma effect of the drug was attributed to the morphological distortion of the tachyzoites as evident by the ultrastructure examination and suppressed the egress of tachyzoites. L/R also induced changes that suggest apoptosis and autophagy of tachyzoites. The parasitophorous vacuole membrane was disrupted and vesiculated. The nanotubular networks inside the parasitophorous vacuole were disrupted. Therefore, the present work opens a new possible way for the approved HIV-PIs as an alternative treatment against acute toxoplasmosis. Furthermore, it increases the list of the opportunistic parasites that can be treated by this drug. The successful in vivo effect of HIV-PIs against Toxoplasma gondii suggests that this parasite may be a target in HIV treated patients, thus decrease the possibility of toxoplasmic encephalitis development.

Identification of novel parasitophorous vacuole proteins in P. falciparum parasites using BioID

Malaria blood stage parasites develop within red blood cells where they are contained in a vacuolar compartment known as the parasitophorous vacuole (PV). This compartment holds a key role in the interaction of the parasite with its host cell. However, the proteome of this compartment has so far not been comprehensively analysed. Here we used BioID in asexual blood stages of the most virulent human malaria parasite Plasmodium falciparum to identify new proteins of the PV. The resulting proteome contained many of the already known PV proteins and validation by GFP-knock-in of 10 previously in P. falciparum uncharacterised hits revealed 5 new PV proteins and two with a partial PV localisation. This included proteins peripherally attached to the inner face of the PV membrane as well as proteins anchored in the parasite plasma membrane that protrude into the PV. Using selectable targeted gene disruption we generated mutants for 2 of the 10 candidates. In contrast we could not select parasites with disruptions for another 3 candidates, strongly suggesting that they are important for parasite growth. Interestingly, one of these included the orthologue of UIS2, a protein previously proposed to regulate protein translation in the parasite cytoplasm but here shown to be an essential PV protein. This work extends the number of known PV proteins and provides a starting point for further functional analyses of this compartment.

LC3-association with the parasitophorous vacuole membrane of Plasmodium berghei liver stages follows a noncanonical autophagy pathway.

Eukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P.berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P.berghei-infected ATG5-/- host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3-independent pathway.

Proteases in Mosquito Borne Diseases: New Avenues in Drug Development.

Mosquito borne diseases continue to propagate and cause millions of deaths annually. They are caused either by protozoan parasites such as Plasmodium, Toxoplasma or by flaviviruses including Dengue and Zika. Among the proteome of such parasitic organisms, proteases play essential roles in events such as host invasion, hemoglobin hydrolysis, replication and immune evasion. Plasmepsin V (PMV), an endoplasmic reticulum resident aspartic protease of Plasmodium spp., is involved in the export of ~400 proteins containing the conserved Plasmodium Export Element motif (PEXEL). Interactions and cleavage of PEXEL proteins by PM V is necessary for export to and across the parasitophorous vacuole membrane. Protease System: Similarly in flaviviruses, a two-component protease system consisting of nonstructural proteins, NS2B and NS3, interacts with other non-structural proteins and plays a major role in viral replication, polyprotein cleavage and virion particle assembly. Thus, proteases involved in indispensable roles in pathogen machinery can be considered as attractive drug targets. Inhibitors against proteases are being used in clinical trials for other communicable and non-communicable diseases. Currently, hydroxyethylamine based inhibitors targeting the catalytic site of PM V with picomolar inhibitory concentrations have been tested in vitro. For recently characterized disease such as Zika, no known treatments exist while compound such as Policresulen has high affinity for Dengue NS2B/NS3 complex. Understanding proteases structure-function relationship and protease-inhibitor interactions can provide new insights for novel chemotherapeutic strategies.

Sarcocystis pantherophisi n. sp., from Eastern Rat Snakes (Pantherophis alleghaniensis) as Definitive Hosts and Interferon Gamma Gene Knockout Mice as Experimental Intermediate Hosts.

Here, we report a new species, Sarcocystis pantherophisi n. sp., with the Eastern rat snake (Pantherophis alleghaniensis) as natural definitive host and the interferon gamma gene knockout (KO) mouse as the experimental intermediate host. Sporocysts (n = 15) from intestinal contents of the snake were 10.8 × 8.9 μm. Sporocysts were orally infective to KO mice but not to laboratory-raised albino outbred house mice (Mus musculus). The interferon gamma KO mice developed schizont-associated neurological signs, and schizonts were cultivated in vitro from the brain. Mature sarcocysts were found in skeletal muscles of KO mice examined 41 days postinoculation (PI). Sarcocysts were slender, up to 70 μm wide and up to 3.5 mm long. By light microscopy, sarcocysts appeared thin-walled (<1 μm) without projections. By transmission electron microscopy, the sarcocyst wall was a variant of "type 1" (type 1i, new designation). The parasitophorous vacuolar membrane (pvm) had approximately 100-nm-wide × 100-nm-long bleb-like evaginations interspersed with 100-nm-wide × 650-nm-long elongated protrusions at irregular distances, and invaginations into the ground substance layer (gs) for a very short distance (6 nm). The gs was smooth, up to 500 nm thick, without tubules, and contained a few vesicles. Longitudinally cut bradyzoites at 54 days PI were banana-shaped, 7.8 × 2.2 μm (n = 5). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes indicated a close relationship with other Sarcocystis parasites that have snake-rodent life cycles. The parasite in the present study was molecularly and biologically similar to a previously reported isolate (designated earlier as Sarcocystis sp. ex Pantherophis alleghaniensis) from P. alleghaniensis, and it was structurally different from other Sarcocystis species so far described.

Evaluation of protective immunity induced by DNA vaccination with genes encoding Toxoplasma gondii GRA17 and GRA23 against acute toxoplasmosis in mice

Toxoplasma gondii, an obligatory intracellular protozoan, can cause serious public health problems and economic losses worldwide. Two novel dense granule proteins (GRA17 and GRA23) were recently identified as T. gondii-secreted proteins which are localized to the parasitophorous vacuole membrane (PVM) and can mediate the movement of small molecules between the host cell and parasitophorous vacuole (PV). In the present study, we evaluated the protective immunity induced by DNA vaccination with genes encoding GRA17 and GRA23 against acute toxoplasmosis in mice. Eukaryotic expressing plasmids pVAX-TgGRA17 and pVAX-TgGRA23 were constructed. Then, BALB/c mice were intramuscularly immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 followed by challenge infection with the highly virulent RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii were examined by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival time after challenge. Our results showed that mice immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 induced specific humoral and cellular responses, with higher level of IgG antibody, increased levels of Th1-type cytokines IFN-γ and IL-12 (p70), and CD3+CD4+CD8− and CD3+CD8+CD4− T cells, as well as prolonged survival time (9.1 ± 0.32 days for pVAX-TgGRA17, 10.8 ± 0.79 days for pVAX-TgGRA23, and 12.6 ± 2.55 days for pVAX-TgGRA17 + pVAX-TgGRA23) compared to the blank control (7.11 ± 0.33 days), PBS control (7.22 ± 0.44 days), and pVAX I control (7.11 ± 0.33 days). These results demonstrated that both TgGRA17 and TgGRA23 are potential vaccine candidates, TgGRA23 has a better immunogenicity, and co-immunization of pVAX-TgGRA17 and pVAX-TgGRA23 induces better protective efficacy.

MHC I presentation of Toxoplasma gondii immunodominant antigen does not require Sec22b and is regulated by antigen orientation at the vacuole membrane.

The intracellular Toxoplasma gondii parasite replicates within a parasitophorous vacuole (PV). T. gondii secretes proteins that remain soluble in the PV space, are inserted into PV membranes or are exported beyond the PV boundary. In addition to supporting T. gondii growth, these proteins can be processed and presented by MHC I for CD8+ T-cell recognition. Yet it is unclear whether membrane binding influences the processing pathways employed and if topology of membrane antigens impacts their MHC I presentation. Here we report that the MHC I pathways of soluble and membrane-bound antigens differ in their requirement for host ER recruitment. In contrast to the soluble SAG1-OVA model antigen, we find that presentation of the membrane-bound GRA6 is independent from the SNARE Sec22b, a key molecule for transfer of host endoplasmic reticulum components onto the PV. Using parasites modified to secrete a transmembrane antigen with opposite orientations, we further show that MHC I presentation is highly favored when the C-terminal epitope is exposed to the host cell cytosol, which corresponds to GRA6 natural orientation. Our data suggest that the biochemical properties of antigens released by intracellular pathogens critically guide their processing pathway and are valuable parameters to consider for vaccination strategies.

Shedding of host autophagic proteins from the parasitophorous vacuolar membrane of Plasmodium berghei

The hepatic stage of the malaria parasite Plasmodium is accompanied by an autophagy-mediated host response directly targeting the parasitophorous vacuolar membrane (PVM) harbouring the parasite. Removal of the PVM-associated autophagic proteins such as ubiquitin, p62, and LC3 correlates with parasite survival. Yet, it is unclear how Plasmodium avoids the deleterious effects of selective autophagy. Here we show that parasites trap host autophagic factors in the tubovesicular network (TVN), an expansion of the PVM into the host cytoplasm. In proliferating parasites, PVM-associated LC3 becomes immediately redirected into the TVN, where it accumulates distally from the parasite’s replicative centre. Finally, the host factors are shed as vesicles into the host cytoplasm. This strategy may enable the parasite to balance the benefits of the enhanced host catabolic activity with the risk of being eliminated by the cell’s cytosolic immune defence.

Morphological and molecular characterization of Sarcocystis arctica-like sarcocysts from the Arctic fox (Vulpes lagopus) from Alaska, USA.

The muscles of herbivores commonly harbor sarcocysts of parasites belonging to species in the genus Sarcocystis, but such muscle parasites are rare in carnivores. Here, we report Sarcocystis arctica-like sarcocysts in muscles of Arctic foxes (Vulpes lagopus) from Alaska, USA, for the first time. The tongues of 56 foxes were examined for Sarcocystis infection using several methods. Sarcocystis bradyzoites were detected in pepsin digests of 13 (23.2%), and sarcocysts were found in histological sections stained with hematoxylin and eosin (HE) of 9 (16.0%). By light microscopy, sarcocysts were up to 4mm long and up to 245μm wide. In HE-stained sections, the sarcocyst wall appeared smooth and up to 1.5μm thick without visible protrusions. By transmission electron microscopy, the sarcocyst wall had a wavy parasitophorous vacuolar membrane (pvm) folded as pleomorphic villar protrusions (vp), sometimes with anastomoses of villar tips. The vp and the ground substance (gs) layer were smooth and without microtubules. The gs was up to 2.0μm thick. The total width of the wall including vp and the gs was up to 4.0μm. The vp were up to 3.0μm long and most closely resembled "type 9c." All sarcocysts were mature and contained numerous 8.1×2.1μm sized bradyzoites. Molecular characterization (at 18S rDNA, 28S rDNA, ITS-1, and cox1) showed the highest affinity for S. arctica of the Arctic fox (V. lagopus) from Norway. In the present investigation, we provide evidence that sarcocysts are common in tongues of Alaskan Arctic foxes suggesting that these carnivores are serving as intermediate hosts, and we also provide ultrastructure of S. arctica from the Arctic fox for the first time.

The Coxiella Burnetii type IVB secretion system (T4BSS) component DotA is released/secreted during infection of host cells and during in vitro growth in a T4BSS-dependent manner.

Coxiella burnetii is a Gram-negative intracellular pathogen and is the causative agent of the zoonotic disease Q fever. To cause disease, C. burnetii requires a functional type IVB secretion system (T4BSS) to transfer effector proteins required for the establishment and maintenance of a membrane-bound parasitophorous vacuole (PV) and further modulation of host cell process. However, it is not clear how the T4BSS interacts with the PV membrane since neither a secretion pilus nor an extracellular pore forming apparatus has not been described. To address this, we used the acidified citrate cysteine medium (ACCM) along with cell culture infection and immunological techniques to identify the cellular and extracellular localization of T4BSS components. Interestingly, we found that DotA and IcmX were secreted/released in a T4BSS-dependent manner into the ACCM. Analysis of C. burnetii-infected cell lines revealed that DotA colocalized with the host cell marker CD63 (LAMP3) at the PV membrane. In the absence of bacterial protein synthesis, DotA also became depleted from the PV membrane. These data are the first to identify the release/secretion of C. burnetii T4BSS components during axenic growth and the interaction of a T4BSS component with the PV membrane during infection of host cells.

Manipulation of the Host Cell Membrane during Plasmodium Liver Stage Egress.

ABSTRACTA crucial step in the life cycle of Plasmodium parasites is the transition from the liver stage to the blood stage. Hepatocyte-derived merozoites reach the blood vessels of the liver inside host cell-derived vesicles called merosomes. The molecular basis of merosome formation is only partially understood. Here we show that Plasmodium berghei liver stage merozoites, upon rupture of the parasitophorous vacuole membrane, destabilize the host cell membrane (HCM) and induce separation of the host cell actin cytoskeleton from the HCM. At the same time, the phospholipid and protein composition of the HCM appears to be substantially altered. This includes the loss of a phosphatidylinositol 4,5-bisphosphate (PIP2) reporter and the PIP2-dependent actin-plasma membrane linker ezrin from the HCM. Furthermore, transmembrane domain-containing proteins and palmitoylated and myristoylated proteins, as well as glycosylphosphatidylinositol-anchored proteins, lose their HCM localization. Collectively, these findings provide an explanation of HCM destabilization during Plasmodium liver stage egress and thereby contribute to our understanding of the molecular mechanisms that lead to merosome formation.

Evaluation of the immune response in BALB/c mice induced by a novel DNA vaccine expressing GRA14 against Toxoplasma gondii.

Toxoplasma gondii can cause severe and even fatal disease in human beings and animals. Effective vaccines may contribute to control toxoplasmosis. GRA14, a novel secreted dense granule protein of T.gondii, has been proposed as a vaccine candidate due to its intervacuolar transport and unique topology in the parasitophorous vacuole membrane. In this study, we constructed a DNA vaccine encoding GRA14 of T.gondii. BALB/c mice were immunized intramuscularly three times at 2week intervals and challenged with T.gondii RH strain 5weeks later. The immune responses were evaluated using lymphocyte proliferation assay, cytokine and antibody measurements. In addition, the survival times and parasite load of mice challenged with the virulent T.gondii RH strain were evaluated. The results showed that the mice immunized with pcGRA14 induced both enhanced specific humoral and Th1 cellular immune responses, and also mice immunized with the pcGRA14 showed an increased survival time and decreased parasite load compared with control groups (P<.05). The results indicated, for the first time, that the GRA14 is a potential DNA vaccine against toxoplasmosis.

Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.

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.