Parasitophorous Vacuole Membrane Research Articles

Coxiella burnetiiexploits host cAMP-dependent protein kinase signalling to promote macrophage survival

Intracellular bacterial pathogens often subvert apoptosis signalling to regulate survival of their host cell, allowing propagation of the bacterial population. Coxiella burnetii, the intracellular agent of human Q fever, inhibits host cell apoptosis through several mechanisms, including prevention of mitochondrial cytochrome c release, triggering of an anti-apoptotic transcriptional programme, and activation of pro-survival kinases. To control host cell survival, C. burnetii delivers effector proteins to the eukaryotic cytosol using a specialized Dot/Icm type IV secretion system (T4SS). Effectors are predicted to regulate activity of pro-survival host signalling proteins, such as Akt and cAMP-dependent protein kinase (PKA), to control infection. Here, we show that host PKA activity is required for C. burnetii inhibition of macrophage apoptosis. PKA is activated during infection and inhibits activity of the pro-apoptotic protein Bad via phosphorylation. Bad is also phosphorylated at an Akt-specific residue, indicating C. burnetii uses two kinases to fully inactivate Bad. Additionally, Bad and the tethering protein 14-3-3β colocalize at the C. burnetii parasitophorous vacuole (PV) membrane during infection, an event predicted to alter Bad promotion of apoptosis. Inhibiting PKA activity prevents Bad recruitment to the PV, but the protein is retained at the membrane during induction of apoptosis. Finally, PKA regulatory subunit I (RI) traffics to the PV membrane in a T4SS-dependent manner, suggesting a C. burnetii effector(s) regulates PKA-dependent activities. This study is the first to demonstrate subversion of host PKA activity by an intracellular bacterial pathogen to prevent apoptosis and survive within macrophages.

Two putative protein export regulators promote Plasmodium blood stage development in vivo

Protein export is considered an essential feature of malaria parasite blood stage development. Here, we examined five components of the candidate Plasmodium translocon of exported proteins (PTEX), a complex thought to mediate protein export across the parasitophorous vacuole membrane into the host cell. Using the murine malaria model parasite Plasmodium berghei, we succeeded in generating parasite lines lacking PTEX88 and thioredoxin 2 (TRX2). Repeated attempts to delete the remaining three translocon components failed, suggesting essential functions for EXP2, PTEX150, and heat shock protein 101 (HSP101) during blood stage development. To analyze blood infections of the null-mutants, we established a flow cytometry-assisted intravital competition assay using three novel high fluorescent lines (Bergreen, Beryellow, and Berred). Although blood stage development of parasites lacking TRX2 was affected, the deficit was much more striking in PTEX88 null-mutants. The multiplication rate of PTEX88-deficient parasites was strongly reduced resulting in out-competition by wild-type parasites. Endogenous tagging revealed that TRX2::tag resides in distinct punctate organelles of unknown identity. PTEX88::tag shows a diffuse intraparasitic pattern in blood stage parasites. In trophozoites, PTEX88::tag also localized to previously unrecognized extensions reaching from the parasite surface into the erythrocyte cytoplasm. Together, our results indicate auxiliary roles for TRX2 and PTEX88 and central roles for EXP2, PTEX150, and HSP101 during P. berghei blood infection.

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 virulence-related rhoptry protein 5 (ROP5) of Toxoplasma Gondii is a novel vaccine candidate against toxoplasmosis in mice

Infections with the intracellular protozoan parasite Toxoplasma gondii pose a serious public health problem and are of great economic importance worldwide. The parasite rhoptry protein 5 (ROP5) has been implicated as a major virulence factor that reduces the accumulation of immunity-related GTPases (IRG) in parasitophorous vacuole membrane (PVM), which maintains PVM integrity and evades IFNγ-mediated killing by intracellular parasites. To study the immunoprotective value of ROP5, BALB/c mice were immunized with a recombinant form of the protein administered alone or in combination with another promising vaccine antigen, rSAG1. All mice vaccinated with the recombinant antigens developed a high level of specific antibody responses against soluble tachyzoite antigens (STAg), a statistically significant increase of the splenocyte proliferation response, and significant levels of IFN-γ and IL-2 production. In contrast to rSAG1, which only stimulated the release of IFN-γ and IL-2, rROP5 induced the specific production of IL-10, the Th2-type cytokine, in addition to IFN-γ and IL-2. These results demonstrated that rROP5 could induce significant cellular and humoral (Th1/Th2) immune responses. Moreover, mice immunized with rROP5 displayed a prolonged survival time against a lethal challenge with the T. gondii RH strain. Additionally, vaccination with the mixture of rROP5+rSAG1 resulted in higher levels of T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge compared to immunization with rROP5 or rSAG1 alone. Our studies show that recombinant ROP5 antigen may be a promising vaccine candidate against toxoplasmosis. To our knowledge, this is the first report to evaluate the immunoprotective value of ROP5.

Remodeling of human red cells infected with Plasmodium falciparum and the impact of PHIST proteins

In an infected erythrocyte (iRBC), renovation and decoration are crucial for malarial parasite survival, pathogenesis and reproduction. Host cell remodeling is mediated by an array of diverse parasite-encoded export proteins that traffic within iRBC. These remodeling proteins extensively modify the membrane and cytoskeleton of iRBC and help in formation of parasite-induced novel organelles such as 'Maurer's Cleft (MC), tubulovesicular network (TVN) and parasitophorous vacuole membrane (PVM) inside the iRBC. The genome sequence of Plasmodium falciparum shows expansion of export proteins, which suggests a complex requirement of these export proteins for specific pathogenesis and erythrocyte remodeling. Plasmodium helical intersperse sub-telomeric (PHIST) is a family of seventy-two small export proteins and many of its recently discovered functional characteristics suggest an intriguing putative role in modification of an iRBC. This review highlights the recent advances in parasite genomics, proteomics, and cell biology studies unraveling the host cell modification; providing a speculation on the impact of PHIST proteins in modification of the iRBC.

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

BackgroundToxoplasma 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.ResultsThrough 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.ConclusionsWe 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.

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials.

The ETRAMP Family Member SEP2 Is Expressed throughout Plasmodium berghei Life Cycle and Is Released during Sporozoite Gliding Motility

The early transcribed membrane proteins ETRAMPs belong to a family of small, transmembrane molecules unique to Plasmodium parasite, which share a signal peptide followed by a short lysine-rich stretch, a transmembrane domain and a variable, highly charged C-terminal region. ETRAMPs are usually expressed in a stage-specific manner. In the blood stages they localize to the parasitophorous vacuole membrane and, in described cases, to vesicle-like structures exported to the host erythrocyte cytosol. Two family members of the rodent parasite Plasmodium berghei, uis3 and uis4, localize to secretory organelles of sporozoites and to the parasitophorous membrane vacuole of the liver stages. By the use of specific antibodies and the generation of transgenic lines, we showed that the P. berghei ETRAMP family member SEP2 is abundantly expressed in gametocytes as well as in mosquito and liver stages. In intracellular parasite stages, SEP2 is routed to the parasitophorous vacuole membrane while, in invasive ookinete and sporozoite stages, it localizes to the parasite surface. To date SEP2 is the only ETRAMP protein detected throughout the parasite life cycle. Furthermore, SEP2 is also released during gliding motility of salivary gland sporozoites. A limited number of proteins are known to be involved in this key function and the best characterized, the CSP and TRAP, are both promising transmission-blocking candidates. Our results suggest that ETRAMP members may be viewed as new potential candidates for malaria control.

Experimental Transmission ofSarcocystis muris(Apicomplexa: Sarcocystidae) Sporocysts from a Naturally Infected Cat (Felis catus) to Immunocompetent and Immunocompromised Mice

Cats serve as definitive hosts for zoonotic Toxoplasma gondii , a protozoan that threatens human reproductive health, but they also excrete sporocysts of related protozoan that pose no known human health risk. Here we provide the first definitive evidence for natural infection with the enzootic parasite Sarcocystis muris, one such enzootic parasite. Sporulated Sarcocystis sp. sporocysts were found in rectal contents of an adult feral cat ( Felis catus ) in Giza, Egypt. After these sporocysts were orally inoculated into 2 Swiss Webster mice, sarcocysts were found to have developed in skeletal muscles 114 days later. As observed through transmission electron microscopy, the cyst wall corresponded to Type 1, and the parasitophorous vacuolar membrane had tiny outpocketing of blebs (<200 nm thick) that were not invaginated into the interior of the cyst; these structures were identical to the sarcocyst wall described for a Costa Rican isolate of S. muris that has served as an experimental model for nearly 4 decades. Two parasite-free cats fed sarcocyst-infected muscles developed patent infections; fully sporulated sporocysts (10-11 × 7.0 μm) were found in the lamina propria of small intestines of cats killed 6 and 7 days postinoculation (PI). Interferon gamma gene knockout (KO) mice were orally inoculated with sporocysts from experimentally infected cats, and their tissues were examined histologically; sarcocysts were found in 5 KO mice killed 87, 115, 196, 196, 196 days PI, but no stages were seen in 5 KO mice 10, 14, 14, 18, and 39 days PI. Bradyzoites were released from intramuscular sarcocysts of a KO mouse killed 115 days PI and orally inoculated into 5 KO mice. No stage of Sarcocystis was found in any organ (including intestinal lamina propria) of KO mice killed 4, 8, 81, 190, and 190 days PI, confirming that the definitive host is required to complete the life cycle even in the case of immunodeficient mice. This is the first confirmation of S. muris infection in a naturally infected cat anywhere.

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Recent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells

BackgroundGTPases are the family of hydrolases that bind and hydrolyze guanosine triphosphate. The large Immunity-related GTPases and the small GTPase ADP-ribosylation factor-6 in host cells are known to accumulate on the parasitophorous vacuole membrane (PVM) of Toxoplasma gondii and play critical roles in this parasite infection, but these GTPases cannot explain the full extent of infection.ResultsIn this research, RhoA and Rac1 GTPases from the host cell were found to accumulate on the PVM regardless of the virulence of the T. gondii strains after T. gondii invasion, and this accumulation was dependent on their GTPase activity. The real-time micrography of T. gondii tachyzoites invading COS-7 cells overexpressing CFP-RhoA showed that this GTPase was recruited to the PVM at the very beginning of the invasion through the host cell membrane or from the cytosol. Host cell RhoA and Rac1 were also activated after T. gondii tachyzoites invasion, which was needed for host cell cytoskeleton reorganization to facilitate intracellular pathogens invasion. The decisive domains for the RhoA accumulation on the PVM included the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box, respectively, which were related to the binding of GTP for enzymatic activity and mDia for the regulation of microtubules. The recruited CFP-RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, unlike the unassociated RhoA in the host cell cytosol that migrated to the cell membrane towards the EGF activation spot. This result supported the hypothesis that the recruited RhoA or Rac1 on the PVM were in the GTP-bound active form. Wild-type RhoA or Rac1 overexpressed cells had almost the same infection rates by T. gondii as the mock-treated cells, while RhoA-N19 or Rac1-N17 transfected cells and RhoA, Rac1 or RhoA + Rac1 siRNA-treated cells showed significantly diminished infection rates compared to mock cells.ConclusionsThe accumulation of the RhoA and Rac1 on the PVM and the requisite of their normal GTPase activity for efficient invasion implied their involvement and function in T. gondii invasion.

A member of the Plasmodium falciparum PHIST family binds to the erythrocyte cytoskeleton component band 4.1

BackgroundPlasmodium falciparum parasites export more than 400 proteins into the cytosol of their host erythrocytes. These exported proteins catalyse the formation of knobs on the erythrocyte plasma membrane and an overall increase in erythrocyte rigidity, presumably by modulating the endogenous erythrocyte cytoskeleton. In uninfected erythrocytes, Band 4.1 (4.1R) plays a key role in regulating erythrocyte shape by interacting with multiple proteins through the three lobes of its cloverleaf-shaped N-terminal domain. In P. falciparum-infected erythrocytes, the C-lobe of 4.1R interacts with the P. falciparum protein mature parasite-infected erythrocyte surface antigen (MESA), but it is not currently known whether other P. falciparum proteins bind to other lobes of the 4.1R N-terminal domain.MethodsIn order to identify novel 4.1R interacting proteins, a yeast two-hybrid screen was performed with a fragment of 4.1R containing both the N- and α-lobes. Positive interactions were confirmed and investigated using site-directed mutagenesis, and antibodies were raised against the interacting partner to characterise it’s expression and distribution in P. falciparum infected erythrocytes.ResultsYeast two-hybrid screening identified a positive interaction between the 4.1R N- and α-lobes and PF3D7_0402000. PF3D7_0402000 is a member of a large family of exported proteins that share a domain of unknown function, the PHIST domain. Domain mapping and site-directed mutagenesis established that it is the PHIST domain of PF3D7_0402000 that interacts with 4.1R. Native PF3D7_0402000 is localized at the parasitophorous vacuole membrane (PVM), and colocalizes with a subpopulation of 4.1R.DiscussionThe function of the majority of P. falciparum exported proteins, including most members of the PHIST family, is unknown, and in only a handful of cases has a direct interaction between P. falciparum-exported proteins and components of the erythrocyte cytoskeleton been established. The interaction between 4.1R and PF3D7_0402000, and localization of PF3D7_0402000 with a sub-population of 4.1R at the PVM could indicate a role in modulating PVM structure. Further investigation into the mechanisms for 4.1R recruitment is needed.ConclusionPF3D7_0402000 was identified as a new binding partner for the major erythrocyte cytoskeletal protein, 4.1R. This interaction is consistent with a growing body of literature that suggests the PHIST family members function by interacting directly with erythrocyte proteins.

Malaria Parasite cGMP-dependent Protein Kinase Regulates Blood Stage Merozoite Secretory Organelle Discharge and Egress

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites

Clinical malaria is associated with proliferation of blood-stage parasites. During the blood stage, Plasmodium parasites invade host red blood cells, multiply, egress and reinvade uninfected red blood cells to continue the life cycle. Here we demonstrate that calcium-dependent permeabilization of host red blood cells is critical for egress of Plasmodium falciparum merozoites. Although perforin-like proteins have been predicted to mediate membrane perforation during egress, the expression, activity and mechanism of action of these proteins have not been demonstrated. Here, we show that two perforin-like proteins, perforin-like protein 1 and perforin-like protein 2, are expressed in the blood stage. Perforin-like protein 1 localizes to the red blood cell membrane and parasitophorous vacuolar membrane in mature schizonts following its Ca(2+)-dependent discharge from micronemes. Furthermore, perforin-like protein 1 shows Ca(2+)-dependent permeabilization and membranolytic activities suggesting that it may be one of the effector proteins that mediate Ca(2+)-dependent membrane perforation during egress.

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network

Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.

Host Cell Subversion by Toxoplasma GRA16, an Exported Dense Granule Protein that Targets the Host Cell Nucleus and Alters Gene Expression

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

A perforin-like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes.

Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin-like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore-forming toxin, we conclude that rupture of the erythrocyte membrane is blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin-like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.

A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine

Recently it has been shown in rodent malaria models that immunisation with genetically attenuated Plasmodium parasites can confer sterile protection against challenge with virulent parasites. For the mass production of live attenuated Plasmodium parasites for vaccination, safety is a prerequisite. Knockout of a single gene is not sufficient for such a strategy since the parasite can likely compensate for such a genetic modification and a single surviving parasite is sufficient to kill an immunised individual. Parasites must therefore be at least double-attenuated when generating a safe vaccine strain. Genetic double-attenuation can be achieved by knocking out two essential genes or by combining a single gene knockout with the expression of a protein toxic for the parasite. We generated a double-attenuated Plasmodium berghei strain that is deficient in fatty acid synthesis by the knockout of the pdh-e1α gene, introducing a second attenuation by the liver stage-specific expression of the pore-forming bacterial toxin perfringolysin O. With this double genetically attenuated parasite strain, a superior attenuation was indeed achieved compared with single-attenuated strains that were either deficient in pyruvate dehydrogenase (PDH)-E1 or expressed perfringolysin O. In vivo, both single-attenuated strains resulted in breakthrough infections even if low to moderate doses of sporozoites (2,000–5,000) were administered. In contrast, the double genetically attenuated parasite strain, given at moderate doses of 5,000 sporozoites, did not result in blood stage infection and even when administered at 5- to 20-fold higher doses, only single and delayed breakthrough infections were observed. Prime booster immunisation with the double genetically attenuated parasite strain completely protected a susceptible mouse strain from malaria and even a single immunisation conferred protection in some cases and lead to a markedly delayed onset of blood stage infection in others. Importantly, premature rupture of the parasitophorous vacuole membrane by liver stage-specific perfringolysin O expression did not induce host cell death and soluble parasite proteins, which are released into the host cell cytoplasm, have the potential to be processed and presented via MHC class I molecules. This, in turn, might support immunological responses against Plasmodium-infected hepatocytes.

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL-like motif at the N-terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin-tagged versions of these uncharacterized proteins show co-localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM.

Spatial association with PTEX complexes defines regions for effector export into Plasmodium falciparum-infected erythrocytes

Export of proteins into the infected erythrocyte is critical for malaria parasite survival. The majority of effector proteins are thought to export via a proteinaceous translocon, resident in the parasitophorous vacuole membrane surrounding the parasite. Identification of the Plasmodium translocon of exported proteins and its biochemical association with exported proteins suggests it performs this role. Direct evidence for this, however, is lacking. Here using viable purified Plasmodium falciparum merozoites and three-dimensional structured illumination microscopy, we investigate remodelling events immediately following parasite invasion. We show that multiple complexes of the Plasmodium translocon of exported proteins localize together in foci that dynamically change in clustering behaviour. Furthermore, we provide conclusive evidence of spatial association between exported proteins and exported protein 2, a core component of the Plasmodium translocon of exported proteins, during native conditions and upon generation of translocation intermediates. These data provide the most direct cellular evidence to date that protein export occurs at regions of the parasitophorous vacuole membrane housing the Plasmodium translocon of exported proteins complex.