Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca
-dependent events. Little is known about TPCs in ...unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca
indicator targeted to the apicoplast, we show that Ca
signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca
uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.
Ookinete invasion of the mosquito midgut is an essential step for the development of the malaria parasite in the mosquito. Invasion involves recognition between a presumed mosquito midgut receptor ...and an ookinete ligand. Here, we show that enolase lines the ookinete surface. An antienolase antibody inhibits oocyst development of both Plasmodium berghei and Plasmodium falciparum, suggesting that enolase may act as an invasion ligand. Importantly, we demonstrate that surface enolase captures plasminogen from the mammalian blood meal via its lysine motif (DKSLVK) and that this interaction is essential for midgut invasion, because plasminogen depletion leads to a strong inhibition of oocyst formation. Although addition of recombinant WT plasminogen to depleted serum rescues oocyst formation, recombinant inactive plasminogen does not, thus emphasizing the importance of plasmin proteolytic activity for ookinete invasion. The results support the hypothesis that enolase on the surface of Plasmodium ookinetes plays a dual role in midgut invasion: by acting as a ligand that interacts with the midgut epithelium and, further, by capturing plasminogen, whose conversion to active plasmin promotes the invasion process.
Regulated exocytosis allows the timely delivery of proteins and other macromolecules precisely when they are needed to fulfil their functions. The intracellular parasite Toxoplasma gondii has one of ...the most extensive regulated exocytic systems among all unicellular organisms, yet the basis of protein trafficking and proteolytic modification in this system is poorly understood. We demonstrate that a parasite cathepsin protease, TgCPL, occupies a newly recognized vacuolar compartment (VAC) that undergoes dynamic fragmentation during T. gondii replication. We also provide evidence that within the VAC or late endosome this protease mediates the proteolytic maturation of proproteins targeted to micronemes, regulated secretory organelles that deliver adhesive proteins to the parasite surface during cell invasion. Our findings suggest that processing of microneme precursors occurs within intermediate endocytic compartments within the exocytic system, indicating an extensive convergence of the endocytic and exocytic pathways in this human parasite.
Toxoplasma gondii and Neospora caninum, which cause the diseases toxoplasmosis and neosporosis, respectively, are two closely related apicomplexan parasites. They have similar heteroxenous life ...cycles and conserved genomes and share many metabolic features. Despite these similarities, T. gondii and N. caninum differ in their transmission strategies and zoonotic potential. Comparative analyses of the two parasites are important to identify the unique biological features that underlie the basis of host preference and pathogenicity. T. gondii and N. caninum are obligate intravacuolar parasites; in contrast to T. gondii, events that occur during N. caninum infection remain largely uncharacterized. We examined the capability of N. caninum (Liverpool isolate) to interact with host organelles and scavenge nutrients in comparison to that of T. gondii (RH strain). N. caninum reorganizes the host microtubular cytoskeleton and attracts endoplasmic reticulum (ER), mitochondria, lysosomes, multivesicular bodies, and Golgi vesicles to its vacuole though with some notable differences from T. gondii. For example, the host ER gathers around the N. caninum parasitophorous vacuole (PV) but does not physically associate with the vacuolar membrane; the host Golgi apparatus surrounds the N. caninum PV but does not fragment into ministacks. N. caninum relies on plasma lipoproteins and scavenges cholesterol from NPC1-containing endocytic organelles. This parasite salvages sphingolipids from host Golgi Rab14 vesicles that it sequesters into its vacuole. Our data highlight a remarkable degree of conservation in the intracellular infection program of N. caninum and T. gondii. The minor differences between the two parasites related to the recruitment and rearrangement of host organelles around their vacuoles likely reflect divergent evolutionary paths.
The close-knit group of apicomplexan parasites displays a wide variety of cell division modes, which differ between parasites as well as between different life stages within a single parasite ...species. The beginning and endpoint of the asexual replication cycles is a 'zoite' harboring the defining apical organelles required for host cell invasion. However, the number of zoites produced per division round varies dramatically and can unfold in several different ways. This plasticity of the cell division cycle originates from a combination of hard-wired developmental programs modulated by environmental triggers. Although the environmental triggers and sensors differ between species and developmental stages, widely conserved secondary messengers mediate the signal transduction pathways. These environmental and genetic input integrate in division-mode specific chromosome organization and chromatin modifications that set the stage for each division mode. Cell cycle progression is conveyed by a smorgasbord of positively and negatively acting transcription factors, often acting in concert with epigenetic reader complexes, that can vary dramatically between species as well as division modes. A unique set of cell cycle regulators with spatially distinct localization patterns insert discrete check points which permit individual control and can uncouple general cell cycle progression from nuclear amplification. Clusters of expressed genes are grouped into four functional modules seen in all division modes: 1. mother cytoskeleton disassembly; 2. DNA replication and segregation (D&S); 3. karyokinesis; 4. zoite assembly. A plug-and-play strategy results in the variety of extant division modes. The timing of mother cytoskeleton disassembly is hard-wired at the species level for asexual division modes: it is either the first step, or it is the last step. In the former scenario zoite assembly occurs at the plasma membrane (external budding), and in the latter scenario zoites are assembled in the cytoplasm (internal budding). The number of times each other module is repeated can vary regardless of this first decision, and defines the modes of cell division: schizogony, binary fission, endodyogeny, endopolygeny.
Proteases regulate key events during infection by the pervasive intracellular parasite Toxoplasma gondii. Understanding how parasite proteases mature from an inactive zymogen to an active enzyme is ...expected to inform new strategies for blocking their actions. Herein, we show that T. gondii cathepsin B protease (TgCPB) does not undergo self-maturation but instead requires the expression of a second papain-family cathepsin protease, TgCPL. Using recombinant enzymes we also show that TgCPL is capable of partially maturing TgCPB in vitro. Consistent with this interrelationship, antibodies with validated specificity detected TgCPB in the lysosome-like vacuolar compartment along with TgCPL. Our findings also establish that TgCPB does not localize to the rhoptries as previously reported. Accordingly, rhoptry morphology and rhoptry protein maturation are normal in TgCPB knock-out parasites. Finally, we show that although maturation of TgCPL is independent of TgCPB, it may involve an additional protease(s) in conjunction with self-maturation.
Background:Toxoplasma gondii expresses several cathepsin proteases, but little is known about how they are activated.
Results:Toxoplasma cathepsin B protease localizes to lysosome-like compartment, and its maturation requires a parasite cathepsin L protease.
Conclusion: Maturation of Toxoplasma cathepsin B is unconventionally nonself-dependent.
Significance: The unusual maturation of Toxoplasma cathepsin B suggests a novel mechanism of protease activation within the endolysosomal system.
Toxoplasma gondii tachyzoites infect host cells by an active invasion process leading to the formation of a specialized compartment, the parasitophorous vacuole (PV). PVs resist fusion with host cell ...endosomes and lysosomes and are thus distinct from phagosomes. Because the parasite remains sequestered within the PV, it is unclear how T. gondii-derived antigens (Ag's) access the major histocompatibility complex (MHC) class I pathway for presentation to CD8(+) T cells. We demonstrate that recruitment of host endoplasmic reticulum (hER) to the PV in T. gondii-infected dendritic cells (DCs) directly correlates with cross-priming of CD8(+) T cells. Furthermore, we document by immunoelectron microscopy the transfer of hER components into the PV, a process indicative of direct fusion between the two compartments. In strong contrast, no association between hER and phagosomes or Ag presentation activity was observed in DCs containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite-derived Ag in actively infected cells was blocked when hER retrotranslocation was inhibited, indicating that the hER serves as a conduit for the transport of Ag between the PV and host cytosol. Collectively, these findings demonstrate that pathogen-driven hER-PV interaction can serve as an important mechanism for Ag entry into the MHC class I pathway and CD8(+) T cell cross-priming.
Apicomplexan protozoan pathogens avoid destruction and establish a replicative niche within host cells by forming a nonfusogenic parasitophorous vacuole (PV). Here we present evidence for ...lysosome-mediated degradation of Toxoplasma gondii after invasion of macrophages activated in vivo. Pathogen elimination was dependent on the interferon gamma inducible-p47 GTPase, IGTP, required PI3K activity, and was preceded by PV membrane indentation, vesiculation, disruption, and, surprisingly, stripping of the parasite plasma membrane. Denuded parasites were enveloped in autophagosome-like vacuoles, which ultimately fused with lysosomes. These observations outline a series of mechanisms used by effector cells to redirect the fate of a classically nonfusogenic intracellular pathogen toward a path of immune elimination.
In Plasmodium, meiosis occurs in diploid zygotes as they develop into haploid motile ookinetes inside the mosquito. Further sporogonic development involves transformation of ookinetes into oocysts ...and formation of infective sporozoites.
Reverse genetics was employed to examine the role of the meiotic specific recombinase Dmc1, a bacterial RecA homolog during sporogony in Plasmodium berghei. PbDmc1 knockout (KO) parasites showed normal asexual growth kinetics compared to WT parasites; however oocyst formation in mosquitoes was reduced by 50 to 80%. Moreover, the majority of oocysts were retarded in their growth and were smaller in size compared to WT parasites. Only a few Dmc1 KO parasites completed maturation resulting in formation of fewer sporozoites which were incapable of infecting naive mice or hepatocytes in vitro. PbDmc1 KO parasites were shown to be approximately 18 times more sensitive to Bizelesin, a DNA alkylating drug compared to WT parasites as reflected by impairment of oocyst formation and sporogonic development in the mosquito vector.
Our findings suggest that PbDmc1 plays a critical role in malaria transmission biology.
The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In an attempt to identify novel ...parasite secreted proteins that influence host cell activity, we identified and characterized a transmembrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrated that GRA64 appears to interact with components of the host endosomal sorting complexes required for transport (ESCRT). Genetic disruption of GRA64 does not affect acute
virulence or encystation in mice, as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of Δ
tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic
infection.
Toxoplasma gondii is a widespread foodborne parasite that causes congenital disease and life-threatening complications in immunocompromised individuals. Part of this parasite's success lies in its ability to infect diverse organisms and host cells and to persist as a latent infection within parasite-constructed structures called tissue cysts. In this study, we characterized a protein that is secreted by T. gondii into its parasitophorous vacuole during intracellular infection, which we dub GRA64. On the vacuolar membrane, this protein is exposed to the host cell cytosol and interacts with specific host ESCRT proteins. Parasites lacking the GRA64 protein exhibit ultrastructural changes in tissue cysts during chronic infection. This study lays the foundation for future studies on the mechanics and consequences of host ESCRT-parasite protein interactions.
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