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  Actin inhibitors such as phalloidin and DNase I also inhibit DdPPK2-mediated synthesis of polyP. [...]this particular actin-related protein complex is an enzyme that can polymerize into an actin-like filament concurrent with its synthesis of a polyP chain in a fully reversible reaction [15].\\n It was also shown that polyPs have a potent modulatory activity on blood coagulation [24] and inflammation [25]. Role of PolyP in Pathogenesis PolyP, which in bacteria is mainly of long-chain type (>300 and up to 1,000 Pi residues), has been reported to be important for virulence of different bacteria, such as Salmonella spp., Shigella flexneri, Vibrio cholerae, Neisseria meningitides, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, but the mechanism involved is not known [30].

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca 2+ -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 2+ indicator targeted to the apicoplast, we show that Ca 2+ 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 2+ 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. Two-pore channels (TPCs) are cation channels that localize to acidic organelles to regulate Ca 2+ dependent events. Here, Li et al. characterize a TPC from Toxoplasma gondii (TgTPC) that localizes to the apicoplast, is critical for maintaining its integrity and relevant for Ca 2+ uptake from the ER through stabilizing inter-organelle contact.

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate.

Ca 2+ signaling in cells begins with the opening of Ca 2+ channels in either the plasma membrane (PM) or endoplasmic reticulum (ER), leading to a sharp increase in the physiologically low (<100 nM) cytosolic Ca 2+ level. The temporal and spatial regulation of Ca² + is crucial for the precise activation of key biological processes. In the apicomplexan parasite Toxoplasma gondii , which infects approximately one-third of the global population, Ca² + signaling governs essential aspects of the parasite’s infection cycle. T. gondii relies on Ca² + signals to regulate pathogenic traits, with several Ca² + -signaling components playing critical roles. Ca 2+ entry from the extracellular environment has been demonstrated in T. gondii for both, extracellular parasites, exposed to high Ca 2+ , and intracellular parasites, which acquire Ca² + from host cells during host Ca² + signaling events. Active egress, an essential step of the parasite’s infection cycle, is preceded by a large increase in cytosolic Ca 2+ , most likely initiated by release from intracellular stores. However, extracellular Ca 2+ is also necessary to reach a cytosolic Ca 2+ threshold required for timely egress. In this study, we investigated the mechanism of intracellular Ca² + store replenishment and identified a central role for the SERCA-Ca 2+ -ATPase in maintaining Ca² + homeostasis within the ER and in other organelles. We demonstrate mitochondrial Ca 2+ uptake, which occurs by transfer of Ca 2+ from the ER, likely through membrane contact sites. Our findings suggest that the T. gondii ER plays a key role in sequestering and redistributing Ca² + to intracellular organelles following Ca² + influx at the PM.

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca 2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca 2+ -responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca 2+ -responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca 2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca 2+ -responsive proteins, such as parasite Ca 2+ -dependent protein kinases. Our approach also identified numerous Ca 2+ -responsive proteins that are not predicted to bind Ca 2+ , yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca 2+ -responsive enzyme that relocalized to the parasite apex upon Ca 2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca 2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca 2+ -regulated serine/threonine phosphatase activity in apicomplexan parasites.

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like organelle, termed the \"Vacuolar Compartment/Plant-Like Vacuole\" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that contribute to the maturation of invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that the TgCRT protein plays a key role in maintaining the integrity of the parasite's endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in volume by ~15-fold and overlaps with adjacent endosome-like compartments. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in ΔTgCRT parasites. Expression of subtilisin protease 1 is significantly reduced, which impedes trimming of microneme proteins, and significantly decreases parasite invasion. Chemical or genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring integrity of the endolysosomal system, microneme protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in substrate transport that impacts VAC volume and the integrity of the endolysosomal system in T. gondii.

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.

The lytic cycle of Toxoplasma gondii is critical for parasite dissemination and disease progression in the host. Calcium signaling plays a crucial role in driving these processes; however, the molecules that control calcium storage and release remain poorly understood. The endoplasmic reticulum, likely the largest calcium reservoir in T. gondii , has been understudied in the context of calcium signaling. Here, we uncover a direct link between ER redox regulation and calcium homeostasis, showing that ER redox activity can influence calcium signaling events that govern microneme protein maturation and secretion, parasite invasion, and replication. Our findings indicate that redox-dependent calcium regulation in the ER contributes to control of the parasite lytic cycle and reveals a previously unrecognized mechanism that may influence parasite virulence.

Transient receptor potential (TRP) channels participate in calcium ion (Ca 2+ ) influx and intracellular Ca 2+ release. TRP channels have not been studied in Toxoplasma gondii or any other apicomplexan parasite. In this work, we characterize TgGT1_310560, a protein predicted to possess a TRP domain (TgTRPPL-2), and determined its role in Ca 2+ signaling in T. gondii , the causative agent of toxoplasmosis. TgTRPPL-2 localizes to the plasma membrane and the endoplasmic reticulum (ER) of T. gondii . The ΔTgTRPPL-2 mutant was defective in growth and cytosolic Ca 2+ influx from both extracellular and intracellular sources. Heterologous expression of TgTRPPL-2 in HEK-3KO cells allowed its functional characterization. Patching of ER-nuclear membranes demonstrates that TgTRPPL-2 is a non-selective cation channel that conducts Ca 2+ . Pharmacological blockers of TgTRPPL-2 inhibit Ca 2+ influx and parasite growth. This is the first report of an apicomplexan ion channel that conducts Ca 2+ and may initiate a Ca 2+ signaling cascade that leads to the stimulation of motility, invasion, and egress. TgTRPPL-2 is a potential target for combating toxoplasmosis.

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.