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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Acidocalcisomes are acidic organelles containing calcium and a high concentration of phosphorus in the form of pyrophosphate (PPi ) and polyphosphate (poly P). Organelles with these ...characteristics have been found from bacteria to human cells implying an early appearance and persistence over evolutionary time or their appearance by convergent evolution. Acidification of the organelles is driven by the presence of vacuolar proton pumps, one of which, the vacuolar proton pyrophosphatase, is absent in animals, where it is substituted by a vacuolar proton ATPase. A number of other pumps, antiporters, and channels have been described in acidocalcisomes of different species and are responsible for their internal content. Enzymes involved in the synthesis and degradation of PPi and poly P are present within the organelle. Acidocalcisomes function as storage sites for cations and phosphorus, and participate in PPi and poly P metabolism, calcium homeostasis, maintenance of intracellular pH, and osmoregulation. Experiments in which the acidocalcisome Ca2+ -ATPase of different parasites were downregulated or eliminated, or acidocalcisome Ca2+ was depleted revealed the importance of this store in Ca2+ signaling needed for host invasion and virulence. Acidocalcisomes interact with other organelles in a number of organisms suggesting their association with the endosomal/lysosomal pathway, and are considered part of the lysosome-related group of organelles.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. Like most apicomplexans, T. gondii possesses several ...plant-like features, such as the chloroplast-like organelle, the apicoplast. We describe and characterize a novel organelle in T. gondii tachyzoites, which is visible by light microscopy and possesses a broad similarity to the plant vacuole. Electron tomography shows the interaction of this vacuole with other organelles. The presence of a plant-like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, a cathepsin L-like protease (TgCPL), an aquaporin (TgAQP1), as well as Ca²⁺/H⁺ and Na⁺/H⁺ exchange activities, supports similarity to the plant vacuole. Biochemical characterization of TgVP1 in enriched fractions shows a functional similarity to the respective plant enzyme. The organelle is a Ca²⁺ store and appears to have protective effects against salt stress potentially linked to its sodium transport activity. In intracellular parasites, the organelle fragments, with some markers colocalizing with the late endosomal marker, Rab7, suggesting its involvement with the endocytic pathway. Studies on the characterization of this novel organelle will be relevant to the identification of novel targets for chemotherapy against T. gondii and other apicomplexan parasites as well.
Phosphogypsum wastes from phosphate fertilizer industries are stockpiled in stacks with high contamination potential. An assessment of the environmental impact, including the use of geochemical ...tracers such as rare earth elements (REE) and Cl/Br ratios, was carried out in the phosphogypsum stack located at the Estuary of Huelva (SW Spain). Inside the pile, highly polluted acid pore-waters flows up to the edge of the stack, emerging as small fluvial courses, known as edge outflows, which discharge directly into the estuary. The disposal area is divided into four zones; two unrestored zones with surface ponds of industrial process water and two a priori already-restored zones. However, an extensive sampling of edge outflows conducted in the perimeter of the four zones demonstrates the high potential of contamination of the whole stack, including those zones that were supposedly restored. These solutions are characterized by a pH of 1.9 and concentrations of 6100mg/L for P, 1970mg/L for S, 600mg/L for F, 200mg/L for NH4+, 100mg/L for Fe, 10–30mg/L for Zn, As and U, and 1–10mg/L for Cr, Cu and Cd. Preliminary restoration actions and those planned for the future prioritize removal of ponded process water and cover of the phosphogypsum with artificial topsoil. These actions presuppose that the ponded process water percolates through the porous medium towards the edge up to reach the estuary. However, geochemical tracers rule out this connection and point to an estuarine origin for these leachates, suggesting a possible tidal-induced leaching of the waste pile in depth. These findings would explain the ineffectiveness of preliminary restoration measures and should be considered for the development of new action plans.
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•Acidity and contaminants from phosphogypsum leaching are released to an estuary.•Already-restored zones act as a pollution source just as unrestored zones.•Cl/Br ratios and REE patterns were suitable to assess the restoration inefficiency.•Changes in the remediation strategies of the phosphogypsum stack are hence necessary.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The ...apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance.
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