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Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi.

ABSTRACT: The contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease, collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress; it also has a role in cell shrinking after hyperosmotic stress. Here, we report that, in addition to its role in osmoregulation, the CVC of T. cruzi has a role in the biogenesis of acidocalcisomes. Expression of dominant-negative mutants of the CVC-located small GTPase Rab32 (TcCLB.506289.80) results in lower numbers of less-electron-dense acidocalcisomes, lower content of polyphosphate, lower capacity for acidocalcisome acidification and Ca(2+) uptake that is driven by the vacuolar proton pyrophosphatase and the Ca(2+)-ATPase, respectively, as well as less-infective parasites, revealing the role of this organelle in parasite infectivity. By using fluorescence, electron microscopy and electron tomography analyses, we provide further evidence of the active contact of acidocalcisomes with the CVC, indicating an active exchange of proteins between the two organelles.

SUBMITTER: Niyogi S

PROVIDER: S-EPMC4487017 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi.

Niyogi Sayantanee S Jimenez Veronica V Girard-Dias Wendell W de Souza Wanderley W Miranda Kildare K Docampo Roberto R

Journal of cell science 20150511 12

The contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease, collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress; it also has a role in cell shrinking after hyperosmotic stress. Here, we report that, in addition to its role in osmoregulation, the CVC of T. cruzi has a role in the biogenesis of acidocalcisomes. Expression of dominant-negative mutants of the CVC-located small GTPase Rab32 (TcCLB.506289.80) resu ...[more]

PMID: 25964650

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Project description:The use of digitonin to permeabilize the plasma membrane of Trypanosoma cruzi allowed the identification of a non-mitochondrial nigericin- or bafilomycin A1-sensitive Ca(2+)-uptake mechanism. Proton uptake, as detected by ATP-dependent Acridine Orange accumulation, was also demonstrated in these permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles. This latter process was inhibited (and reversed) by bafilomycin A1, nigericin and NH4Cl in different stages of T. cruzi. Ca2+ released Acridine Orange from permeabilized cells, suggesting that the dye and Ca2+ were being accumulated in the same acidic compartment and that Ca2+ was taken up in exchange for protons. Addition of bafilomycin A1 (5 microM), nigericin (1 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 microM) to fura 2-loaded epimastigotes increased their intracellular Ca2+ concentration ([Ca2+]i). Although this effect was more noticeable in the presence of extracellular Ca2+, it was also observed in its absence. Addition of NH4Cl (10-40 mM) to different stages of T. cruzi, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, increased both [Ca2+]i in fura 2-loaded cells, and intracellular pH (pHi) in 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF)-loaded cells. Treatment of the cells with the Ca2+ ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase in [Ca2+]i and a significantly higher increase in [Ca2+]i after addition of NH4Cl, nigericin or bafilomycin A1, all agents which increase the pH of acidic compartments and make ionomycin more effective as a Ca(2+)-releasing ionophore. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that most of the Ca2+ stored in different stages of T. cruzi is present in the acidic compartment thus identified. Taken together, these results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which we have named the 'acidocalcisome' and which appears to be a unique organelle present in trypanosomatids.

Project description:BackgroundTSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties.Methods/principle findingsHere we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the 'adhesive' TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to trypomastigote internalization. These motifs are not conserved in the 'non-adhesive' TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation.Conclusion/significanceIn this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by parasite strains.

Project description:BACKGROUND: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. METHODOLOGY/PRINCIPAL FINDINGS: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. CONCLUSIONS/SIGNIFICANCE: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

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Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi.

Publications

Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi.

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