Inactive trans-Sialidase Expression in iTS-null Trypanosoma cruzi Generates Virulent Trypomastigotes.

Disclosing virulence factors from pathogens is required to better understand the pathogenic mechanisms involved in their interaction with the host. In the case of Trypanosoma cruzi several molecules are associated with virulence. Among them, the trans-sialidase (TS) has arisen as one of particular relevance due to its effect on the immune system and involvement in the interaction/invasion of the host cells. The presence of conserved genes encoding for an inactive TS (iTS) isoform is puzzlingly restricted to the genome of parasites from the Discrete Typing Units TcII, TcV, and TcVI, which include highly virulent strains. Previous in vitro results using recombinant iTS support that this isoform could play a different or complementary pathogenic role to that of the enzymatically active protein. However, direct evidence involving iTS in in vivo pathogenesis and invasion is still lacking. Here we faced this challenge by transfecting iTS-null parasites with a recombinant gene that allowed us to follow its expression and association with pathological events. We found that iTS expression improves parasite invasion of host cells and increases their in vivo virulence for mice as shown by histopathologic findings in heart and skeletal muscle.

Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease. In immunosuppressed individuals, as it occurs in the coinfection with human immunodeficiency virus (HIV), the central nervous system may be affected. In this regard, reactivation of Chagas disease is severe and often lethal, and it accounts for meningoencephalitis. Astrocytes play a crucial role in the environment maintenance of healthy neurons; however, they can host HIV and T. cruzi. In this report, human astrocytes were infected in vitro with both genetically modified-pathogens to express alternative fluorophore. As evidenced by fluorescence microscopy and flow cytometry, HIV and T. cruzi coexist in the same astrocyte, likely favoring reciprocal interactions. In this context, lower rates of cell death were observed in both T. cruzi monoinfected-astrocytes and HIV-T. cruzi coinfection in comparison with those infected only with HIV. The level of HIV replication is significantly diminished under T. cruzi coinfection, but without affecting the infectivity of the HIV progeny. This interference with viral replication appears to be related to the T. cruzi multiplication rate or its increased intracellular presence but does not require their intracellular cohabitation or infected cell-to-cell contact. Among several Th1/Th2/Th17 profile-related cytokines, only IL-6 was overexpressed in HIV-T. cruzi coinfection exhibiting its cytoprotective role. This study demonstrates that T. cruzi and HIV are able to coinfect astrocytes thus altering viral replication and apoptosis.

trans-Sialidase neutralizing antibody detection in Trypanosoma cruzi-infected domestic reservoirs.

The detection of Trypanosoma cruzi infection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected with Leishmania spp. We used a trans-sialidase inhibition assay (TIA) for T. cruzi diagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinant trans-sialidase, an enzyme that is not detected in the coendemic Leishmania species or Trypanosoma rangeli parasites. T. cruzi infection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development of trans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence of trans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool for T. cruzi detection in the main domestic reservoirs.

Estimation of sensitivity and specificity of several Trypanosoma cruzi antibody assays in blood donors in Argentina.

BACKGROUND: The absence of a gold standard test for Trypanosoma cruzi antibodies represents a problem not only for the evaluation of screening tests, but also for appropriate blood donor counseling. The aim of this study was to estimate the sensitivity and specificity of multiple blood donor screening tests for T. cruzi antibodies in Argentina. STUDY DESIGN AND METHODS: From June 2006 to March 2007 a sample of 1455 blood donors was recruited from two blood banks in Chaco province, an area of Argentina with highly endemic T. cruzi infection. Samples were tested by three epimastigote lysate enzyme immunoassays (EIAs), one recombinant antigen EIA, two indirect hemagglutination assay (IHA) tests, a particle agglutination assay (PA), and a research trans-sialidase inhibition assay (TIA). Sensitivity and specificity were estimated using latent class analysis (LCA). RESULTS: LCA estimated the consensus prevalence of T. cruzi infection at 24.5%. Interassay correlation was higher among the four EIA tests and TIA compared to IHA tests. Assay sensitivities varied from 96 to 99.7 for different EIAs, 91% for TIA, 84% for PA, and 66 to 74% for IHA tests. Relative to the LCA, assay specificities were from 96% to almost 100%. CONCLUSION: Based on the comparison of several tests in a large population from an endemic area for T. cruzi infection, our data showed an adequate sensitivity for EIA tests in contrast to PA and IHA assays. The latter tests should no longer be used for blood donor screening.

The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation.

The trans-sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans-sialidase-mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans-sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans-sialidase-apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans-sialidase on the immune system are due to the sialylation of endogenous acceptor molecules.

Differential expression of a virulence factor, the trans-sialidase, by the main Trypanosoma cruzi phylogenetic lineages.

The clinical outcome of Chagas disease is highly variable, mainly because of the heterogeneity of Trypanosoma cruzi, a parasite for which 2 major phylogenetic groups (I and II) were recently defined. Epidemiological and immunological data indicate that the prevalence of T. cruzi II in patients living in the southern cone of South America correlates with the alterations caused by Chagas disease. We report here that infection with T. cruzi II isolates induces 100% mortality in mice, in contrast to infection with T. cruzi I isolates, in which almost all mice enter the chronic phase even when a 1000-fold higher inoculum is administered. Trypomastigotes from T. cruzi II strains express and shed significantly higher amounts of trans-sialidase than do those from the T. cruzi I lineage. Disorganization of the thymus histoarchitecture associated with the circulating enzyme was observed after infection with T. cruzi II strains, in contrast to transient thymus lesions found in mice infected with T. cruzi I strains. Therefore, trans-sialidase becomes the first T. cruzi virulence factor identified that is differentially expressed by the main parasite groups and that contributes to their contrasting behaviors.

Evaluation of Brucella abortus phosphoglucomutase (pgm) mutant as a new live rough-phenotype vaccine.

Brucella abortus S19 is the vaccine most frequently used against bovine brucellosis. Although it induces good protection levels, it cannot be administered to pregnant cattle, revaccination is not advised due to interference in the discrimination between infected and vaccinated animals during immune-screening procedures, and the vaccine is virulent for humans. Due to these reasons, there is a continuous search for new bovine vaccine candidates that may confer protection levels comparable to those conferred by S19 but without its disadvantages. A previous study characterized the phenotype associated with the phosphoglucomutase (pgm) gene disruption in Brucella abortus S2308, as well as the possible role for the smooth lipopolysaccharide (LPS) in virulence and intracellular multiplication in HeLa cells (J. E. Ugalde, C. Czibener, M. F. Feldman, and R. A. Ugalde, Infect. Immun. 68:5716-5723, 2000). In this report, we analyze the protection, proliferative response, and cytokine production induced in BALB/c mice by a deltapgm deletion strain. We show that this strain synthesizes O antigen with a size of approximately 45 kDa but is rough. This is due to the fact that the deltapgm strain is unable to assemble the O side chain in the complete LPS. Vaccination with the deltapgm strain induced protection levels comparable to those induced by S19 and generated a proliferative splenocyte response and a cytokine profile typical of a Th1 response. On the other hand, we were unable to detect a specific anti-O-antigen antibody response by using the fluorescence polarization assay. In view of these results, the possibility that the deltapgm mutant could be used as a vaccination strain is discussed.

Thymocyte depletion in Trypanosoma cruzi infection is mediated by trans-sialidase-induced apoptosis on nurse cells complex.

Trypanosoma cruzi, the causative agent of Chagas' disease, induces transient thymic aplasia early after infection-a phenomenon that still lacks a molecular explanation. The parasite sheds an enzyme known as trans-sialidase (TS), which is able to direct transfer-sialyl residues among macromolecules. Because cell-surface sialylation is known to play a central role in the immune system, we tested whether the bloodstream-borne TS is responsible for the thymic alterations recorded during infection. We found that recombinant TS administered to naive mice was able to induce cell-count reduction mediated by apoptosis, mimicking cell subsets distribution and histologic findings observed during the acute phase of the infection. Thymocytes taken after TS treatment showed low response to Con A, although full ability to respond to IL-2 or IL-2 plus Con A was conserved, which resembles findings from infected animals. Alterations were found to revert several days after TS treatment. The administration of TS-neutralizing Abs to T. cruzi-infected mice prevented thymus alterations. Results indicate that the primary target for the TS-induced apoptosis is the so-called "nurse cell complex". Therefore, we report here supporting evidence that TS is the virulence factor from T. cruzi responsible for the thymic alterations via apoptosis induction on the nurse cell complex, and that TS-neutralizing Abs elicitation during infection is associated with the reversion to thymic normal parameters.