Oral infectivity through carnivorism in murine model of Trypanosoma cruzi infection.

Introduction: Oral transmission of T. cruzi is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat from animals infected with T. cruzi may be responsible for transmitting the infection. Therefore, the general objective of this study was to investigate host-pathogen interactions between the parasite and gastric mucosa and the role of meat consumption from infected animals in the oral transmission of T. cruzi. Methods: Cell infectivity assays were performed on AGS cells in the presence or absence of mucin, and the roles of pepsin and acidic pH were determined. Moreover, groups of five female Balb/c mice were fed with muscle tissue obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by a parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacities. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed, and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies and by LC-MS/MS and bioinformatics analysis. Results: Trypomastigote migration in the presence of mucin was reduced by approximately 30%, whereas in the presence of mucin and pepsin at pH 3.5, only a small proportion of parasites were able to migrate (∼6%). Similarly, the ability of TCTs to infect AGS cells in the presence of mucin is reduced by approximately 20%. In all cases, 60-100% of the animals were fed meat from mice infected in the acute phase or infected with trypomastigotes or amastigotes developed high parasitemia, and 80% died around day 40 post-infection. The adhesion assay showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS and bioinformatics analysis, also confirmed that transialidase, cysteine proteinases, and gp63 may be involved in TCTs attachment or invasion of human stomach cells because they can potentially interact with different proteins in the human stomach mucosa. In addition, several human gastric mucins have cysteine protease cleavage sites. Discussion: Then, under our experimental conditions, consuming meat from infected animals in the acute phase allows the T. cruzi infection. Similarly, trypomastigotes and amastigotes could infect mice when administered orally, whereas cysteinyl proteinases and trans-sialidase appear to be relevant molecules in this infective process.

Trypanosoma cruzi pathogenicity involves virulence factor expression and upregulation of bioenergetic and biosynthetic pathways.

The molecular repertoire of Trypanosoma cruzi effects its virulence and impacts the clinical course of the resulting Chagas disease. This study aimed to determine the mechanism underlying the pathogenicity of T. cruzi. Two T. cruzi cell lines (C8C3hvir and C8C3lvir), obtained from the clone H510 C8C3 and exhibiting different virulence phenotypes, were used to evaluate the parasite's infectivity in mice. The organ parasite load was analysed by qPCR. The proteomes of both T. cruzi cell lines were compared using nLC-MS/MS. Cruzipain (Czp), complement regulatory protein (CRP), trans-sialidase (TS), Tc-85, and sialylated epitope expression levels were evaluated by immunoblotting. High-virulence C8C3hvir was highly infectious in mice and demonstrated three to five times higher infectivity in mouse myocardial cells than low-virulence C8C3lvir. qPCR revealed higher parasite loads in organs of acute as well as chronically C8C3hvir-infected mice than in those of C8C3lvir-infected mice. Comparative quantitative proteomics revealed that 390 of 1547 identified proteins were differentially regulated in C8C3hvir with respect to C8C3lvir. Amongst these, 174 proteins were upregulated in C8C3hvir and 216 were downregulated in C8C3lvir. The upregulated proteins in C8C3hvir were associated with the tricarboxylic acid cycle, ribosomal proteins, and redoxins. Higher levels of Czp, CRP, TS, Tc-85, and sialylated epitopes were expressed in C8C3hvir than in C8C3lvir. Thus, T. cruzi virulence may be related to virulence factor expression as well as upregulation of bioenergetic and biosynthetic pathways proteins.

Demodex folliculorum (Trombidiformes: Demodicidae) and Demodex brevis Prevalence in an Extreme Environment of Chile.

The prevalence of mites of the genus Demodex and their associations with host age, gender, workplace, and comorbid skin and ocular conditions were studied in participants in Antofagasta, Chile, which is in a region with an extreme environment. We examined 680 participants aged 18-88 yr using standardized surface skin biopsies. Among them, Demodex had a prevalence of 13.5 % (95% confidence interval, 10.88-16.17). A slightly higher prevalence was observed in males (51.1%; 95% confidence interval, 40.9-61.3; nonsignificant) and participants in the 69-88 yr age group (50.0%; 95% confidence interval, 23.8-76.2; P < 0.05). Regarding the species involved, Demodex folliculorum was found in 89.1% (CI 82.7-95.5) of cases, while D. brevis was found in the remaining 10.9% of cases. A higher prevalence (25.0% CI 16.1-33.91) was observed in participants who worked indoors in generally enclosed and dust-rich environments (such as theaters, libraries, and administrative offices). There was also a strong association between Demodex prevalence and conjunctival hyperemia, with 35.9% (95% confidence interval, 9.1-35; OR 17.9) of the Demodex positive participants having this pathology compared to 10.3% of the noninfested participants. In summary, the prevalence of Demodex in Antofagasta, Chile (13.5%) was toward the lower end of the range reported among other regions around the world. Environmental factors such as exposure to the sun (including ultraviolet rays) or environmental pollution may affect the mites. In addition, Demodex genetics (related to virulence) and the ocular or skin microbiota may positively or negatively influence infestation and pathology.

Decreased cruzipain and gp85/trans-sialidase family protein expression contributes to loss of Trypanosoma cruzi trypomastigote virulence.

Two cell lines derived from a single Trypanosoma cruzi clone by long-term passaging generated a highly virulent (C8C3hvir) and a low virulent (C8C3lvir) cell line. The C8C3hvir cell line was highly infective and lethal to Balb/c mice, and the C8C3lvir cell line was three- to five-fold less infective to mouse cardiomyocytes than C8C3hvir. The highly virulent T. cruzi cell line abundantly expressed the major cysteine proteinase cruzipain (Czp), complement regulatory protein (CRP) and trans-sialidase (TS), all of which are known to act as virulence factors in this parasite. The in vitro invasion capacity and in vivo Balb/c mouse infectiveness of the highly virulent strain was strongly reduced by pre-treatment with antisense oligonucleotides targeting TS or CRP or with E64d. Based on these results, we conclude that decreased levels of TS, CRP and Czp expression could contribute to loss of T. cruzi trypomastigote virulence.

Role of the Ubiquitin-Proteasome Systems in the Biology and Virulence of Protozoan Parasites.

In eukaryotic cells, proteasomes perform crucial roles in many cellular pathways by degrading proteins to enforce quality control and regulate many cellular processes such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein-quality control, and development. The catalytic heart of these complexes, the 20S proteasome, is highly conserved in bacteria, yeast, and humans. However, until a few years ago, the role of proteasomes in parasite biology was completely unknown. Here, we summarize findings about the role of proteasomes in protozoan parasites biology and virulence. Several reports have confirmed the role of proteasomes in parasite biological processes such as cell differentiation, cell cycle, proliferation, and encystation. Proliferation and cell differentiation are key steps in host colonization. Considering the importance of proteasomes in both processes in many different parasites such as Trypanosoma, Leishmania, Toxoplasma, and Entamoeba, parasite proteasomes might serve as virulence factors. Several pieces of evidence strongly suggest that the ubiquitin-proteasome pathway is also a viable parasitic therapeutic target. Research in recent years has shown that the proteasome is a valid drug target for sleeping sickness and malaria. Then, proteasomes are a key organelle in parasite biology and virulence and appear to be an attractive new chemotherapeutic target.

Aromatic glycosyl disulfide derivatives: evaluation of their inhibitory activities against Trypanosoma cruzi.

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(ß-D-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC50 values ranging from 4 to 11 µM at 37 °C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.

Virulence factors of Trypanosoma cruzi: who is who?

The aim of this review is to gather the current knowledge of Trypanosoma cruzi's virulence factors described to date in an integrative way, relating these with the parasite's life cycle and trying to elucidate their importance in each process. Several aspects relevant for the parasite's survival, such as invasion, resistance to oxidative damage, escape from the phagolysosomal vacuole and differentiation, among others, will be discussed. However, there is still a lot to learn about what virulence really means in T. cruzi and which parasite molecules are absolutely required to make T. cruzi one of the most successful pathogens to invade, survive and persist in a mammalian host.

A protein phosphatase 1 gamma (PP1γ) of the human protozoan parasite Trichomonas vaginalis is involved in proliferation and cell attachment to the host cell.

In this work, evidence for a critical role of Trichomonas vaginalis protein phosphatase 1 gamma (TvPP1γ) in proliferation and attachment of the parasite to the mammalian cell is provided. Firstly, proliferation and attachment of T. vaginalis parasites to HeLa cells was blocked by calyculin A (CA), a potent PP1 inhibitor. Secondly, it was demonstrated that the enzyme activity of native and recombinant TvPP1γ proteins was inhibited by CA. Thirdly, reverse genetic studies confirmed that antisense oligonucleotides targeted to PP1γ but not PP1α or ß inhibited proliferation and attachment of trichomonads CA-treated parasites underwent cytoskeletal modifications, including a lack of axostyle typical labelling, suggesting that cytoskeletal phosphorylation could be regulated by a CA-sensitive phosphatase where the role of PP1γ could not be ruled out. Analysis of subcellular distribution of TvPP1γ by cell fractionation and electron microscopy demonstrated the association between TvPP1γ and the cytoskeleton. The expression of adhesins, AP120 and AP65, at the cell surface was also inhibited by CA. The concomitant inhibition of expression of adhesins and changes in the cytoskeleton in CA-treated parasites suggest a specific role for PP1γ -dependent dephosphorylation in the early stages of the host-parasite interaction. Molecular modelling of TvPP1γ showed the conservation of residues critical for maintaining proper folding into the gross structure common to PP1 proteins. Taken together, these results suggest that TvPP1γ could be considered a potential novel drug target for treatment of trichomoniasis.

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome.

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the alpha5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.

A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi.

Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 microM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.

Niveles de anticuerpos anti-Gal en personas infectadas y no-infectadas por trypanosoma cruzi: probable inducción por bacterias y por el parásito / Anti-Gal antibodies levels in trypanosoma cruzi infected and non-infected persons: probable induction by bacteria and by the parasite

Se estudiaron los niveles de anticuerpos contra epítopes Gal Ó 1,3 Gal en 407 sueros humanos chagásicos (92) y no chagásicos (315), mediante la reacción de hemaglutinación con eritrocitos de conejo; con inmunoelectrotransferencia se investigó la reactividad de sueros con altos títulos de anticuerpos anti-Gal frente a antígenos de escherichia coli y serratia marcescens. Finalmente, utilizando un anticuerpo anti-Gal purificado se identificó epítopes Gal Ó 1,3 Gal en formas metacíclicas de 12 cepas altoandinas chilenas de trypanosoma cruzi. Entre los 92 sueros chagásicos, se demostró que en el 68,5 por ciento (63) de los menos chagásicos se detectó anticuerpos anti-Gal a títulos ò 1:1.600, mientras que entre los sueros no chagásicos, sólo el 15,6 por ciento (49) mostró respuesta anti-Gal a títulos similares. Estos datos sugieren que la determinación de estos anticuerpos podría contribuir a complementar el diagnóstico de la infección, especialmente cuando se establezcan títulos de corte ò 1:3.200. La inmunoelectrotransferencia mostró que sueros de personas infectadas con T. cruzi reconocen varios antígenos presentes en E. coli y S. marcescens, lo que refuerza la idea de que a lo menos en parte estas bacterias serían capaces de estimular estas respuestas. El análisis autorradiográfico utilizando anticuerpo anti-Gal purificado, mostró diferencias en la expresión de los epítopes Gal Ó 1,3 Gal en las diferentes cepas de T. cruzi. Estos resultados sugieren que los anticuerpos anti-Gal podrían tener real significado en los mecanismos de inmunidad natural y protección de la infección en chilenos infectados con T. cruzi