Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion.

BACKGROUND: The question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells. METHODOLOGY/PRINCIPAL FINDINGS: Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS++, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS++ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion. CONCLUSION/SIGNIFICANCE: Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion.

Host cell invasion and oral infection by Trypanosoma cruzi strains of genetic groups TcI and TcIV from chagasic patients.

BACKGROUND: Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. METHODS: Trypanosoma cruzi metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were incubated with metacyclic forms and the number of internalized parasites was counted. RESULTS: All TcI and TcIV T. cruzi strains were poorly infective by the oral route. Parasites were either undetectable or were detected in small numbers in the mouse stomach four days post oral administration. Replicating parasites were found in the stomach and/or in the heart 30 days post-infection. As compared to TcI lineage, the migration capacity of TcIV parasites through the gastric mucin-coated filter was higher but lower than that exhibited by TcVI metacyclic forms previously shown to be highly infective by the oral route. Expression of pepsin-resistant gp90, the surface molecule that downregulates cell invasion, was higher in TcI than in TcIV parasites and, accordingly, the invasion capacity of TcIV metacyclic forms was higher. Gp90 molecules spontaneously released by TcI metacyclic forms inhibited the parasite entry into host cells. TcI parasites exhibited low intracellular replication rate. CONCLUSIONS: Our findings indicate that the poor capacity of TcI lineage, and to a lesser degree of TcIV parasites, in invading gastric epithelium after oral infection of mice may be associated with the inefficiency of metacyclic forms, in particular of TcI parasites, to migrate through the gastric mucus layer, to invade target epithelial cells and to replicate intracellularly.

Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease.

Acetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD(+)-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruzi at concentrations that did not affect host cell viability. In addition, in vivo infection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coli with a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 µM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi.

Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D (1)H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.