The 70-kDa heat-shock protein is a major antigenic determinant in human Trypanosoma cruzi/Leishmania braziliensis braziliensis mixed infection.

Five sera from Bolivian individuals chronically infected by Trypanosoma cruzi, and suffering an active Leishmania braziliensis braziliensis metastatic mucocutaneous lesion were characterized. They reacted with the T. cruzi recombinant antigens that are currently used as Chagas diagnostic reagents, and with several L. b. braziliensis proteins as assessed by Western blot. These sera showed an intense reaction with a T. cruzi and an L. b. braziliensis polypeptide of about 70 kDa. Expression cloning techniques demonstrated that the target of this immunologic reaction was a cross-reactive antigen, the 70-kDa heat-shock protein (HSP 70). High levels of anti-HSP 70 reactivity and positive reactions with all or some of the T. cruzi recombinant antigens JL7, JL8, and JL5, defined a serologic pattern that was characteristic of the T. cruzi/L. b. braziliensis mixed infection.

Humoral autoimmune response in Chagas' disease: Trypanosoma cruzi ribosomal antigens as immunizing agents.

Molecular expression cloning techniques have revealed that patients with chronic Chagas' heart disease (cChHD) present a strong humoral response against the cloned C-terminal portions of the four Trypanosoma cruzi ribosomal P proteins TcP1, TcP2 alpha (TcP2b), TcP2 beta (TcPJL5), and TcP0. This protein family presents several features that may be important in the immunopathology of Chagas disease. Their exposed location on the ribosome, and the amplification of their parasite-specific, Ser free C-terminal domain, generate a strong anti-parasite P response that may induce anti-P autoimmunity. Evidences indicate that the serological pattern of the anti-P response from chagasic patients may be the consequence of a chronic immunization with T. cruzi ribosomal antigens.

Relevance of hemin for in vitro differentiation of Trypanosoma cruzi.

Simple culture conditions that allow good growth and high yields of trypomastigotes are described. The proportion of metacyclic trypomastigotes increases with the concentration of hemin in the culture medium, reaching a peak of 80% after 10 days with 20 mg hemin/liter.

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton.

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.

Regulation of phospholipase C-gamma(1) by the actin-regulatory protein villin.

The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-gamma(1) (PLC-gamma(1)) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-gamma(1) activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-gamma(1). Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-gamma(1). The catalytic activity of PLC-gamma(1) was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 microM. Villin inhibited PLC-gamma(1) activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)), since increasing concentrations of PIP(2) reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-gamma(1) activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP(2). In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-gamma(1) and activates PLC-gamma(1) catalytic activity. Villin regulates PLC-gamma(1) activity by modifying its own ability to bind PIP(2). This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-gamma(1) by villin.