Abstract
BackgroundThe unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.Methodology/Principal FindingsViable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.Conclusions/SignificanceHerein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.
Highlights
The identification of molecules involved in the multiple interactions of Trypanosoma cruzi with host cells, as well as their resulting signaling pathways, is fundamental to the understanding of the mammalian infection by the parasite
Inhibitor Cocktail, anti-mouse-IgG antibodies conjugated to Peroxidase, anti-phospho-Serine and anti- a-tubulin antibodies were purchased from SIGMA-ALDRICH; human plasma fibronectin from Gibco; DeStreak rehydration solution from GE Healthcare Life Science; PRO-Q Diamond Phosphoprotein Gel Stain, anti-phospho-Threonine and anti-phospho-Tyrosine antibodies from Invitrogen; and anti-phospho ERK antibody from Cell Signaling
Differences in the distribution of phosphorylated proteins in trypomastigotes incubated with fibronectin or laminin were detected by immunofluorescence, as compared to parasites incubated with Bovine serum albumin (BSA), when a mixture containing anti-pS, -pT and -pY antibodies was employed (Fig. 1)
Summary
The identification of molecules involved in the multiple interactions of Trypanosoma cruzi with host cells, as well as their resulting signaling pathways, is fundamental to the understanding of the mammalian infection by the parasite. The parasites are released in the circulation upon rupture of the host cells and may invade new cells or be ingested by the insect vector where they differentiate into epimastigotes [1]. During this complex life cycle, the parasite is exposed to different environments and has to respond adequately in order to survive. Members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored
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