Characterization of Trypanosoma cruzi Microvesicles as Potential Chemotherapeutic Targets

Chagas disease (CD), a major cause of heart failure in the Americas, is caused by Trypanosoma cruzi. There are few chemotherapeutic options to prevent or treat chronic CD, which can be sustained for decades. One possible contributing factor to long-term CD infection is the microvesicles secreted by the infective T. cruzi trypomastigote forms of the parasites (TcVes). It has been hypothesized that TcVes contain immunomodulatory proteins that allow the parasites to evade the host immune response as well as facilitate host-cell infection. To analyze the TcVes proteome, parasites were allowed to shed microvesicles in various media (i.e., DMEM-FBS, DMEM-BSA, DMEM-Glc, and DMEM-Glc/BSA) at different time points over a 24h period. TcVes were harvested by differential ultracentrifugation and analyzed by SDS-PAGE/silver stain gels and Western blotting, as well as by liquid chromatography-tandem mass spectrometry (LC-MS/MS). By nanoparticle tracking analysis, the mean diameter of TcVes obtained under different experimental conditions varied from 137 to 227 nm. By SDS-PAGE/silver stain, we observed a similar array of proteins with increasing concentrations for up to 8 hours shedding time in DMEM-Glc, which correlates to approximately 95% viable parasites. Furthermore, it was evident that DMEM-Glc, but not -FBS or -BSA, is the most appropriate medium for proteomic analysis of TcVes due to the absence of contaminant non-T. cruzi proteins. The protein composition of the microvesicles is currently being assessed by LC-MS/MS.  We anticipate the identification of several immunomodulatory proteins in trypomastigote microvesicles. Therefore, chemotherapies that target TcVes may be a viable option to combat Chagas disease.