On the one hand, apart from the nine proteins specifically involved in vesicle transport and trafficking, we identified 13 out of the 22 most common exosome proteins. On the see more other hand, we found very few proteins from intracellular compartments (1% of the secretome). The viability of Trypanosoma tested with flow cytofluorometric analysis and microscopic analysis CB-839 order suggests that the nonspecific release of material from lysed cells is modest and the yield of secreted proteins is not correlated to viability but is strain-specific (for example, Biyamina produced

4 times less secreted proteins than other strains). Moreover, the comparison between the total proteome and the secretome showed in this study

also suggests that contaminations from nonspecific release would be relevant only if the kinetics of release is highly protein-specific. In addition, ubiquitin seems to play a key role in the sorting of proteins into exosomes [70], and we identified ubiquitin and 25 related proteins of the ubiquitin/proteasome pathway. Thus, the overall picture of the Trypanosoma secretome shows homologies with exocytosis occurring in the flagellar pocket Stattic chemical structure and with exosome-related proteomes. Interestingly, we have successfully demonstrated for the first time the presence of vesicles at the trypanosome surface using electron microscopy and further shown that similar vesicles are present in the secretion medium. Moreover, proteomic analysis of TIRSP confirmed the presence of a set of proteins that is very similar (71%, 46/65) to ESPs purified from isolated parasites. Thus, both approaches converge to strengthen the hypothesis of a new secretion pathway in Trypanosoma. Indeed, the size of the vesicle-like structure observed on electronic microscopy pictures fits with microvesicles

(50-100 nm). This situation seems to be shared with Leishmania, a close relative of Trypanosoma, where the absence of transit peptides in secreted proteins and the presence of microvesicles at the promastigote surface were recently demonstrated [20]. This differs from the case of P. falciparum, where a specific host-targeting motif was described for secreted proteins [71]. This could be hypothesized to present several advantages for Trypanosoma, in comparison to the Erastin cell line classical secretory pathway: it may deliver an avalanche of new epitopes to overwhelm the host immune system or to communicate between trypanosomes themselves by exchanging receptors in the form of non-protein cytosolic compounds or even potentially genomic information. As such, microvesicles could be a flexible way for Trypanosoma to reversibly adapt its machinery and to homogenize the survival strategy at the population level. Conclusions This study provides the first overview of proteins secreted by Trypanosoma brucei.