Proteomic Analysis of Trypanosoma cruzi Secretome: Characterization of Two Populations of Extracellular Vesicles and Soluble Proteins

Abstract

Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells.

Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells

“…Several members of this gene family were mainly found in the membrane bound fractions of YuYu-EVs, while most of them were found in the soluble fractions of Y-EVs. Those data are in agreement with previous secretome analysis [37], showing the presence of soluble and membrane bound TS from T. cruzi trypomastigotes [13,38]. …”

“…It is also possible that these differential host cell–parasite interactions induced by trypomastigote-derived vesicles could be determined by distinct EV subpopulations (i.e. exosomes and ectosomes), containing very diverse composition of virulence factors and other host-cell modulators, as previously described for metacyclic trypomastigotes [13]. Future expression and functional proteomics of different EV subpopulations could shed some light on these disparate modulatory activities of trypomastigotes vesicles on various host cells types.…”

“…The ensuing supernatant contained soluble, secreted proteins released by the parasite, and the pellet total EVs. For proteomic analysis, pooled preparations of purified total EVs or VF fractions were subjected to (LC-MS/MS), as previously described [10,13], with some modifications (see below). The experimental workflow including the purification and characterization of EVs and VF fraction is depicted in Figure 1.

10.1080/20013078.2018.1463779-F0001Figure 1.Workflow employed for the production, fractionation, and characterization of Trypanosoma cruzi EVs and vesicle-free fraction from Y and YuYu strains.

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The languages of parasite communication

“…This parasite releases membrane-bound vesicles by at least two different mechanisms [38]. Larger vesicles (ectosomes) bud from the plasma membrane, while smaller vesicles (exosomes) are found in the flagellar pocket.…”

“…Larger vesicles (ectosomes) bud from the plasma membrane, while smaller vesicles (exosomes) are found in the flagellar pocket. Vesicles purified from epimastigotes, the proliferative forms in the insect vector, and metacyclics, which are the infective forms for mammals, showed differences in both their protein and small RNA contents, suggesting that they have specialised functions [38,39]. Pretreatment of mice with parasite-derived vesicles can alter the outcome of infection, resulting in an increased amastigote burden and an intensified inflammatory response in the heart (the latter being manifested by increased levels of IL4 and IL10 mRNA and infiltration by macrophages and CD4 + lymphocytes).…”