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.

BackgroundThe Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites.Methodology/Principal FindingsThe genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (José-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively.ConclusionsDespite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.

BackgroundLysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway.Methodology/Principal FindingsHEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells.Conclusions/SignificanceThe above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.

The cell surface of Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These molecules are involved in a variety of interactions between this parasite and its mammalian and insect hosts. Here, using the neutral detergent Triton X-114, we obtained fractions rich in GPI-anchored and other membrane proteins from insect developmental stages of T. cruzi. These fractions were analyzed by twodimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS), resulting in the identification of 98 proteins of metacyclic trypomastigotes and 280 of epimastigotes. Of those, approximately 65% (n=245) had predicted lipid posttranslational modification sites (i.e., GPI-anchor, myristoylation, or prenylation), signal-anchor sequence, or transmembrane domains that could explain their solubility in detergent solution. The identification of some of these modified proteins was also validated by immunoblotting. We also present evidence that, in contrast to the noninfective proliferative epimastigote forms, the infective nonproliferative metacyclic trypomastigote forms express a large repertoire of surface glycoproteins, such as GP90 and GP82, which are involved in adhesion and invasion of host cells. Taken together, our results unequivocally show stage-specific protein profiles that appear to be related to the biology of each T. cruzi insectderived developmental form.