Trypanosoma cruzi, the causative agent of the Chagas disease, has a complex life cycle alternating between replicative and noninfective forms with nonreplicative and infective forms of the parasite. Metacyclogenesis is a process that takes place in the invertebrate host, comprising morphogenetic transformation from a noninfective form to an infective form, such that parasites acquire the ability to invade human cells. We analyze here the metacyclogenesis process by 2D electrophoresis coupled to MALDI-TOF MS. A large proportion of unique proteins expressed during metacyclogenesis were observed. Interestingly, 50% of the spots were found to differ between epimastigotes and trypomastigotes. We provide a 2D map of the infective metacyclic trypomastigotes. Sixty six protein spots were successfully identified corresponding to 43 different proteins. We analyzed the expression profiles for the identified proteins along metacyclogenesis and classified them into three groups according to their maximal level of expression. We detected several isoforms for a number of proteins, some displaying differential expression during metacyclogenesis. These results suggest that posttranslational modifications may be a fundamental part of the parasite's strategy for regulating gene expression during differentiation. This study contributes to the identification of relevant proteins involved in the metacyclogenesis process. The identification and molecular characterization of these proteins will render vital information about the steps of the parasite differentiation into the infective form.

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In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity.

The genus Phytomonas comprises trypanosomatids that can parasitize a broad range of plant species. These flagellates can cause diseases in some plant families with a wide geographic distribution, which can result in great economic losses. We have demonstrated previously that Phytomonas serpens 15T, a tomato trypanosomatid, shares antigens with Trypanosoma cruzi, the agent of human Chagas disease. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to identify proteins of P. serpens 15T that are recognized by sera from patients with Chagas disease. After 2D-electrophoresis of whole-cell lysates, 31 peptides were selected and analyzed by tandem mass spectrometry. Twentyeight polypeptides were identified, resulting in 22 different putative proteins. The identified proteins were classified into 8 groups according to biological process, most of which were clustered into a cellular metabolic process category. These results generated a collection of proteins that can provide a starting point to obtain insights into antigenic cross reactivity among trypanosomatids and to explore P. serpens antigens as candidates for vaccine and immunologic diagnosis studies.