Functional genomic analysis of the Trypanosoma cruzi submitted the different types of stress.
The differentiation of epimastigotes into metacyclic trypomastigotes (metacyclogenesis) involves the transformation of a replicative, non-infectious form of T. cruzi into a non-replicative, infectious stage. An essential event in metacyclogenesis that can be mimicked in vitro, is the nutritional stress faced by the parasites at the mid-gut of the insect host. Important changes in the gene expression program occur during this process and it is likely that they play an important role in the physiological and morphological changes observed during the metacyclogenesis. However, little information is available concerning the ability of different stress conditions in triggering metacyclogenesis and also, about the genes involved in the regulation of these responses. In this work, it was performed a comparative analysis of different stress conditions (temperature increase, pH decrease and nutritional stress) in triggering metacyclogenesis in vitro. It was observed that parasites subjected to the three stress conditions were able to differentiate. However, the parasites that underwent pH and temperature stresses showed a delay in the onset of metacyclogenesis, but this difference was minimized by the end of the process. To compare the biological properties of metacyclic trypomastigotes obtained from the three sources, infection assays were carried on with Vero cells and Swiss mice. All trypomastigotes were able to infect Vero cells, as well as infect mice. To investigate the gene expression profiles of the parasites under the stress conditions microarray technology has been used. A set of 32 genes was selected that are specifically expressed under stress conditions. The microarray data for the 32 genes selected were confirmed by quantitative real-time PCR (qPCR) showing a concordance of results of more than 90%. Due to the fact that the selected genes have in general showed a similar response in different tresses studied, a specific microarray analysis was performed to select genes that are expressed significantly different among the stresses. Concomitantly, shotgun proteome LC-MS/MS analysis have been carried out in order to gain further insight into the relative abundance of T. cruzi proteins when the different stress conditions were established. After data analysis, 173 proteins were selected as differentially expressed under the stress conditions when compared to unstressed epimastigote form. Proteomic data have shown a more complex profile (of differential expression) than that observed from the microarray data. All these data together allowed us to infer the potential importance of post-translational regulation of gene expression in triggering epimastigote differentiation to the infective forms.
Advisor:Marco Aurélio Krieger
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Trypanosoma cruzi Proteômica Proteomics Heat-Shock Proteins In Vitro
Date of Publication:04/17/2008