Caracterização de genes de Trypanosoma cruzi envolvidos na via de biossíntese de glicosilfosfatidilinositol

Abstract

Glycosylphosphatidylinositol (GPI) is the predominant structure that Trypanosoma cruzi uses to anchor proteins on its surface, many of them essential for the virulence of the parasite or its escape from the host immune response. Furthermore, T. cruzi GPI anchors exert immunostimulatory and regulatory activities, including the induction of pro-inflammatory cytokines synthesis by host macrophages. Therefore, genes encoding enzymes and other factors involved in the T. cruzi GPI biosynthetic pathway are potentially attractive targets for the development of new drugs against the disease caused by parasite, known as Chagas disease. The aim of this project was to investigate the T. cruzi GPI biosynthetic pathway, through biochemical and functional characterization of genes encoding components of this pathway. Thus, DNA sequences obtained from T. cruzi genome database were analyzed and their gene products evaluated by (i) heterologous complementation in conditional lethal mutants of Saccharomyces cerevisiae, (ii) gene expression assays as well as cellular localization in the parasite, and (iii) analysis of the phenotype of knockout parasites for some of these genes. In silico analyses of sequences available in the T. cruzi genome database (TriTrypDB) resulted in the identification of 18 genes encoding GPI biosynthesis proteins as well as inositol phosphorylceramide synthase gene (IPCS). T. cruzi sequences corresponding to TcDPM1, TcGPI3, and TcGPI12 genes, expressed in epimastigotes as GFP-fusion proteins, showed a cellular localization compatible with the endoplasmic reticulum. Analyses of RNA levels of TcGPI8 and TcGPI10 showed increased expression in epimastigotes and amastigotes, the two proliferative stages of the parasite. Yeasts defective in various genes of the GPI biosynthetic pathway, transformed with the respective T. cruzi genes were analyzed. We showed that TcDPM1, TcGPI10, and TcGPI12 genes were able to restore the growth of yeast mutants in non-permissive conditions, indicating that these proteins are homologous to yeast proteins. Recombinant proteins were also obtained in bacteria with the objective of developing studies about their structures. To investigate the role of GPI anchored proteins of T. cruzi, we disrupt the TcGPI8 gene, which encodes the complex catalytic subunit that catalyzes the last step of the pathway. Although we were able to generate parasites presenting one TcGPI8 allele deleted, in which TcGPI8 mRNA levels were reduced, several attempts to delete the second TcGPI8 allele resulted in parasites with the insertion of drugs resistance genes in the two alleles, but showing genome rearrangements and, consequently, still expressing the TcGPI8 mRNA. The characterization of these TcGPI8 mutant parasites showed that, although they present only minor changes in the glycocalyx composition, the effects of these changes on their infective capacity were quite evident. Therefore, besides deepening the knowledge about the components of the GPI anchors biosynthesis, which appears to be essential for the parasite survival, this work resulted in the generation of yeast mutants complemented with T. cruzi orthologous genes, which now became valuable tools to be used in high throughput screening assays that may led to the discovery of new drugs for the treatment of Chagas disease.