Cloning of Trypanosoma cruzi Ciclophilin A gene in prokaryotic and eukaryotic cells

POTENZA, MARIANA., BUA, J. AND RUIZ

San Diego, Estados Unidos

Congreso; Current Topics in Gene Expression and Proteomics Meeting; 2003

Invitrogen Corporation

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas’ disease. It is difficult to control parasite transmission in certain remote rural areas, thus the need of therapeutic drugs is fundamental. The currently used trypanocidal drugs are very effective on the parasite but have limiting side effects in humans. A better knowledge of the biological and biochemical features of the parasite is necessary for the discovery of new drug targets. Cyclosporin A (CsA) and its non-immunosuppressive derivatives have shown anti-parasitic activity against Trypanosoma cruzi (Potenza, M. et al. Biocell, 26, 2002), though its mechanism of action is not known. Cyclophilins (CyPs), the target molecules for CsA, are involved in protein folding events. The immunosuppressive effects of CsA in humans are mediated by the complex CyP-CsA, that binds a calcineurine molecule, which inhibits the proliferation of T cells. In some parasites, the CyP-CsA complex binds a Glycoprotein P. We have identified and characterized seven genes that code for cyclophilins in T. cruzi. We have cloned, expressed and purified three of these seven genes using differents combinations of expression vectors and bacterial strains in order to reach high performance in recombinant protein production. We have tested these recombinant proteins for appropiate folding and enzymatic activity. The most abundantly expressed T. cruzi cyclophilin gene codes for TcCyP19. Recombinant TcCyP19 has shown an enzymatic activity that could be inhibited by CsA and its derivatives. We intend to analyse the proteins that bind TcCyP19-CsA complex to elucidate the way CsA exerts its anti-parasitic activity. For fulfilling the study of these protein complexes we cloned TcCyP19 in a multi affinity tag vector pAff2c (Nilsson,J. Jour. Mol. Rec. 9, 1996, 585-594), which facilitates protein purification. This cassette with poly-histidine tag, biotin and BSA binding peptide from pAff2c was succefully inserted into pRibotex vector (Martínez,S. Gene199, 1997, 71-76) for transfection into the parasite. Once we get stable transfectants, parasites will be lysed and the functional organisation of the complex components will be identified and analysed using proteomic tools. This protein component analysis will provide deeper understanding of the role of CyPs in the parasite and the mechanism of action of CsA against Trypanosoma cruzi. The results obtained from this studies, in addition to the evaluation of the non-immunosuppressive derivatives effects on the parasite, may be useful for the development of possible new drugs against  Chagas’ disease.