THE INTRASPECIFIC GENETIC DIVERSITY OF TRYPANOSOMA CRUZI CELL SURFACE ANTIGENS. USING A NOVEL PCR APPROACH

Abstract

Trypanosoma cruzi the causative agent of Chagas disease has a vast collection of surface antigen genesclassified into families. These families are mucins, transialidases and MASPS which constitute half of theparasites genome. The members of these families are characterized by conserved sequence regions at their 5'and 3' ends that do encode a signal peptide and GPI anchor sequence. Their central regions constitutehypervariable regions some of which are marked with repeat sequences. A simple PCR technique that wouldform the basis to a pilot study for capturing the intragenomic diversity of the T.cruzi surface antigen genes isneeded as a step towards more advanced techniques.Degenerate primers for targeting mucins, trans-sialidaseand MASP genes were designed from conserved nucleotide sequences at the 5' and 3' regions of the genes andused in highly stringent PCR reactions to amplify a whole library of surface antigen genes. Purified PCRproducts were cloned into pGEM-T Easy vectors sequenced by Sanger methodology. Generated sequenceswere aligned against surface antigen gene sequences from the CL-Brener genome accessed through tritrypDBdatabase. Sequence analysis of the nucleotide sequence from PCR products were phylogenetically analysed todetermine their diversity. Degenerate mucin primers were able to produce PCR products from genomic DNA ofT. cruzi from all DTUs some of which were confirmed to mucins. The generated sequences were diverse fromeach other with half of the sequences showing similarities to a cosmid C71. A non-surface antigen gene,histone deacetylase, was also discovered and found to share similarity with 7 of the generated sequences.Exploration of traditional southern, western and northern blots to microarray and next generation sequencingtechniques for study of parasite surface antigen are recommended.