Mapping the genome and characterization of an acetyltransferase of Trypanosoma cruzi

Abstract: Trypanosoma cruzi (T.cruzi) is a flagellate protozoan parasite that causes Chagas' disease and is most common in Latin American countries. The disease currently infects over 13 million persons and about 100 million more are at risk. Despite basic research for many years, there is neither a vaccine nor useful drugs available to combat the disease. We were involved in sequencing the genome of this parasite, launched by WHO in order to increase knowledge on the molecular biology of the parasite and disease pathology. In the first part of this thesis, we tried to elucidate the molecular karyotype (actual numbers and sizes of chromosomes in a cell) of T.cruzi, which was an essential part of the genome project, newly published in Science together with two other kinetoplastid genomes. For the karyotyping study, three strains were used. CL-Brener (lineage IIe), chosen as a reference strain in the genome project and two T. cruzi lineage 1 strains, Sylvio X 10/7 and CAI/72 for comparative purposes. Since the T cruzi chromosomes do not condense during cell division, we separated the intact chromosomes by PFGE using three different running conditions. We hybridized 239 cDNA markers (probes) to the PFGE separated chromosomes. Using densitometry analysis- the total number of chromosomes for CL-Brener was estimated to be 55 and 57 in the two other strains. Size differences between homologous chromosomes, especially for CL-Brener are very high, up to 173%, and may be due to chromosome rearrangements. Forty markers distributed into 15 linkage groups, were found to identify specific chromosomes or chromosome pairs, hence showing conservation of gene order (synteny) between strains. This data provides valuable information for the finishing of the CL- Brener genome sequence. In the second part of this thesis, a Trypanosoma cruzi acetyltransferase (TcAT) protein that was annotated in our laboratory during the genome sequencing was characterized as a first step to evaluate its future potential as a drug target. Acetyltransferases (ATs) in general are enzymes that transfer acetyl-Coenzyme A (AcCo A) to the Nterminal of a protein or a peptide. In principle, acetylation is a posttranslational modification present in a majority of eukaryotes, altering the properties of proteins in different ways. The consequences of acetylation include effects on protein stability, protein-protein interaction and DNA binding. Our analysis of TcAT shows that the gene is single copy. The TcAT motifs match the GCN5-related acetyltransferase (GNAT) family. Orthologous proteins are present in the other two kinetoplastids (Trypanosoma brucei and Leishmania). The protein appears to be more closely related to N-terminal acetyltransferases (ATs) than to histone acetyltransferases (HATs). The native protein has a cytosolic location. It is expressed in the three different life-cycle stages of the parasite, determined by western blot. The recombinant protein has an autoacetylation activity. The 3D topology model prediction for TcAT is typical of the GNAT super family of ATs. The N-terminal of the human ortholog (HYPO-HUMAN) is elongated and there are three extra alpha helices predicted. Interestingly, kinetoplastids also have three extra helices but not in the same positions as in human. Some of the predicted functional sites where Acetyl CoA binds, and ligand binding sites were conserved in Kinetoplastids compared to other organisms including human. Proteins that interact with TcAT, native substrate specificity and metabolic pathway are not yet known. Despite that fact, the predicted protein structures of Kinetoplastids, amino acid, functional and ligand binding sites seem to differ from that of human counterpart and could be exploited and used in drug design against Trypanosomes and Leishmania.