Comparative genomics and molecular characterization of N-alpha acetyltransferase in trypanosomes for drug target identification

Abstract

Trypanosoma cruzi and Trypanosoma brucei are flagellated protozoan parasites that cause Chagas disease and African trypanosomiasis in Latin American and African countries, respectively. Currently, over 8 million people are infected with T. cruzi and about 25 million more are at risk. About half a million people are affected by T. brucei. Trypanosome species share many peculiar biological and biochemical features, such as RNA editing. In contrast, they exhibit profound differences at the level of host-parasite interaction and disease pathology. Both parasites are transmitted to their host via different insect vectors. There are no available vaccines, and the current treatments have severe adverse effects. We were involved in sequencing the T. cruzi genome, an initiative launched by the WHO to increase our knowledge of the molecular basis of the parasite.

The aim of this thesis was to participate in the sequencing and analysis of the T. cruzi genome, and use the data to investigate acetyltransferase enzymes, presumably linked to important metabolic pathways, as possible drug targets. In Papers I and II, we describe genome sequencing and analysis of two distinct T. cruzi strains. One of the selected strains, CL Brener, was found to be a genetic hybrid of two divergent strains; and it contains about 22 000 genes, encoded on 700 scaffolds with a total genome size of 110 Mb. About 50% of the genes are of unknown function, and lack homology to other sequenced eukaryotes. Large numbers of members of surface molecule gene families, such as trans-sialidase, mucin, mucin-associated protein, and GP63 were found. Comparative analyses revealed that TcI had a smaller genome by up to about 11 Mb. The genome size difference was linked to genes encoding surface molecules and to other repeats and repeated genes. Additionally, six reading frames present in TcVI were not detected in TcI. Genetic polymorphisms such as, indels, microsatellites and SNPs were identified and analyzed. Many genes were found to be under different selective pressures in T. cruzi, indicating differential evolutionary rates, signifying their importance to parasite biology. Within syntenic regions, the two genomes have the same gene complement. Identified features warrant sequencing of further T. cruzi strains, and findings from our studies offer opportunities for more targeted functional studies as well as tools for epidemiology.

In the second part of this thesis, Papers III to V, a Trypanosoma cruzi acetyltransferase gene family, identified in the genome project, was chosen for functional characterization as a first step to evaluate its potential as drug target.

Acetyltransferases are responsible for protein acetylation, where an acetyl molecule is transferred from acetyl-Coenzyme A to lysine residues in a protein sequence, N-epsilon acetylation, and to N-termini of proteins or stability, protein-protein interaction, localization to organelles and acts as degradation signals. The impact of this post-translational modification in parasite is not known.

We have identified T. cruzi NatC and A, and show that they are expressed in the three life cycle stages (epimastigote, trypomastigote, and amastigote). The catalytic and auxiliary subunits form a complex in vivo. Additionally, they partially co-sediment with the ribosome and may have both co-translational and post-translational protein acetylation functions. In epimastigote, the catalytic subunit of T. cruzi NatA was localized both in the nuclear periphery and in cytoplasm, whereas NatC was predominantly assigned to the cytoplasm. The auxiliary subunit of NatA was mainly confined to the cytoplasm with cytoskeletal-like labelling, whereas NatC showed a punctate profile. Interestingly, the staining patterns of the different subunits analysed for NatA and NatC differ between the life cycle stages, which suggests differential regulation and expression.

The native substrates for NatC and predicted NatA, are similar to those described in yeast and humans,suggesting evolutionary conserved functions. The proteins appear to acetylate a large number of proteins Nterminally, suggesting that manipulation of the enzymes may simultaneously affect many cellular functions and thereby could interfere with or abolish infection. Additionally, our data indicate that NatC and A, may have both Nalpha and N-epsilon acetylation potential.

Collectively, the genome analyses presented here have provided more molecular insights into the parasite's biology, and have narrowed the gaps between scientific communities working on parasite research. The identification of Nats and native substrates has hopefully laid a solid foundation for future study of Nats, which could provide chemo- therapeutic targets for parasitic diseases.