Genome Sequencing and Analysis
Genome sequences represent an invaluable resource for scientists working to understand the biology of various pathogens. The analysis of genome contents undoubtedly constitute a first important step toward the development of better drugs, vaccines and methods for pathogen control. Such projects have been an important activity of my laboratory since 1998. Using the latest sequencing and bioinformatics tools, we have decoded and annotated large portions of the African and American trypanosome genomes, causative agents of African sleeping sickness and Chagas disease, respectively. Both of these flagellated protozoan parasites are deadly pathogens, invariably fatal to infected humans (and domestic animals). They possess several highly unusual molecular mechanisms including trans-splicing of polycistronic transcripts, editing of mitochondrial RNA transcripts, and apparent lack of RNA polymerase II promoters for most protein-encoding genes. They also possess complex mechanisms for antigenic variation, and unique organelles such as the kinetoplast and the glycosome. We have also recently completed the sequencing of the Schistosoma mansoni and Perkinsus marinus genomes. S. mansoni is a trematode parasite that causes schistosomiasis (Bilharzia), a highly prevalent tropical disease and leading cause of severe morbidity in many developing countries. P. marinus is a facultative intracellular parasite of the eastern oyster.

Comparative Genomics of Human Pathogens
A special area of interest within my group is the comparative genomics of pathogens with an emphasis on the study of the evolution of genome architecture and content in pathogens, as well as the identification of genes that are phylogenetically restricted or that have been transferred horizontally. Our recent investigations have focused on three trypanosomatid parasites. The three pathogens (Trypanosoma brucei, Trypanosoma cruzi and Leishmania major) are distantly related but have distinctive life cycles, different insect vectors, and, perhaps most importantly, different disease characteristics.
Despite high levels of divergence at the sequence level, the three species exhibit a striking conservation of gene order, suggesting that selection has maintained gene order among the trypanosomatids over hundreds of millions of years of evolution. Computation of clusters of orthology allows us to i) examine global synteny as well as the selective and mutational forces that act on chromosomal and genome structure, ii) dissect insertion/deletion events, gene duplication between species, and comparative chromosomal architecture for better insights into the evolutionary relationship of the trypanosomatids, and iii) define the ‘core’ proteome of a trypanosomatid and during that process, identify genes that appear to be restricted to each of the species. Such gene products could provide targets for drug design as well as vaccine candidates.
Our current comparative efforts encompass a variety of pathogenic and non-pathogenic trypanosomatid strains for which sequencing projects are underway. Comparative analyses of these genomes will not only reveal great insights into the evolution of trypanosomatids, but will allow us to identify subsets of genes specifically linked to virulence and survival mechanisms.

Functional Proteomics - Elucidation of kinetoplastid interactomes and host-pathogen protein-protein interactions
We have initiated a large-scale project aimed at investigating protein-protein interactions (PPIs) in trypanosomatids using the yeast two-hybrid (Y2H) methodology. In order to validate a variety of platforms for high-throughput testing of PPIs in kinetoplastids, we carried out a comprehensive evaluation of the physical interactions between subunits of Trypanosoma brucei proteasome 26S. As this project expands to characterize the full interactomes, we expect it to result in a significant improvement of the annotation of predicted proteins (particularly hypothetical proteins) and the identification of kinetoplastid-specific hubs of interacting proteins that can be exploited as drug targets. We are also making progress towards investigating the trypanosomatid ‘infectome’, by examining the interactions selected subset of T. brucei, T. cruzi and L. major proteins against the human ORFeome.

Functional Genomics - Characterization of a novel surface protein family (MASP) in Trypanosoma cruzi
Comparison of the gene content of the three parasites (El-Sayed et al., 2005) revealed a conserved core of ~6200 genes. Many of the species-specific genes encode for large families of surface proteins. In T. cruzi, those include trans-sialidases, mucins and a novel large gene family that we named mucin-associated surface protein or MASP. The MASP family contains 1377 members corresponding to ~6% of the T.cruzi diploid genome and is characterized by conserved N- and C-terminal domains and a central highly variable region. Despite the large size of the MASP family in T.cruzi and its likely location on the parasite surface, it has remained undetected. We are using combination of approaches to functionally characterize the MASP family. Our specific goals are as follows: 1) Investigate the expression profile of MASP throughout the T.cruzi life cycle and in distinct host cells; 2) Analyze the cellular location of MASP; 3) Investigate a potential role for MASP in host cell attachment and invasion; 4) Test by ELISA whether MASP is recognized by sera from chagasic patients; and 5) Investigate the mechanisms of control of MASP expression throughout the parasite’s life cycle. This work is being done in close collaboration with Dr. Daniella Bartholomeu at the Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

The Human Microbiome and Disease