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 are currently sequencing the genomes of the Schistosoma mansoni and Perkinsus marinus. 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
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. My 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 is allowing 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.
While my future comparative efforts will not be limited to trypanosomatids, I will continue my research in this area. There are multiple Trypanosoma sequencing projects underway, including strains that are non-pathogenic to humans or animals all together. Comparative analyses of these genomes will not only reveal great insights into the evolution of trypanosomatids, but may also allow us to identify subsets of genes specifically linked to different pathogeneses.

Functional Genomics
1) Assessment of gene function – RNA interference
Investigation of gene function is rapidly developing into a new focus of investigation within my group. In the past two years, we have established a RNA interference knockdown system in T. brucei and created several RNAi mutant cell lines for genes of interest, including a putative DICER. My future goals in this area include the design of a high throughput application of RNAi to assess gene function in T. brucei. The preliminary results generated from our current studies have provided the basis for a new grant application (R21) that was submitted to NIH on October 1, 2005, in which we are seeking support for the i) construction and validation of a partial T. brucei ORFeome RNAi library RNA interference (RNAi); and ii) use of genome-wide oligo microarrays as well as proteomic profiling to analyze the RNAi response in wild-type and RNAi-deficient cells. This work is being performed with Dr. Appolinaire Djikeng, senior staff scientist in my group.

2) Functional characterization of a novel surface protein family in the T. cruzi intracellular parasite – a more directed study
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 have begun to use a 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 using northern and western blot analyses; and characterize the repertoire of expressed MASPs by generating expressed sequence tags (ESTs) from multiple cDNA libraries; 2) Analyze the cellular location of MASP using several complementary approaches; 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.

3) Transcriptional analysis of gene expression networks in archeal methanogens and T. brucei
In a pilot study, we are investigating the utility of ORF and oligonucleotide microarrays for examining 1) the molecular events of life-cycle differentiation of African trypanosomes and 2) the underlying mechanisms of the sensitivity to human serum among strains of T. brucei using pairs of human-resistant and serum sensitive clones of T. b. rhodensiense. Microarrays containing cDNAs, genomic fragments, and most recently T. brucei chromosome II ORFs have been constructed and screened with RNA populations from various developmental stages of Trypanosoma brucei. In a first TIGR publication that includes microarray data, we reported the successful construction and use of 400-element microarray to study parasite differentiation. Our early studies and lobbying efforts have resulted in the placement of T. brucei arrays on the list of top priorities for production and distribution through TIGR’s PFGRC.
Through two different grants from the DOE, my group has also been responsible for the construction and validation of microarrays for the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum. We have completed two large-scale expression studies on these organisms under a variety of cell culture conditions (including temperature and pressure) in collaboration with Douglas Clark at University of California, Berkeley and John Reeve at Ohio State University. These studies are providing invaluable information of the genetic basis for encoding the metabolic capacity to synthesize de novo all of the building blocks essential for cellular life forms from inorganic constituents.