Trypanosoma brucei is the protozoan parasite responsible for Human African Trypanosomiasis (HAT), otherwise known as African Sleeping Sickness. There is an urgent need for new control strategies for this disease, as there is no effective vaccine. Many of the drugs used to treat HAT are toxic and ineffective. A complete understanding of the parasite's life cycle will potentially reveal novel targets for therapeutic intervention. The life cycle of the parasite requires growth and differentiation in both the tsetsfe fly and mammalian host. The ability of the parasites to adapt and replicate in each unique environment is dependent on the ability to regulate the presence of specific proteins throughout the life cycle. Our bioinformatic studies suggest that African trypanosomes contain a modified base called 5-methylcytosine, and this modified base is required for life cycle-dependent protein expression by repressing gene transcription. We will test this hypothesis using a combination of molecular, biochemical and genetic strategies. First, the subcellular localization and developmental regulation of 5-methylcytosine will be determined by probing purified nuclear and mitochondrial DNA from T. brucei parasites with an antibody specific to 5-methylcytosine. Next, we will identify the enzymatic machinery responsible for 5-methycytosine generation in T. brucei. The putative T. brucei cytosine-5 DNA methyltransferase will be produced and purified from E. coli. In vitro DNA methylation reactions will be used to determine if this protein truly has the ability to synthesize 5-methylcytosine, and determine the biochemical requirements for this reaction. Last, we will determine the relationship between 5-methlycytosine and transcriptional repression. Parasites lacking 5- methylcytosine will be constructed using RNA interference technology, and the effect on transcriptional silencing of target genes will be analyzed using northern blots. Our studies will potentially improve control of HAT. We predict that the 5-methylcytosine pathway is required for the parasite's ability to regulate surface antigen expression and evade the human immune system. Thus, drugs targeting this DNA modification will likely compromise parasite transmission or pathogenicity. 4.4 Item 7. Project Narrative There is an urgent need for new therapeutics to prevent and treat Human African Trypanosomiasis, as there is currently no effective vaccine and drug treatments are toxic and unreliable. Our studies of cytosine DNA methylation in Trypanosoma brucei may reveal the mechanism by which the parasites regulate expression of surface antigens and evade the human immune system. Drugs that target cytosine DNA methylation could possibly eliminate parasite immune system evasion or transmission. ? ? ?
|Militello, Kevin T; Chen, Leanne M; Ackerman, Sarah E et al. (2014) A map of 5-methylcytosine residues in Trypanosoma brucei tRNA revealed by sodium bisulfite sequencing. Mol Biochem Parasitol 193:122-6|
|Militello, Kevin T; Simon, Robert D; Qureshi, Mehr et al. (2012) Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli. FEMS Microbiol Lett 328:78-85|
|Militello, Kevin T; Wang, Ping; Jayakar, Sangeeta K et al. (2008) African trypanosomes contain 5-methylcytosine in nuclear DNA. Eukaryot Cell 7:2012-6|