The protistan parasite Trypanosoma brucei is transmitted by the tsetse vector, lives freely in the bloodstream and causes the lethal disease African Sleeping Sickness in humans and the similar disease Nagana in various livestock. There are only four drugs for this disease, and the most effective drug, melarsoprol, is highly toxic and parasite resistance to this drug is on the rise. Thus, it becomes increasingly important to find new anti-parasitic targets and develop new therapeutic strategies. The parasite's interface to its hosts is a dense glycoprotein coat on the cell surface which consists of a single type of variant surface glycoprotein (VSG) in the form parasitizing the mammalian bloodstream and of procyclin in the form multiplying in the fly midgut. The coat protects the parasite from lytic host components and antigenic variation of the VSG coat is the parasite's means to evade the immune response. The complete VSG coat is expressed with extreme efficiency from a single VSG gene. This expression level is crucial for the parasite because silencing VSG expression leads to rapid cessation of parasite growth in culture and effective clearance of trypanosomes from infected mice. T. brucei has evolved a unique multifunctional RNA polymerase (pol) I system to effectively transcribe both VSG and Procyclin genes (class I transcription). In other eukaryotes, this efficient enzyme transcribes exclusively the large ribosomal gene unit whereas in T. brucei, RNA pol I is recruited to four structurally different promoters, involved in development-dependent transcriptional regulation, and sequestered into two distinct subnuclear compartments. Hence, it is most likely that this unprecedented versatility requires essential proteins, protein domains and protein-protein interactions that are unique to the parasite and absent in the hosts. As parasite-specific features, we have thus far characterized the novel, multi-subunit transcription factor CITFA that is indispensable for class I transcription and for RNA pol I, the novel and essential subunit RPA31, an unusual N-terminal extension domain in subunit RPA2, and a variant set of the common subunits RPB5, RPB6 and RPB10 that is specific to RNA pol I. As a prerequisite for small molecule inhibition studies, we will proceed to functionally characterize these unique proteins and analyze their protein-protein interactions. In addition, we will exploit our newly developed protein purification technology to identify new class I transcription factors. Public Health Statement The proposed studies of this application will explore transcription factors that are indispensable for the lethal parasite Trypanosoma brucei to express its major cell surface antigens and to survive in its human and vector hosts. Accordingly, these factors are essential for parasite growth. Since some of them appear to be specific to the parasite, they are potential new targets for chemotherapy. Such targets are urgently needed be- cause drugs to cure a T. brucei infection are few and toxic and parasite resistance to these drugs is on the rise.
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