Project Summary This application focuses on the protozoan parasite Trypanosoma brucei, which is transmitted among mammalian hosts by tsetse-flies and, due to effective immune evasion, causes chronic and lethal infections, specifically African sleeping-sickness in humans and nagana in cattle. Whereas human African trypanosomiasis has been declining recently, livestock infections remain prevalent and have a profound effect on economic development in sub-Saharan Africa. There are no vaccines and therapeutic drugs have serious side effects and decreasing efficacy. Thus, there is a pressing need for research to better understand the biology of these pathogens and the mechanisms they use to survive within their hosts. T. brucei undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. To cope with the changing environment, the parasite transitions through distinct life cycle forms that have evolved to assure survival and successful transmission to the next host. For instance, when residing in the mammalian host, T. brucei expresses a variant surface glycoprotein (VSG) coat, which is the paradigm for antigenic variation. Although there are hundreds of VSG genes in the genome, bloodstream-form VSG expression is restricted to 1 of about 15 specialized telomeric bloodstream expression sites (BES). In the salivary glands of the insect vector metacyclic trypanosomes are covered by a specific and small subset of VSGs, the metacyclic VSGs (mVSGs), which enable transmission to a vertebrate host. mVSG expression is triggered by an unknown mechanism and in vitro can be achieved in the absence of tsetse tissues by induced expression of the RNA-binding protein 6 (RBP6). One major goal of this application will be to examine how trypanosomes receive instructions to begin synthesizing the mVSG coat, and when each cell begins expressing a single mVSG. We will use single-cell RNA-Seq and long-term live-cell imaging to answer mechanistic questions. In addition, our novel finding that mVSG genes encode an exonic splicing enhancer exposed a completely new aspect of VSG regulation and may explain the tightly-controlled extremely high output of mature mRNAs.