African Trypanosome Genes Specifically Required for Fitness in vivo
This report details the initial validation of hits from a genome-scale RNAi fitness screen to reveal genes that showed a specific loss of fitness in vivo. Analyses of African trypanosome biology are often carried out in HMI culture medium which is rich in nutrients and may not necessarily be representative of the environments encountered in vivo. We reasoned that parasites in vivo are likely to display increased dependence upon certain nutritional pathways as well as defences against oxidative stress and innate immune attack. Prior to this project, the lab ran a genome-scale RNAi fitness-profiling screen in rats in order to address this question. Analysis of the resulting list of genes that displayed a specific loss-of-fitness in vivo showed two genes linked to phosphatidylinositol metabolism, providing validation for the screen. Other hits included genes linked to folate metabolism and to DNA repair. This report details the initial analysis and validation of the hits from the screen and the development of systems for more detailed follow-up. One particularly promising approach described here is the growth of T. brucei in a simple serum system, which we believe effectively mimics aspects of the in vivo environment. Two genes were successfully validated, one of which has since been independently confirmed in the literature, and initial investigations into the underlying biology behind their phenotypes are described. The appendices to this report provide further detail regarding the hits from the in vivo screen, as well as describing two other projects which I worked on during my time in David Horn’s laboratory.
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