Characterisation of uptake and sub-cellular transit of human serum trypanolytic factors by Trypanosoma brucei

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African trypanosomes are protozoan parasites that cause a range of important human and animal diseases. Human African Trypanosomaiasis (HAT) is typically fatal without drug treatment. Transmission is via the tsetse fly and parasites then circulate in the bloodstream and tissue fluids of their mammalian hosts. The public health situation has improved recently with increased monitoring and therapy averting more than 1.3 million DALYs (Disability-Adjusted Life Years) during 2000 and cases estimated at less than 70,000 in 2006. Humans have an innate immunity to infection by many of the African trypanosomes, mediated by two components of human serum, termed trypanolytic factor (TLF) 1 and 2. The mode of action depends on the ability of apolipoprotein-L1, a component of both trypanolytic factors, to form pores in the lysosome, an organelle responsible for protein degradation. The human infective forms, Trypanosoma brucei gambiense and T. b. rhodesiense, are able to evade this innate immunity by reduced TLF1 uptake or expression of the serum resistance antigen, respectively. There are several outstanding questions: how does TLF2 enter the cell; how do TLF1 and TLF2 transit to the lysosome; which parasite proteins are essential to this process; what are their contributions to TLF efficacy; and, what other routes to human serum resistance exist? Answers to these questions will enable the development of diagnostic tools for the characterisation of wild isolates of African trypanosomes whose human serum sensitivity status is unknown. This is particularly important given the prevalence of African trypanosomes amongst wild and domestic mammals in sub-Saharan Africa and their likely interaction with the human population. The proposal aims to take advantage of recent developments by the applicant, which allow the whole trypanosome genome to be screened for factors that interact with the trypanolytic components of human serum. This approach has already been used to identify the trypanosome factors that interact with and can contribute to resistance to the available anti-HAT drugs. An RNAi library containing fragments corresponding to >99% of the parasite's genes was selected with human serum. Only cells containing RNAi fragments targeting genes whose knockdown reduces sensitivity to human serum survive, and sequencing of these fragments allows the identification of genes and their corresponding proteins that may interact with the trypanolytic components of human serum. An initial analysis identified a surface receptor known to be responsible for the uptake of TLF1 and a lysosomal protein known to influence trypanosome sensitivity to human serum, so validating the approach. Four other proteins were also identified, including one other lysosomal protein, one likely responsible for regulating lysosomal pH, and two other proteins of unknown function. It is now necessary to carry out high throughput sequencing to identify all the genes being targeted by the human serum-selected RNAi library. Characterisation of these genes, their surrounding DNA sequence and their protein products will have several consequences. Firstly, an understanding of the proteins required for trypanolytic factor transit to and action in the lysosome. Secondly, the identification of trypanosome proteins that may contribute to human serum resistance in the field. Thirdly, translation of these findings will lead to the development of tools for the diagnosis and characterisation of wild African trypanosome isolates. This work will present opportunities for the development of diagnostic tools and is also likely to present new opportunities for anti-trypanosomal toxin delivery.