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

非洲锥虫是一种原生动物寄生虫，可引起一系列重要的人类和动物疾病。人类非洲锥虫病(HAT)在没有药物治疗的情况下通常是致命的。通过采采蝇和寄生虫传播，然后在它们的哺乳动物宿主的血液和组织液中循环。公共卫生状况最近有所改善，加强了监测和治疗，在2000年期间避免了130多万个伤残调整寿命年，2006年估计减少了不到70000个病例。人类对许多非洲锥虫的感染有一种天生的免疫力，这种免疫力是由人血清的两种成分--胰溶因子(TLF)1和2介导的。作用方式取决于载脂蛋白-L1在溶酶体中形成孔的能力，载脂蛋白-L1是这两种锥虫因子的组成部分，溶酶体是负责蛋白质降解的细胞器。人类感染形式布氏锥虫和罗德氏锥虫分别能够通过减少TLF1对血清耐药抗原的摄取或表达来逃避这种先天免疫。有几个悬而未决的问题：TLF2是如何进入细胞的；TLF1和TLF2是如何转运到溶酶体的；哪些寄生虫蛋白在这个过程中是必不可少的；它们对TLF疗效的贡献是什么；以及，还有哪些其他途径导致人类血清抵抗？对这些问题的回答将有助于开发诊断工具，以确定人类血清敏感性状况未知的非洲锥虫野生分离株的特征。鉴于非洲锥虫在撒哈拉以南非洲野生和家养哺乳动物中的普遍存在以及它们可能与人类种群相互作用，这一点尤其重要。该提案旨在利用申请者最近的发展，允许对整个锥虫基因组进行筛选，以寻找与人血清中的锥虫分解成分相互作用的因素。这种方法已经被用来确定与现有抗HAT药物相互作用并可能导致耐药性的锥虫因子。用人血清筛选RNAi文库，该文库包含与寄生虫99%基因相对应的片段。只有含有靶向基因的RNAi片段的细胞才能存活，这些基因的敲除降低了对人血清的敏感性，对这些片段的测序可以识别可能与人血清的胰酶降解成分相互作用的基因及其相应的蛋白质。初步分析确定了一种已知负责TLF1摄取的表面受体和一种已知影响锥虫对人血清敏感性的溶酶体蛋白，从而验证了该方法。还鉴定了其他四种蛋白质，包括另一种溶酶体蛋白，一种可能负责调节溶酶体pH的蛋白质，以及另外两种功能未知的蛋白质。现在有必要进行高通量测序，以确定人血清选择的RNAi文库所针对的所有基因。对这些基因、它们周围的DNA序列和它们的蛋白质产物的特征将产生几个后果。首先，了解胰酶溶解因子转运到溶酶体并在其中起作用所需的蛋白质。其次，鉴定可能导致现场人类血清抵抗力的锥虫蛋白。第三，这些发现的翻译将导致开发诊断和鉴定非洲锥虫野生分离株的工具。这项工作将为诊断工具的开发提供机会，也可能为抗锥虫毒素的传递提供新的机会。

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.