权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

布氏锥虫对人血清锥虫分解因子的摄取和亚细胞转运的表征

基本信息

批准号：
MR/K011987/1
负责人：
Sam Alsford
金额：
$ 46.66万
依托单位：
London School of Hygiene & Tropical Medicine
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2013
资助国家：
英国
起止时间：
2013 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FK011987%2F1
关键词：
Characterisation uptake sub cellular transit

项目摘要

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.

非洲锥虫是原生动物寄生虫，可引起一系列重要的人类和动物疾病。人类非洲锥虫病（HAT）通常是致命的，没有药物治疗。传播是通过采采蝇和寄生虫，然后循环在血液和组织液的哺乳动物宿主。公共卫生状况最近有所改善，2000年加强了监测和治疗，避免了130多万残疾调整生命年，2006年估计病例不到70 000例。人类对许多非洲锥虫的感染具有先天免疫力，由人血清的两种成分介导，称为锥虫溶解因子（TLF）1和2。作用模式取决于载脂蛋白-L1（两种锥虫溶解因子的组分）在溶酶体（负责蛋白质降解的细胞器）中形成孔的能力。人类感染型布氏冈比亚锥虫和T. B. Rhodesiense，能够分别通过减少TLF 1摄取或血清抗性抗原的表达来逃避这种先天免疫。有几个悬而未决的问题：TLF 2如何进入细胞; TLF 1和TLF 2如何转运到溶酶体;哪些寄生虫蛋白质对这一过程至关重要;它们对TLF功效的贡献是什么;以及存在哪些其他途径导致人类血清耐药性？这些问题的答案将有助于开发诊断工具，用于表征人类血清敏感性状态未知的非洲锥虫野生分离株。鉴于非洲锥虫在撒哈拉以南非洲的野生和家养哺乳动物中的流行及其与人类的可能相互作用，这一点尤为重要。该提案旨在利用申请人最近的发展，这些发展允许对整个锥虫基因组进行筛选，以寻找与人血清的锥虫分解成分相互作用的因子。这种方法已经被用于识别与现有抗HAT药物相互作用并导致其耐药性的锥虫因子。用人血清选择含有对应于>99%寄生虫基因的片段的RNAi文库。只有含有靶向基因的RNAi片段的细胞存活，所述基因的敲低降低了对人血清的敏感性，并且这些片段的测序允许鉴定可能与人血清的锥虫分解组分相互作用的基因及其相应的蛋白质。初步分析确定了已知负责TLF 1摄取的表面受体和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，包括一种其他溶酶体蛋白质，一种可能负责调节溶酶体pH，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鉴定人血清选择的RNAi文库靶向的所有基因。对这些基因、它们周围的DNA序列和它们的蛋白质产物的表征将产生几个结果。首先，了解锥虫溶解因子转运到溶酶体并在溶酶体中起作用所需的蛋白质。第二，在该领域鉴定可能有助于人血清抗性的锥虫蛋白。第三，这些发现的翻译将导致开发工具，用于诊断和表征野生非洲锥虫分离株。这项工作将为开发诊断工具提供机会，也可能为抗锥虫毒素提供新的机会。