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

• 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

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.

非洲锥虫是引起一系列重要人类和动物疾病的原生动物寄生虫。人类非洲锥虫病（HAT）通常是致命的，而没有药物治疗。通过采采蝇传播，然后寄生虫在其哺乳动物宿主的血液和组织液中循环。最近，由于监测和治疗的增加，公共卫生状况最近有所改善，2000年的监测和治疗避免了超过130万达利（残疾调整后的生活年），2006年估计的病例估计不到70,000。载脂蛋白L1是两种锥溶因子的一个成分，它在溶酶体中形成毛孔，这是负责蛋白质降解的细胞器。人类感染性形式，锥虫brucei gambiense和T. b。 Rhodesiense，能够分别减少TLF1摄取或血清抗性抗原的表达来逃避这种先天的免疫力。有几个出色的问题：TLF2如何进入单元格； TLF1和TLF2如何转移到溶酶体；哪种寄生虫蛋白对于此过程至关重要；他们对TLF功效的贡献是什么？而且，还有哪些其他通往人血清抗性的路线？这些问题的答案将使诊断工具的开发用于表征人类血清敏感性状态的非洲锥虫的野生隔离株。鉴于撒哈拉以南非洲的野生和家庭哺乳动物中非洲锥虫的流行及其与人口的可能相互作用，这一点尤其重要。该提案旨在利用申请人的最新发展，这使整个锥虫基因组可以筛选与人血清的锥虫分量相互作用的因素。这种方法已被用来识别与可用抗帽子药物相互作用的锥虫因子。与人血清一起选择了含有寄生虫基因> 99％的片段的RNAi文库。只有含有RNAi片段的细胞靶向基因降低对人血清的敏感性的基因，并且对这些片段的测序可以鉴定基因及其相应的蛋白质，这些蛋白可能与人血清的锥虫分量相互作用。初始分析确定了已知的表面受体，该表面受体负责TLF1的摄取和已知会影响锥虫对人血清敏感性的溶酶体蛋白，因此验证了该方法。还鉴定了另外四种蛋白质，包括另一种溶酶体蛋白，一种可能导致溶酶体pH的负责，还有另一种功能未知的蛋白质。现在有必要进行高通量测序，以识别人血清选择的RNAi文库靶向的所有基因。这些基因的表征，周围的DNA序列及其蛋白质产物将产生几种后果。首先，了解试剂液溶液因子转移到溶酶体中所需的蛋白质。其次，鉴定可能有助于人类血清抗性的锥虫蛋白。第三，这些发现的翻译将导致开发用于诊断和表征野生非洲锥虫分离株的工具。这项工作将为开发诊断工具提供机会，也可能为抗肌体毒素提供新的机会。

项目成果

Genetic dissection of drug resistance in trypanosomes.

• DOI: 10.1017/s003118201300022x
• 发表时间: 2013-10
• 期刊: Parasitology
• 影响因子: 2.4
• 作者: Alsford S;Kelly JM;Baker N;Horn D
• 通讯作者: Horn D

Cathepsin-L can resist lysis by human serum in Trypanosoma brucei brucei.

• DOI: 10.1371/journal.ppat.1004130
• 发表时间: 2014-05
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Alsford S;Currier RB;Guerra-Assunção JA;Clark TG;Horn D
• 通讯作者: Horn D

Decoding the network of Trypanosoma brucei proteins that determines sensitivity to apolipoprotein-L1.

• DOI: 10.1371/journal.ppat.1006855
• 发表时间: 2018-01
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Currier RB;Cooper A;Burrell-Saward H;MacLeod A;Alsford S
• 通讯作者: Alsford S

Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy

• DOI: 10.1016/j.pt.2013.03.004
• 发表时间: 2013-05-01
• 期刊: TRENDS IN PARASITOLOGY
• 影响因子: 9.6
• 作者: Alsford, Sam;Field, Mark C.;Horn, David
• 通讯作者: Horn, David

Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei.

• DOI: 10.1096/fj.201700311r
• 发表时间: 2017-10
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Macedo JP;Currier RB;Wirdnam C;Horn D;Alsford S;Rentsch D
• 通讯作者: Rentsch D