Identification of specific metabolites in mycolactone producing mycobacteria and Buruli ulcer infection: diagnostic biomarkers through metabolomic

布鲁里溃疡是一种严重的、进展缓慢的皮肤感染，它破坏组织，由环境中发现的称为溃疡分枝杆菌的细菌引起。尽管加纳、多哥、贝宁、科特迪瓦、喀麦隆、墨西哥、巴布亚新几内亚、东南亚和澳大利亚等30多个国家描述了这种疾病，但它仍被列为一种被忽视的热带疾病，主要影响居住在受感染地区的农村社区的个人。世界卫生组织的全球布鲁里溃疡倡议已将开发简单的诊断工具确定为控制疾病的优先领域之一。代谢组学是分析科学和生物学中一个不断发展的领域，它寻求识别能够区分患病和非患病个体的新的生物标志物。在一项由剑桥-非洲卓越研究伙伴关系(CAPREx)资助的试点研究中，我们试图确定最佳样本类型，以鉴定只能在布鲁里溃疡患者中发现的细菌代谢物。我们收集了28例确诊的布鲁里溃疡患者和21例非布鲁里溃疡的热带溃疡患者的组织活检、拭子和细针抽吸物。这些样品在加纳大学生物化学、细胞和分子生物学系进行了前处理，以提取剑桥大学气相色谱-质谱仪(GC-MS)所需的成分。我们确定组织活检和细针抽吸物是鉴定代谢物的最佳标本，而不是病变拭子。有趣的是，在两组患者中发现的代谢物包括身体碱、腐胺、品尼醇、棕榈酸酯、萘、毒死磷和奥司匹罗。前两种代谢物很有趣，因为它们将所有样本归类为含有退化组织。脂肪酸棕榈酸酯是人体组织中常见的代谢物。Pinitol和后三种确定的代谢物是化学残留物，可能存在于患者用作非正统药物的治疗膏剂中。在这项提案中，我们正寻求进一步开展这项试点研究，在我们观察的基础上，从更大的患者样本群中收集组织活检和细针抽吸物，以使我们能够对细菌和感染组织中发现的化合物进行更详细的分析。我们还打算扩大受影响个人的生物样本的范围，将尿样包括在我们的分析中，这可能会提供更方便的生物流体进行采样。为了更多地了解我们确定的生物标记物的新颖性，我们将包括另一个阶段的实验，涉及在溃烂分枝杆菌和其他类似的感染加纳人的细菌菌株中发现的化合物的特征，包括伪热分枝杆菌、利夫兰迪分枝杆菌和海洋分枝杆菌。该项目产生的数据将对确定新的诊断方法很重要，并使我们了解感染期间分枝杆菌的生物学，这可能会为未来的治疗带来更好的前景。

Buruli ulcer is a severe, slow progressing skin infection which destroys tissue and is caused by bacteria found in the environment called Mycobacterium ulcerans. The disease is classified as a neglected tropical disease despite being described in over 30 countries including Ghana, Togo, Benin, Côte d'Ivoire, Cameroon, Mexico, Papua New Guinea, South East Asia and Australia, and mostly affects individuals in rural communities living in infected areas. The World Health Organisation's Global Buruli Ulcer Initiative has identified the development of simple diagnostic tools as one of the priority areas in the control of the disease. Metabolomics is a growing field in analytical science and biology which seeks to identify novel biomarkers that can distinguish between diseased and non-diseased individuals. In a pilot study, funded under the Cambridge-Africa Partnership for Research Excellence (CAPREx), we sought to determine the best types of sample for the identification of metabolites from the bacteria that can be found only in Buruli ulcer patients. We collected tissue biopsy, swabs and fine needle aspirates from 28 Buruli ulcer confirmed patents and 21 patients with tropical ulcers that were not Buruli ulcer. The samples were pre-processed at the Department of Biochemistry, Cell and Molecular Biology at the University of Ghana, to extract the required components for gas chromatography-mass spectrometry (GC-MS) at the University of Cambridge. We identified tissue biopsy and fine needle aspirates as the best specimen for the identification of metabolites in preference to lesion swabs. Interestingly, metabolites identified in both groups of patients included cadaverine, putrescine, pinitol, palmitate, naphthalene, chlopyrifos and oxaspiro. The former two metabolites are interesting because they classify all the samples as containing degenerating tissue. The fatty acid palmitate is a common metabolite present in human tissue. Pinitol and the later three identifies metabolites are chemical residues that may have been present in the treatment poultice used as unorthodox remedies by the patients. In this proposal, we are seeking to further this pilot study, building on our observations and collecting the tissue biopsies and fine needle aspirates from a larger patient sample group to allow us to perform a more detailed analysis of the compounds found within the bacteria and also within infected tissue. We also intend to expand the scope of the biological samples from affected individuals by including urine samples in our analysis which may provide a more convenient biofluid to sample. To gain more insight into the novelty of the biomarkers we identify, we will include another phase of experimentation which involves the characterization of the compounds found in M. ulcerans and other similar bacteria strains that infect people in Ghana including M. pseudishottsii, M. liflandii and M. marinum DL. Data generated from this project will be important in the identification of new diagnostics and also give us insight to the biology of the mycobacterium during infection which may lead to better prospects for future treatments.