Dissecting fatty acid metabolism in livestock trypanosomes

非洲动物锥虫病（AAT）又称那加纳病，是一种影响撒哈拉以南非洲各地牛和其他牲畜的毁灭性疾病，每年有6 000多万头牛面临风险，300多万头牲畜死亡，对农业社区产生巨大的社会经济影响。AAT主要由单细胞寄生虫刚果锥虫引起。目前还没有治疗这种疾病的疫苗，由于对用于治疗AAT的药物产生耐药性，治疗失败很常见。我们迫切需要新药，但阻碍新药开发的一个主要问题是缺乏对刚果锥虫的生物学理解。相反，我们目前对锥虫的大部分知识来自相关物种布鲁氏锥虫，其变体导致人类疾病昏睡病。发现抗AAT药物的一个潜在途径是了解寄生虫细胞代谢，以及药物如何在不影响宿主的情况下杀死病原体。非洲锥虫在新陈代谢方面是独一无二的，虽然以前认为刚果锥虫和布鲁氏锥虫在生物学上非常相似，但我最近获得的数据强调了这些物种之间的几个代谢差异。例如，虽然已知布氏绦虫几乎完全依赖宿主来源的葡萄糖来满足其能量需求，但刚果锥虫似乎使用较少的葡萄糖，并以较低的速度代谢葡萄糖。此外，布鲁氏体能够利用葡萄糖代谢的一些产物产生一类称为脂肪酸的代谢物，脂肪酸是细胞结构的重要分子群。相比之下，我们没有发现刚果锥虫中葡萄糖衍生脂肪酸的证据。我最近发现，虽然布氏体能够合成脂肪酸，但刚果锥虫更喜欢从宿主的血液中清除这些脂肪酸。特别是，我已经确定了刚果锥虫在实验室生长所需的四种脂肪酸。非洲锥虫的这一要求以前没有报道过，因此刚果锥虫使用这四种脂肪酸的方式对锥虫生物学来说是新的。事实上，这种寄生虫的脂肪酸代谢尚未被研究过。至关重要的是，脂肪酸使用的这些机制也可能与宿主代谢不同，这可以用于药物开发。在这项研究中，我建议调查这些脂肪酸是如何被刚果锥虫利用和代谢的。为此，我将使用质谱等尖端技术来详细绘制刚果螺旋体如何代谢这四种脂肪酸，以及它们是否是这种寄生虫的潜在能量来源。我将把这种方法与生物信息学技术和高分辨率质谱成像相结合，研究刚果螺杆菌所拥有的酶以及这些酶在细胞中的位置，以便准确确定脂肪酸代谢的主要代谢途径。然后，我将研究这些途径中的活性如何随着环境的变化而变化，这取决于外源脂肪酸的存在和缺失，以了解这些途径对寄生虫是否必需，以及是否确实是药物发现的可行途径。最后，我将选择关键酶，并使用新获得的遗传工具来操纵它们在细胞中的存在，以确定对细胞生长的总体影响。结合已知抑制剂的测试，这些目标将提供刚果锥虫脂肪酸代谢的非常详细的图片，让我们深入了解这种未被研究的病原体的生物学，并潜在地确定候选药物靶点。

Animal African Trypanosomiasis (AAT), also known as Nagana, is a devastating disease affecting cattle and other livestock across sub-Saharan Africa, with more than 60 million cattle at risk and in excess of 3 million deaths per year, with a great socio-economic impact on agricultural communities. AAT is principally caused by the single-celled parasite Trypanosoma congolense. There are no vaccines to treat the disease, and treatment failure is common due to resistance against the drugs used to treat AAT. New drugs are sorely needed, but a major problem hindering their development is a lack of biological understanding of T. congolense. Instead, most of our current knowledge on trypanosomes derives from the related species T. brucei, variants of which cause the human disease Sleeping Sickness. One potential avenue to drug discovery against AAT is by gaining an understanding of parasite cell metabolism, and how drugs may affect this in ways that kill the pathogen without impacting the host.African trypanosomes are unique in their metabolism, and whilst it was previously thought that T. congolense and T. brucei were very similar in their biology, recent data I have generated has highlighted several metabolic distinctions between these species. For example, whilst T. brucei is known to rely almost entirely on host-derived glucose for its energy demands, T. congolense appears to use less glucose, and metabolise it at a reduced rate. In addition, T. brucei is able to use some of the products of glucose metabolism to generate a class of metabolites called fatty acids, an important group of molecules for cellular structure. In contrast, we found no evidence of glucose-derived fatty acids in T. congolense.I recently found that whilst T. brucei is able to synthesise fatty acids, T. congolense prefers to scavenge these from the bloodstream of the host. In particular, I have identified four fatty acids that T. congolense requires to grow in the laboratory. This requirement has not previously been reported for African trypanosomes, and the ways in which these four fatty acids are used by T. congolense are therefore novel to trypanosome biology. Indeed, fatty acid metabolism is unexplored in this parasite species. Crucially, these mechanisms of fatty acid usage may also differ from host metabolism, and this could be exploited for drug development. In this study, I propose to investigate how these fatty acids are used, and metabolised by T. congolense. To do this, I will use cutting edge technologies such as mass spectrometry to map in detail how the four fatty acids are metabolised by T. congolense, and whether they are a potential source of energy for this parasite species. I will combine this approach with bioinformatic techniques and high-resolution mass spectrometry imaging to investigate the enzymes T. congolense possesses and where these are localised in the cell, in order to accurately determine the major metabolic pathways involved in fatty acid metabolism. I will then investigate how the activity in these pathways changes in response to the environment, depending on the presence and absence of exogenous fatty acids, in order to understand whether these pathways are essential to the parasite, and indeed a viable avenue for drug discovery. Finally, I will select key enzymes and use newly-available genetic tools to manipulate their presence in the cell in order to determine the overall effect on cell growth. Combined with the testing of known inhibitors, these objectives will give a very detailed picture of fatty acid metabolism in T. congolense, giving us an insight into the biology of this understudied pathogen and potentially identifying candidate drug targets.