Dissecting fatty acid metabolism in livestock trypanosomes

非洲动物锥虫病(AAT)，又称Nagana，是一种影响整个撒哈拉以南非洲的牛和其他牲畜的破坏性疾病，有6000多万头牛处于危险之中，每年有300多万人死亡，对农业社区产生了重大的社会经济影响。AAT主要由单细胞寄生虫锥虫引起。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，治疗失败的情况很常见。人们迫切需要新药，但阻碍它们发展的一个主要问题是缺乏对弓形虫的生物学理解。相反，我们目前关于锥虫的大部分知识都来自相关物种布鲁氏锥虫，其变异会导致人类疾病睡眠病。发现治疗AAT的药物的一个潜在途径是了解寄生虫细胞的新陈代谢，以及药物如何通过在不影响宿主的情况下杀死病原体的方式影响这一代谢。非洲锥虫在它们的新陈代谢中是独特的，虽然以前人们认为贡戈氏锥虫和布鲁氏锥虫在生物学上非常相似，但我最近产生的数据突出了这两个物种之间的几个代谢差异。例如，虽然已知布鲁氏支原体的能量需求几乎完全依赖于宿主来源的葡萄糖，但贡氏支原体似乎使用较少的葡萄糖，并以较低的速度代谢它。此外，布氏毛滴虫能够利用葡萄糖代谢的一些产物产生一类代谢物，称为脂肪酸，这是细胞结构的一组重要分子。相比之下，我们在龙眼蜂中没有发现葡萄糖衍生的脂肪酸的证据。我最近发现，虽然布鲁氏毛滴虫能够合成脂肪酸，但龙眼蜂更喜欢从宿主的血液中清除这些脂肪酸。特别是，我已经确定了龙须草在实验室中生长所需的四种脂肪酸。这一要求以前在非洲锥虫中还没有报道，因此这四种脂肪酸被弓形锥虫利用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还没有被研究过。至关重要的是，这些脂肪酸的使用机制也可能不同于宿主的新陈代谢，这可以被用于药物开发。在这项研究中，我建议调查这些脂肪酸是如何被弓形虫利用和代谢的。为了做到这一点，我将使用尖端技术，如质谱学，详细绘制出这四种脂肪酸是如何被弓形虫新陈代谢的，以及它们是否是这种寄生虫物种的潜在能源。我将把这一方法与生物信息学技术和高分辨率质谱学成像相结合，研究贡氏毛虫所拥有的酶以及这些酶在细胞中的定位，以便准确地确定参与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是如何随着环境的变化而变化的，这取决于外源脂肪酸的存在和不存在，以了解这些途径是否对寄生虫是必要的，甚至是一个可行的药物发现途径。最后，我将选择关键的酶，并使用最新可用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。结合对已知抑制剂的测试，这些目标将给出一幅非常详细的关于弓形虫脂肪酸代谢的图景，使我们能够深入了解这种未被研究的病原体的生物学，并有可能确定候选的药物靶点。

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.