Trans-signalling: A novel mechanism of Leishmania host cell immune evasion through the release of parasite signalling proteins

细胞内寄生是最成功和致命的人类病原体的主要标志，特别是如果免疫细胞被用作宿主。在这里，我们建议使用原生动物寄生虫利什曼原虫（L.）donovani作为模型系统来研究病原体衍生的信号分子对寄生虫生存力、毒力和宿主细胞表达谱的影响。L.杜氏利什曼病是内脏利什曼病的病原体，内脏利什曼病是全球主要的被忽视的热带疾病，并且由于全球变暖而被认为是欧洲最重要的新兴寄生虫病。尽管细胞内利什曼原虫感染与全球死亡率和发病率相关，但令人惊讶的是，人们对这些微生物如何重新编程其宿主细胞以建立允许生存的条件知之甚少。TranSig联盟专注于分泌的利什曼原虫信号蛋白，其可以反式作用以调节宿主细胞表型。我们的项目出现从一系列先前发表的观察显示（i）细胞内利什曼原虫的巨噬细胞免疫信号转导和抗微生物活性的失活，（ii）释放的寄生虫酪蛋白激酶同源物CK1.2和伴侣HSP 90到宿主细胞质，和（iii）直接相互作用的CK1.2与宿主免疫蛋白。我们假设CK1.2通过外泌体以HSP 90依赖的方式释放到宿主细胞质中，在那里它通过宿主蛋白的磷酸化调节信号传导，以建立细胞内寄生虫存活的容许条件。TranSig通过三个互补和多学科的任务来研究这一创新的工作假设，通过应用遗传学和显微镜方法来深入了解利什曼原虫胞外激酶CK1.2的功能，定位和相互作用（任务1），通过使用化学遗传学方法来功能分析HSP 90磷酸化对伴侣蛋白功能和定位的作用（任务2），以及通过使用RNAseq技术通过转录谱分析研究CK1.2和Hsp90及其相互作用伴侣之间的调节关系，以及激酶和伴侣蛋白活性对宿主细胞表型的影响（任务3）。我们的最终目标是将我们的研究结果转化为新的有效的抗利什曼原虫疗法，通过干扰寄生虫蛋白质的释放，从而恢复宿主细胞的抗微生物潜力。TransSig联盟动员并协同两个世界知名的传染病和寄生虫学中心，法国巴斯德研究所（合作伙伴1）和德国伯恩哈德·诺赫特热带医学研究所（合作伙伴2）。值得注意的是，这两个合作伙伴在利什曼原虫应激信号传导方面有着共同的兴趣，德国合作伙伴是寄生虫热休克蛋白和伴侣生物学方面的专家，法国合作伙伴提供寄生虫激酶生物学和应激诱导蛋白磷酸化方面的专业知识。

Intracellular parasitism is a major hallmark of the most successful and deadly human pathogens, especially if immune cells are exploited as hosts. Here we propose to use the protozoan parasite Leishmania (L.) donovani as model system to investigate the impact of pathogen-derived signalling molecules on parasite viability, virulence, and host cell expression profile. L. donovani is the causative agent of visceral leishmaniasis which is a major Neglected Tropical Disease worldwide and is regarded as the most significant emerging parasitic disease in Europe due to global warming. Despite the relevance of intracellular Leishmania infection in global mortality and morbidity, surprisingly little is known on how these microbes reprogram their host cell to establish permissive conditions for survival. The TranSig consortium is focused on secreted Leishmania signalling proteins that may act in trans to modulate the host cell phenotype. Our project emerges from a series of previously published observations showing (i) inactivation of macrophage immune signalling and anti-microbial activities by intracellular Leishmania, (ii) release of the parasite casein kinase homolog CK1.2 and the chaperone HSP90 into the host cell cytoplasm, and (iii) direct interaction of CK1.2 with host immune proteins. We hypothesize that CK1.2 is released through exosomes into the host cell cytoplasm in a HSP90-dependent manner, where it modulates signalling by phosphorylation of host proteins in order to establish permissive conditions for intracellular parasite survival. TranSig investigates this innovative working hypothesis through three complementary and multi-disciplinary tasks by applying genetic and microscopic approaches to gain insight into the function, localization, and interactions of the Leishmania ecto-kinase CK1.2 (Task 1), by using a chemical-genetics approach to functionally analyse the role of HSP90 phoshorylation on chaperone function and localization (Task 2), and by investigating the regulatory relationship between CK1.2 and Hsp90 and their interaction partners, and the impact of both kinase and chaperone activities on the host cell phenotype by transcript profiling using RNAseq technology (Task 3). Our ultimate goal is to translate our research findings into novel potent anti-leishmanial therapies by interfering with parasite protein release thus restoring the host cell anti-microbial potential. The TranSig consortium mobilizes and synergizes two world-renowned centers in infectious diseases and parasitology, the Institut Pasteur in France (Partner 1) and the Bernhard Nocht Institute for Tropical Medicine in Germany (Partner 2). Significantly, both partners have a common interest in Leishmania stress signaling, with the German partner being an expert in parasite heat shock protein and chaperone biology, and the French partner providing expertise in parasite kinase biology and stress-induced protein phosphorylation.