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最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的话。在这里，我们建议使用原生动物寄生虫利什曼原虫(Leishmania(L.)多诺瓦尼作为模型系统，研究病原体衍生的信号分子对寄生虫活性、毒力和宿主细胞表达谱的影响。杜诺氏利什曼原虫是内脏利什曼病的病原体，是世界范围内一种被忽视的主要热带疾病，被认为是全球变暖导致的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有关，但令人惊讶的是，人们对这些微生物如何重新编程宿主细胞以建立允许的生存条件知之甚少。TRANSEG联合体专注于分泌的利什曼原虫信号蛋白，这些蛋白可能通过反式作用来调节宿主细胞的表型。我们的项目源于先前发表的一系列观察结果，显示(I)细胞内利什曼原虫使巨噬细胞免疫信号和抗微生物活性失活，(Ii)寄生虫酪蛋白激酶同系物CK1.2和伴侣HSP90释放到宿主细胞细胞质中，以及(Iii)CK1.2与宿主免疫蛋白直接相互作用。我们假设CK1.2以一种HSP90依赖的方式通过外体释放到宿主细胞细胞质中，在这种方式中，它通过宿主蛋白的磷酸化来调节信号转导，以便为寄生虫在细胞内的生存建立允许的条件。TRANSPG通过三个互补和多学科的任务来研究这一创新的工作假说，方法是应用遗传和显微方法来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)，使用化学遗传学的方法从功能上分析HSP90磷酸化对伴侣功能和定位的作用(任务2)，以及通过使用RNAseq技术(任务3)研究CK1.2和HSP90及其相互作用伙伴之间的调控关系，以及激酶和伴侣活性对宿主细胞表型的影响(任务3)。我们的最终目标是通过干扰寄生虫蛋白的释放，将我们的研究成果转化为新的有效的抗利什曼疗法，从而恢复宿主细胞的抗微生物潜力。TRANSPOG联盟动员和协同两个世界知名的传染病和寄生虫学中心，法国的巴斯德研究所(合作伙伴1)和德国的伯恩哈德·诺希特热带医学研究所(合作伙伴2)。值得注意的是，两个合作伙伴都对利什曼原虫应激信号有共同的兴趣，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.