Functional profiling of microbial proteases that modulate host-bacterial interactions in the gut

调节肠道宿主-细菌相互作用的微生物蛋白酶的功能分析

• 批准号: 450273105
• 负责人: Dr. Markus Lakemeyer
• 金额: --
• 依托单位: Department of Pathology
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/450273105?language=en
• 关键词: Functional; profiling; microbial; proteases; modulate

Ulcerative colitis and Crohn’s disease are forms of inflammatory bowel disease (IBD) that affect more than 6.8 million patients worldwide. Because no cure is available to date, treatment is limited to easing the typical IBD symptoms such as severe diarrhea, weight loss, fatigue and pain. Although the exact mechanisms are unclear, excessive proteolysis and dysregulated signaling of protease-activated receptors (PARs) have been linked to IBD and additional gastrointestinal (GI) diseases. PARs are a unique class of four eukaryotic G-protein coupled receptors (GPCRs) that are directly regulated by proteases. In contrast to classical GPCRs, PARs are not activated by binding of soluble ligands. Instead, proteolytic cleavage of a peptide sequence in the extracellular N-terminal domain (NTD) reveals a neo-N-terminus that acts as a tethered activating ligand. As many GI diseases are accompanied with a dysbiosis of the human microbiome, proteases derived from commensal bacteria are likely to be important regulators of gut homeostasis and pathogenesis, thus making them potential therapeutic targets. Nevertheless, no study so far has globally characterized which proteases are produced by commensal strains and how these enzymes affect health and disease by proteolysis of co-localized human PARs. Beneficial commensal bacteria might counteract excessive inflammation in the gut by basal activation of PARs or by proteolytic desensitization of the receptor, while pathobiont species could secrete proteases that promote IBD via increased PAR activation. Preliminary studies in the group of Prof. Matthew Bogyo have already revealed three commensal strains that activate PAR2 via secreted proteases, thus demonstrating the need to further study these interactions. In this work I aim to globally characterize proteases from commensal bacteria that modulate human PAR signaling in the gut. First, I will develop an in vitro high-throughput assay for monitoring proteolysis of the NTDs of all four human PARs and for direct analysis of the respective cleavage sites. After its establishment, I will screen individual cultures of 250 gut commensal strains for proteolytic PAR-processing. Next, I will identify the responsible proteases using a multi-step approach of biochemical fractionation and mass-spectrometry based chemoproteomic techniques. I will validate that proteolytic cleavage also occurs in the context of full length, membrane-bound receptor and investigate how these events impact downstream signaling in mammalian cell lines. Finally, I will apply multicellular and organoid models of intestinal barrier integrity to study the biological impact of microbial PAR-processing on intestinal permeability. Ultimately, success in these aims will not only lead to the characterization of novel microbial proteases and their role in host-microbe interaction, but also lay the foundation for future microbiome-based treatment options.

溃疡性结肠炎和克罗恩病是炎症性肠病（IBD）的一种形式，影响全球680多万患者。由于迄今为止没有治愈方法，治疗仅限于缓解典型的IBD症状，如严重腹泻，体重减轻，疲劳和疼痛。虽然确切的机制尚不清楚，但过度的蛋白水解和蛋白酶激活受体（PAR）的信号转导失调与IBD和其他胃肠道（GI）疾病有关。PAR是一类独特的四种真核G蛋白偶联受体（GPCR），直接受蛋白酶调节。与经典的GPCR相反，PAR不被可溶性配体的结合激活。相反，胞外N-末端结构域（NTD）中的肽序列的蛋白水解裂解揭示了充当栓系活化配体的新-N-末端。由于许多胃肠道疾病伴随着人体微生物组的生态失调，因此来源于肠道细菌的蛋白酶可能是肠道稳态和发病机制的重要调节剂，从而使其成为潜在的治疗靶点。然而，到目前为止，还没有研究全面表征哪些蛋白酶是由大肠杆菌菌株产生的，以及这些酶如何通过共定位的人PAR的蛋白水解影响健康和疾病。有益的肠道细菌可能通过PAR的基础激活或通过受体的蛋白水解脱敏来抵消肠道中的过度炎症，而致病菌物种可以分泌蛋白酶，通过增加PAR激活来促进IBD。Matthew Bogyo教授小组的初步研究已经揭示了三种通过分泌蛋白酶激活PAR 2的真菌菌株，从而表明需要进一步研究这些相互作用。在这项工作中，我的目标是在全球范围内表征蛋白酶从肠道细菌调节人类PAR信号。首先，我将开发一种体外高通量测定法，用于监测所有四种人PAR的NTD的蛋白水解，并直接分析各自的切割位点。在其建立之后，我将筛选250个肠球菌菌株的单独培养物用于蛋白水解PAR加工。接下来，我将使用基于生化分级和质谱的化学蛋白质组学技术的多步骤方法来鉴定负责的蛋白酶。我将验证蛋白水解切割也发生在全长，膜结合受体的背景下，并研究这些事件如何影响哺乳动物细胞系的下游信号。最后，我将应用肠屏障完整性的多细胞和类器官模型来研究微生物PAR加工对肠通透性的生物学影响。最终，这些目标的成功不仅将导致新型微生物蛋白酶的表征及其在宿主-微生物相互作用中的作用，还将为未来基于微生物组的治疗方案奠定基础。