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

溃疡性结肠炎和克罗恩病是炎症性肠病（IBD）的两种形式，影响着全球680多万患者。由于迄今为止还没有治愈方法，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，但过度的蛋白质水解和蛋白酶激活受体（PARs）信号失调与IBD和其他胃肠道（GI）疾病有关。PARs是一类独特的真核g蛋白偶联受体（gpcr），由蛋白酶直接调节。与经典的gpcr相比，PARs不受可溶性配体结合的激活。相反，细胞外n端结构域（NTD）的肽序列的蛋白水解裂解揭示了一个新n端，它作为拴链激活配体。由于许多胃肠道疾病都伴随着人类微生物群的生态失调，来自共生菌的蛋白酶可能是肠道内稳态和发病机制的重要调节因子，从而使其成为潜在的治疗靶点。然而，到目前为止，还没有研究在全球范围内确定共生菌株产生哪些蛋白酶，以及这些酶如何通过共定位的人类PARs蛋白水解影响健康和疾病。有益的共生菌可能通过PAR的基础激活或受体的蛋白水解脱敏来抵消肠道中的过度炎症，而病原体物种可以通过增加PAR的激活来分泌促进IBD的蛋白酶。Matthew Bogyo教授小组的初步研究已经揭示了三种共生菌株通过分泌蛋白酶激活PAR2，因此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来自共生菌的蛋白酶，它们调节肠道中人类PAR信号。首先，我将开发一种体外高通量测定方法，用于监测所有四种人类PARs的ntd的蛋白质水解，并直接分析各自的裂解位点。在它建立之后，我将筛选250个肠道共生菌株的个体培养物进行蛋白水解par处理。接下来，我将使用基于生化分离和质谱的化学蛋白质组学技术的多步骤方法来确定负责的蛋白酶。我将验证蛋白水解裂解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传导。最后，我将应用肠道屏障完整性的多细胞和类器官模型来研究微生物par加工对肠道通透性的生物学影响。最终，这些目标的成功不仅将导致新的微生物蛋白酶及其在宿主-微生物相互作用中的作用的表征，而且还将为未来基于微生物组的治疗选择奠定基础。

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.