Chemoproteomic strategies for probing outer membrane vesicles and their role in gut microbial interactions

人类肠道菌群是一个复杂而动态的微生物生态系统，对健康和疾病有着深远的影响。肠道菌群失衡，即生态失调，与一系列胃肠道疾病有关，包括炎症性肠病（IBD）和结直肠癌。宿主或微生物蛋白酶在肠道中过度的蛋白质水解已被认为是IBD的主要诱因，但其潜在机制仍不清楚。最初的报告表明，拟杆菌目细菌分泌大量的外膜囊泡（OMVs），这是一种源自革兰氏阴性细菌外膜的小球形结构。omv作为膜结合和封装货物的远程载体，因此可能在微生物-微生物以及微生物-宿主通信中发挥关键作用。有趣的是，所研究的拟杆菌门omv具有大量的膜锚定脂蛋白，特别是蛋白酶，它们位于表面，从而允许它们直接与环境相互作用。这使得拟杆菌门蛋白酶成为微生物相互作用的潜在强大介质。然而，对于哪些蛋白质通过单个拟杆菌菌株的omv运输，以及外部因素如何调节omv内的组成和蛋白质定位，我们仍然知之甚少。此外，尚不清楚细菌蛋白酶如何影响肠道微生物群的组成，从而调节发病机制。在本项目中，我们将开发和应用化学工具来全面表征拟杆菌属omv及其在微生物相互作用中的功能。首先，我们将使用基于活性的蛋白质谱来识别导致最强OMV分泌的细菌菌株和条件。接下来，我们将应用化学蛋白质组学方法来表征omv的蛋白质组学，特别关注丝氨酸水解酶和预测脂蛋白的脂化状态。选择在omv中发现的蛋白酶将被重组生产并进行生化表征。随后，我们将合成针对单个蛋白酶定制的荧光底物，共价抑制剂和化学探针。有了这些强大的化学工具，我们将阐明omv及其相关蛋白酶如何影响单个菌株和细菌共培养。最后，我们将利用荧光化学探针在荧光显微镜下研究omv的动态生物发生和运输。总的来说，这些目标的成功将导致对细胞外微生物蛋白的全面理解，新酶的表征，以及定制工具的开发，以探测omv在微生物相互作用中的作用。

The human gut microbiota is a complex and dynamic ecosystem of microorganisms that profoundly affects health and disease. An imbalance of the gut microbiota, known as dysbiosis, has been linked to a range of gastrointestinal diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Excessive proteolysis in the gut by host or microbial proteases has been suggested as a major contributor to IBD, but the underlying mechanisms remain largely unclear. Initial reports have suggested that bacteria from the order Bacteroidales excrete significant amounts of outer membrane vesicles (OMVs), small spherical structures that are derived from outer membranes of Gram-negative bacteria. OMVs act as long-range vehicles for membrane-bound and encapsulated cargo and thus might play crucial roles in microbe-microbe as well as microbe-host communication. Intriguingly, the studied Bacteroidales OMVs featured high numbers of membrane-anchored lipoproteins, particularly proteases, that are located at the surface, thus allowing them to directly interact with their environment. This makes Bacteroidales proteases potentially powerful mediators for microbial interactions. However, we still have limited knowledge on which proteins are trafficked via OMVs from individual Bacteroidales strains and how external factors modulate the composition and the protein localization within OMVs. Moreover, it is unknown how bacterial proteases affect the composition of the gut microbiota and thereby modulate pathogenesis. In this project, we will develop and apply chemical tools to comprehensively characterize Bacteroidales OMVs and their function in microbial interactions. First, we will use activity-based protein profiling to identify bacterial strains and conditions that result in strongest OMV secretion. Next, we will apply chemoproteomic methods to characterize the proteomes of OMVs with a particular focus on serine hydrolases and the lipidation status of predicted lipoproteins. Select proteases found in OMVs will be recombinantly produced and biochemically characterized. Subsequently, we will synthesize fluorogenic substrates, covalent inhibitors and chemical probes tailored towards individual proteases. Having these powerful chemical tools in hand, we will elucidate how OMVs and their associated proteases affect individual strains and bacterial co-cultures. Ultimately, we will apply fluorescent chemical probes to study the dynamic biogenesis and trafficking of OMVs via fluorescence microscopy. Overall, success in these aims will result in a comprehensive understanding of extracellular microbial proteins, the characterization of novel enzymes, and the development of tailored tools to probe the role of OMVs in microbial interactions.