Mucin-derived sialic acid metabolism in gut bacteria

胃肠道是由不同的微生物群落（称为肠道微生物群）定植的，它们的组成对人类健康有深远的影响。人体肠道微生物群的组成受到肠道中复杂膳食和宿主碳水化合物降解的极大影响。与肠道内层有关的细菌有能力以覆盖胃肠道的黏液层提供的糖链为食。唾液酸或n -乙酰神经氨酸（Neu5Ac）是存在于粘蛋白碳水化合物链末端的一种丰富的糖残基，是肠道细菌的关键靶点。唾液酸的细菌唾液苷酶分解代谢释放游离的Neu5Ac的粘蛋白，可在粘膜环境驱动肠道炎症和感染。例如，在抗生素治疗期间和之后，肠道中游离唾液酸水平的升高会促进艰难梭菌和沙门氏菌的扩张，因为这些细菌缺乏唾液酸酶（但拥有允许细菌利用游离Neu5Ac的机制），因此依赖于肠道微生物群成员从粘蛋白中释放的游离Neu5Ac。我们最近在肠道共生细菌中发现了一种不寻常的唾液酸酶活性，它不像水解唾液酸酶那样释放游离的Neu5Ac，而是从粘蛋白中产生转糖基化产物2,7-无水Neu5Ac，这将该酶归类为分子内反式唾液酸酶（it -唾液酸酶）。该提案的目的是在分子水平上表征肠道细菌如何利用2,7-无水Neu5Ac，并测试it -唾液酸酶将i)通过使肠道细菌以自私的方式从粘蛋白中产生和利用2,7-无水Neu5Ac，从而为肠道细菌提供体内主要的营养优势，ii)通过减少Neu5Ac的可用性来限制肠道病原体的生长（结果是使它们挨饿）。具体来说，这个项目旨在回答以下问题：1。鼠体内2,7-无水neu5ac的代谢途径是什么？哪些肠道细菌能够利用2,7-无水neu5ac作为唯一的营养来源？it -唾液酸酶是否赋予肠道细菌在体内的竞争优势？gnavus菌株对肠道游离唾液酸水平的影响是什么？产唾液酸酯酶的菌株能损害鼠伤寒沙门氏菌在体内的定植吗？该项目分为三个目标来解决这些问题。在第一个目标中，我们将利用我们的重组it -唾液酸酶酶合成2,7-无水neu5ac（非市售），以适当的量来生物化学研究我们的模式生物R. gnavus的代谢途径，鉴定和表征参与这一过程的蛋白质。然后，我们将通过结合生物信息学分析、稳定同位素探测（SIP）和体外实验验证，将这项工作扩展到肠道微生物群，以确定来自人类肠道的哪些其他共生细菌能够利用2,7-无水neu5ac。基于我们的体外数据（已发表的和初步的），我们将在小鼠模型中进行实验，以确定表达it唾液酸酶的微生物对其在粘膜层定殖、调节肠道唾液酸水平和减少沙门氏菌感染能力的影响。这一基本知识对于探索新的抗感染方法作为抗生素的替代品非常重要，这些方法通过调节粘膜环境而不是针对病原体本身来减轻抗菌素耐药性（AMR）的风险。

The gastrointestinal (GI) tract is colonized by a diverse community of microbes (called the gut microbiota) whose composition has a profound impact on human health. The composition of the human gut microbiota is greatly influenced by the degradation of complex dietary and host carbohydrates in the gut. Bacteria associated with the lining of the gut have the ability to forage on sugar chains provided by the mucus layer covering the GI tract. Sialic acid or N-acetylneuraminic acid (Neu5Ac) is an abundant sugar residue found in terminal location of mucin carbohydrate chains and a key target of intestinal bacteria. Sialic acid catabolism by bacterial sialidases releases free Neu5Ac from mucins which availability in the mucosal environment drives intestinal inflammation and infection. For example, elevated levels of free sialic acid in the gut, during and post antibiotic treatments, promote the expansion of Clostridium difficile and Salmonella, as these bacteria lack a sialidase (but possess the machinery allowing the bacteria to utilise free Neu5Ac) and thus rely on free Neu5Ac released from mucins by members of the gut microbiota. We recently discovered an unusual sialidase activity in gut commensal bacteria, which instead of releasing free Neu5Ac as in the case of hydrolytic sialidases, produces a transglycosylation product, 2,7-anhydro-Neu5Ac from mucins, which classifies this enzyme as an intramolecular trans-sialidase (IT-sialidase). The aim of the proposal is to characterise at the molecular level how 2,7-anhydro-Neu5Ac is utilised by gut bacteria and test the hypothesis that IT-sialidase will i) provide gut bacteria with a major nutritional advantage in vivo by enabling them to produce and utilise 2,7-anhydro-Neu5Ac from mucins in a selfish manner and ii) limit enteric pathogens outgrowth by reducing the availability of Neu5Ac (and starving them as a result). Specifically, this project aims to answer the following questions:1. What is the pathway for 2,7-anhydro-Neu5Ac metabolism in R. gnavus?2. Which gut bacteria are able to utilise 2,7-anhydro-Neu5Ac as sole source of nutrient?3. Do IT-sialidases confer gut bacteria with a competitive advantage in vivo?4. What is the impact of R. gnavus strains on the level of free sialic acid in the gut?5. Can IT-sialidase producing strains impair S. Typhimurium colonisation in vivo?The project is divided into 3 objectives to address these questions.In the first objective, we will exploit our recombinant IT-sialidase to enzymatically synthesise 2,7-anhydro-Neu5Ac (not commercially available) in suitable amount to biochemically study the metabolic pathways in our model organism R. gnavus, identify and characterise the proteins involved in this process.We will then expand this work to the gut microbiota by using a combination of bioinformatics analyses coupled with stable isotope probing (SIP) and experimental validation in vitro to identify which other commensal bacteria from the human gut are able to utilise 2,7-anhydro-Neu5Ac.Building from our in vitro data (published and preliminary), we will then perform experiments in mouse models to determine the impact of the IT-sialidase-expressing microbes on their ability to colonise the mucosal layer, modulate the level of sialic acid in the gut, and reduce Salmonella infection.This basic knowledge is important to explore novel anti-infective approaches as alternatives to antibiotics, which alleviate the risk of antimicrobial resistance (AMR) by modulating the mucosal environment rather than targeting the pathogen per se.