Mucin-derived sialic acid metabolism in gut bacteria

肠道细菌中粘蛋白衍生的唾液酸代谢

• 批准号: BB/P008895/1
• 负责人: Nathalie Juge
• 金额: $ 70.03万
• 依托单位: Quadram Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP008895%2F1
• 关键词: Mucin; derived; sialic; acid; metabolism

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.

胃肠道（GI）由多种微生物群落（称为肠道微生物群）定殖，其组成对人类健康具有深远影响。人类肠道微生物群的组成受到肠道中复杂饮食和宿主碳水化合物降解的极大影响。与肠道内壁相关的细菌能够在覆盖胃肠道的粘液层提供的糖链上觅食。唾液酸或N-乙酰神经氨酸（Neu 5Ac）是在粘蛋白糖链的末端位置发现的丰富的糖残基，并且是肠道细菌的关键靶标。由细菌唾液酸酶催化的唾液酸从粘蛋白释放游离Neu 5Ac，其在粘膜环境中的可用性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升高，促进艰难梭菌和沙门氏菌的扩增，因为这些细菌缺乏唾液酸酶（但具有允许细菌利用游离Neu 5Ac的机制），因此依赖于由肠道微生物群成员从粘蛋白释放的游离Neu 5Ac。我们最近发现了一种不寻常的唾液酸酶活性在肠道细菌，而不是释放游离的Neu 5Ac的水解唾液酸酶的情况下，产生转糖基化产物，2，7-脱水-Neu 5Ac从粘蛋白，它分类这种酶作为分子内转唾液酸酶（IT-唾液酸酶）。该提案的目的是在分子水平上验证肠道细菌如何利用2，7-脱水-Neu 5Ac，并测试以下假设：IT-唾液酸酶将i）通过使肠道细菌能够产生和利用2，7-脱水-Neu 5Ac，ii）通过减少Neu 5Ac的可用性来限制肠道病原体的生长（结果是饿死他们）。具体而言，本项目旨在回答以下问题：1。2，7-脱水-Neu 5Ac在R. gavus？2.哪些肠道细菌能够利用2，7-脱水Neu 5Ac作为唯一的营养来源？3. IT-唾液酸酶赋予肠道细菌体内竞争优势吗？4. R的影响是什么？gavus菌株对肠道中游离唾液酸水平的影响？5.产IT-唾液酸酶菌株能损害S.鼠伤寒杆菌体内定殖？本项目主要分为三个目标：第一个目标是利用我们的重组IT-唾液酸酶，酶促合成适量的2，7-脱水Neu 5Ac（目前尚未上市），以生物化学的方法研究我们的模式生物R. gnavus，识别和鉴定参与这一过程的蛋白质。然后，我们将通过使用生物信息学分析与稳定同位素探测（SIP）和体外实验验证相结合，将这项工作扩展到肠道微生物群，以识别来自人类肠道的其他肠道细菌能够利用2，7-脱水-Neu 5Ac。（已发表和初步），然后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它们在粘膜层定殖、调节肠道中唾液酸水平减少沙门氏菌感染。这些基础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，其通过调节粘膜环境而不是靶向病原体本身来减轻抗微生物剂耐药性（AMR）的风险。

项目成果

Ruminococcus gnavus: friend or foe for human health.

• DOI: 10.1093/femsre/fuad014
• 发表时间: 2023-03-10
• 期刊: FEMS microbiology reviews
• 影响因子: 11.3

Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.

• DOI: 10.1074/jbc.ra120.014454
• 发表时间: 2020-10-02
• 期刊: The Journal of biological chemistry
• 作者: Bell A;Severi E;Lee M;Monaco S;Latousakis D;Angulo J;Thomas GH;Naismith JH;Juge N
• 通讯作者: Juge N

Glycobiology of Host‐Microbe Interactions in the Gut

肠道宿主-微生物相互作用的糖生物学

• 发表时间: 2019
• 期刊: The FASEB Journal
• 作者: N. Juge

Mucosal glycan degradation of the host by the gut microbiota.

• DOI: 10.1093/glycob/cwaa097
• 发表时间: 2021-06-29
• 期刊: Glycobiology
• 影响因子: 4.3
• 作者: Bell A;Juge N
• 通讯作者: Juge N

A special sugar: how sialic acid impacts on metabolism, health and disease

一种特殊的糖：唾液酸如何影响新陈代谢、健康和疾病

• 发表时间: 2019
• 期刊: Microbiology Today
• 作者: Bell A