Mucin-derived sialic acid metabolism in gut bacteria

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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.