Mucin-derived sialic acid metabolism in gut bacteria

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.

The mammalian intestinal tract is protected by a mucus layer, which contains heavily glycosylated mucin proteins that comprise up to 80% carbohydrate and are often terminated by sialic acid residues. The most abundant sialic acid is N-acetylneuraminic acid (Neu5Ac) which can serve as an energy source by commensal and pathogenic bacteria. The availability of sialic acid in the mucosal compartment is particular important for the proliferation of enteropathogenic bacteria during infection. We recently discovered an usual sialidase activity in Ruminococcus gnavus, which instead of releasing free sialic acid, produces a trans-glycosylation product, 2,7-anhydro-Neu5Ac specifically from alpha-2-3-linked sialylated substrates, which classifies this enzyme as an intramolecular trans-sialidase and recently confirmed that 2,7-anhydro-Neu5Ac was used as a substrate by R. gnavus. Our bioinformatics analysis showed that IT-sialidases are predicted to occur in 11% of bacterial metagenomes examined, suggesting that bacterial IT-sialidases may play a key role in driving symbiotic host associations. This project will (1) examine the mechanisms and occurrence of IT-sialidase mediated sialic acid metabolism in gut bacteria, (2) test the hypothesis that it provides a competitive nutritional advantage to the bacteria and (3) investigate how this directly affects the level of sialic acid in the gut and impact on pathogens in vivo. Outcomes of the project will provide an enhanced understanding of sialic acid metabolism in the intestinal ecosystem and a molecular route to devise alternative strategies to antibiotics for reducing or preventing enteric infection and antimicrobial resistance.

Public Health
Infectious diseases are a growing public health issue due to increasing global anti-microbial resistance (AMR). In humans, microbial infection typically starts with bacteria interacting with the mucosal surfaces which are more exposed and prone to infection. Elevated levels of sialic acid are induced by antibiotic therapy and pathogen infection. Therefore, reducing the amount of sialic acid in the mucosal environment is a novel strategy to reduce microbial infection. Currently much effort is centred upon developing new anti-microbial agents. This work will provide a completely novel approach to alleviate the risk of AMR by modulating the mucosal environment rather than targeting the pathogen per se. Within the timeframe of this study, we will focus on Salmonella for which antimicrobial resistance to cephalosporin has greatly increased. Other antibiotic-associated enteric pathogens include Clostridium difficile, an ubiquitous organism that recently emerged in animals and in humans as the main cause of nosocomial diarrhoea. In the last decade incidences of C. difficile infections (CDI) increased markedly, partly due to the use of antimicrobials, in particular cephalosporins. Future applications could target multi-drug resistant pathogens targeting other mucosal sites in the body such as Staphylococcus aureus, an ubiquitous bacterial pathogen that also uses sialic acid as nutrient source, and respiratory pathogens including Hemophilus influenzae, Streptococcus pneumoniae, or Pseudomonas aeruginosa which induce expression of sialic acid as adhesion sites. The longer-term application of this research will be designing and testing suitable routes and pharmaceutical formulations for IT-sialidase mucosal delivery depending on the site of the infection. These could include the use of pre/probiotic/synbiotic approaches in the gut or aerosol in the airways.

Carbohydrate/Enzyme biotech companies
The outcomes of this work will be of direct interest to companies commercializing novel glycoenzymes (e.g. trans-sialidases) (e.g. Prozomix, Megazyme, Nzytech) and those developing effective bioassays, glycosidase inhibitors, and carbohydrate-based diagnostics and therapeutics (e.g. Ludger; Iceni Diagnosis), or speciality carbohydrates such as sialylated oligosaccharides (e.g. Glycom, Carbosynth, Inbiose) for potential application as neutraceutical, pharmaceutical and cosmetic ingredients.

Biopharmaceutical/drug companies
The research may also be of interest to biopharmaceutical companies (e.g. GlaxoSmithKline) developing novel sialidase (or neuraminidase) inhibitors incl. Influenza neuraminidase inhibitors.

Food and Healthcare companies (Probiotics/Microbiome)
This research may also be of interest to companies interested in modulating the gut microbiome via pro/prebiotic approaches (e.g. Danone, Nestle, Yakult) but also an increasing number of biotech startups operating in the emerging gut microbiome space (e.g. MicroBiome Therapeutics, Enterome Bioscience).

Early career researcher
This project will provide a unique training opportunity for a post-doctoral scientist to work in a multidisciplinary environment spanning research Labs at IFR and UEA (BIO and ENV). There will also be opportunities for undergraduate students (from UEA) and visiting scientists to gain research experience through short-term placements. The PDRA will receive expert training in anaerobic microbiology, heterologous expression, carbohydrate biosynthesis and analysis (HPLC, MS- and NMR-based methods), enzymatic assays, bioinformatics (TGAC) and statistical analyses. The techniques are well-established and there is considerable expertise in conducting research on glycobiology and gut microbiology. There will be opportunities to collaborate with chemists and structural biologists. The PDRA will benefit from the established international network of academic and industrial collaborations of the NRP Labs.