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To protect or not to protect; that is the mucus. Determining the co-operation between commensals and pathogens in cross-feeding of intestinal mucus
Secondary Supervisor(s): Dr Lucy Crouch
University of Registration: University of Birmingham
BBSRC Research Themes: Understanding the Rules of Life (Microbiology)
Project Outline
Commensal bacteria within the human gut microbiome provides protection against infection by intestinal pathogens, such as Enteroaggregative Escherichia coli (EAEC). The mucus layer lining the intestinal tract also serves as a barrier separating pathogens from the underlying epithelium. Commensals are in perfect harmony with the mucus layer, encoding specialised enzymes (CAZymes) to metabolise mucus glycan residues as a nutrient source. However, this harmony is disturbed during pathogenic infection. One such pathogen EAEC, encodes a Protein important for colonisation (Pic), which functions as a mucinase that cleaves the polypeptide backbone of mucins within the mucus.
Preliminary work
Using human gut organoids, we have established a mucosoid microbiota infection model. Mucosoid cultures comprise a polarised epithelial layer that is grown on a transwell filter (1). The epithelial cells continually secrete a thick layer of mucus, which can be harvested and used in downstream experimentation.
Dr. Rossiter’s team have validated this model, using combined RNAscope in situ hybridisation and immunohistochemistry.
Dr. Crouch’s team have recently characterised panels of mucin-specific enzymes from a prominent human gut commensal that break pig stomach mucins to their monosaccharide components (2).
Aims
Using this microbiota infection model we will test how the degradation of mucus by CAZymes (from commensals) and Mucinases (from pathogens) modulates colonisation ex vivo.
To do this, we will achieve the following:
1) Generate three-dimensional intestinal organoids from ‘normal’ colon tissue
2) Culture mucosoids from 3D intestinal organoids and co-infect these with a range of intestinal commensal bacteria, such as Akkermansia muciniphila and intestinal pathogen EAEC 042
3) Monitor colonisation of the commensals and pathogen over the course of the infection (up to 7 days), using viability counts, RNAscope ISH and confocal microscopy
4) Harvest the mucus and characterise its composition after infection to determine the bacterial-mediated breakdown of its peptide backbone and glycan components, using established methods (2).
In summary, these insights will provide a mechanistic understanding of how commensals use mucus to provide colonisation resistance to the host and simultaneously for the resolution of an established infection.
References
1. Boccellato F, Woelffling S, et al. (2019). Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection. Gut. 68(3):400-413.
2. Bakshani et al., 2024, BioRxiv, https://doi.org/10.1101/2024.03.27.586211.
3. Paone, P. and Cani, P.D. (2020) Mucus barrier, mucins and gut microbiota: the expected slimy partners? Gut. 69:2232-2243.
