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Title: Investigation of a novel S-layer glycosylation cluster within Clostridium difficile
Author: Richards, Emma Jane Lynda
ISNI:       0000 0004 7657 5804
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Clostridium difficile is a Gram-positive spore-forming, anaerobic bacterium that is the leading cause of gastrointestinal infection acquired after antibiotic therapy. The symptoms of Clostridium difficile infection (CDI) are primarily attributed to the action of two toxins Toxin A (TcdA) and Toxin B (TcdB), which have been extensively studied. However, how C. difficile successfully colonizes the gut remains poorly defined. It is likely that the cell wall of C. difficile plays a key role in the interaction and colonization of host gastrointestinal tissues. The outermost layer of the cell wall, the Surface layer (S-layer) is a paracrystalline array composed of a complex of two S-layer proteins (SLPs); the high molecular weight (HMW) SLP and low molecular weight (LMW) SLP, derived by post-translational cleavage of the precursor protein SlpA. Previous studies concluded that SlpA in a variety of strains is not glycosylated. SlpA shows sequence variation between strains and 12 distinct slpA cassettes have been recently described. Type 11 cassette strains possess an unusual genotype; adjacent to slpA, they possess a cluster of genes similar to those observed in other species. The protein products of these genes collectively and sequentially glycosylate surface proteins.   In this study, SlpA of type 11 cassette strain Ox247 was demonstrated to be glycosylated using a combination of nuclear magnetic resonance, mass spectrometry and genetic analysis. The putative structure of the Ox247 glycan was determined collaboratively using mass spectrometry and nuclear magnetic resonance. The SlpA of Ox247 is glycosylated only on the LMW-SLP via a threonine residue, of which this O-linked glycosylation parallels other bacterial S-layer glycosylation examples including Geobacillus stearothermophilus and Paenibacillus alvei. The cluster of 19 genes adjacent to slpA was genetically characterised as an operon. Insertional inactivation of genes predicted to initiate glycosylation (orf2) and to ligate the glycan to the protein (orf19) extracellularly resulted in a S-layer deficient in glycosylation and migration of the LMW-SLP on SDS-PAGE at its predicted MW of 20 kDa. However, upon glycosylation, the LMW SLP shows an aberrant migration to 50 kDa, representing the addition of sugar mass to the LMW-SLP. Insertional activation of genes predicted to be glycosyltransferases produced several protein species bearing varying length glycans as confirmed by mass spectrometry, and differential migration on SDS PAGE at MWs between 25-40 kDa. The data obtained is consistent with the S-layer glycosylation model proposed in Geobacillus stearothermophilus, where the glycan is synthesised inside the cell, translocated via an ABC transporter through the membrane to the targeted protein and ligated via an O-linkage. Creation of partially glycosylated mutants suggests that the specificity of this ABC transporter is not restricted to the full-length glycan. This study provides the first functional characterisation of S-layer glycosylation in a Gram-positive pathogen. A broad range of in vitro experiments including antibiotic resistance, motility, aggregation, sporulation, biofilm formation, toxin production and adhesion to mammalian cells, coupled with in vivo analysis using a mouse model were performed to analyse the lifestyle and pathogenesis of C. difficile cells with this post-translational modification. Our data indicates that SlpA glycosylation affects cell division, adhesion and biofilm production.
Supervisor: Fairweather, Neil Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral