Structural studies of a novel adhesin involved in hostspecific lactobacillus reuteri biofilm formation in the vertebrate gastrointestinal tract AND Purification method development for structural studies of WaaP: an essential gram-negative bacteria lipopolysaccharide kinase

Sequira, Saannya (2016) Structural studies of a novel adhesin involved in hostspecific lactobacillus reuteri biofilm formation in the vertebrate gastrointestinal tract AND Purification method development for structural studies of WaaP: an essential gram-negative bacteria lipopolysaccharide kinase. Doctoral thesis, University of East Anglia.

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Abstract

Structural Studies of a Novel Adhesin Involved in Host-specific Lactobacillus reuteri Biofilm Formation in the Vertebrate Gastrointestinal Tract
In vertebrates, strains of the gut symbiont, Lactobacillus reuteri, form biofilms on the proximal gastric stratified epithelium in a host-dependent manner. The gene for a novel serine-rich repeat (SRR) adhesin, conserved amongst rat and porcine L. reuteri,
is essential for successful biofilm formation, suggesting initial adhesion to be the most crucial step for colonisation, and a possible driver of host-specificity. High resolution crystal structures of the partial binding regions (BR) of two SRR adhesins, BR55 and BR78, from porcine L. reuteri ATCC 53608 and rodent L. reuteri 100-23 strains, respectively, are reported. Although 50% similar, the BRs were highly conserved in
overall structure and in the orientation of putative binding residues. BR55 and BR78’s right-handed parallel β-sheet solenoid-type fold (typical of pectate lyase like proteins), and their aromatic residue-rich putative binding cleft indicated that glycans were the most likely binding substrates. Potential binding residues were identified, allowing
mutagenesis for functional studies. SAXS analysis of the complete BR55 protein revealed two regions of intrinsic disorder at the N- and C-terminal, beyond the ordered β-solenoid core. Structural information from this investigation provided valuable insights for ongoing biochemical analyses.
Purification Method Development for Structural Studies of WaaP: an Essential Gram-Negative Bacteria Lipopolysaccharide (LPS) Kinase
LPS is a vital component of Gram-negative bacteria outer membrane (OM), playing a crucial role in maintaining OM integrity against host-immune defenses and antibiotics. In turn, phosphorylation of the LPS core oligosaccharide is essential for structural viability of LPS in E.coli, S. enterica and P. aeruginosa. The waaP gene, encoding a Kdo sugar kinase, is a proven virulence factor, essential for phosphorylating LPS.
Furthermore, waaP is conserved amongst many Gram-negative pathogens, and evokes interest as a potential antibiotic target. This thesis reports purification method development of recombinant WaaP for structural studies, and proposes strategies for accessing other WaaP homologues for crystallography. P. aeruginosa WaaP was
successfully purified in adequate amounts for crystallography.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Brian Watkins
Date Deposited: 29 Nov 2017 12:52
Last Modified: 31 Dec 2018 01:38
URI: https://ueaeprints.uea.ac.uk/id/eprint/65623
DOI:

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