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Title: Structural studies of a novel adhesin involved in host-specific 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
Author: Sequira, Saannya
ISNI:       0000 0004 6497 7611
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2016
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available