Identification of host-pathogen interacting molecules of Campylobacter jejuni using phage display technology and in silico sequence analysis

Abruquah, Harry H. (2009) Identification of host-pathogen interacting molecules of Campylobacter jejuni using phage display technology and in silico sequence analysis. PhD thesis, University of Nottingham.

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Abstract

Campylobacter jejuni is the leading cause of human gastroenteritis world-wide, and the antecedent infection in Guillain-Barré syndrome. Although much progress has been made regarding its virulence determinants,our understanding of the molecular basis of C. jejuni pathogenesis still lags behind that of other enteric pathogens.

Bacterial pathogenesis is often dependent on proteinaceous virulence factors transported to the bacterial cell surface or released into the external environment. Understanding the molecular basis of interactions between these proteins and host cells is necessary in understanding and controlling infections.

Background: Previously identified adhesins of C. jejuni NCTC11168 have still not provided us with a total understanding of the pathogenesis of campylobacteriosis. It is hypothesized that as yet unidentified C. jejuni surface proteins interact with host proteins contributing to colonisation and pathogenesis. This study sought to screen the genome of C. jejuni NCTC11168 to identify additional genes that may code for other adhesins using phage display technology and in silico sequence analysis.

Methods: A phage display library was constructed by the insertion of randomly fragmented chromosomal DNA of C. jejuni NCTC11168 into the phagemid vector pG8SAET. Following affinity panning of the library against holo- and apo-lactoferrin, enriched clones were screened with ELISA to identify affinity-binding clones. Several phage clones were randomly selected and their C. jejuni DNA inserts sequenced and analysed with bio-informatic tools.

In order to identify novel autotransporter proteins of C. jejuni, the amino acid sequences of previously described adhesion-associated autotransporters were employed in BLAST searches of the predicted coding sequences of C. jejuni NCTC11168 genome database.

Results: Screening of the phage display library resulted in the identification of C. jejuni NCTC11168 gene, Cj0609c, encoding a putative periplasmic protein, designated LimC, predicted to be a member of the SGNH-family of hydrolases, a diverse family of lipases and esterases.

Searching the genome of C. jejuni NCTC11168, Cj0628 was identified as coding a protein with characteristics of autotransporters, designated CapA. CapA was demonstrated to be surface-exposed, mutants of which had a lowered ability to associate with or invade Caco-2 cells and failed to colonize chicken guts, indicating that CapA plays a role in host association and colonization by Campylobacter.

Conclusion: LimC may be involved in binding of C. jejuni to lactoferrin for iron acquisition in vivo, and/or play further role in adhesion, colonization and internalisation of C. jejuni into host cells. CapA also plays a role in adhesion. Further characterization of these proteins should contribute to our understanding of the pathogenesis of C. jejuni.

Item Type:Thesis (PhD)
Supervisors:Wooldridge, K.G.
Ala'Aldeen, D.A.
Uncontrolled Keywords:Campylobacter jejuni, Phage display, Bacteriophages, Pathogenesis, Campylobacter infections, Host-pathogen interactions
Faculties/Schools:UK Campuses > Faculty of Medicine and Health Sciences > School of Molecular Medical Sciences
ID Code:813
Deposited By:Dr Harry H ABRUQUAH
Deposited On:18 Jan 2010 13:50
Last Modified:18 Jan 2010 13:50

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