Proceedings of The Physiological Society
University of Newcastle (2004) J Physiol 559P, PC12
Inhibition of Pnc adhesion to host mucosa and identification of Host target proteins by Phages isolated by phage display analysis.
Blau, Karin; Shagan, Malou; Kaganovitz, Antonina; Portnoi, Maxim; Ling, Eduard; Dagan, Ron; Mizrachi Nebenzahl , Yaffa;
1. Immunology and microbiology, Ben Gurion University of the Negev, Beer Sheva, Israel. 2. Physiology, Ben Gurion University of the Negev, Beer Sheva, Israel.
Aims: Pnc adhesion to host mucosa is a prerequisite for carriage and disease development. Pnc adhesion is probably mediated by CW-L proteins. Filamentous bacteriophages have been genetically engineered so to incorporate the DNA template corresponding to the peptide displayed on its surface. We used random phage display library screening of Pnc CW-L for identification of their binding peptide in attempt to identify their receptors and develop inhibitors of Pnc adhesion. Methods: Pnc serotype 3 (WU2) CW-L were purified by fetuin affinity chromatography, separated by 2D PAGE and sequenced. 20 proteins were detected. Among them, fructose-biphosphate aldolase (FBA) and elongation factor G (EFG) proteins, previously identified as lectins, were cloned and expressed in E. coli. Anti-FBA and anti-EFG antibodies were produced in mice and rabbits, respectively. The proteins and the antibodies were used for phage display library screening. Results: 15 EFG and 30 FBA binding peptides were found. Five out 15 phages EFG binding peptides and 4 out of 5 phages expressing FBA binding peptide inhibited Pnc adhesion to cultured human HaCat and A549 epithelial cells, respectively. The inhibitory peptides were sequenced. Conclusions: 1) Phage display allows identification of novel peptide inhibitor of Pnc adhesion to host mucosa. 2) We are currently analyzing the significance of these peptides and the existence of host homologous proteins.
Where applicable, experiments conform with Society ethical requirements