Details

Autor(en) / Beteiligte

• Jagadeesan, Balamurugan
• Fleishman Littlejohn, Amy E
• Amalaradjou, Mary Anne Roshni
• Singh, Atul K
• Mishra, Krishna K
• La, David
• Kihara, Daisuke
• Bhunia, Arun K

Titel

N-Terminal Gly.sub.224-Gly.sub.411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60

Ist Teil von

• PloS one, 2011-06, Vol.6 (6), p.e20694

Ort / Verlag

Public Library of Science

Erscheinungsjahr

2011

Link zum Volltext

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• Background Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated. Methods Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met.sub.1 -Pro.sub.223) and N2 (Gly.sub.224 -Gly.sub.411 ), and the ADH region contains C1 (Gly.sub.412 -Val.sub.648) and C2 (Pro.sub.649 -Val.sub.866). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells. Results The N2 subdomain exhibited the greatest affinity for Hsp60 with a K.sub.D of 9.50#177;2.6 nM. The K.sub.D of full-length LAP (7.2#177;0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 [micro]m diameter) coated with N2 subdomain showed significantly (P0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells. Conclusion These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies.