Abstract
Background
Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aidsListeria 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 (Met1–Pro223) and N2 (Gly224–Gly411), and the ADH region contains C1 (Gly412–Val648) and C2 (Pro649–Val866). Each subdomain was cloned and overexpressed inEscherichia 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 KD of 9.50±2.6 nM. The KD of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (PL. 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.
Date of this Version
6-29-2011
DOI
10.1371/journal.pone.0020694
Recommended Citation
Jagadeesan, Balamurugan; Fleishman Littlejohn, Amy E.; Amalaradjou, Mary Ann Roshni; Singh, Atul K.; Mishra, Krishna K.; La, David; Kihara, Daisuke; and Bhunia, Arun K., "N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60." (2011). Department of Food Science Faculty Publications. Paper 5.
http://dx.doi.org/10.1371/journal.pone.0020694
Comments
This is the publisher pdf of Jagadeesan B, Fleishman Littlejohn AE, Amalaradjou MAR, Singh AK, Mishra KK, et al. (2011) N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60. PLoS ONE 6(6): e20694 and is available at: 10.1371/journal.pone.0020694.