Molecular characterization of Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase homologue involved in the adhesion of Listeria monocytogenes to intestinal epithelial cells
Abstract
Listeria adhesion protein (LAP) promotes the adhesion of Listeria monocytogenes to the intestinal epithelial cells by interacting with mammalian receptor heat shock protein 60 (Hsp60). LAP consists of 866 amino acids with an N-terminal aldehyde dehydrogenase and a C-terminal alcohol dehydrogenase region. Though LAP acts as a virulence factor, its homologues are present in all Listeria spp., except L. grayi. Amino acid sequences of LAP of Listeria spp. exhibit a high degree of similarity but are not identical to each other. Thus the goals of this study were (i) determine if LAP is a general or a pathogen specific adhesion factor and understand the involvement of LAP in virulence mechanism; and (ii) identify the LAP domain(s) responsible for the interaction with its receptor Hsp60. The interaction between recombinant LAP (rLAP) from pathogenic and non pathogenic Listeria spp., with its receptor Hsp60 was investigated using a surface plasmon resonance biosensor. The binding kinetics parameters (association rate constant and dissociation equilibrium constant) for rLAP-Hsp60 interaction were similar for both pathogenic and non-pathogenic Listeria spp. In cell culture adhesion experiments using enterocyte-like Caco-2 cells, presence of exogenous Hsp60 competitively inhibited the adhesion of only L. monocytogenes and L. ivanovii but not the other Listeria spp. Furthermore, LAP was detected in the secreted portion of L. monocytogenes protein preparations but not from that of L. innocua. Also, the cell surface expression of LAP in L. monocytogenes was two-fold higher than that of L. innocua . Thus, LAP is secreted and possibly gets re-associated on the cell surface of pathogenic Listeria through an unknown mechanism while secretion or the surface re-association of LAP is impaired in nonpathogenic Listeria. To determine the LAP domain interacting with Hsp60, LAP structure was predicted and defined into 4 regions (N1, N2, C1 and C2) which were capable of interacting with Hsp60. Purified recombinant N1, N2, C1 and C2 domains were used in Far Western and immunofluorescence assay to determine the LAP binding domain to Hsp60. N2 LAP reacted with Hsp60 in Far Western and also showed significantly (P<0.05) higher binding to Hsp60 when compared to other LAP domains in ELISA. Also, the dissociation equilibrium constant and binding to Hsp60 of N2 LAP and full length LAP were similar in nature. Thus, N2 is determined to be responsible for LAP interaction with its mammalian receptor Hsp60. In conclusion, bacterial cell surface LAP mediates adhesion of L. monocytogenes to intestinal epithelial cells through interaction with host cell Hsp60 and N2 domain of LAP is responsible for this interaction.
Degree
Ph.D.
Advisors
Bhunia, Purdue University.
Subject Area
Agronomy|Microbiology
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