Heat Shock Protein 60 Acts as a Receptor for theListeriaAdhesion Protein in Caco-2 Cells

Abstract

The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

LAP, an alcohol acetaldehyde dehydrogenase enzyme in Listeria, promotes bacterial adhesion to enterocyte-like Caco-2 cells only in pathogenic species

“…We previously reported the significance of LAP as a pathogenic factor in L. monocytogenes, promoting bacterial adhesion to intestinal epithelial cells by interacting with mammalian receptor Hsp60 Pandiripally et al, 1999;Wampler et al, 2004). LAP homologues are present in non-pathogenic Listeria species, and thus it was speculated that LAP may serve as a general colonization/adhesion factor in Listeria.…”

“…2a). In our previous study, mammalian cell-associated Hsp60 was shown to be a receptor for LAP (Wampler et al, 2004). In the present study, we employed a competitive assay, where adhesion was examined in bacteria pretreated with purified Hsp60.…”

“…This assay was performed as described before (Kim et al, 2006;Wampler et al, 2004). Briefly, fresh Listeria cells were harvested, washed in PBS and used at an m.o.i.…”

“…Previous research has demonstrated that LAP interacts with the cellular receptor Hsp60 (Wampler et al, 2004), and exhibits preferential affinity towards cells of intestinal origin Kim et al, 2006;Pandiripally et al, 1999). We have recently shown that LAP secretion is essential for L. monocytogenes pathogenesis and that its secretion is SecA2-dependent (Burkholder et al, 2009).…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…The interaction of this alcohol acetaldehyde dehydrogenase with Hsp60, its host cell receptor, promotes bacterial adhesion to intestinal cells. [29][30][31][32] Anaerobic growth induces significant increases in the level of lap transcript and Lap secretion via the accessory secretion system SecA2 (see regulation section). Oral administration of lap-deficient strains to mice confirmed that Lap is essential for full virulence.…”