Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor

Abstract

Listeria adhesion protein (LAP) is an important adhesion factor in Listeria monocytogenes and interacts with its cognate receptor, mammalian heat shock protein 60 (Hsp60). The genetic identity of LAP was determined to be alcohol acetaldehyde dehydrogenase (Aad). A recombinant Escherichia coli strain expressing aad confirmed the involvement of Aad in adhesion to Caco-2 cells. Binding kinetics (ka) of recombinant LAP (rLAP) to Hsp60 was examined in a surface plasmon resonance sensor and was determined to be 5.35 x 10(8) M(-1) s(-1) and it was equivalent to the binding of anti-Hsp60 antibody (ka = 2.15 x 10(9) M(-1) s(-1)) to Hsp60. In contrast, Internalin B, an adhesion/invasion protein from L. monocytogenes, used as a control, had binding kinetics (ka) of only 2.9 x 10(6) M(-1) s(-1). The KD value of rLAP was 1.68 x 10(-8) M, which was significantly lower than Internalin B (KD = 6.5 x 10(-4) M). These results suggest that Hsp60 has significantly higher avidity for anti-Hsp60 antibody and LAP than Internalin B. In summary, LAP is identified as an alcohol acetaldehyde dehydrogenase and binding of recombinant E. coli to Caco-2 cells or rLAP to Hsp60 protein was found to be highly specific.

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

“…Amplified products were cloned into pGEMT-easy vector (Promega) and designated pGEMT-LAP-iva, pGEMT-LAP-wel and pGEMT-LAP-inn, respectively. Products were subcloned into pET28a using BamHI and SacI restriction sites introduced into the forward and reverse primers, resulting in pELAP-iva, pELAP-wel and pELAP-inn (Kim et al, 2006). Finally, all three constructs were transformed into Escherichia coli BL21(DE3) (Novagen) and overexpression of LAP was achieved by adding 1 mM IPTG during exponential growth.…”

“…Finally, all three constructs were transformed into Escherichia coli BL21(DE3) (Novagen) and overexpression of LAP was achieved by adding 1 mM IPTG during exponential growth. Recombinant (r) LAP iva (L. ivanovii), LAP wel (L. welshimeri) and LAP inn (L. innocua) proteins were purified on nickel affinity columns (EMD Chemicals) and examined by Western blotting (Kim et al, 2006).…”

“…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).…”

Unfolding the relationship between secreted molecular chaperones and macrophage activation states

“…While an unusual proposal, it must be seen against the background of the emerging evidence for the diverse moonlighting actions of cell stress proteins. To highlight this, the prototypic molecular chaperone, Hsp60, has recently been identified as: (1) a human cell surface protein involved in sperm capacitation (Asquith et al 2004); (2) an insect neurotoxin (Yoshida et al 2001) and (3) a human protein acting as a tethering ligand for the alcohol acetaldehyde dehydrogenase of Listeria monocytogenes, thus allowing this bacterium to adhere to human cells and colonise humans (Kim et al 2006). This is a remarkably diverse set of functions for any protein and suggests many more surprises are in store for researchers working with chaperonin 60 and presumably with all other stress response proteins.…”