GROEL HEAT-SHOCK-PROTEIN OF HAEMOPHILUS-DUCREYI - ASSOCIATION WITH CELL-SURFACE AND CAPACITY TO BIND TO EUKARYOTIC CELLS

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protei n (Hsp), is a dominant protein produced not only in response to heat s tress hut also under in vitro growth conditions. Extracellular localiz ation of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supe rnatants of washed bacteria by immunoblotting with a Haemophilus ducre yi GroEL-specific mouse monoclonal antibody (BB11). To investigate bin ding of the Hsp to eukaryotic cells, the 58.5-kDa Bsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 c ells, HeLa cells, and human fibroblasts; and then analyzed by immunobl otting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results . In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to t he eukaryotic cells tested but exerted only limited (about 20%) inhibi tion af H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducrey i adherence to HEp-2 sells, indicating possible involvement of the 58. 5-kDa Hsp in the attachment of bacteria to host cells and to each othe r.