SURFACE-STRUCTURE, HYDROPHOBICITY, PHAGOCYTOSIS, AND ADHERENCE TO MATRIX PROTEINS OF BACILLUS-CEREUS CELLS WITH AND WITHOUT THE CRYSTALLINESURFACE PROTEIN LAYER

Nonopsonic phagocytosis of Bacillus cereus by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface prope rties and structure was studied. Two reference strains (ATCC 14579(T) and ATCC 4342) and two clinical isolates (OH599 and OH600) from period ontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinoge n, One-day-old cultures of strains OH599 and OH600 were readily ingest ed by PMNs in the absence of opsonins, while cells from 6-day-old cult ures were resistant. Both young and old cultures of the reference stra ins of B, cereus were resistant to PMN ingestion, Preincubation of PMN s with the phagocytosis-resistant strains of B, cereus did not affect the phagocytosis of the sensitive strain, Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline prote in layer on the cell surface. In thin-sectioned cells of older culture s (3 to 6 days old), the S-layer was observed to peel off from the cel ls. No S-layer was detected on the reference strains. Extraction of ce lls,vith detergent followed by sodium dodecyl sulfate-polyacrylamide g el electrophoresis revealed a major 97-kDa protein from the strains OH 599 and OH600 but only a weak 97-kDa band from the reference strain AT CC 4342, One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and f ibronectin, In contrast, reference strains (hydrophobicity, -1.0 to 4. 2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins. Gold-labell ed biotinylated fibronectin was localized on the S-layer on the cell s urface as well as on fragments of S-layer peeling oi the cells of a 6- day-old culture of B, cereus OH599, Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not t o fibronectin, Heating the cells at 84 degrees C totally abolished bin ding to both proteins. Benzamidine, a noncompetitive serine protease i nhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased. Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of B, cereus cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane p roteins. Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active prot ease evidently attached to the S-layer.