RESUMEN
It has been shown that Fusobacterium nucleatum PK1594 coaggregates with Prophyromonas gingivalis PK1924 through a galactose-binding adhesin. In the present study, attachment of F. nucleatum PK1594 to a variety of mammalian cells was characterized. F. nucleatum PK1594 attached to all eukaryotic cells tested, including human buccal epithelial cells, gingival and periodontal ligament fibroblasts, HeLa cells and murine lymphocytes, macrophages, and polymorphonuclear leukocytes. These attachments were (i) inhibited by galactose, lactose and N-acetylgalactosamine and (ii) inhibited by monoclonal antibody specific for the galactose-binding adhesin of F. nucleatum PK1594. In addition, a coaggregation-defective mutant of F. nucleatum PK1594 (PK2172), which does not exhibit galactose binding activity, did not attach to the mammalian cells. Coaggregation of F. nucleatum PK1594 with P. gingivalis PK 1924 and Actinobacillus actinomycetemcomitans JP2, but not with other bacteria, showed a similar pattern with sugars, monoclonal antibody, and the adhesin-deficient mutant. The results suggest that the attachment of F. nucleatum PK1594 to mammalian cells and its coaggregation with periodontal pathogens are mediated by the same galactose-binding adhesin.
Asunto(s)
Adhesión Bacteriana , Fusobacterium nucleatum/fisiología , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Bacterias Anaerobias/fisiología , Adhesión Bacteriana/efectos de los fármacos , Adhesión Bacteriana/fisiología , Células Epiteliales/microbiología , Galactosa/metabolismo , Galactosa/fisiología , Células HeLa/microbiología , Humanos , Leucocitos/microbiología , Ratones , Mucosa Bucal/citología , Mucosa Bucal/microbiología , Periodoncio/citología , Periodoncio/microbiologíaRESUMEN
Fusobacterium nucleatum has been identified as significantly associated with sites with active periodontal disease and, as a group, the oral fusobacteria coaggregate with members of all oral bacteria genera tested. Monoclonal antibodies were prepared and used in conjunction with other potential inhibitors, such as simple sugars and amino acids, to characterize coaggregation interactions, of F. nucleatum PK1594. Four unique monoclonal antibodies, 5H11, 14C7, 19F2 and 29C12, were obtained by their ability to inhibit coaggregation of F. nucleatum PK1594 with Actinomyces israelii PK16. They were also capable of inhibiting other coaggregations including Streptococcus oralis H1, S. oralis J22, Capnocytophaga ochracea ATCC33596, Prevotella denticola PK1277 and Prevotella intermedia PK1511. All of these interactions were completely inhibited by N-acetylneuraminic acid. Neither N-acetylneuraminic acid nor monoclonal antibody 5H11 had any inhibitory effect on other F. nucleatum PK1594 interactions, including all galactose-inhibitable coaggregations. The results indicate that F. nucleatum PK1594 expresses upon its surface a distinct type of adhesin that mediates coaggregation interactions that are inhibited by N-acetylneuraminic acid.
Asunto(s)
Adhesinas Bacterianas/inmunología , Fusobacterium nucleatum/fisiología , Ácido N-Acetilneuramínico/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Adhesión Bacteriana , Relación Dosis-Respuesta Inmunológica , Fusobacterium nucleatum/inmunología , Bacterias Gramnegativas/fisiología , Hibridomas/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB CRESUMEN
Attachment of Fusobacterium nucleatum to various oral surfaces is mediated by several adhesins anchored on its outer surface. Monoclonal antibodies (MAbs) were prepared and used to identify the putative galactose-binding adhesin of F. nucleatum PK1594. Four unique MAbs, 8G7, 26B9, 28G11, and 29D4, were isolated on the basis of their ability to inhibit coaggregation of F. nucleatum PK1594 with Porphyromonas gingivalis PK1924. All four MAbs were also capable of inhibiting galactose-inhibitable interactions of F. nucleatum PK1594 with other oral gram-negative bacteria and with erythrocytes. Preincubation of F. nucleatum PK1594 with MAb 26B9 or its Fab fragments at concentrations lower than 1 microg/ml resulted in complete inhibition of coaggregation with P. gingivalis PK1924 or hemagglutination. F. nucleatum PK1594 surface components prepared by mild sonication or by extracting whole cells with detergents were subjected to Western blot analysis. None of the MAbs were able to recognize any polypeptide in these experiments. Therefore, detergent extracts of F. nucleatum PK1594 surface components were subjected to three experimental procedures: (i) separation by ion-exchange chromatography and testing of fractions for reaction with MAb 26B9 in an enzyme-linked immunosorbent assay (ELISA), (ii) lactose-Sepharose affinity chromatography and testing of the lactose eluate in ELISA with MAb 26B9, and (iii) immunoseparation with either MAb 26B9 or 8G7. Collectively, the results suggest that the putative adhesin is a 30-kDa outer membrane polypeptide which mediates the coaggregation with P. gingivalis PK1924 as well as other galactose-sensitive interactions of F. nucleatum PK1594.