RESUMO
Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti-Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock-out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10-fold more than the wild-type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)-8, IL-6 and tumour necrosis factor (TNF)alpha in epithelial cells, and induced IL-1beta and TNFalpha production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.
Assuntos
Aggregatibacter actinomycetemcomitans/patogenicidade , Proteínas da Membrana Bacteriana Externa/metabolismo , Fatores de Virulência/metabolismo , Infecções por Actinobacillus/microbiologia , Aggregatibacter actinomycetemcomitans/metabolismo , Aggregatibacter actinomycetemcomitans/ultraestrutura , Animais , Aderência Bacteriana/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Células Cultivadas , Fator H do Complemento/metabolismo , Citocinas/genética , Citocinas/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Feminino , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Doenças Periodontais/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Virulência/genéticaRESUMO
The interaction between epithelial cells and microorganisms is the most important step in bacterial infections. Actinobacillus actinomycetemcomitans was suggested to play a significant role in the initiation of periodontitis because of its bacteriological characteristics. Prostaglandins (PG) mediate the inflammatory response. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide and contributes to innate immunity. E-cadherin is responsible for an epithelial intercellular junction. In this study, we investigated the syntheses of PGE2 and E-cadherin and the expression of hBD-2 in human gingival epithelial cells (HGEC) following exposure to A. actinomycetemcomitans. The levels of PGE2 and cyclooxygenase-2, which are responsible for an increase in PGE2, were increased depending on bacteria exposure time. hBD-2 mRNA was induced by A. actinomycetemcomitans, while HGEC exposed to A. actinomycetemcomitans showed a decrease in E-cadherin levels. Etodolac, a selective cyclooxygenase-2 inhibitor reinforced the increase in hBD-2 mRNA levels by A. actinomycetemcomitans. Furthermore, the etodolac suppressed the decrease in E-cadherin levels. Thus, endogenous PGE2 is involved in the hBD-2 and E-cadherin responses of HGEC to A. actinomycetemcomitans. These findings suggest that the inflammatory and antimicrobial response of gingival epithelial cells to A. actinomycetemcomitans is involved in the initiation of periodontal inflammation. A. actinomycetemcomitans may destroy the mechanical epithelial barrier by destroying E-cadherin.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Caderinas/biossíntese , Dinoprostona/biossíntese , Gengiva/microbiologia , beta-Defensinas/genética , Células Cultivadas , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Regulação da Expressão Gênica/imunologia , Gengiva/patologia , Humanos , Isoenzimas/genética , Proteínas de Membrana , Periodontite , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/análiseRESUMO
We have identified six major sarcosyl-insoluble outer membrane proteins (Omp) of Actinobacillus actinomycetemcomitans Y4, and designated them as Omp100, Omp64, Omp39, Omp29, Omp18 and Omp16 according to the molecular mass. A similar N-terminal sequence was found in the first 15 amino acid residues of Omp16 and Omp18. The N-terminal sequence of Omp29 matched perfectly with the sequence previously identified. We cloned and determined the DNA sequences of three complete genes encoding Omp100, Omp64 and Omp18/16, and one incomplete gene encoding Omp39. Each Omp revealed homologies with some bacterial virulence factors responsible for adhesion, invasion, serum resistance, or protein antigenicity. Serum from patients with periodontitis suspected to be related to A. actinomycetemcomintans infection strongly reacted with Omp100, Omp29 and Omp16 as did serum from mice immunized with A. actinomycetemcomitans Y4 whole bacteria. These findings suggest that Omps of A. actinomycetemcomitans can be associated with periodontal disease.