RESUMO
BACKGROUND AND OBJECTIVE: Periodontopathogens experience several challenges in the oral cavity that may influence their transcription profile and resulting phenotype. This study evaluated the effect of environmental changes on phenotype and gene expression in a serotype b Aggregatibacter actinomycetemcomitans isolate. MATERIAL AND METHODS: Cultures in early exponential phase and at the start of stationary growth phase in microaerophilic and anaerobic atmospheres were evaluated. Cell hydrophobic properties were measured by adherence to n-hexadecane; in addition, adhesion to, and the ability to invade, KB cells was evaluated. Relative transcription of 12 virulence-associated genes was determined by real-time reverse transcritption quantitative PCR. RESULTS: The culture conditions tested in this study were found to influence the phenotypic and genotypic traits of A. actinomycetemcomitans. Cells cultured in microaerophilic conditions were the most hydrophobic, reached the highest adhesion efficiency and showed up-regulation of omp100 (which encodes an adhesion) and pga (related to polysaccharide synthesis). Cells grown anaerobically were more invasive to epithelial cells and showed up-regulation of genes involved in host-cell invasion or apoptosis induction (such as apaH, omp29, cagE and cdtB) and in adhesion to extracellular matrix protein (emaA). CONCLUSION: Environmental conditions of different oral habitats may influence the expression of factors involved in the binding of A. actinomycetemcomitans to host tissues and the damage resulting thereby, and thus should be considered in in-vitro studies assessing its pathogenic potential.
Assuntos
Aggregatibacter actinomycetemcomitans/genética , Meio Ambiente , Interação Gene-Ambiente , Aggregatibacter actinomycetemcomitans/patogenicidade , Alcanos/farmacologia , Apoptose/genética , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Técnicas Bacteriológicas , Células Epiteliais/microbiologia , Proteínas da Matriz Extracelular/genética , Regulação Bacteriana da Expressão Gênica/genética , Genótipo , Humanos , Células KB/microbiologia , N-Glicosil Hidrolases/genética , Fenótipo , Polissacarídeos Bacterianos/genética , Subunidades Proteicas/genética , Transcrição Gênica/genética , Fatores de Virulência/genéticaRESUMO
Periodontal diseases result from the interaction of bacterial pathogens with the host's gingival tissue. Gingival epithelial cells are constantly challenged by microbial cells and respond by altering their transcription profiles, inducing the production of inflammatory mediators. Different transcription profiles are induced by oral bacteria and little is known about how the gingival epithelium responds after interaction with the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. In the present study, we examined the transcription of genes involved in signaling transduction pathways in gingival epithelial cells exposed to viable A. actinomycetemcomitans. Immortalized gingival epithelial cells (OBA-9) were infected with A. actinomycetemcomitans JP2 for 24 h and the transcription profile of genes encoding human signal transduction pathways was determined. Functional analysis of inflammatory mediators positively transcribed was performed by ELISA in culture supernatant and in gingival tissues. Fifteen of 84 genes on the array were over-expressed (P < 0.01) after 24 h of infection with viable A. actinomycetemcomitans. Over-expressed genes included those implicated in tissue remodeling and bone resorption, such as CSF2, genes encoding components of the LDL pathway, nuclear factor-κB-dependent genes and other cytokines. The ELISA data confirmed that granulocyte-macrophage colony-stimulating factor/colony-stimulating factor 2, tumor necrosis factor-α and intercellular adhesion molecule-1 were highly expressed by infected gingival cells when compared with control non-infected cells, and presented higher concentrations in tissues from patients with aggressive and chronic periodontitis than in tissues from healthy controls. The induction in epithelial cells of factors such as the pro-inflammatory cytokine CSF2, which is involved in osteoclastogenesis, may help to explain the outcomes of A. actinomycetemcomitans infection.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Periodontite Agressiva/genética , Periodontite Crônica/genética , Citocinas/biossíntese , Gengiva/microbiologia , Transdução de Sinais/genética , Periodontite Agressiva/metabolismo , Apoptose , Aderência Bacteriana , Estudos de Casos e Controles , Linhagem Celular Transformada , Periodontite Crônica/metabolismo , Fatores Estimuladores de Colônias/biossíntese , Meios de Cultivo Condicionados , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Gengiva/citologia , Gengiva/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Transcrição Gênica , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Adhesion to and invasion of epithelial cells by the periodontopathogen Porphyromonas gingivalis is promoted by the major fimbriae, encoded by fimA. The microorganism can be classified in six genotypes, based on fimA sequence, and genotype II strains are more prevalent than others in periodontitis patients. This study aimed to determine the adhesive and invasive abilities on KB cells of different fimA allelic variants of P. gingivalis isolates. Twenty-two isolates and six reference strains representing the six fimA genotypes and non-typeable strains were screened for their adhesion and invasion abilities on KB cells, using standard methods. All strains were able to adhere and, except for one, to invade KB cells. However, these properties were not homogeneous among strains belonging to the same genotype. There was no correlation between adhesion and invasion efficiencies. Isolate KdII 865 (fimA genotype II) was the most invasive and the second most adhesive strain, whereas reference strain ATCC 33277 (fimA I) showed a low adhesion ability but was highly invasive. These data indicated that fimA genotypes of P. gingivalis are not related to the adhesion and invasion abilities on KB cells, suggesting that the increased prevalence and proportion of certain genotypes may be attributed to other characteristics besides FimA variation.
Assuntos
Células Epiteliais/microbiologia , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/fisiologia , Porphyromonas gingivalis/fisiologia , Adesão Celular/genética , Endocitose , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Variação Genética , Genótipo , Humanos , Células KB , Porphyromonas gingivalis/genéticaRESUMO
Fimbria encoded by the gene fimA is considered one of the main factors in the colonization of the oral cavity by Porphyromonas gingivalis. Allelic variation in fimA led to the classification of strains of P. gingivalis into six genotypes. The occurrence of P. gingivalis was determined by polymerase chain reaction using 16S rRNA primers in 302 subgingival samples obtained from 102 Brazilian subjects exhibiting different periodontal conditions. Distribution of fimA genotypes was assessed in 146 P. gingivalis positive samples by polymerase chain reaction using primers pairs homologous to the different fimA genes. P. gingivalis was detected in 51 of 57 (89.4%) patients with periodontal attachment loss, in six of 20 gingivitis patients (30.0%) and in two of 25 (8.0%) subjects with a healthy periodontium. Variant type II was the only type detected in 53 sites (39.3%), distributed among 19 periodontitis patients (37.3%) and in one patient with no periodontal destruction. Type Ib was the second most prevalent genotype in periodontitis patients (19.6%). Genotype V was not detected in the studied population. Type IV was the most commonly type found among gingivitis patients, either alone or in combination with other genotypes. Multiple genotypes were detected in nine sites (6.1%). A fimA genotype was not identified in 26 sites (17.8%) of 146 sites positive for P. gingivalis, suggesting that other alleles of fimA not yet sequenced may be prevalent in this population. These data demonstrated that P. gingivalis type II strains followed by type Ib are more prevalent in periodontitis patients from a multiracial population in Brazil, suggesting an increased pathogenic potential of these types.