Influence of Aae Autotransporter Protein on Adhesion and Biofilm Formation by Aggregatibacter actinomycetemcomitans

Abstract The periodontopathogen Aggregatibacter actinomycetemcomitans colonizes oral cavity by binding to and invading epithelial cells as well as by participating in biofilms formed on hard surfaces. Aae, an autotransporter protein, is implicated in bacterial adhesion to epithelial cells. Due to the multiple functions of bacterial autotransporter proteins, this study aimed to evaluate the role of aae in A. actinomycetemcomitans ability to adhere to both saliva-coated hydroxyapatite (SHA) and biofilm. An aae null mutant was constructed. Its hydrophobic properties as well as its ability to adhere to epithelial cells, SHA and to form biofilm were evaluated and compared with the parental strain, A. actinomycetemcomitans VT1169. The aae null mutant showed reduced hydrophobicity, as well as decreased binding to SHA and biofilm formation compared to the parental strain. These data suggest that aae mediates A. actinomycetemcomitans adhesion to epithelial cells and may be involved in biofilm formation and interaction with adsorbed salivary proteins.

Resumo O peridontopatógeno Aggregatibacter actinomycetemcomitans coloniza a cavidade oral aderindo e invadindo as células epiteliais e participando da formação de biofilme em superfícies duras. Aae, uma proteína autotransportadora está relacionada com a adesão bacteriana às células epiteliais. Devido às múltiplas funções desempenhadas por proteínas bacterianas autotransportadoras, este estudo teve como objetivo avaliar o papel de aae de A. actinomycetemcomitans tanto na capacidade de aderir à hidroxiapatita recoberta por saliva (SHA), quanto a de formar biofilme. Um mutante nulo aae foi construído. Suas propriedades hidrofóbicas, bem como a sia capacidade para aderir às células epiteliais, à SHA e para formar biofilme foram avaliadas e comparadas com a cepa -mãe, A. Actinomycetemcomitans VT1169. O mutante nulo aae apresentou redução de hidrofobicidade, assim como diminuição da adesão à SHA e na formação de biofilme, quando comparado à cepa parental. Estes dados sugerem que aae media a adesão de A. Actinomycetemcomitans às células epiteliais e pode também estar envolvida na formação de biofilme e na interação com proteínas salivares adsorvidas.

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production.Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the differentA. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation.Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the differentA. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels.Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b.Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b ofA. actinomycetemcomitans.

Distribution of serotype-specific genotypes of aggregatibacter actinomycetemcomitans in Brazilian patients with down syndrome with different periodontal conditions / Distribución de los serotipos genotipos específicos de aggregatibacter actinomycetemcomitans en los pacientes brasileños con síndrome de down con diferentes condiciones periodontales

This study evaluated the occurrence of highly or minimally leukotoxic strains of Aggregatibacter actinomycetemcomitans (Aa) from patients with Down syndrome and the distribution of the different serotype-specific genotypes of this microorganism. Sixty-seven patients with Down syndrome were subjected to dental, periodontal and radiographic evaluations. Samples of subgingival biofilm were collected and plated onto TSBV agar and characteristic colonies of A. actinomycetemcomitans were identified by biochemical methods. The occurrence of this bacterium was also evaluated directly in the clinical specimens by PCR. The presence of 530 bp deletion in the promoter region was also determined by PCR in order to evaluate distribution of highly or minimally leukotoxic strains. A. actinomycetemcomitans was detected in 11.1 percent by culture and 22.2 percent by PCR from periodontally healthy subjects, 100 percent of the patients with Down syndrome with aggressive periodontitis, 50 percent and 75 percent of patients with chronic periodontitis by culture and PCR respectively. Only two patients with aggressive periodontitis were colonized by highly leukotoxic Aa. Serotype-specific genotypes a and c were the most prevalent. The results suggest the role of peculiar characteristics of Aa and patients with Down syndrome in the development of periodontitis and the influence of peculiar characteristics of the population in this process.

Este estudio evaluó la presencia de cepas altamente o mínimamente toxicas de Aggregatibacter actinomycetemcomitans (Aa) de los pacientes con síndrome de Down y la distribución de los serotipos genotipos específicos de este microrganismo por cultivo y PCR. Sesenta y siete pacientes con síndrome de Down fueron sometidos a un tratamiento dental y evaluaciones clínicas. Las muestras de biofilme subgingival fueron recogidas y cultivadas en agar TSBV y colonias características de Aa fueran identificadas mediante métodos bioquímicos. La presencia de esta bacteria se evaluó también directamente en las muestras clínicas por PCR. Los aislados y las muestras clínicas también se probaron con el fin de evaluar la distribución de serotipos de genotipos específicos por PCR, mientras que la presencia de delección de 530 bp en la región promotora del gen ltxC también fue determinado por PCR con el fin de evaluar de distribución de las cepas altamente o mínimamente toxicas. Aa fue aislado en 11,1 por ciento y 22,2 por ciento por PCR de pacientes periodontalmente sanos; en todos los pacientes con síndrome de Down con periodontitis agresiva, y en 50 por ciento y 75 por ciento de los pacientes con periodontitis crónica por cultivo y PCR, respectivamente. Sólo dos pacientes con periodontitis agresiva fueron colonizados por cepas altamente tóxicas. Los serotipos y genotipos específicos a y c fueron los más frecuentes. Los resultados sugieren una asociación de las peculiares características de Aa con las características de los pacientes con síndrome de Down en el desarrollo de la periodontitis.

Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis / Distribuição de biótipos e atividade leucotóxica de Aggregatibacter actinomycetemcomitans isolados de pacientes brasileiros com periodontite crônica

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18 percent) of the periodontal patients and one (2 percent) healthy subject. However, by PCR, this organism was detected in 44 percent of the periodontal patients and in 16 percent of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases

Aggregatibacter actinomycetemcomitans é um importante agente etiológico da periodontite e produz infecções extra-bucais. Neste estudo, foram detectados os biótipos, o gene ltxA associado à produção de leucotoxina e o promotor ltx em A. actinomycetemcomitans de pacientes com e sem periodontite. A atividade leucotóxica sobre células HL-60 também foi avaliada. A atividade leucotóxica foi determinada através do método de exclusão do azul de tripam. A deleção de 530 bp no promotor ltx foi avaliada usando-se o par de iniciadores PRO. A. actinomycetemcomitans foi detectado por cultura e por PCR. Por cultura, A. actinomycetemcomitans foi detectado em nove pacientes com periodontite (18 por cento) e em um indivíduo sadio (2 por cento). Por PCR esse microrganismo foi detectado em 44 por cento dos pacientes com periodontite e em 16 por cento dos saudáveis. Verificou-se diferença estatística entre a detecção do promotor do operon ltx, por PCR, diretamente do biofilme subgengival e do DNA bacteriano. Somente uma amostra clínica apresentou A. actinomycetemcomitans altamente leukotóxico. O biótipo II foi o mais prevalente e não foi observada correlação biótipo-atividade leucotóxica. A expressão da leucotoxina por A. actinomycetemcomitans na doença periodontal e outras doenças infecciosas necessita ser avaliado.

Analysis of the presence of pathogens which predict the risk of disease at peri-implant sites through polymerase chain reaction (PCR)

A presença dos ADN de Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis e Prevotella intermedia em amostras coletadas de sulco periimplantar de 19 pacientes parcialmente desdentados foi analisada pela reação em cadeia da polimerase (PCR). Dentre esses 19 pacientes, dez apresentavam histórico de doença periodontal e nove não apresentavam antecedentes. Os resultados obtidos nesta análise foram relacionados com a profundidade do sulco periimplantar, o sangramento à sondagem e o provável risco de doença. Constatou-se que houve a amplificação do ADN das bactérias-alvo em sete amostras, sendo quatro de pacientes sem histórico de periodontopatia. Este resultado sugere que mesmo na ausência de sinais inflamatórios significantes, essa detecção qualitativa pode indicar risco de periimplantite, requerendo manutenção pós-operatória mais rigorosa.

DNA probe analysis in smoker and non smoker rapidly progressive periodontitis patients--a pilot study.

Smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease. The bacteria A. actinomycetemcomitans, P. gingivalis and P. intermedius as indicated as the potential pathogens associated with periodontal disease. Since the bacteria mentioned as well as smoking are factors associated with periodontitis it is of importance to elucidate the interrelationship between these factors. The purpose of this study was to investigate the prevalence of A. actinomycetemcomitans, P. gingivalis and P. intermedius in subgingival plaque samples obtained form healthy and diseased sites of patients with rapidly progressive periodontitis who were smokers and non smokers along with other clinical parameters.