Gene expression and phenotypic traits of Aggregatibacter actinomycetemcomitans in response to environmental changes.

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.

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates.

BACKGROUND AND OBJECTIVE: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. MATERIAL AND METHODS: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. RESULTS: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. CONCLUSION: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates.

INTRODUCTION: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. METHODS: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. RESULTS: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. CONCLUSION: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Genotypic and phenotypic analysis of Streptococcus mutans from different oral cavity sites of caries-free and caries-active children.

INTRODUCTION: Streptococcus mutans exhibits extensive genotypic diversity, but the role of this variation is poorly understood. This study aimed to determine the number and distribution of genotypes of S. mutans isolated from caries-active and caries-free children and to evaluate some of their phenotypic traits. METHODS: Stimulated saliva, tongue surface and biofilms over sound and carious teeth surfaces were sampled from 10 caries-free and 11 caries-active children aged 5-8 years. A total of 339 isolates of S. mutans were genotyped by arbitrarily primed polymerase chain reaction using OPA2 primer. One isolate from each genotype was tested for its acid susceptibility and its ability to form a biofilm. RESULTS: Fifty-one distinct genotypes were determined, one to three genotypes in each oral sample. A single genotype was detected in seven children, whereas the remaining 14 children exhibited two to seven genotypes. There were no significant differences in the number of genotypes detected in caries-free and caries-active children. No correlation was observed between the number of genotypes and the mutans streptococci salivary levels. Five of the six high biofilm-forming genotypes were obtained from caries-active children, although the differences in biofilm formation between isolates from caries-free and caries-active children were not statistically significant. Genotypes with low susceptibility to acid challenge were statistically more frequent among isolates from caries-active children than among those from caries-free children. CONCLUSION: The present data suggested that there were differences in the distribution of genotypes of S. mutans according to the oral site and that S. mutans populations differ in their acid susceptibility and ability to form biofilms, factors allowing their colonization of sucrose-rich environments.

Nuevos blancos terapéuticos en leucemia aguda pediatrica: caracterización de las mutaciones del gen FLT3 / Newer therapeutic targets in acute limphoblastic leukemia: characterization of mutations of the gene FLT3

El tratamiento de elección para las leucemias agudas pediátricas es la quimioterapia convencional, que ha permitido obtener tasas de sobrevida que actualmente parecen difíciles de superar. En los últimos años se han intensificado las investigaciones dirigidas a descubrir nuevos blancos terapéuticos, entre los que se encuentra el receptor FLT3. Los blastos leucémicos puede presentar formas mutadas de dicho receptor, siendo las más frecuentes mutaciones internas en tándem (FLT3 ITD) y mutaciones puntuales en la zona de activación (FLT3 ALM). Objetivos: Poner a punto la detección de mutacoines de FLT3, analizar su prevalencia en nuestra población de pacientes con diagnóstico de Leucemia Mieloblástica Aguda (LMA) o de Leucemia Linfoblática Aguda en infantes (LLA I), y evaluar su asociación con parámetros clínicos y de laboratorio. Pacientes y Método: El estudio de las mutaciones se realizó por RT PCR, en un total de 122 pacientes (92 LMA y 30 LLA 1). Resultados: Se detectaron mutaciones en el 15,2 de las LMA y en 10 de las LLA -1. La prevalencia de las FLT3 ITD mostró un aumento gradual con la edad de los pacientes, y la media de edad fue significativamente mayor. Con respecto a asociaciones con recuentos leucocitarios, alteraciones genéticas, subtipos FAB y valor pronóstico, si bien hubo difrencias éstas no furon significtivas. Conclusiones: Este es el primer estuido de mutaciones en FLT3 realizado en población pediátrica en nuestro país. La detección de estas mutaciones permitirá individualizar, en el futuro, a los niños candidatos a recibir drogas inhibidoras de FLT3, actualmente en desarrollo.

Adhesion and invasion to epithelial cells by fimA genotypes of Porphyromonas gingivalis.

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.

Determination of mutacin activity and detection of mutA genes in Streptococcus mutans genotypes from caries-free and caries-active children.

Relationships between genetic diversity, mutacin production and sensitivity to mutacins in Streptococcus mutans were evaluated in 19 clinical isolates from caries-free and caries-active children. Mutacin production was tested against 30 indicator strains; results showed significant variations in the inhibitory spectra of the clinical isolates. There was no association between the inhibitory spectrum of the infecting strain and the caries experience or the level of mutans streptococci infection of the host. Homology to the mutA gene coding for mutacin II was detected in one clinical isolate; none of the clinical isolates showed homology to the mutA genes coding for mutacins I or III. Genotyping by random amplified polymorphic DNA (RAPD) reactions grouped the isolates into three clusters, but no correlation was found between any of the clusters and mutacin activity, caries experience or level of mutans streptococci in the host.