| Grant number: | 03/08598-0 |
| Support Opportunities: | Research Projects - Thematic Grants |
| Duration: | June 01, 2004 - May 31, 2009 |
| Field of knowledge: | Health Sciences - Dentistry - Periodontology |
| Principal Investigator: | Marcia Pinto Alves Mayer |
| Grantee: | Marcia Pinto Alves Mayer |
| Host Institution: | Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil |
Abstract
Actinobacillus actinomycetemcomitans is associated to Aggressive periodontitis mostly in its localized form (UP) and to some forms of chronic periodontitis. There is a great diversity among strains of this organism, which can reflect on the virulence of different clones. Genotyping based on ISs mar be an important toll to differentiate the organism, since the hybridization patterns obtained with mobile elements such as IS 150-like is able to detect small differences, and it was indicated to small epidemiological studies of specific subpopulations of A. actinomycetemcomitans. On the other hand, the analysis using a more stable IS would provide a phylogenic analysis. Pulse Field gel electrophoresis is considered the gold standard among methods used to evaluate the phylogenetic structure of a population. The first part of this study aims to analyze the structure of A. actinomycetemcomitans by genotyping a collection of strains isolated in different geographicallocations, including isolates from Brazil, obtained from patients with different periodontal conditions. The phylogenetic structure will be evaluated by PFGE and the relation between the subpopulations will be analyzed through RFLP using ISs probes. The genetic diversity will be related to the polymorphism of a series ofvirulence-associated genes and to the phenotype. Cytolethal distending toxin, encoded by the cdt operon in Actinobacillus actinomycetemcomitans (Mayer et aI, 1999), is the only known bacterial toxin able to induce DNA damage on target cells. We have previousIy shown that the CD toxic titers vary among strains of Actinobacillus actinomycetemcomitans (FABRIS et al, 2002). In addition, a deletion in the promoter of the operon ltx increases the expression of the leukotoxin, and these strains are strongly associated to LJP (Bueno et al, 1998), mostly among patients of African origino the strains will be characterized by the promoter structure of the ltx and cdt operon, and to the polymorphism of the genes aae and apah, involved in adherence and invasion of epithelial cells respectively. Since neither the immune response nor the in vivo production of CDT are currently known, we also aim to purify the three proteins forming the CDT complex (CDTA, CDTB and CDTC) and to determinate the antibodies titles in sera against these proteins in sera from patients exhibiting different periodontal conditions. We could recently demonstrate that the crude extract of a recombinant E. coli expressing AaCDT was able to inhibit NO synthesis by peritoneal macrophages (FERNANDES, 2002). In order to confirm this observation, the effect of CDT proteins on NO production, celI cycle inhibition and cellular distension will be also evaluated. Actinobacillus actinomycetemcomitans presents several outer surfaces proteins, related to virulence. The antibodies titers against the proteins OMP29, OMP 1000 and Aae, those recognized as viru1ence factors will be analyzed in sera of patients with different periodontal conditions. The titers against OMP29 and OMP 100 were associated to infection by the organism. Are was recently described as an adhesin of the bacteria, enabling adhesion to epithelial cells. It is homologous to Hap, produced by Haemophilus influenzae, but some of the properties presented by Hap were not studied in Aae, such as immunogenicity, or adhesion to matrix components as collagen and fibronectin. The gene aae encoding Aae presents variation mostly in number of repeats among strains, which may reflect on the adhesive ability of different strains as well as in its resistance to inhibition by lactoferrin. A knockout mutant of A actinomycetemcomitans deficient in Aae production will be produced and the ability of the mutant to adhere to extracellular matrix compounds will be evaluated and compared to the wild type. Regulation of virulence-associated genes in A. actinomycetemcomitans is very little understood. We aim to produce a knock-out mutant in arcB, a gene that encodes the sensor protein of PREDOX and signs to an OMPR-like (arcA) protein that regulates transcription Quorum sensing system and possibly the Arc system regulate transcription of the operon ltx in A. actinomycetemcomitans. The wild type and the mutant strains will be submitted to various conditions in order to evaluate factors such as adherence, hydrophobicity, production of toxins and outer membrane proteins profile. Transcription of different genes will be evaluated by RT -PCR and a differential display method (RAP-PCR) in the wild type and the mutant strain. (AU)
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