The invasive gram-negative bacterium, A. actinomycetemcomitans ,is the causative agent of localized aggressive periodontitis but the intracellular processes this microorganism utilizes for pathogenicity are poorly defined. Our laboratory has demonstrated the interaction between A. actinomycetemcomitans and host cell microtubules, which may provide the means for intracellular movement of this non-motile organism. Objective: To identify the bacterial receptors which facilitate the interaction with tubulin. Methods: A transposon mutant library frozen in 96-well microtiter plates was screened for defects in tubulin adhesion by ELISA using purified A. actinomycetemcomitans specific antibodies. The transposon integration site in the chromosome was identified by inverse PCR and DNA sequencing. The resulting sequence was compared with the genomic sequence of the prototypic strain,HK1651, and protein coding sequences derived. Results: Mutants with binding defects of greater than 30% of the wild type adhesion were further studied. Protein homologs were identified in the NCBI database and were grouped into 4 classes of proteins: global regulators of gene transcription (Crp, Pta), an operon for serotype specific antigen (extracellular polysaccharides), an extracellular matrix protein adhesin (EmaA) and an unknown outer membrane protein (Omp141). omp141 codes for putative 141 kDa protein with a putative signal sequence and contains a single transmembrane domain located at the amino terminus. The gene is part of an operon coding for another outer membrane protein upstream and an exopolyphosphatase downstream of omp141. A nitrate/nitrite response regulator homolog is predicted to be proximate to this operon. Conclusion: Candidate genes involved in tubulin interaction have been identified from the library screen. The gene product of omp141 is a putative adhesin mediating the interaction of A. actinomycetemcomitans with microtubules. Biochemical and genetic studies are underway to characterize this protein interaction with tubulin. This work was supported by NIH -NIDCR grant R01-DE09760.

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