Gene profiling of periodontal pathogens in periodontal lesion

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1.Host-induced genes of A.actinomycetemcomitansActinobacillus actinomycetemcomitans possess many virulence factors. However, the potential roles of the virulence factors are not well characterized. In addition, many unknown virulence factors are considered to be involved in the pathogenesis. A.actinomycetemcomitans grows as a rough colony on primary isolates (R-type). The colony converts to a smooth phenotype (S-type) through repeated subculture. Since the phenotypic alteration reflects different gene expressions in response to environmental changes from the in vivo to the in vitro, isolation of genes induced in the R-type strain turns out to be an isolation of host-induced genes, including the virulence factors. In the current study, we identified genes induced in the R-type of A.actinomycetemcomitans using the cDNA subtractive hybridization technique.Three genes, mip,prx and ompA were identified as R-type specific genes. Attention was focused on the mip, and a recombinant Mip-like protein and the deficient mutant were created. The recombinant protein reacted with the patients sera, suggesting the production of the Mip-like protein in periodontal lesions. The mutant was used for an invasion assay and demonstrated impaired ability for the invasion of the host cells. The expression of the mip, prx and ompA may be enhanced in the host, and the Mip may play an important role for invasion of A.actinomycetemcomitans.2.New system for microbiological examination.Microflora of subgingival plaque is a very complicated community. Therefore, microbiological examination is required for the analysis of periodontal pathogens and virulence factors in vivo. In the current study, we established a new system for the examination. Loop-mediated isothermal amplification (LAMP) method was employed for the system. Primers were designed from the nucleotide sequence of 16S ribosomal RNA gene, and the LAMP method detected periodontal pathogens with high sensitivity, specificity and rapidity.