To study anti-bacterial immunity and bacterial antigens associated with juvenile periodontitis (JP), we screened the genomic library of Actinobacillus actinomycetemcomitans (Aa), a G(-) anaerobic microorganism associated with human periodontitis, by expression cloning using JP-associated CD4+T cells from HuPBL-engrafted NOD/SCID mice followed by transfection with IL-2 promoter with Lac-Z for activation and visualization. Such cloning yielded several novel genes, one of which was cloned and designated cagE homologue (i.e., cagE), which encodes a putative bacterial type-IV secretion system with homology, mainly in C-terminus, to Helicobacter pylori cagE and Agrobacterium tumefaciens virB4 (BBRC 303: 1086, 2003). Our recent studies show that CagE of Aa is involved in inducing apoptosis of human cells, including oral epithelia, endothelia, osteoblasts and lymphocytes, based on the biochemical, genetic and morphological studies. Objectives: To assess the clinical significance of CagE-homologue in periodontal infections, serum, gingival crevicular fluid (GCF) and HuPBL samples (n=12) were collected from Aa-infected JP patients. Methods: ELISA, Immuno-blots and in-vitro T-cell proliferation were employed to assess the immuno-reactivity and profiles of CagE-homologue in Aa-infected JP subjects. Results: The results show that approximately >75% (9/12) of Aa-infected subjects developed positive immuno-reactivity higher than that from Aa-free healthy subjects, where there were statistically significant higher levels of: i) CagE-mediated hCD4+T cell proliferation (p<0.03); ii) CagE-specific hIgG titers in serum and GCF samples (p<0.001). The results of our recent immuno-gold staining by using electron-microscopy and CagE-specific scFv-MoAbs show that CagE-homologue is located in the bacterial cytoplasm, not elsewhere, and is secreted by Aa in culture. Conclusions: These data suggest CagE-homologue is a soluble virulence factor and is involved in the microbial pathogenesis and subsequent periodontal immunity and tissue destruction associated with JP. Its molecular mechanisms for disease initiation and progression will require future study. This project is supported by grants from CIHR-MOP37960, Canada and NIDCR, DE14473.

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