Satsuki Kato

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin.

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti‐vesicle antibodies and a FITC‐labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti‐vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP‐DiOC18. In contrast, incubation of cells with free SP‐DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle‐associated toxin in a time and dose‐dependent manner. However, cells became reactive with anti‐vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin‐mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin‐deficient strain of A. actinomycetemcomitans JP2 were non‐toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.

Human epithelial cell death caused by Actinobacillus actinomycetemcomitans infection.

The gingival sulcus is the shallow crevice around the tooth, and its epithelium is a gateway for initial bacterial infection in periodontal disease. Recent studies have shown that Actinobacillus actinomycetemcomitans invades an epithelial cell line, KB cells, in vitro. The aim of the present study was to clarify the changes in KB cells after A. actinomycetemcomitans infection. The cytotoxic effects of A. actinomycetemcomitans on KB cells were determined at 72, 96 and 120 h after infection by an MTT assay. Nuclear morphological changes were observed by staining with Hoechst 33258. Cytoplasmic histone-associated DNA fragmentation in the infected KB cells was determined by ELISA. A. actinomycetemcomitans was cytotoxic on KB cells, and condensation and degradation of the nuclei were observed. DNA fragmentation was increased after the infection. In addition, A. actinomycetemcomitans showed similar cytotoxic effects on human gingival epithelial cells. The present study demonstrated that A. actinomycetemcomitans induces apoptotic cell death of oral epithelial cells in an in-vitro culture system. This induced apoptosis might be involved in the initiation and progression of periodontitis.