Novel Apoptosis-Inducing Activity in Bacteroides forsythus : a Comparative Study with Three Serotypes of Actinobacillus actinomycetemcomitans

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Abstract
Bacteroides forsythus , which has been reported to be associated with periodontitis but has not been recognized as a key pathogen, was found to induce cytolytic activity against HL-60 and other human leukemic cells. This cytolytic activity was demonstrated according to three different criteria: (i) loss of both mitochondrial membrane potential and membrane integrity in cells treated with bacterial extracts and then with Rh123 and propidium iodide, respectively, as demonstrated by flow cytometry; (ii) damage to cytoplasmic membrane, as revealed by scanning electron microscopy (SEM); and (iii) DNA ladder formation and activation of caspase-3. These results indicate that B. forsythus produced an apoptosis-inducing factor(s) found to be composed of protein as judged by heat and trypsin sensitivity. In addition to extracts from B. forsythus , the culture supernatant of this bacterium has the ability to induce a cytolytic effect against peripheral white blood cells, especially lymphocytes. For comparison with B. forsythus , the same analyses were applied to two strains with different serotypes of Actinobacillus actinomycetemcomitans , serotypes a (ATCC 43717) and c (ATCC 43719), in addition to previously reported apoptosis-inducing serotype b (ATCC 43718), which was used as a positive control. The strains of A. actinomycetemcomitans serotypes a and b induced apoptosis in HL-60 cells as judged by the above three criteria but to a slightly lesser extent than did B. forsythus , while the serotype c strain produced apoptosis to a negligible extent. Detailed SEM images showed that the A. actinomycetemcomitans serotype a strain induced large-pore formation and the serotype b strain produced small pores with typical blebbing, while B. forsythus induced severe membrane ruffling. Further DNA ladder formation and caspase-3 activation were observed in the serotype a and b strains but not in the serotype c strain. The present paper is the first report of a protein factor(s) from B. forsythus and the A. actinomycetemcomitans serotype a strain which induces apoptotic cell death.