Tannerella forsythensis (previously named Bacteroides forsythus) is a Gram-negative, anaerobic, fusiform bacterium that is a primary or secondary aetiological agent in periodontal disease in humans. T. forsythensis expresses several putative virulence factors, including a sialidase; however, there has been no molecular genetic characterization of this enzyme. A sialidase clone (pHI-1) was screened from a total of 455 recombinant clones of a genomic DNA library using the 29-(4-methylumbelliferyl)-AE-D-N-acetylneuraminic acid (MUNeuAc) filter-paper spot assay. The sialidase gene ORF (siaHI) consists of a 1395 bp coding sequence and encodes a protein with 465 amino acids with an overall molecular mass of 52 kDa. The sialidase does not have sequence similarity to any other bacterial sialidase. The entire sialidase ORF was expressed in Escherichia coli. Furthermore, the sialidase was purified from the type strain of T. forsythensis and from a recombinant clone, pHI-1 : 1, and was analysed using a non-denaturing gel, revealing that the enzyme preparations were respectively separated as two major bands and as a single band. Southern blot hybridization analysis revealed similar patterns of siaHI-hybridizing bands among clinical isolates of T. forsythensis from periodontitis patients. This is the first study on the cloning and expression of a T. forsythensis sialidase gene and the purification of the SiaHI enzyme from T. forsythensis ATCC 43037 T and recombinant E. coli.

Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-B translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-B-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-

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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.Periodontitis, one of the most common human infectious diseases, is an acute or chronic infectious condition that can result in the inflammatory destruction of periodontal tissues such as periodontal ligaments and alveolar bone. Furthermore, recent studies have indicated that periodontitis might contribute to serious systemic diseases, such as cardiovascular diseases (18). Among more than 300 species of bacteria in the oral cavity, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans are the major periodontopathic bacteria, and several virulence factors related to the pathogenesis of periodontitis have been reported (reviewed in references 3a, 8, and 26). In addition to these two bacteria, Bacteroides forsythus, a gramnegative, anaerobic, and fusiform bacterium (24), has recently been recognized as one of the agents associated with periodontitis. It has been reported that the presence of B. forsythus in subgingival flora was significantly associated ...

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