[Cloning and sequence analysis of a sialidase gene (siaHI) from Bacteroides forsythus ATCC 43037].

Bacteroides forsythus is known as a periodontopathogen associated with periodontitis, and it produces a tripsin-like protease, cell-death inducing factor, and sialidase (neuraminidase), as putative virulence factors. The purpose of this study was to clone the sialidase gene from B. forsythus ATCC 43037, and to analyze the biological characteristics. A positive clone (pHI-1) was successfully isolated, among a total of 455 recombinant clones, using a filter paper sialidase assay with the fluorogenic sialidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeuNAc). Sequencing of the inserted DNA of pHI-1(3.2 kbp) was carried out, and analysis of the sequence with DNASIS software revealed that the ORF (designated siaHI: 1.4 kbp) would code for the protein with a deduced molecular mass of 52 kDa and a pI of 6.60. Furthermore, we confirmed that siaHI gene was contained in chromosomal DNA from B. forsythus ATCC 43037 and the 3 clinical isolates of B. forsythus. The highest amino acid sequence homology was observed between siaHI gene and a part of sialidase gene from Streptococcus pnumoniae. This is the first report on the cloning and expression of the B. forsythus sialidase gene.

Isolation and identification of a cytopathic activity in Tannerella forsythia.

Interactions between pathogens and host induce human disorders including periodontitis, disintegration of the tooth supporting tissues. Tannerella forsythia has been linked to the periodontitis and several cytopathic reagents have been found in the bacterium; however, its contribution to the disease remains unclear. Biochemical approach to explore the cytopathic effect revealed two distinct activities in T. forsythia (ATCC 43037) extract; one detaches adherent cells from substratum and another arrests cells at G2. An executor of former activity, forsythia detaching factor (FDF) was identified; its genomic sequence and peptidase activity revealed that FDF is a substantial form of putative PrtH; prtH gene was hypothetically identified directly from a DNA fragment of the bacterium and its native product has never been shown. Since FDF was found in the bacterial culture supernatant, its activity implies a contribution to the disintegration of tissues although the mechanism how FDF disturbs cellular anchors remains elusive.

Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease.

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-kappaB 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-kappaB-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-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.