Isolation of the Cytocidal toxin (CCT) from periodontopathic bacteria and antibody preparation against CCT: with a view to clinical application.

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Tannetella forsythensis: one of the important periodontpathic bacteria which possesses the cytocidal toxin (TfCCT), lipoprotein, sialidase as a putative virulence factor. Among these, TfCCT might play an important role in initiation and progression of periodontitis and therefore it was of interest of further characterizing this factor.1. Purification of the cytocidal toxin (TfCCT): TfCCT was purified from T.forsythensis extract by using DEAE Sepharose column and size-exclusion chromatography, and cytocidal activity was detected in two peak fractions corresponding to 28 and over 400kDa. Comparing the absorbance at 254 nm suggest that this toxin may interact with DNA. This toxin is a DNA binding protein, further purification was carried out by heparmn column.2. Preparation of anti-cytocidal toxin monoclonal antibodies: Anti-cytocidal toxin monoclonal antibodies that are capable of removing cytocidal toxicity in Tforsythensis sonic extract with protein G-beads were established. As a resul … More t, anti-TfCCT antibody (Me-24) was identified.3. Cloning of CCT-producing clone: The T.forsythensis genoniic library was constructed by using ZAP Express predigested Gigapack cloning kits. For screening, the oligonucleotide degenerative DNA based on the N-terminal amino acid sequence of purified CCT protein was used as a probe. Sequencing of this clone revealed that the size of the insert fragment was 2.1 kbp and there were putative four ORFs (CCT1, 2, 3, and 4). There were high homology in sequence between CCT and prtH4. Evaluation of mechanisms of cytocidal activity: the effects to cell cycle: The recombinant proteins were isolated in vitro and were subjected to analyze the effect of cell cycle arrest or the levels of CyclinBi and p53 at each cell stage. Stained cells were subjected to flowcytometry. High levels of p53 and CyclinB1 were observed at G2 phase in cells treated with this toxin.5. Application for clinical use: This bacterium which has been supposed to have the cytocidal toxin increased in periodontitis patients. We are attempt to analyze the relationship between the levels of antibody titer against CCT or those of CCT protein and the clinical status of periodontitis for the purpose of establishing the simple examination of periodontitis (ELISA, Less