Analyses for the cytopathic mechanism of cytocidal toxin from periodontpathic bacteria

[I]我们从表现出细胞致死膨胀毒素(CDT)样活性的牙周病细菌的提取物中检测到一种新的Tannerella forsyhtensis CCT(Tf-CCT)活性。为了鉴定什么是Tf-CCT，并探讨Tf-CCT引起细胞病变的机制，我们根据纯化蛋白的氨基酸序列克隆了Tf-CCT的基因，并证实了所克隆的基因产物具有细胞病变活性。此外，我们估计了一个与TF-CCT细胞病变有关的区域。报告的详细内容如下：(A)从连翘提取物中纯化的Tf-CCT蛋白的鉴定。经阴离子交换、大小排斥、肝素亲和等步骤纯化的Tf-CCT蛋白，用为中和连翘叶提取物细胞病变而建立的单抗BE-24进行鉴定。选择Tf-Cct的28.5kDa片段进行N-端氨基酸序列分析，并制备简并引物用于…得到更多的序列APQNMDVLL。序列分析表明，Tf-CCT与PrtH蛋白水解酶(PrtH)同源。然而，由于翻译起始点位于报告位点的上游，因此在纯化的组份中检测到的天然Tf-CCT的分子尺寸大于估计的pT-Il-I的大小。(B)为了确定翻译起始点，分别选择TIRST(没有核糖体结合基序；RBS)、第5个(有RBS的报告位点)和第8个(有更典型的RBS)作为第一个ATG构建重组基因。尽管对不同类型的Tf-CCT的表达载体进行了广泛的试验，但没有重组Tf-CCT在大肠杆菌中表达，而体外表达系统如RTS和PURESYSTEM检测到了显著的表达。(C)从第5个ATG(与已报道的prtH相同)表达的重组Tf-CCT(RTF-CCT)表现出最大的细胞病变。(D)蛋白质印迹显示，Tf-CCT除了在C端与人膀胱运输相关蛋白slylp的第三个结构域有明显的同源性外，与任何报道的CDT都没有相似之处。因此，Tf-CCT的细胞病变很可能是通过与调节细胞膜融合的蛋白质相互作用而表现出来的。(E)为了获得RTF-CCT在体内的表达，我们尝试将重组蛋白与几种蛋白标签融合，最终发现C端的STEP-Tag融合在大肠杆菌中实现了RTF-CCT的高效表达。(F)建立了检测牙周炎患者牙龈沟体液中抗Tf-CCT抗体的ELISA体系。AAA-CdtB Vas的氨基酸序列与放线菌血清型(B)相同，而DNA序列包含两个替换。同时，我们还建立了检测牙周炎患者牙龈沟中抗AA-CDtB蛋白抗体的酶联免疫吸附试验系统。较少

[I] We had detected a novel activity of Tannerella forsyhtensis cytocidal toxin (Tf-CCT) from an extract of the periodontopathic bacteria that exhibited Cytolethal distending toxin (CDT)-like activity. To identify what is Tf-CCT, and to investigate the mechanism how Tf-CCT exhibits a cytopathy, we cloned its gene on the basis of amino acid sequence of purified protein, and subsequently, we confirmed the cytopathic activity of the cloned gene product. Moreover, we estimated a domain which contributes to the cytopathy of Tf-CCT. Details of the report are following;(a) Identification of Tf-CCT protein purified from T. forsytliensis extract. Tf-CCT protein purified by anion-exchange, size-exclusion and heparin-affinity was identified by the monoclonal antibody Be-24 that was established for neutralization against the cytopathy of T. forsydiensis extract. An 28.5 kDa fragment of Tf-CCT was chosen for the N-terminal amino acid sequence analysis, and a degenerative primer was prepared for the … More resulted sequence APQNMDVLL. The sequence analysis for both PCR product and cloned gene obtained from T.forsythensis genomic DNA library revealed that Tf-CCT was identical with prtH protease (prtH). However, the translation initiation site resides in upstream of the reported site hence the molecular size of natural Tf-CCT detected in the purified fraction was larger than estimated size of pt-Il-I.(b) To identify the translation initiation site, tirst (without ribosome binding motif; RBS), fifth (reported site with RBS) and eighth (with more typical RBS) were chosen as the first ATG to construct the recombinant gene, respectively. Albeit the extensive trial with different types of expression vectors for Tf-CCT was examined, no recombinant Tf-CCT was expressed in Esclmerichia coli whereas the significant expression was detected by in vitro expression system such as RTS and PURESYSTEM.(c) The recombinant Tf-CCT (rTf-CCT) expressed from fifth ATG (identical with reported prtH) exhibited the greatest cytopathy.(d) The protein BLAST revealed that Tf-CCT shares no similarity with any reported CDT besides a distinct homology with third domain of human vesicle transport-related protein slylp in the C-terminal side. Thus, the cytopathy of Tf-CCT is likely to exhibit through interaction with proteins that contribute regulation of intracellular membrane fusion.(e) To get the expression of rTf-CCT in vivo, we tried to fuse recombinant with several kinds of protein-tags, and eventually we found that C-terminal strep-tag fusion achieved sufficient expression of rTf-CCT in E. coli.(f) An ELISA system was examined for detection of anti-Tf-CCT antibody contained by humor in gingival sulcus from the patients of periodontitis.[II] We cloned and identified and cdtB gene of Actinobacillus actinomycetemcomilans serotype(a) (Aaa-cdtB gene). The amino acid sequence of Aaa-CdtB vas revealed to be identical with that of A. actinomycetemcotnitans serotype (b), while the DNA sequence contains two substitutions. We also examined an ELISA system for detection of anti-Aa-CdtB protein antibody contained by humor in gingival sulcus from the patients of periodontitis. Less