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Study of the virulence factors from periodontopathic bacteria

牙周病细菌毒力因子的研究

基本信息

批准号：
06671832
负责人：
KATO Tetsuo
金额：
$ 1.34万
依托单位：
TOKYO DENTAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Porphyromonas gingivalis, a gram-negative anaerobic rod, has been implicated as a periodontal pathogen. Black-pigmented anaerobic rods such as P.gingivalis show especially high resistance to kanamycin (KM) as compared with the other anaerobic bacteria. A gene for KM-inactivating protein has been isolated from P.gingivalis strain 16-1 in Escheichia coli, utilizing the plasmid vector pTZ18R.The resultant kanamycin resistant E.coli clone, harboring plasmid pPG16 with a P.gingivalis DNA insert, expressed KM-inactivating activity, which was enhanced by hte addition of acetyl CoA,indicating that the KM was inactivated by acetylation. Southern blot analysis indicated that the gene is conserved among all P.gingivalis strains tested. Rabbit antiserum to P.gingivalis 16-1 whole cells reacted with a band of ca.35kDa when a sonic extract of an E.coli JM109 containing pPG16 was prepared for SDS-PAGE.These results indicate that P.gingivalis possesses KM-inactivating activity and that a gene coding for KM-inactivating activity has been isolated from P.gingivalis.Actinobacillus actinomycetemcomitans is associated with human periodontal diseases. Recent studies have reported detectable a hemolytic activity of this organism on blood agar plates. Here we report the isolation and characterization of recombinant clone from a genomic library of A.actinomycetemcomitans ATCC 43718 that confer on E.coli the ability to lyse horse red blood cells. DNA hybridization analysis indicates that the gene was conserved among all A.actinomycetemcomitans strains tested. E.coli transformed with this gene lysed horse.sheep and human erythrocytes but not those of rabbit. Heating and treatment with proteinase K resulted in the complete hindrance of the hemolytic activity. Further study will help in understanding the function of A.actinomycetemcomitans hemolytic activity as a periodontal virulence factor.

牙龈卟啉单胞菌是一种革兰氏阴性厌氧杆菌，被认为是牙周致病菌。与其他厌氧菌相比，黑色厌氧杆菌（如牙龈卟啉单胞菌）对卡那霉素（KM）表现出特别高的抗性。利用质粒载体pTZ 18 R从牙龈卟啉单胞菌16-1菌株中克隆到一个KM失活蛋白基因，构建了一个卡那霉素抗性克隆pPG 16，该克隆表达KM失活蛋白，乙酰辅酶A的加入增强了KM的失活活性，表明KM是通过乙酰化而失活的。Southern杂交分析表明，该基因在所有供试牙龈卟啉单胞菌菌株中是保守的。用兔抗牙龈卟啉单胞菌16-1全细胞的抗血清制备含pPG 16的大肠杆菌JM 109的声波提取物，经SDS-PAGE检测，可得到一条分子量约为35 kDa的带，表明牙龈卟啉单胞菌具有Km失活活性，并从牙龈卟啉单胞菌中分离到了Km失活基因。最近的研究报告了可检测的溶血活性，这种生物体的血琼脂平板。本文报道了从伴放线菌ATCC 43718的基因组文库中分离并鉴定重组克隆，该克隆赋予大肠杆菌裂解马红细胞的能力。DNA杂交分析表明，该基因在所有供试的伴放线菌菌株中是保守的。用该基因转化的大肠杆菌能裂解马、羊和人的红细胞，但不裂解兔的红细胞。加热和用蛋白酶K处理导致溶血活性的完全阻碍。进一步的研究将有助于了解伴放线菌溶血活性作为牙周致病因子的作用。