Extracellular proteolytic activities expressed by Bacillus pumilus isolated from endodontic and periodontal lesions
Extracellular proteolytic activities expressed by Bacillus pumilus isolated from endodontic and periodontal lesions
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DOI：
10.1099/jmm.0.47754-0
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发表时间：
2008-05-01
影响因子：
3
通讯作者：
Mayo, John A.
中科院分区：
文献类型：
--
作者：
Johnson, Blair T.;Shaw, Lindsey N.;Mayo, John A.
本研究的目的是鉴定从需要牙髓治疗的牙齿的根管和严重边缘牙周炎的牙周袋中获得的12株芽孢杆菌，并确定这些菌株是否具有细胞外蛋白分解活性，以及通过体外检测，是否有这种活性能够降解与牙髓和牙周病变的产生有关的病理生理底物。通过对菌株BJ0055的16S rRNA基因序列测定，对所有菌株进行了生化和碳水化合物发酵模式的鉴定。所有菌株产生胞外蛋白水解酶活性的筛选都是在普通的蛋白酶底物上进行的。所有的菌株都被鉴定为短小芽孢杆菌，并且都具有胞外蛋白水解活性。利用荧光团标记的弹性蛋白和胶原蛋白以及几种发色多肽，对菌株BJ0055产生的胞外蛋白酶的可能的病理生理相关性进行了评估。使用特定类别的抑制剂研究作用于每种底物的可能类别的蛋白水解酶。测定了不同pH值下缓冲液中的活性-pH曲线。胞外活性有酪蛋白、弹性蛋白、胶原酶、谷氨酰内肽酶和丙氨酰三肽酶样活性。未检测到类胰蛋白酶活性。检测到丝氨酸和类胰凝乳酶的丝氨酸蛋白酶活性，在中性和碱性，但不是酸性的pH下观察到活性。从牙髓和牙周病变分离的短小芽孢杆菌菌株显示出降解弹性蛋白、胶原和其他底物的胞外活性。这些活性可能是导致根尖周炎和严重边缘牙周炎组织损伤的致病因子。
The purpose of the present study was to identify 12 Bacillus isolates that had been obtained from root canals of teeth requiring endodontic therapy and from periodontal pockets in severe marginal periodontitis, and to determine whether these isolates exhibited extracellular proteolytic activity and, using in vitro assays, whether any such activity could degrade substrates that would be pathophysiologically relevant with regard to the production of endodontic and periodontal lesions. Biochemical and carbohydrate fermentation patterns were used in the identification of all strains, which was confirmed by determination of the 16S rRNA gene sequence for strain BJ0055. Screening for production of extracellular proteolytic activity by all strains was done with a general proteinase substrate. All isolates were identified as representing Bacillus pumilus and all exhibited extracellular proteolytic activity. The putative pathophysiological relevance of extracellular proteinase production in strain BJ0055 was assessed using fluorophore-labelled elastin and collagen and several chromogenic peptides. Probable classes of proteinases acting on each substrate were investigated using class-specific inhibitors. Activity-pH profiles were determined in buffers at different pH values. Extracellular activities that were caseinolytic, elastinolytic, collagenolytic, glutamyl endopeptidase-like, and alanyl tripeptidyl peptidase-like were observed. No trypsin-like activities were detected. Serine- and chymotrypsin-like serine proteinase activities were detected, with activity observed at neutral and alkaline, but not acidic, pH. B. pumilus strains isolated from endodontic and periodontal lesions exhibited extracellular activities that degrade elastin, collagen and other substrates. These activities may be virulence factors that contribute to tissue damage in apical periodontitis and severe marginal periodontitis.