Yotis W W, Sharma V K, Gopalsami C, Chegini S, McNulty J, Hoerman K, Keene J, Simonson L G
Department of Microbiology, Loyola University Medical Center, Maywood, Illinois 60153.
J Clin Microbiol. 1991 Jul;29(7):1397-406. doi: 10.1128/jcm.29.7.1397-1406.1991.
The outer membranes (OMs) from serovars a, b, and c of Treponema denticola, originally isolated from periodontal patients, were prepared. Dialysis of the OMs against 20 mM MgCl2 yielded the aggregable (A) and the nonaggregable (NA) moieties of the OMs. The absence of muramic acid, adenosine triphosphatase, hexokinase, and nucleic acid as well as electron microscopy indicated that the OM preparations were homogeneous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the A and NA moieties of the OMs showed approximately 25 Coomassie brilliant blue R-250 stain-positive bands or 47 silver-stained polypeptides. The relative molecular masses ranged between 14 and 97 kDa. The electrophoretic polypeptide profiles of the A and NA moieties shared many similarities among serovars a, b, and c. However, they exhibited variation in the overall pattern, intensity, or location of the polypeptide stained zones. This was especially true for serovar b. Two-dimensional electrophoretic studies showed an excess of 100 silver-stained spots with isoelectric points of 4.6 to 7.0 and relative molecular masses in the 14- to 97-kDa range. The OMs contained simple proteins, glycoproteins, and lipoproteins. The NA moieties of the OMs contained 4 to 6, 10 to 12, and 4 to 6 glycopeptides as well as two, seven, and two lipoprotein bands for serovars a, b, and c, respectively. The A moieties of the OMs showed 7 to 9, 11 to 13 and 5 to 6 glycopeptides as well as four, five, and three lipoprotein bands for serovars a, b, and c, respectively. Lipopolysaccharide was detected in the OMs of the three serovars following removal of proteins with proteinase K, pronase and silver staining of sodium dodecyl sulfate-polyacrylamide gels, or removal of lipopolysaccharide from the OMs by hot phenol extraction. The 66- and 53-kDa bands were present in serovars b and c, while a band with a relative molecular mass of 45 kDa was present only in serovar c. Endotoxin-like activity was also shown in the OMs of the three serovars by the Limulus amebocyte clotting assay and the chick embryo lethality test. This is the first report on selected biochemical properties of the OM macromolecules of three known serovars of T. denticola.
制备了最初从牙周病患者中分离出的齿垢密螺旋体血清型a、b和c的外膜（OMs）。将OMs对20 mM MgCl₂进行透析，得到了OMs的可聚集部分（A）和不可聚集部分（NA）。缺乏胞壁酸、三磷酸腺苷酶、己糖激酶和核酸以及电子显微镜检查表明OM制剂是均一的。对OMs的A和NA部分进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，显示出约25条考马斯亮蓝R - 250染色阳性条带或47条银染多肽。相对分子质量在14至97 kDa之间。血清型a、b和c的A和NA部分的电泳多肽图谱有许多相似之处。然而，它们在多肽染色区的整体模式、强度或位置上表现出差异。血清型b尤其如此。二维电泳研究显示有超过100个银染斑点，其等电点为4.6至7.0，相对分子质量在14至97 kDa范围内。OMs包含简单蛋白质、糖蛋白和脂蛋白。血清型a、b和c的OMs的NA部分分别含有4至6、10至12和4至6个糖肽以及两条、七条和两条脂蛋白带。血清型a、b和c的OMs的A部分分别显示7至9、11至13和5至6个糖肽以及四条、五条和三条脂蛋白带。在用蛋白酶K、链霉蛋白酶去除蛋白质并对十二烷基硫酸钠 - 聚丙烯酰胺凝胶进行银染后，或通过热酚提取从OMs中去除脂多糖后，在三种血清型的OMs中检测到脂多糖。66 kDa和53 kDa的条带出现在血清型b和c中，而相对分子质量为45 kDa的条带仅出现在血清型c中。通过鲎试剂变形细胞凝集试验和鸡胚致死试验，在三种血清型的OMs中也显示出类内毒素活性。这是关于齿垢密螺旋体三种已知血清型的OM大分子的选定生化特性的首次报道。
