Murphy T F, Loeb M R
Department of Medicine, State University of New York, Buffalo.
Microb Pathog. 1989 Mar;6(3):159-74. doi: 10.1016/0882-4010(89)90066-1.
The emergence of Branhamella catarrhalis as an important human pathogen has stimulated interest in investigations of the outer membrane (OM) of the bacterium. In this study, the OM of B. catarrhalis was isolated and partially characterized. Radiolabelled cells were lysed and fractionated by isopycnic centrifugation in a continuous sucrose gradient. Five fractions were identified. Fraction A consisted of OM fragments of varying density. Fractions B and C were OM of a discrete density containing some cytoplasmic membrane. Fraction D was cytoplasmic membrane and Fraction E contained smaller less dense fragments of cytoplasmic membrane. The protein composition of the Branhamella OM is typical for that of Gram-negative bacteria in that approximately 10 to 20 proteins were present with six to eight of these proteins predominating. Having isolated and partially characterized the OM by sucrose density centrifugation, five simpler techniques for isolating OM were employed and the preparations compared to OM isolated on the gradient. Techniques that are based on differential detergent solubility of OM and cytoplasmic membrane were ineffective in isolating OM of B. catarrhalis. By contrast, techniques that involved collection of OM vesicles were successful in isolating OM of B. catarrhalis. Collection of vesicles from broth culture supernatants and EDTA-heat-induced vesicles were identified as convenient and reliable methods for isolating OM. Isolating and partially characterizing the OM of B. catarrhalis represents an initial step in a systematic study of outer membrane antigens of the bacterium.
卡他布兰汉菌作为一种重要的人类病原体的出现，激发了人们对该细菌外膜（OM）研究的兴趣。在本研究中，卡他布兰汉菌的外膜被分离并进行了部分特性分析。用放射性标记的细胞经连续蔗糖梯度等密度离心裂解并分级分离。鉴定出五个组分。组分A由不同密度的外膜片段组成。组分B和C是具有离散密度且含有一些细胞质膜的外膜。组分D是细胞质膜，组分E含有较小、密度较低的细胞质膜片段。卡他布兰汉菌外膜的蛋白质组成是革兰氏阴性菌的典型组成，大约有10到20种蛋白质，其中6到8种蛋白质占主导。通过蔗糖密度离心分离并部分表征了外膜后，采用了五种更简单的外膜分离技术，并将制备物与在梯度上分离的外膜进行比较。基于外膜和细胞质膜不同洗涤剂溶解度的技术在分离卡他布兰汉菌外膜方面无效。相比之下，涉及收集外膜囊泡的技术成功地分离出了卡他布兰汉菌的外膜。从肉汤培养上清液中收集囊泡以及EDTA热诱导囊泡被确定为分离外膜的方便可靠方法。分离并部分表征卡他布兰汉菌的外膜是对该细菌外膜抗原进行系统研究的第一步。
