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Lipopolysaccharide Complexes with Pasteurella haemolytica Leukotoxin

机译：溶血巴斯德氏菌毒素的脂多糖复合物

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The presence of lipopolysaccharide (LPS) in gram-negative bacterial repeats-in-toxin (RTX) toxin preparations, as well as the harsh conditions required to remove it, suggests that LPS may complex with RTX toxins. Concentrated culture supernatant (CCS) preparations of the RTX toxin Pasteurella haemolytica leukotoxin (LKT) contained LKT and LPS as the most prominent components, with LKT and LPS constituting ≈30 and 50% of the density of the silver-stained fraction on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. CCS LKT contained 3.69 ± 0.46 mg of LPS per mg of protein, which was estimated to indicate an LPS/LKT molar ratio of ≈60:1. Subjection of the CCS LKT to preparative SDS-PAGE resulted in separation of LPS from LKT as detected by silver-stained analytical SDS-PAGE; however, the LKT fraction (SDS-PAGE LKT) contained significant endotoxin activity as detected by the Limulusamebocyte lysate assay. Subjection of the SDS-PAGE LKT to a second preparative SDS-PAGE run resulted in a reduction of the LPS/LKT molar ratio to 1:20. The target cell specificity of LKT for bovine leukocytic cells was retained by the SDS-PAGE LKT, and isolated LPS at comparable concentrations to that in CCS LKT exhibited no leukolytic activity. Addition of isolated LPS back to SDS-PAGE LKT resulted in reconstitution of an LPS-LKT complex. Immediately following reconstitution of the LPS-LKT complex, there was minimal change in leukolytic activity of the complex, but following 9.5 h at temperatures from ?135 to 37°C, the LPS-LKT complex exhibited increased leukolytic activity and thermal stability compared to SDS-PAGE LKT. Therefore, it appears that LPS complexes with LKT, resulting in enhanced and stabilized leukolytic activity.
机译：革兰氏阴性细菌重复毒素（RTX）毒素制品中脂多糖（LPS）的存在，以及去除脂多糖所需的苛刻条件，表明LPS可能与RTX毒素复合。 RTX毒素溶血巴斯德氏菌白细胞毒素（LKT）的浓缩培养上清液（CCS）制剂中LKT和LPS是最突出的成分，其中LKT和LPS构成银的≈30％和50％十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上分别染色的级分。 CCS LKT每毫克蛋白质包含3.69±0.46毫克LPS，据估计表明LPS / LKT摩尔比约为60：1。 CCS LKT受到制备型SDS-PAGE的影响，通过银染的分析性SDS-PAGE检测到，LPS与LKT分离。然而，通过 Li 嗜血红细胞裂解液检测发现，LKT级分（SDS-PAGE LKT）含有明显的内毒素活性。 SDS-PAGE LKT进行第二次制备性SDS-PAGE运行，导致LPS / LKT摩尔比降低至1:20。 SDS-PAGE LKT保留了LKT对牛白细胞的靶细胞特异性，并且与CCS LKT中浓度相当的分离LPS没有显示出白细胞溶解活性。将分离的LPS加回到SDS-PAGE LKT导致LPS-LKT复合物的重建。重组LPS-LKT复合物后，该复合物的溶白活性几乎没有变化，但在约135°C至37°C的温度下9.5小时后，与SDS相比，LPS-LKT复合物显示出更高的溶白活性和热稳定性。页LKT。因此，似乎LPS与LKT复合，导致增强和稳定的白细胞溶解活性。

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