van Leengoed L A, Dickerson H W
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602.
Infect Immun. 1992 Feb;60(2):353-9. doi: 10.1128/iai.60.2.353-359.1992.
In vitro production of a secreted hemolytic cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of calcium in culture media. This is not the case with Escherichia coli hemolysins, however, where calcium has been shown to be required only for activation and binding to target cells. Because the cytolysins of A. pleuropneumoniae have structural and functional similarities to those of hemolytic E. coli, we sought to reexamine the role that calcium plays in the secretion and activity of A. pleuropneumoniae cytolysins. A. pleuropneumoniae hemolytic strain S4074 secreted two major proteins into culture supernatants independent of the presence of calcium in growth medium. These proteins were identified with murine monoclonal antibodies as the 105-kDa cytolysin I and the 103-kDa cytolysin II. It was found that both cytolysins required calcium for binding to erythrocyte membranes. Culture fluids from bacteria grown with calcium lysed porcine erythrocytes even after free calcium in the fluid was removed prior to the hemolytic assay. When bacteria were grown in medium depleted of calcium, no lysis of erythrocytes was detected unless calcium was added to assay buffers. Culture supernatants from A. pleuropneumoniae nonhemolytic strain 1421 grown with or without calcium contained two predominant proteins, which were identified with mouse monoclonal antibodies as the 103-kDa cytolysin II and the 120-kDa cytolysin III. Binding to erythrocytes (without hemolysis) by cytolysin II was dependent on calcium. Cytolysin III did not bind to erythrocytes. These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of cytolysin I. Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of calcium. When calcium was depleted, cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all. The data suggest that regardless of the target cell involved, calcium plays an integral role in the function but not the production of A. pleuropneumoniae cytolysins.
据报道，胸膜肺炎放线杆菌分泌性溶血素的体外产生依赖于培养基中钙的存在。然而，对于大肠杆菌溶血素而言并非如此，在大肠杆菌中，钙仅被证明是激活和与靶细胞结合所必需的。由于胸膜肺炎放线杆菌的溶血素与溶血性大肠杆菌的溶血素在结构和功能上具有相似性，我们试图重新研究钙在胸膜肺炎放线杆菌溶血素的分泌和活性中所起的作用。胸膜肺炎放线杆菌溶血菌株S4074将两种主要蛋白质分泌到培养上清液中，这与生长培养基中钙的存在无关。这些蛋白质被鼠单克隆抗体鉴定为105 kDa的溶血素I和103 kDa的溶血素II。发现两种溶血素都需要钙来结合红细胞膜。即使在溶血试验前去除了培养液中的游离钙，用含钙培养基培养的细菌的培养液仍能裂解猪红细胞。当细菌在缺钙的培养基中生长时，除非在测定缓冲液中添加钙，否则未检测到红细胞裂解。用含钙或不含钙培养基培养的胸膜肺炎放线杆菌非溶血菌株1421的培养上清液含有两种主要蛋白质，它们被鼠单克隆抗体鉴定为103 kDa的溶血素II和120 kDa的溶血素III。溶血素II与红细胞的结合（不发生溶血）依赖于钙。溶血素III不与红细胞结合。这些结果表明，菌株S4074裂解猪红细胞的能力（以及菌株1421不具备这种能力）与溶血素I的存在直接相关。溶血素I、II和III仅在有钙的情况下才与猪中性粒细胞和纯化的中性粒细胞膜结合。当钙被耗尽时，菌株S4074的溶血素I结合能力降低，而菌株1421的溶血素II和III根本不结合。数据表明，无论涉及何种靶细胞，钙在胸膜肺炎放线杆菌溶血素的功能而非产生过程中起着不可或缺的作用。
