Atapattu Dhammika N, Czuprynski Charles J
Department of Pathobiological Sciences, University of Wisconsin, School of Veterinary Medicine, 2015 Linden Dr. West, Madison, WI 53706, USA.
Infect Immun. 2005 Sep;73(9):5504-13. doi: 10.1128/IAI.73.9.5504-5513.2005.
Mannheimia haemolytica is a key pathogen in the bovine respiratory disease complex. It produces a leukotoxin (LKT) that is an important virulence factor, causing cell death in bovine leukocytes. The LKT binds to the beta(2) integrin CD11a/CD18, which usually activates signaling pathways that facilitate cell survival. In this study, we investigated mechanisms by which LKT induces death in bovine lymphoblastoid cells (BL-3). Incubation of BL-3 cells with a low concentration of LKT results in the activation of caspase-3 and caspase-9 but not caspase-8. Similarly, the proapoptotic proteins Bax and BAD were significantly elevated, while the antiapoptotic proteins Bcl-2, Bcl(XL) and Akt-1 were downregulated. Following exposure to LKT, we also observed a reduction in mitochondrial cytochrome c and corresponding elevation of cytosolic cytochrome c, suggesting translocation from the mitochondrial compartment to the cytosol. Consistent with this observation, tetramethylrhodamine ethyl ester perchlorate staining revealed that mitochondrial membrane potential was significantly reduced. These data suggest that LKT induces apoptosis of BL-3 cells via a caspase-9-dependent mitochondrial pathway. Furthermore, scanning electron micrographs of mitochondria from LKT-treated BL-3 cells revealed lesions in the outer mitochondrial membrane, which are larger than previous reports of the permeability transition pore through which cytochrome c is usually released.
溶血曼氏杆菌是牛呼吸道疾病综合征中的一种关键病原体。它产生一种白细胞毒素（LKT），这是一种重要的毒力因子，可导致牛白细胞死亡。LKT与β(2)整合素CD11a/CD18结合，而CD11a/CD18通常会激活促进细胞存活的信号通路。在本研究中，我们调查了LKT诱导牛淋巴母细胞（BL-3）死亡的机制。用低浓度LKT孵育BL-3细胞会导致caspase-3和caspase-9激活，但不会导致caspase-8激活。同样，促凋亡蛋白Bax和BAD显著升高，而抗凋亡蛋白Bcl-2、Bcl(XL)和Akt-1则下调。暴露于LKT后，我们还观察到线粒体细胞色素c减少，而胞质细胞色素c相应升高，这表明细胞色素c从线粒体区室转移到了胞质溶胶中。与此观察结果一致，四甲基罗丹明乙酯高氯酸盐染色显示线粒体膜电位显著降低。这些数据表明，LKT通过caspase-9依赖的线粒体途径诱导BL-3细胞凋亡。此外，对经LKT处理的BL-3细胞的线粒体进行扫描电子显微镜观察发现，线粒体外膜存在损伤，这些损伤比之前报道的通常通过其释放细胞色素c的通透性转换孔要大。
