Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa
Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa
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DOI：
10.1074/jbc.m603088200
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发表时间：
2007-03-02
影响因子：
4.8
通讯作者：
Ito, Makoto
中科院分区：
文献类型：
--
作者：
Okino, Nozomu;Ito, Makoto
我们先前报道了来自铜绿假单胞菌菌株AN 17的中性神经酰胺酶的纯化、分子克隆和表征（Okino，N.，Tani，M.，Imayama，S.，Ito，M.（1998）J.Biol.Chem.273，14368 -14373; Okino，N.，Ichinose，S.，Omori，A.，Imayama，S.，中村，T.，Ito，M.（1999）J.Biol.Chem.274，36616 -36622）。有趣的是，编码该酶的基因与铜绿假单胞菌基因组中编码溶血性磷脂酶C（plcH）的基因相邻，铜绿假单胞菌是众所周知的机会性感染病原体。我们在这里报告，同时生产的PlcH和神经酰胺酶诱导的几种脂质和PlcH诱导的溶血显着增强的神经酰胺酶的作用。当该菌株与鞘磷脂或磷脂酰胆碱一起培养时，两种酶的产生急剧增加，导致无细胞培养上清液中溶血活性的增加。神经酰胺和鞘氨醇也有效地促进神经酰胺酶的产生，但不是PlcH。此外，我们发现，溶血活性的蜡状芽孢杆菌鞘磷脂酶显着增强通过添加重组假单胞菌神经酰胺酶。红细胞的TLC分析表明，由鞘磷脂酶从鞘磷脂产生的神经酰胺被神经酰胺酶部分转化为鞘氨醇。神经酰胺酶无效突变株引起的绵羊红细胞溶血比野生型菌株少得多。在与野生型菌株共培养的红细胞中检测到鞘氨醇，但在与突变型菌株共培养的红细胞中检测不到鞘氨醇。最后，我们发现，增强PlcH诱导的溶血神经酰胺酶不仅发生在绵羊，但也人的红细胞。这些结果可能表明神经酰胺酶增强了PlcH诱导的细胞毒性，并为鞘脂降解酶在铜绿假单胞菌致病性中的作用提供了新的见解。
We previously reported the purification, molecular cloning, and characterization of a neutral ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273,14368-14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274,36616-36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the ceramidase. A ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcH-induced hemolysis by the ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.