Terada L S, Johansen K A, Nowbar S, Vasil A I, Vasil M L
University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Infect Immun. 1999 May;67(5):2371-6. doi: 10.1128/IAI.67.5.2371-2376.1999.
Pseudomonas aeruginosa is a persistent pathogen in the airways of patients with cystic fibrosis or bronchiectasis from other causes and appears to have evolved strategies to survive the inflammatory response of the host. We hypothesized that the secreted hemolytic phospholipase C (PLC) of P. aeruginosa (PlcHR) would decrease neutrophil respiratory burst activity. We found that while intact wild-type P. aeruginosa cells stimulated moderate respiratory burst activity from human neutrophils, an isogenic mutant pseudomonas (DeltaHR strain) containing a targeted deletion of the plcHR operon induced a much more robust oxidative burst from neutrophils. In contrast, a second pseudomonas mutant (DeltaN) containing a disruption in the gene encoding the nonhemolytic PLC (PlcN) was not different from the wild type in stimulating neutrophil O2.- production. Readdition of purified PlcHR to the DeltaHR strain suppressed neutrophil O2.- production to levels stimulated by wild-type bacteria. Interestingly, purified PlcHR decreased phorbol myristate acetate (PMA)- but not formyl methionyl-leucyl-proline (fMLP)-induced respiratory burst activity, suggesting interference by PlcHR with a protein kinase C (PKC)-specific signaling pathway. Accordingly, the PKC inhibitor bisindolylmaleimide inhibited the oxidative burst induced by either PMA or intact pseudomonas, but not by fMLP, whereas the p38 kinase inhibitor SB-203580 fully inhibited the respiratory burst induced by fMLP or the PlcHR-replete wild-type bacteria, but not PMA or the PlcHR-deficient DeltaHR bacterial mutant. We conclude that expression of PlcHR by P. aeruginosa suppresses bacterium-induced neutrophil respiratory burst by interfering with a PKC-dependent, non-p38 kinase-dependent pathway.
铜绿假单胞菌是囊性纤维化患者或其他原因导致的支气管扩张患者气道中的一种持续性病原体，并且似乎已经进化出在宿主炎症反应中存活的策略。我们推测铜绿假单胞菌分泌的溶血磷脂酶C（PLC）（PlcHR）会降低中性粒细胞的呼吸爆发活性。我们发现，完整的野生型铜绿假单胞菌细胞刺激人中性粒细胞产生适度的呼吸爆发活性，而一个靶向缺失plcHR操纵子的同基因假单胞菌突变体（DeltaHR菌株）诱导中性粒细胞产生更强有力的氧化爆发。相比之下，第二个假单胞菌突变体（DeltaN），其编码非溶血PLC（PlcN）的基因发生了破坏，在刺激中性粒细胞产生超氧阴离子方面与野生型没有差异。将纯化的PlcHR重新添加到DeltaHR菌株中，可将中性粒细胞产生超氧阴离子的水平抑制到野生型细菌刺激的水平。有趣的是，纯化的PlcHR降低了佛波酯肉豆蔻酸酯乙酸酯（PMA）诱导的呼吸爆发活性，但没有降低甲酰甲硫氨酰亮氨酰脯氨酸（fMLP）诱导的呼吸爆发活性，这表明PlcHR干扰了蛋白激酶C（PKC）特异性信号通路。因此，PKC抑制剂双吲哚马来酰亚胺抑制了PMA或完整假单胞菌诱导的氧化爆发，但不抑制fMLP诱导的氧化爆发，而p38激酶抑制剂SB - 203580完全抑制了fMLP或含有PlcHR的野生型细菌诱导的呼吸爆发，但不抑制PMA或缺乏PlcHR的DeltaHR细菌突变体诱导的呼吸爆发。我们得出结论，铜绿假单胞菌表达的PlcHR通过干扰PKC依赖性、非p38激酶依赖性途径抑制细菌诱导的中性粒细胞呼吸爆发。
