E-coli virulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosislysis and lung injury in a rat pneumonia model
E-coli virulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosislysis and lung injury in a rat pneumonia model
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DOI：
10.1152/ajplung.00482.2004
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发表时间：
2005-08-01
影响因子：
4.9
通讯作者：
Knight, PR
中科院分区：
文献类型：
--
作者：
Russo, TA;Davidson, BA;Knight, PR
肠道革兰氏阴性杆菌，例如大肠杆菌，是院内肺炎的最常见原因。在本研究中，在体外和革兰氏阴性肺炎大鼠模型中评估了野生型肠外致病性大肠杆菌 (ExPEC) (CP9) 和缺乏溶血素 (Hly) 和细胞毒性坏死因子 (CNF) 的同基因衍生物，以检验这些毒力因子诱导中性粒细胞凋亡和/或坏死/溶解的假设。通过体外 caspase-3/7 和 LDH 活性以及中性粒细胞形态确定，Hly 在较低的大肠杆菌滴度 (1 x 10(5-6) cfu) 下介导中性粒细胞凋亡，在较高滴度 (>= 1 x 10(7) cfu) 下介导坏死/裂解。数据表明，CNF 促进细胞凋亡，但不促进坏死或裂解。我们还证明，膜联蛋白 V/7-氨基-放线菌素 D 染色对于使用活大肠杆菌进行的细胞凋亡评估是不可靠的。 caspase-3/7和LDH活性以及中性粒细胞形态的使用支持了这样的观点：在我们使用活大肠杆菌的体内革兰氏阴性肺炎模型中，坏死而不是细胞凋亡是影响中性粒细胞的主要机制。此外，体内研究表明 Hly 介导肺损伤。当用纯化的脂多糖攻击动物时，没有观察到中性粒细胞坏死，这证明了使用活细菌的重要性。这些发现证实，Hly 通过介导中性粒细胞毒性来促进 ExPEC 毒力，其中坏死/溶解是 Hly 对体内中性粒细胞和肺损伤的主要影响。 Hly 介导的肺损伤是否是由于中性粒细胞坏死、Hly 的直接作用或两者兼而有之，尚不清楚。
Enteric gram-negative bacilli, such as Escherichia coli are the most common cause of nosocomial pneumonia. In this study a wild-type extraintestinal pathogenic strain of E. coli (ExPEC)(CP9) and isogenic derivatives deficient in hemolysin (Hly) and cytotoxic necrotizing factor (CNF) were assessed in vitro and in a rat model of gram-negative pneumonia to test the hypothesis that these virulence factors induce neutrophil apoptosis and/or necrosis/lysis. As ascertained by in vitro caspase-3/7 and LDH activities and neutrophil morphology, Hly mediated neutrophil apoptosis at lower E. coli titers (1 x 10(5-6) cfu) and necrosis/lysis at higher titers (>= 1 x 10(7) cfu). Data suggest that CNF promotes apoptosis but not necrosis or lysis. We also demonstrate that annexin V/7-amino-actinomycin D staining was an unreliable assessment of apoptosis using live E. coli. The use of caspase-3/7 and LDH activities and neutrophil morphology supported the notion that necrosis, not apoptosis, was the primary mechanism by which neutrophils were affected in our in vivo gram-negative pneumonia model using live E. coli. In addition, in vivo studies demonstrated that Hly mediates lung injury. Neutrophil necrosis was not observed when animals were challenged with purified lipopolysaccharide, demonstrating the importance of using live bacteria. These findings establish that Hly contributes to ExPEC virulence by mediating neutrophil toxicity, with necrosis/lysis being the dominant effect of Hly on neutrophils in vivo and by lung injury. Whether Hly-mediated lung injury is due to neutrophil necrosis, a direct effect of Hly, or both is unclear.