Departments of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita, Osaka, 565-0871, Japan; Departments of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka, 565-0871, Japan.
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan, and.
J Biol Chem. 2012 Mar 23;287(13):10472-10481. doi: 10.1074/jbc.M111.280321. Epub 2012 Jan 18.
Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia throughout the world, with high morbidity and mortality rates. A major feature of pneumococcal pneumonia is abundant neutrophil infiltration. In this study, we identified S. pneumoniae α-enolase as a neutrophil binding protein in ligand blot assay and mass spectrometry findings. Scanning electron microscopic and fluorescence microscopic analyses also revealed that S. pneumoniae α-enolase induces formation of neutrophil extracellular traps, which have been reported to bind and kill microbes. In addition, cytotoxic assay results showed that α-enolase dose-dependently increased the release of extracellular lactate dehydrogenase from human neutrophils as compared with untreated neutrophils. Furthermore, an in vitro cell migration assay using Chemotaxicell culture chambers demonstrated that α-enolase possesses neutrophil migrating activity. Interestingly, bactericidal assay findings showed that α-enolase increased neutrophil extracellular trap-dependent killing of S. pneumoniae in human blood. Moreover, pulldown assay and mass spectrometry results identified myoblast antigen 24.1D5 as an α-enolase-binding protein on human neutrophils, whereas flow cytometric analysis revealed that 24.1D5 was expressed on human neutrophils, but not on human monocytes or T cells. Together, our results indicate that α-enolase from S. pneumoniae increases neutrophil migrating activity and induces cell death of human neutrophils by releasing neutrophil extracellular traps. Furthermore, we found that myoblast antigen 24.1D5, which expressed on the surface of neutrophils, bound to α-enolase of S. pneumoniae.
肺炎链球菌是世界范围内社区获得性肺炎最常见的病原体，具有较高的发病率和死亡率。肺炎链球菌性肺炎的一个主要特征是大量中性粒细胞浸润。在这项研究中，我们通过配体印迹分析和质谱发现，肺炎链球菌α-烯醇酶是一种中性粒细胞结合蛋白。扫描电子显微镜和荧光显微镜分析也表明，肺炎链球菌α-烯醇酶诱导中性粒细胞胞外诱捕网的形成，据报道，这些诱捕网可以结合并杀死微生物。此外，细胞毒性测定结果表明，与未经处理的中性粒细胞相比，α-烯醇酶可剂量依赖性地增加人中性粒细胞释放细胞外乳酸脱氢酶。此外，使用 Chemotaxicell 培养室的体外细胞迁移测定表明，α-烯醇酶具有中性粒细胞迁移活性。有趣的是，杀菌测定结果表明，α-烯醇酶增加了人血液中中性粒细胞胞外诱捕网依赖的肺炎链球菌杀伤作用。此外，下拉测定和质谱结果鉴定了肌母细胞抗原 24.1D5 是人中性粒细胞上的α-烯醇酶结合蛋白，而流式细胞分析显示 24.1D5 表达于人中性粒细胞上，但不表达于人单核细胞或 T 细胞上。总之，我们的结果表明，肺炎链球菌的α-烯醇酶通过释放中性粒细胞胞外诱捕网增加中性粒细胞的迁移活性，并诱导人中性粒细胞的细胞死亡。此外，我们发现表达于人中性粒细胞表面的肌母细胞抗原 24.1D5 与肺炎链球菌的α-烯醇酶结合。
