Wang Shiwei, Shen Aimee, Setlow Peter, Li Yong-qing
Department of Physics, East Carolina University, Greenville, North Carolina, USA.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.
J Bacteriol. 2015 Jul;197(14):2361-73. doi: 10.1128/JB.00200-15. Epub 2015 May 4.
The Gram-positive spore-forming anaerobe Clostridium difficile is a leading cause of nosocomial diarrhea. Spores of C. difficile initiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individual C. difficile spores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar to Bacillus spores, individual C. difficile spores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca(2+) and dipicolinic acid (CaDPA) at a heterogeneous time T1, rapidly released CaDPA at Tlag, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlysis. T1 and Tlag values for individual spores were heterogeneous, but ΔTrelease periods (Trelease - Tlag) were relatively constant. In contrast to Bacillus spores, heat treatment did not stimulate spore germination in the two C. difficile strains tested. C. difficile spores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate. C. difficile spores did not germinate with CaDPA, in contrast to B. subtilis and C. perfringens spores. However, the detergent dodecylamine induced C. difficile spore germination, and rates were increased by spore coat removal although cortex hydrolysis did not follow Trelease, in contrast with B. subtilis. C. difficile spores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the few sleC spores that partially germinated. Overall, these findings indicate that C. difficile and B. subtilis spore germination exhibit key differences.
Spores of the Gram-positive anaerobe Clostridium difficile are responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts, C. difficile spores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individual C. difficile spores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events in C. difficile spores differs from that in Bacillus spores and provide new insights into C. difficile spore germination.
革兰氏阳性产芽孢厌氧菌艰难梭菌是医院获得性腹泻的主要病因。艰难梭菌的芽孢在胃肠道中被胆盐触发萌发生长时引发感染。我们使用拉曼光谱和微分干涉对比（DIC）显微镜分析了单个艰难梭菌芽孢的萌发生长动力学。与芽孢杆菌属的芽孢相似，单个艰难梭菌芽孢在牛磺胆酸盐加甘氨酸存在的情况下开始萌发生长，在异质时间T1时开始缓慢泄漏约15%浓度的钙离子和吡啶二羧酸螯合物（CaDPA），在Tlag时快速释放CaDPA，在Trelease时完成CaDPA释放，并在Tlysis时完成肽聚糖皮层水解。单个芽孢的T1和Tlag值是异质的，但ΔTrelease期（Trelease - Tlag）相对恒定。与芽孢杆菌属的芽孢不同，热处理并未刺激所测试的两种艰难梭菌菌株的芽孢萌发生长。艰难梭菌芽孢单独在牛磺胆酸盐或甘氨酸存在的情况下不会萌发生长，不同的胆盐对芽孢萌发生长的促进作用不同，其中牛磺胆酸盐和牛磺脱氧胆酸盐的促进效果最佳。将芽孢短暂暴露于牛磺胆酸盐加甘氨酸中足以使单个芽孢开始萌发生长。与枯草芽孢杆菌和产气荚膜梭菌的芽孢不同，艰难梭菌芽孢在CaDPA存在的情况下不会萌发生长。然而，去污剂十二烷基胺可诱导艰难梭菌芽孢萌发生长，去除芽孢衣可提高萌发生长速率，不过与枯草芽孢杆菌不同的是，皮层水解并不跟随Trelease发生。缺乏皮层溶解酶SleC的艰难梭菌芽孢萌发生长极差，在少数部分萌发生长的sleC芽孢中未观察到皮层水解现象。总体而言，这些发现表明艰难梭菌和枯草芽孢杆菌的芽孢萌发生长存在关键差异。
革兰氏阳性厌氧菌艰难梭菌的芽孢是这种重要的医院病原体引发感染的原因。当暴露于牛磺胆酸盐等萌发剂时，艰难梭菌芽孢在胃肠道中通过萌发生长恢复活性并引发疾病，但其萌发生长仅通过全群体测量进行过研究。在这项工作中，我们使用拉曼光谱和DIC显微镜监测单个艰难梭菌芽孢的萌发生长动力学、芽孢对萌发生长的启动作用以及萌发剂类型和浓度、亚致死热休克和去除芽孢衣对萌发生长的影响。我们的数据表明艰难梭菌芽孢萌发生长事件的顺序与芽孢杆菌属芽孢不同，并为艰难梭菌芽孢萌发生长提供了新的见解。
