Pizarro-Guajardo Marjorie, Calderón-Romero Paulina, Castro-Córdova Pablo, Mora-Uribe Paola, Paredes-Sabja Daniel
Gut Microbiota and Clostridia Research Group, Departamento de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile.
Gut Microbiota and Clostridia Research Group, Departamento de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile
Appl Environ Microbiol. 2016 Feb 5;82(7):2202-2209. doi: 10.1128/AEM.03410-15.
The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact of C. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863-3875, 2013, http://dx.doi.org/10.1128/JB.00369-13; J. Phetcharaburanin et al., Mol Microbiol 92:1025-1038, 2014, http://dx.doi.org/10.1111/mmi.12611), there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore's outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface of C. difficile spores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets.
厌氧芽孢形成菌艰难梭菌是发达国家和发展中国家医院内抗生素相关性腹泻的主要病因。代谢休眠的芽孢形式被认为是传播、感染和持续存在的形态类型，最外层的芽孢衣层在艰难梭菌芽孢与宿主首次接触期间的芽孢-宿主相互作用以及感染复发期间的芽孢持续存在中可能起主要作用。尽管已经对芽孢衣的生物学进行了一些研究（J. Barra-Carrasco等人，《细菌学杂志》195:3863-3875，2013年，http://dx.doi.org/10.1128/JB.00369-13；J. Phetcharaburanin等人，《分子微生物学》92:1025-1038，2014年，http://dx.doi.org/10.1111/mmi.12611），但关于该层的超微结构变异性和稳定性的信息却很缺乏。在这项工作中，我们利用透射电子显微镜照片分析了不同菌株中芽孢最外层的变异性，发现菌株内部和菌株之间芽孢衣的超微结构形态存在明显的变异性。通过透射电子显微镜照片，我们观察到尽管该层在芽孢纯化过程中是稳定的，但在室温下储存6个月后会部分丢失。这些观察结果通过间接免疫荧光显微镜得到了证实，在间接免疫荧光显微镜下观察到两种芽孢衣标记物BclA1的N端结构域和CdeC的水平显著下降。同样值得注意的是，从艰难梭菌生物膜获得的芽孢上芽孢衣标记物CdeC的存在取决于生物膜培养条件和所用菌株。总的来说，这些结果提供了关于艰难梭菌芽孢芽孢衣表面异质性和稳定性的信息。这些发现具有直接影响，在开发以芽孢衣蛋白为靶点诊断和/或清除艰难梭菌芽孢的新方法时应予以考虑。
