Proteases and sonication specifically remove the exosporium layer of spores of Clostridium difficile strain 630
Proteases and sonication specifically remove the exosporium layer of spores of Clostridium difficile strain 630
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DOI：
10.1016/j.mimet.2013.01.016
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发表时间：
2013-04-01
影响因子：
2.2
通讯作者：
Paredes-Sabja, Daniel
中科院分区：
文献类型：
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作者：
Escobar-Cortes, Karina;Barra-Carrasco, Jonathan;Paredes-Sabja, Daniel
艰难梭菌孢子是这种厌氧病原体在医院表面和宿主体内持续存在的手段。人们对难辨梭菌孢子表面的蛋白质缺乏了解，主要是由于缺乏有效分离最外层（外孢子）的既定方法。在这项工作中，我们提出了通过蛋白酶消化或超声处理去除艰难梭菌孢子外孢子层的方法，使孢子外壳结构保持完整。透射电镜观察结果显示，萨克齐菌和蛋白酶K （SPk）处理艰难梭菌孢子后，孢子的外孢子囊完全脱落，而胰蛋白酶和超声波处理后孢子的外孢子囊大部分脱落，只留下附着在孢子外皮上的薄薄的外孢子囊层。艰难梭菌孢子的疏水性测量表明，SPk完全去除外孢子产生的孢子疏水性约为1%（即孢子处于有机相的百分比），而用胰蛋白酶或超声处理，留下一层薄薄的外孢子，产生的孢子疏水性约为10%。去除外孢子囊增加了艰难梭菌孢子形成菌落的能力。这些外孢子提取方法应该有助于进一步研究鉴定难辨梭菌孢子表面的蛋白质，这些蛋白质可能在感染的初始阶段发挥作用。(C) 2013 Elsevier B.V.版权所有
Clostridium difficile spores are the means through which this anaerobic pathogen may persist in hospital surfaces and in the host. There is a lack of knowledge in the proteins that localize to the surface of C difficile spores primarily due to the lack of established methods to efficiently separate the outermost layer, the exosporium. In this work, we propose methods to remove the exosporium layer of C. difficile spores through either protease digestion or sonication treatment leaving the spore coat structure intact. Transmission electron microscopy micrographs show that the treatment of C. difficile spores with sarkosyl and proteinase K (SPk) completely removed the exosporium, while trypsin and sonication removed most of the exosporium but left a thin exosporium layer attached to the spore coat. Measurement of hydrophobicity of C. difficile spores shows that complete removal of the exosporium by SPk yields spores with an hydrophobicity of similar to 1% (i.e., percentage of the spores in the organic phase), while treatments with trypsin or sonication, which leave a thin layer of exosporium, yield spores with an hydrophobicity of similar to 10%. Removal of the exosporium increased C. difficile spore's ability to form colonies. These exosporium extraction methods should aid in further research to identify proteins localized on the spore surfaces of C difficile that might play a role on the initial stages of infection. (C) 2013 Elsevier B.V. All rights reserved.