Proteases and sonication specifically remove the exosporium layer of spores of Clostridium difficile strain 630

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 ~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 ~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. ► The exosporium layer of Clostridium difficile spores is stable. ► Proteases and sonication can remove the exosporium layer leaving the spore coat intact. ► The hydrophobicity of the spore surface can be used as a marker for exosporium removal.