The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli.

is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC's ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC's self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of spores. The endospore of is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against infection.