Abstract
Clostridioides difficile 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 C. difficile 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 C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli 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 C. difficile spores.IMPORTANCE The endospore of Clostridioides difficile 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 C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly's molecular bases may be essential to developing new therapies against C. difficile infection.
Highlights
Clostridioides difficile is an obligately anaerobic, spore-forming, Grampositive 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 C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host [8,9,10]
Genetic studies of CdeC and CdeM have shown that both are essential for the assembly of the exosporium layer, CdeC has pleiotropic roles in the biology of C. difficile spores, including spore coat assembly, spore resistance, and spore germination [8, 21]
Summary
Clostridioides difficile is an obligately anaerobic, spore-forming, Grampositive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. 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 C. difficile spores. The cysteine-rich proteins CdeC and CdeM have been identified, with 9% and 8.7% of the amino acid content, respectively, which have a predominant role in the exosporium function and assembly [8, 19]. CdeA exhibits a DUF1540 domain, but to our knowledge, no function has been assigned to this family; its distinctive signature is the presence of four conserved cysteines
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