Investigating the Roles of CwlD and GerS in <i>Clostridioides difficile</i> Spore Cortex Modification

Abstract

Clostridioides difficile is a notable nosocomial pathogen distinguished by its capacity to produce spores, essential for its survival and dissemination. The spore cortex, consisting of a modified peptidoglycan layer, is essential for dormancy and germination. This work examines the enzyme CwlD, which facilitates the transformation of N-acetylmuramic acid (NAM) into muramic-δlactam (MAL)—a change crucial for adequate cortex production and germination. The lipoprotein GerS is essential for CwlD function in C. difficile; however, no GerS homolog is present in Bacillus subtilis, a well-established model for spore biology. To assess the species requirements for CwlD and GerS, we expressed FLAG-tagged versions of C. difficile CwlD, both in the presence and absence of GerS, in a B. subtilis ΔcwlD background. Despite the effective assembly and integration of all genetic constructs, no expression of FLAG-tagged protein was seen. The data indicates that factors such as translational inefficiency or protein degradation may impede the expression of these heterologous proteins in B. subtilis. This contrasts with prior research demonstrating observable production of variants even though they remained non-functional in B. subtilis. These findings highlight the significance of the native cellular environment for the functional expression of CwlD–GerS and enhance the comprehension of species-specific regulatory mechanisms in spore life. These results may guide future therapy strategies aimed at targeting C. difficile germination by inhibiting CwlD-GerS-mediated MAL production. This would impede spore germination, presenting a potential approach to avoid infection initiation and diminish recurrence by maintaining remaining spores in a dormant state.