N-Deacetylases required for muramic-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance

Abstract

Spores are produced by many organisms as a survival mechanism activated in response to several environmental stresses. Bacterial spores are multilayered structures, one of which is a peptidoglycan layer called the cortex, containing muramic-lactams that are synthesized by at least two bacterial enzymes, the muramoyl-L-alanine amidase CwlD and the N-deacetylase PdaA. This study focused on the spore cortex of Clostridium difficile, a Gram-positive, toxin-producing anaerobic bacterial pathogen that can colonize the human intestinal tract and is a leading cause of antibiotic-associated diarrhea. Using ultra-HPLC coupled with high-resolution MS, here we found that the spore cortex of the C. difficile 630erm strain differs from that of Bacillus subtilis. Among these differences, the muramic-lactams represented only 24% in C. difficile, compared with 50% in B. subtilis. CD630_14300 and CD630_27190 were identified as genes encoding the C. difficile N-deacetylases PdaA1 and PdaA2, required for muramic-lactam synthesis. In a pdaA1 mutant, only 0.4% of all muropeptides carried a muramic-lactam modification, and muramic-lactams were absent in the cortex of a pdaA1–pdaA2 double mutant. Of note, the pdaA1 mutant exhibited decreased sporulation, altered germination, decreased heat resistance, and delayed virulence in a hamster infection model. These results suggest a much greater role for muramic-lactams in C. difficile than in other bacteria, including B. subtilis. In summary, the spore cortex of C. difficile contains lower levels of muramic-lactams than that of B. subtilis, and PdaA1 is the major N-deacetylase for muramic-lactam biosynthesis in C. difficile, contributing to sporulation, heat resistance, and virulence.