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-δ-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 630Δerm strain differs from that of Bacillus subtilis Among these differences, the muramic-δ-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-δ-lactam synthesis. In a pdaA1 mutant, only 0.4% of all muropeptides carried a muramic-δ-lactam modification, and muramic-δ-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-δ-lactams in C. difficile than in other bacteria, including B. subtilis In summary, the spore cortex of C. difficile contains lower levels of muramic-δ-lactams than that of B. subtilis, and PdaA1 is the major N-deacetylase for muramic-δ-lactam biosynthesis in C. difficile, contributing to sporulation, heat resistance, and virulence.
Highlights
Spores are produced by many organisms as a survival mechanism activated in response to several environmental stresses
Tetrapeptides were the most frequent stem peptide encountered (43.5% of all muropeptides), the analysis surprisingly showed that 21.9% of muropeptides were not substituted with a stem peptide of any kind, not even with the single L-alanine as described in B. subtilis [16]
These results suggest that the C. difficile spore cortex is different compared with other published cortex analyses
Summary
Cortex from pure spore samples were extracted and analyzed through UHPLC (ultra-HPLC) coupled to HRMS (high-resolution MS), as described under “Experimental procedures.” This analysis of the 630⌬erm spores constitutes the first detailed analysis of the cortex structure of C. difficile (Fig. 1). Hexasaccharides and octasaccharides represented a higher relative abundance in the CD630_14300 mutant (11.6 and 5.6% of all muropeptides, respectively, compared with 1.80 and 0.10% in the parental strain) These results suggest that CD630_14300 is the major N-deacetylase responsible for muramic-␦-lactam synthesis and was renamed pdaA1. Because the TEM experiment suggested a potential defect in spore assembly, sporulation percentages were investigated for the parental strain harboring the empty control plasmid pMTL84151, the pdaA1 mutant harboring pMTL84151, and the complemented pdaA1 mutant harboring pCH67, in sporulating cultures after 72 h (Fig. 5) as described previously [33]. Ethanol-resistant and heat-resistant spores produced by the complemented strain represented 87% (3.11 ϫ 106 CFU/ml) and 79% (2.86 ϫ 106 CFU/ml), respectively, of total cell titers These results suggest that the pdaA1 spores have a decreased heat resistance compared with the parental strain (p Ͻ 0.005), and the phenotype is restored in the complemented strain. These results indicate that the pdaA1 mutant has a significantly delayed virulence compared with the parental strain (Kaplan-Meier log rank, p ϭ 0.002286)
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