N-Deacetylases required for muramic-δ-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-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.

Keywords: Clostridium difficile; Gram-positive bacteria; N-deacetylase; acetylation; bacteria; bacterial pathogenesis; germination; infectious disease; microbiology; muramic-δ-lactams; peptidoglycan; spore cortex; sporulation.

Figure 1.

Muropeptide analysis of spore peptidoglycan by HRMS-coupled UHPLC. A–D, relative abundance (%) of muropeptides for the 630Δerm strain (A), ΔCD630_27190 (B), ΔCD630_14300 (C), and double ΔCD630_14300ΔCD630_27190 (D) mutants. Numerical peaks refer to peaks identified in the parental strain. Alphabetical peaks refer to new peaks identified in the mutant profiles. Peak identification refers to Table 1.

Figure 2.

Spores lacking muramic-δ-lactam undergo a delayed germination. Spore germination was monitored for the optical density assay at 600 nm after the addition of 0.1% sodium taurocholate in BHISG, for 60 min (A) or 24 h (B), for the 630Δerm(pMTL84151) parental strain in blue, ΔpdaA1(pMTL84151) strain in green, and the complemented pdaA1 mutant strain (ΔpdaA1(pCH67)) in gray. Results are expressed as the ratio of OD_{600 nm} observed at time point (T) over the initial OD_{600 nm} at T = 0 (T0). Assessment of germination delay in solid BHI supplemented with horse blood and taurocholates was performed for the 630Δerm and the ΔpdaA1 strains (C). Colony sizes were measured (D) and presented in blue for the 630Δerm strain and in green for the ΔpdaA1 strain. *, Student's t test, p < 0.005. Results are expressed as the average values and standard deviations of at least three independent experiments, except for the assessment of germination delay in solid BHI (C) which is representative of three independent experiments.

Figure 3.

pdaA1 mutant spore morphology. EM of 630Δerm spores (A) and endospores (C) was compared with ΔpdaA1 mutant spores (B) and endospores (D). The spore layers identified are indicated in the inset between A and B. The legends are indicated by the bars in the bottom right corner, representing 200 nm for the spores and 500 nm for the endospores. The white arrow indicates detached structures.

Figure 4.

Modification of cortex structure and spore morphology does not alter chemical resistance. Spore measurements (A) were obtained for pure spore suspensions of the parental (blue) and pdaA1 mutant strain (green). Solid bars indicate the measured length (left axis, nm), and the dotted bars indicate the calculated volumes (right axis, nm³). Chemical and heat sensitivities of purified spore suspensions (B) were obtained for three independent biological replicates of pure spore suspensions: 630Δerm(pMTL84151) spores in blue, ΔpdaA1(pMTL84151) spores in green, and the complemented mutant spores ΔpdaA1(pCH67) strain in gray. *, Student's t test, p < 0.05.

Figure 5.

pdaA1 mutant has a defect in sporulation and spore heat resistance. Quantification of sporulation titers after 72 h incubation in SM broth (A), percentage of spores recovered after ethanol treatment (ET), heat shock (HS), and heat-resistant spores (heat^R) (B) of 630Δerm(pMTL84151) strain in blue, ΔpdaA1(pMTL84151) strain in green, and the complemented strain ΔpdaA1(pCH67) strain in gray are presented. Results are expressed as the average values and standard deviations of at least three independent experiments. *, Student's t test, p < 0.005.

Figure 6.

pdaA1 mutant strain has a delayed virulence. Kaplan-Meier survival curve (p = 0.002286) depicts hamster survival (A) and average time to mortality (B) for the strain 630Δerm in blue and ΔpdaA1 mutant in green. *, Student's t test, p < 0.005.