Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases

Abstract

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile, σ^V is required for lysozyme resistance, and σ^V is activated in response to lysozyme. Once activated, σ^V, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σ^V regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find that pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficileIMPORTANCEClostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Keywords: cell envelope; gene expression; signal transduction; stress response; σ factors.

FIG 1

(A) A model of the C. difficile cell envelope. The cell envelope includes a crystalline S-layer, peptidoglycan (PG), and cell wall polysaccharides. (B) Organization of the csfV operon in C. difficile strain R20291. The csfV operon carries 7 genes, including pdaV, encoding a peptidoglycan deacetylase, prsA2, encoding a putative peptidyl-prolyl isomerase, csfV, encoding an ECF σ factor, rsiV, encoding an anti-σ factor, lbpA, encoding a lysozyme-binding protein, cdr1410, encoding a putative dehydrogenase accessory protein, and cdr1411, encoding a conserved hypothetical protein.

FIG 2

Induction of the csfV operon by lysozyme is csfV dependent. The wild-type (GMK208) or ΔcsfV (GMK211) strain containing a P_pdaV-rfp reporter plasmid was grown to an OD₆₀₀ of 0.3, incubated with lysozyme for 1 h, and then fixed and removed from the anaerobic chamber. Samples were exposed to air overnight to allow for maturation of the chromophore. Fluorescence was measured via a plate reader. Experiments were performed in biological triplicate, with the mean and standard deviation shown. Data were analyzed by a two-way analysis of variance with Sidak’s multiple-comparison test. *, P < 0.05; ****, P < 0.0001 (compared to the wild-type strain with the corresponding lysozyme concentration).

FIG 3

Preincubation with a subinhibitory concentration of lysozyme increases resistance level. (A) Overnight cultures of the wild-type or ΔcsfV (CDE2966) strain were subcultured and grown for ∼8 h; 20 μg/ml lysozyme was added and left for the duration indicated prior to setup of the lysozyme MIC plates. Experiments were performed in biological triplicate, with the mean and standard deviation shown. Data were analyzed by a two-way analysis of variance with Sidak’s multiple-comparison test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (compared to the wild-type strain at the corresponding time point). (B) Overnight cultures were subcultured and grown to an OD₆₀₀ of 0.3; various subinhibitory concentrations of lysozyme were added as indicated, and cultures were incubated for ∼5 h prior to setup of MIC plates. Experiments were performed in biological triplicate, with the mean and standard deviation shown. Data were analyzed by a two-way analysis of variance with Sidak’s multiple-comparison test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (compared to the wild-type strain with the corresponding lysozyme concentration).

FIG 4

Peptidoglycan deacetylases are necessary for lysozyme resistance. (A) Overnight cultures were subcultured into TY medium and grown to an OD₆₀₀ of 0.3; 20 μg/ml lysozyme was added and incubated for 5 h prior to setup of MIC plates (wild type [WT], R20291; ΔpgdA, GMK241; ΔpdaV, GMK152; ΔcsfV operon, GMK157; ΔcsfV operon ΔpgdA, GMK243; ΔpdaV ΔpgdA, GMK301). Experiments were performed in biological triplicate, with the mean and standard deviation shown. Data were analyzed by a one-way analysis of variance with Tukey’s multiple-comparison test. ****, P < 0.0001 compared to the wild-type parent strain, unless indicated by a bar between the compared strains. (B) Strains carrying either P_xyl-pdaV (pCE618) or an empty vector (pAP114) were constructed (ΔcsfV operon ΔpgdA pAP114, GMK312; ΔcsfV operon ΔpgdA pCE618, GMK313; ΔpdaV ΔpgdA pAP114, GMK314; ΔpdaV ΔpgdA pCE618, GMK315; ΔpdaV pAP114, GMK316; ΔpdaV pCE618, GMK317; ΔcsfV operon pAP114, GMK174; ΔcsfV operon pCE618, GMK177). Overnight cultures were subcultured into TY Thi₁₀ medium supplemented with 1% xylose. Cultures were grown to an OD₆₀₀ of 1.0, and a lysozyme MIC plate was set up with 1% xylose. Experiments were performed in biological triplicate, with the mean and standard deviation shown. Data were analyzed by a one-way analysis of variance with Sidak’s multiple-comparison test. ****, P < 0.0001.

FIG 5

PdaV and PdgA are redundant peptidoglycan deacetylases. Peptidoglycan was purified from cultures grown to mid-log phase (OD₆₀₀ = 0.6 to 0.8). Peptidoglycan was digested with mutanolysin. Fragments were separated using reversed-phase HPLC and structures determined using mass spectrometry. (B) Total percentages of deacetylated residues are shown for the indicated strains (WT, R20291; ΔpdaV, GMK152; ΔpgdA, GMK241; ΔpgdA ΔpdaV, GMK301). Experiments were performed in biological triplicate or quadruplicate, with the mean and standard deviation shown. Data were analyzed by a one-way analysis of variance with Tukey’s multiple-comparison test. ****, P < 0.0001 compared to the wild-type parent strain. NAG, N-acetylglucosamine.

FIG 6

RsiV and LbpA act as lysozyme inhibitors. (A) Peptidoglycan from M. lysodeikticus was combined with 10 μg/ml lysozyme and purified RsiV or LbpA. The A₄₅₀ was monitored every minute for 30 min to determine degradation of peptidoglycan by lysozyme. Degradation after 30 min is shown. Data were analyzed by a one-way analysis of variance with Tukey’s multiple-comparison test. ****, P < 0.0001. (B) Overnight cultures were subcultured into TY Thi₁₀ medium supplemented with 1% xylose and grown to an OD₆₀₀ of 1.0, and a lysozyme MIC plate was set up with 1% xylose. Data were analyzed by a one-way analysis of variance with Tukey’s multiple-comparison test. **, P < 0.01.

FIG 7

SlpA and DltABCD are minor contributors to lysozyme resistance. CRISPRi knockdown was used to determine the contribution of slpA and the dltABCD operon to lysozyme resistance in the wild-type and ΔcsfV operon strains. Two different sgRNAs were tested for dltABCD and csfV (wild type, R20291; negative control, LS134; R20291 slpA, GMK344; R20291 dltABCD, GMK345; GMK346; csfV, GMK347; GMK348). In the ΔcsfV operon background, one sgRNA was used (ΔcsfV operon negative control, LS134; ΔcsfV operon dltABCD, GMK362; ΔcsfV operon slpA, GMK363). Overnight cultures grown with 1% xylose were subcultured in TY supplemented with 1% xylose and grown to an OD₆₀₀ of 1.0, and then MICs were determined. Experiments were performed in biological triplicate, with the mean and standard deviation shown. Data were analyzed by a one-way analysis of variance with Tukey’s multiple-comparison test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.