Identification of DraRS in Clostridioides difficile, a Two-Component Regulatory System That Responds to Lipid II-Interacting Antibiotics

Abstract

Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 220,000 infections, 12,000 deaths, and upwards of $1 billion in medical costs in the United States each year. C. difficile is highly resistant to a variety of antibiotics, but we have a poor understanding of how C. difficile senses and responds to antibiotic stress and how such sensory systems affect clinical outcomes. We have identified a spontaneous C. difficile mutant that displays increased daptomycin resistance. We performed whole-genome sequencing and found a nonsense mutation, S605*, in draS, which encodes a putative sensor histidine kinase of a two-component system (TCS). The draS^S605* mutant has an ~4- to 8-fold increase in the daptomycin MIC compared to the wild type (WT). We found that the expression of constitutively active DraR^D54E in the WT increases daptomycin resistance 8- to 16-fold and increases bacitracin resistance ~4-fold. We found that a selection of lipid II-inhibiting compounds leads to the increased activity of the luciferase-based reporter P_draR-sluc^opt, including vancomycin, bacitracin, ramoplanin, and daptomycin. Using RNA sequencing (RNA-seq), we identified the DraRS regulon. Interestingly, we found that DraRS can induce the expression of the previously identified hex locus required for the synthesis of a novel glycolipid produced in C. difficile. Our data suggest that the induction of the hex locus by DraR explains some, but not all, of the DraR-induced daptomycin and bacitracin resistance. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. C. difficile encodes ~50 annotated two-component systems (TCSs); however, only a few have been studied. The function of these unstudied TCSs is not known. Here, we show that the TCS DraRS plays a role in responding to a subset of lipid II-inhibiting antibiotics and mediates resistance to daptomycin and bacitracin in part by inducing the expression of the recently identified hex locus, which encodes enzymes required for the production of a novel glycolipid in C. difficile.

Keywords: cell envelope; gene expression; signal transduction; stress response; two-component regulatory system; two-component regulatory systems.

FIG 1

draS^S605* leads to increased lysozyme resistance and increased expression of P_draR. (A) Graphical representation of DraS membrane topology as predicted by TOPCONS. Transmembrane domains (TM) and histidine kinase domains (HK) are labeled. The location of the S605* mutation is indicated. (B) Lysozyme MICs comparing CRISPRi knockdowns of draRS in the ΔcsfV-operon and ΔcsfV-operon draS^S605* strains containing either a negative sgRNA (Neg sgRNA) (pIA34), draR-targeting sgRNA, or draR-targeting sgRNA 2. Data were analyzed by one-way analysis of variance (ANOVA) with Sidak’s multiple-comparison test. ns, not significant (P > 0.05); ****, P < 0.0001. (C) Lysozyme MICs comparing WT R20291 with either the EV control (pAP114) or P_xyl-draR^D54E (pCE783). Data were analyzed by an unpaired t test. *, P < 0.05. (D) Luminescence assay of the P_draR-sluc^opt (pCE806) reporter in the ΔcsfV-operon and ΔcsfV-operon draS^S605* strains. Luminescence values were normalized to the OD₆₀₀. Data were analyzed by an unpaired t test. ***, P < 0.001. MICs are plotted on a log₂ scale. RLU, relative luminescence units.

FIG 2

Activation of DraRS with lipid II cycle-inhibiting compounds. Shown are the luminescence activities of P_draR-sluc^opt in R20291 WT and ΔdraRS strains with increasing concentrations of daptomycin (A), vancomycin (B), bacitracin (C), and ramoplanin (D). NS, not significant. ****, P < 0.0001.

FIG 3

Overproduction of DraR^D54E leads to increased daptomycin and bacitracin resistance. Shown are daptomycin (A) and bacitracin (B) MICs comparing the EV (empty vector) and the overproduction of DraR^D54E in the R20291 WT and ΔdraRS strains. Data were analyzed using one-way ANOVA with Sidak’s multiple comparisons. ns, not significant (P > 0.05); *, P < 0.05; ***, P < 0.001. MICs are plotted on a log₂ scale.

FIG 4

(A) Overproduction of DraR^D54E leads to increased daptomycin resistance in 630Δerm. Data were analyzed using an unpaired t test. ****, P < 0.0001. (B) P_draR-sluc^opt responds to daptomycin in VPI 10463 and 050-P50-2011 but does not respond to daptomycin in 630Δerm. MICs are plotted on a log₂ scale.

FIG 5

Comparison between the WT and ΔdraRS strains exposed to a concentration gradient of daptomycin with the sluc^opt reporters P₃₀₁₆-sluc^opt, P₂₁₈₈-sluc^opt, P_pgdA-sluc^opt, P₂₃₉₆-sluc^opt, P₂₀₆₆-sluc^opt, and P_hexS-sluc^opt.

FIG 6

(A) Daptomycin MICs comparing CRISPRi knockdowns of genes identified in the DraR regulon. (B) Daptomycin MICs comparing the xylose-inducible overproduction of genes identified in the DraR regulon. MICs are plotted on a log₂ scale.

FIG 7

Daptomycin (A) and bacitracin (B) MICs of P_xyl-DraR^D54E in the WT, ΔhexRK, and ΔhexSDF strains. Data were analyzed using one-way ANOVA with Sidak’s multiple comparisons. ****, P < 0.0001; ***, P < 0.001; *, P < 0.05; ns, not significant (P > 0.05). MICs are plotted on a log₂ scale.