Regulatory Role of Anti-Sigma Factor RsbW in Clostridioides difficile Stress Response, Persistence, and Infection

Abstract

The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhea, faces a variety of stresses in the environment and in the mammalian gut. To cope with these stresses, alternative sigma factor B (σ^B) is employed to modulate gene transcription, and σ^B is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW), in which σ^B is assumed to be "always on," was generated. ΔrsbW did not show fitness defects in the absence of stress but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better compared to the parental strain. ΔrsbW was defective in spore and biofilm formation, but it displayed increased adhesion to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand the unique phenotype of ΔrsbW showed changes in expression of genes associated with stress responses, virulence, sporulation, phage, and several σ^B-controlled regulators, including the pleiotropic regulator sinRR'. While these profiles were distinct to ΔrsbW, changes in some σ^B-controlled stress-associated genes were similar to those reported in the absence of σ^B. Further analysis of ΔrsbW showed unexpected lower intracellular levels of σ^B, suggesting an additional post-translational control mechanism for σ^B in the absence of stress. Our study provides insight into the regulatory role of RsbW and the complexity of regulatory networks mediating stress responses in C. difficile. IMPORTANCE Pathogens like Clostridioides difficile face a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σ^B) enable the bacterium to respond quickly to different stresses. Anti-sigma factors like RsbW control sigma factors and therefore the activation of genes via these pathways. Some of these transcriptional control systems provide C. difficile with the ability to tolerate and detoxify harmful compounds. Here, we investigate the role of RsbW in C. difficile physiology. We demonstrate distinctive phenotypes for a rsbW mutant in growth, persistence, and virulence and suggest alternate σ^B control mechanisms in C. difficile. Understanding C. difficile responses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.

Keywords: Clostridioides difficile; anti-sigma factor RsbW; regulation; sigma factor B; stress responses.

FIG 1

Tolerance of external stresses by ΔrsbW. (A, B) Growth of wild-type (WT), ΔrsbW, and complemented strain ΔrsbW+rsbW as measured by optical density at 600 nm (OD_600nm) was compared in tryptone yeast medium in the presence of 1% oxygen (A) and brain-heart infusion (BHI) with 0.5% (wt/vol) yeast extract and 0.1% (wt/vol) l-cysteine (BHI-S) medium adjusted to pH 5 (B). (C) The zones of inhibition on BHI plates supplemented with 4 and 6 M paraquat were compared for WT, ΔrsbW, and ΔrsbW+rsbW. (D) Colony counts for WT, ΔrsbW, and ΔrsbW+rsbW on BHI-S agar supplemented with and without 1.5 mM sodium nitroprusside (N = 3, with 3 technical replicates/experiment). Error bars indicate standard deviation (SD). Significant differences are indicated by asterisks: *, P < 0.05; **, P < 0.01 as determined by Mann-Whitney U test.

FIG 2

ΔrsbW displays severe defects in sporulation. C. difficile strains WT, ΔrsbW, and ΔrsbW+rsbW were grown on 70:30 sporulation medium. (A) Representative phase-contrast microscopy images were taken after 24 h (N = 3, with 5 representative fields/experiment). (B) Visible spores were enumerated from the microscopy images and compared with total number of cells. (C) Bacteria cultured on 70:30 sporulation medium were diluted to OD_600nm 1.0 and subjected to heat and ethanol treatment. Germination frequency was calculated from colony counts of cultures before and after treatment, on brain-heart infusion with 0.5% (wt/vol) yeast extract and 0.1% (wt/vol) l-cysteine (BHI-S) ± 0.1% taurocholate (N = 3, with 3 technical replicates/experiment). Error bars indicate SD. Significant differences are denoted with asterisks: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 as determined Student’s t test or Mann-Whitney U test.

FIG 3

Biofilm formation is modulated by RsbW. (A) C. difficile strains were cultured in brain-heart infusion with 0.5% (wt/vol) yeast extract and 0.1% (wt/vol) l-cysteine (BHI-S) + 0.1 M glucose for 24 and 72 h in 24-well tissue culture-treated polystyrene plates, and biofilm biomass was quantified by crystal violet at OD_600nm (N = 3, with 3 technical replicates/experiment). (B) 24- and 72-h biofilms grown in chamber slides were stained with FilmTracer LIVE/DEAD. Orthogonal views of the biofilm z-stack depict biofilm thickness (N = 3, with 5 representative images/experiment). Error bars indicate SD. n.s., no significance; **, P < 0.01, as determined Student’s t test or Mann-Whitney U test.

FIG 4

ΔrsbW demonstrates increased cell adhesion but decreased virulence. (A) WT C. difficile, ΔrsbW, and ΔrsbW+rsbW adherence to epithelial cells across 3, 6, 12, and 24 h after infection in an in vitro vertical diffusion chamber (VDC) infection model (N = 3, with 3 technical replicates/experiment). (B) Survival curve of Galleria mellonella infected with C. difficile WT, ΔrsbW, and ΔrsbW+rsbW strains across 24 and 72 h (N = 5, with 8 larvae/strain/time point). (C) Adherent bacterial population recovered from infected G. mellonella gut enumerated with CFU/mL on brain-heart infusion with 0.5% (wt/vol) yeast extract and 0.1% (wt/vol) l-cysteine (BHI-S) agar. Error bars indicate SD. n.s., no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as determined by Mann-Whitney U test and log rank Mantel Cox test for the survival curve.

FIG 5

Bacterial transcriptomics of ΔrsbW reveals extensive differential expression compared with WT. (A) Volcano plot reveals differential gene expression when early stationary phase (10 h) ΔrsbW is compared to the WT (N = 3; P < 0.05). (B, C) Heat map representation of sporulation genes (B) and phage-associated genes (C) that were differentially expressed in ΔrsbW. Blue and red gradients indicate down- and upregulation, respectively, compared to WT.

FIG 6

Immunoblotting shows altered relative concentrations of intracellular σ^B in ΔrsbW. (A) Bacterial whole-cell lysates from C. difficile strains C. difficile WT, ΔrsbW, and ΔrsbW+rsbW grown in tryptose yeast broth ± sodium nitroprusside (SNP) to early exponential phase (5 h) were analyzed by immunoblotting with anti σ^B or anti-R20291 sera (for normalization). The image is representative of three independent experiments. (B) Band intensities were calculated using Fiji (ImageJ) to determine relative intracellular σ^B concentrations at 5 h ±25 μM SNP. Error bars indicate SD. n.s., no significance; *, P < 0.05; **, P < 0.01, as determined by Student’s t test.