Response Regulator CD1688 Is a Negative Modulator of Sporulation in Clostridioides difficile

Abstract

Two-component signal transduction systems (TCSs), consisting of a sensor histidine kinase (HK) and a response regulator (RR), sense environmental stimuli and then modulate cellular responses, typically through changes in gene expression. Our previous work identified the DNA binding motif of CD1586, an RR implicated in Clostridioides difficile strain R20291 sporulation. To determine the role of this RR in the sporulation pathway in C. difficile, we generated a deletion strain of cd1688 in the historical 630 strain, the homolog of cd1586. The C. difficile Δcd1688 strain exhibited a hypersporulation phenotype, suggesting that CD1688 negatively regulates sporulation. Complementation of the C. difficile Δcd1688 strain restored sporulation. In contrast, a nonphosphorylatable copy of cd1688 did not restore sporulation to wild-type (WT) levels, indicating that CD1688 must be phosphorylated to properly modulate sporulation. Expression of the master regulator spo0A, the sporulation-specific sigma factors sigF, sigE, sigG, and sigK, and a signaling protein encoded by spoIIR was increased in the C. difficile Δcd1688 strain compared to WT. In line with the increased spoIIR expression, we detected an increase in mature SigE at an earlier time point, which arises from SpoIIR-mediated processing of pro-SigE. Taken together, our data suggest that CD1688 is a novel negative modulator of sporulation in C. difficile and contributes to mediating progression through the spore developmental pathway. These results add to our growing understanding of the complex regulatory events involved in C. difficile sporulation, insight that could be exploited for novel therapeutic development. IMPORTANCE Clostridioides difficile causes severe gastrointestinal illness and is a leading cause of nosocomial infections in the United States. This pathogen produces metabolically dormant spores that are the major vehicle of transmission between hosts. The sporulation pathway involves an intricate regulatory network that controls a succession of morphological changes necessary to produce spores. The environmental signals inducing the sporulation pathway are not well understood in C. difficile. This work identified a response regulator, CD1688, that, when deleted, led to a hypersporulation phenotype, indicating that it typically acts to repress sporulation. Improving our understanding of the regulatory mechanisms modulating sporulation in C. difficile could provide novel strategies to eliminate or reduce spore production, thus decreasing transmission and disease relapse.

Keywords: Clostridioides difficile; response regulator; sporulation; two-component regulatory systems.

FIG 1

Deletion of the cd1688 gene in C. difficile. (A) cd1688 locus in C. difficile 630 wild-type (WT) and C. difficile Δcd1688 strains. Boxes indicate positions of homology arms used for construction of the deletion strain. (B) PCR confirmation of cd1688 gene deletion in C. difficile Δcd1688. The cd1688 gene deletion removed 661 bp. (C) Abundance of cd1688, cd1689, and cd1685 in the C. difficile Δcd1688 strain relative to the WT strain measured via qRT-PCR. Three biological replicates of each strain were grown in BHIS to an OD₆₀₀ of 1.0. *, P ≤ 0.05.

FIG 2

Transcript abundance of predicted gene targets of CD1688 in the C. difficile WT and Δcd1688 strains. The expression of genes involved in ABC/ion transport, proteolysis, and sporulation was measured from RNA samples isolated from three biological replicates during stationary-growth phase in BHIS medium via qRT-PCR (OD₆₀₀, ~1.0). The expression of each gene in the Δcd1688 strain is measured relative to the expression in the WT strain in the same growth conditions. *, P ≤ 0.05.

FIG 3

Sporulation efficiency of C. difficile WT versus Δcd1688 strains. Phase-contrast microscopy of C. difficile WT (A), Δcd1688 (B), Δcd1688::p1688 (C), and Δcd1688::p1688^D50A (D) grown on 70:30 sporulation agar supplemented with 0.1% xylose for 24 h. Inset numbers represent sporulation efficiency as measured by ethanol resistance assays. *, P ≤ 0.05 as determined by a one-way ANOVA followed by Dunnett’s multiple-comparison test compared to C. difficile WT630.

FIG 4

Transcript abundance of some known regulators of sporulation in the C. difficile WT and Δcd1688 strains. Expression of ccpA (A), codY (B), sinR (C), sinR′ (D), and rstA (E) was measured from RNA samples isolated at 8 h, 10 h, and 12 h postinoculation on 70:30 sporulation media from C. difficile WT and Δcd1688 strains. Expression for each gene is presented relative to the WT sample at 8 h. *, P ≤ 0.05 as determined by Student's t test compared to the C. difficile WT630 strain at the same time point.

FIG 5

Gene expression profile of sporulation-specific genes and levels of phosphorylated Spo0A in C. difficile WT versus the C. difficile Δcd1688 strain. (A) Transcript abundance of spo0A in the Δcd1688 strain at 8 h, 10 h, and 12 h postinoculation on 70:30 medium relative to the WT sample at 8 h. (B) Detection of Spo0A phosphorylation. Cell lysates isolated from C. difficile WT and Δcd1688 strains at 8 h, 10 h, and 12 h postinoculation on 70:30 sporulation agar were resolved via Phos-tag SDS-PAGE and subjected to immunoblotting using anti-Spo0A antibody. (C to F) Transcript abundance of sigF (C), sigE (D), sigG (E), and sigK (F). All gene expression was measured via qRT-PCR from RNA samples isolated at 8 h, 10 h, and 12 h postinoculation on 70:30 sporulation media. *, P ≤ 0.05 as determined by Student's t test compared to the C. difficile WT630 strain at the same time point.

FIG 6

Altered expression of spoIIR and SpoIIR-dependent processing of SigE in C. difficile Δcd1688. (A) Transcript abundance of spoIIR in the C. difficile Δcd1688 strain at 8 h, 10 h, and 12 h postinoculation on 70:30 sporulation media measured via qRT-PCR relative to the WT strain 8 h sample. *, P ≤ 0.05. (B) EMSA analysis indicating in vitro binding between RR CD1586/CD1688 and the spoIIR promoter region. (C) Western blot analysis of cell lysates isolated from the C. difficile WT and Δcd1688 strains at 8 h, 10 h, and 12 h postinoculation on 70:30 sporulation agar. A total of 15 μg of protein was resolved by SDS-PAGE and subjected to immunoblotting using anti-SigE antibody. (D) The amount of total SigE (pro- and mature forms) was determined by quantification of band intensities in the immunoblot using ImageJ. (E) Percentage of processed, mature SigE. The intensity of mature SigE was divided by total intensity of SigE (pro-SigE plus mature SigE) in each sample.

FIG 7

Working model of regulation of sporulation by CD1688. Green arrows represent genes, blue rectangles or circles represent proteins, solid lines indicate known interactions, dashed lines indicate indirect regulation, and red line indicates our proposed regulation via CD1688. Sporulation genes are grouped by their cellular location (either mother cell or forespore). We do not have any evidence that CD1688 is specific to any cellular compartment. Purple circle represents protein phosphorylation. Figure was created using BioRender.com.