Spo0A Suppresses sin Locus Expression in Clostridioides difficile

Abstract

Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR' (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficileIMPORTANCEClostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

Keywords: C. difficile; Clostridioides difficile; SinR; Spo0A; gene regulation; virulence gene regulation.

FIG 1

In the absence of Spo0A, C. difficile produces elevated levels of SinR. (A) qRT-PCR results of sin locus transcripts in C. difficile strains collected at the 10-h time point. The representative results from three independent experiments are shown. The asterisks (***) indicate statistical difference at P < 0.005. (B) Western blot analysis of parent strains (R20291, JIR8094, and UK01) and their respective spo0A mutants and complemented strains using SinR- and Spo0A-specific antibodies, demonstrating upregulated SinR in the absence of Spo0A. The sinR mutant served as a negative control. Glutamate dehydrogenase (GDH) detection using anti-GDH antibodies was used as loading control.

FIG 2

Spo0A suppresses sin locus expression in the 630erm::PtetSpo0A strain in a dose-dependent manner. (A) qRT-PCR analysis of sinR, sinI, and spo0A transcripts from the 630erm::PtetSpo0A strain grown with increasing concentrations of ATc for 10 h. (B) Western blots of protein extracts from the induced cultures. Coomassie-stained gel is provided as a loading control.

FIG 3

Spo0A represses sin locus expression. (A) β-Glucuronidase activity of the Psin-gusA fusions in the parent strain R20291 and R20291::spo0A mutant. Plasmid pBA038 has gusA as the reporter gene fused to 600 bp of sin locus upstream. A plasmid carrying PspoIIAB-gusA (pBA029) and a plasmid carrying a promoterless gusA gene (pBA040) were used as positive and negative controls, respectively. (B) Expression of β-glucuronidase in parent strain R20291 and the spo0A mutant carrying plasmids pBA037 (475-bp Psin-gusA), pBA038 (600-bp Psin-gusA), and pBA009 (340-bp Psin-gusA). The error bars in panels A and B correspond to standard errors of the means of results from 3 biological replicates, where ** and *** indicate P < 0.05 and P < 0.005, respectively (by two-tailed Student's t test). At least three independent experiments were performed.

FIG 4

(A) Spo0A binds to sin locus upstream DNA. Shown are schematics of the 340-bp upstream sin locus denoting the general locations of the M1, M2, R1, and R2 sequences (not to scale) with respect to the translation start (+1) of sinR. The lower lines indicate the location and the sizes of the DNA fragments used for the biotinylated-DNA pulldown assay. (B) Western blot analysis using Spo0A-specific antibody to detect endogenous Spo0A in input and eluate fractions. For the biotin-labeled DNA pulldown assay, the promoter regions of spoIIAB and gluD were used as positive and negative controls, respectively. (C) Three DNA fragments (118, 135, and 140 bp) spanning different regions of the 340-bp sin locus upstream were used independently to carry out the binding, and Spo0A was detected as in panel B. (D) Expression of β-glucuronidase in parent strain R20291 and R20291::spo0A mutant strains carrying plasmids with gusA as the reporter gene fused to the wild-type and mutated promoters of sinR. A strain carrying a promoterless gusA plasmid (pBA040) was used as a control. Data represent the means ± standard errors of the means (n = 3). The asterisks (***) in panel A indicate statistical difference at P < 0.005 (by two-tailed Student's t test). NS, not significant.