Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta-Toxin Production

Abstract

Clostridium perfringens toxin production is often regulated by the Agr-like quorum sensing (QS) system signaling the VirS/VirR two-component regulatory system (TCRS), which consists of the VirS membrane sensor histidine kinase and the VirR response regulator. VirS/VirR is known to directly control expression of some genes by binding to a DNA binding motif consisting of two VirR boxes located within 500 bp of the target gene start codon. Alternatively, the VirS/VirR system can indirectly regulate production levels of other proteins by increasing expression of a small regulatory RNA, VR-RNA. Previous studies demonstrated that C. perfringens beta-toxin (CPB) production by C. perfringens type B and C strains is positively regulated by both the Agr-like QS and the VirS/VirR TCRS, but the mechanism has been unclear. The current study first inactivated the vrr gene encoding VR-RNA to show that VirS/VirR regulation of cpb expression does not involve VR-RNA. Subsequently, bioinformatic analyses identified a potential VirR binding motif, along with a predicted strong promoter, ∼1.4 kb upstream of the cpb open reading frame (ORF). Two insertion sequences were present between this VirR binding motif/promoter region and the cpb ORF. PCR screening of a collection of strains carrying cpb showed that the presence and sequence of this VirR binding motif/promoter is highly conserved among CPB-producing strains. Reverse transcription-PCR (RT-PCR) and a GusA reporter assay showed this VirR binding motif is important for regulating CPB production. These findings indicate that VirS/VirR directly regulates cpb expression via VirS binding to a VirR binding motif located unusually distant from the cpb start codon. IMPORTANCE Clostridium perfringens beta-toxin (CPB) is only produced by type B and C strains. Production of CPB is essential for the pathogenesis of type C-associated infections, which include hemorrhagic necrotizing enteritis and enterotoxemia in both humans and animals. In addition, CPB can synergize with other toxins during C. perfringens gastrointestinal diseases. CPB toxin production is cooperatively regulated by the Agr-like quorum sensing (QS) system and the VirS/VirR two-component regulatory system. This study now reports that the VirS/VirR regulatory cascade directly controls expression of the cpb gene via a process involving a VirR box binding motif located unusually far (∼1.4 kb) upstream of the cpb ORF. This study provides a better understanding of the regulatory mechanisms for CPB production by the VirS/VirR regulatory cascade.

Keywords: Clostridium perfringens; VirR box; VirS/VirR; beta-toxin; gene regulation; insertion sequences; regulatory system; two-component regulatory system.

FIG 1

Comparison of CPB production and VR-RNA (vrr) gene expression between wild-type CN1795 or CN3685, their virS-null mutants, and complemented strains. (A) Western blot analysis of CPB production by wild-type strains CN1795 and CN3685, the CN1795::virS mutant, the CN3685::virS mutant, and the CN1795::virScomp or CN3685::virScomp complemented strains when cultured in TY broth for 5 h. Size of the immunoreactive protein, which matches the molecular mass of CPB, is indicated at left. This CPB Western blot analysis was repeated three times, and a representative of those three blots is shown. (B) Quantitative reverse transcription-PCR (qRT-PCR) analyses of vrr gene expression levels for the same strains listed above. A 500-ng aliquot of RNA purified from a 5-h culture of each isolate grown in TY medium was synthesized to cDNA, and then each cDNA was diluted to 5 ng/μl for qRT-PCR. For this analysis, the relative quantitative of mRNA expression was normalized to the level of constitutive expression of the housekeeping 16S rRNA and calculated by the comparative threshold cycle (2^−ΔΔCT) method. Data are presented as the mean ± standard deviation (SD) of three independent experiments. Error bars indicate standard deviations. *, P < 0.05 versus control.

FIG 2

Construction and characterization of CN1795 and CN3685 vrr-null mutants. (A) PCR assay confirmation of the construction of a vrr isogenic null mutant in wild-type strains CN1795 or CN3685. Using DNA purified from wild-type strains, an ∼200-bp PCR product was amplified using internal primers, whereas the same PCR assay amplified an ∼1.1-bp product using DNA purified from mutant strains due to the insertion of an ∼900-bp intron into the vrr gene. (B) Southern blot hybridization of an intron-specific probe with DNA from the wild-type strains or their vrr null mutants. The purified DNA from each strain was digested with EcoRI and electrophoresed on a 1% agarose gel prior to blotting and hybridization with an intron-specific probe. The size of DNA fragments is indicated on the left. (C) RT-PCR evaluation of vrr gene expression. RT-PCR analysis demonstrating that CN1795::vrr and CN3685::vrr null mutants have lost expression of their vrr gene. The RT-PCR assay was repeated three times, and a representative result of three experiments is shown. (D) Vegetative growth phenotype of vrr isogenic null mutants. All strains were cultured in TY broth, and the optical density at 600 nm (OD₆₀₀) was determined at the designated time points.

FIG 3

Western blot analyses comparing CPA, PFO, CPB, and ETX production by wild-type CN1795 or CN3685 versus that by their isogenic vrr-null mutants. (A) The wild-type strains (CN1795 and CN3685) and vrr-null mutants (CN1795::vrr and CN3685::vrr) were grown in TY broth for 5 h prior to Western blot analysis for the specific toxins. The molecular mass of each toxin is indicated on the left of each blot. (B) TY broth cultures of the same strains shown in panel A after overnight growth and Western blot analysis. The molecular mass of each toxin is indicated on the left. Blots shown are representative results of three repetitions.

FIG 4

Identification of a VirR binding motif upstream of the cpb ORF. (A) Bioinformatic analysis identified the presence of a VirR binding motif ∼1.4 bp upstream of the cpb ORF in both type B strain ATCC 3626 and type C strain NCTC10719. The nucleotide identity of this region was compared with the consensus VirR binding motif described for C. perfringens (29). (B) PCR survey to evaluate the presence of this VirR binding motif upstream of the cpb ORF in other C. perfringens type B and C strains.

FIG 5

RT-PCR analyses of expression from sequences upstream of the CN1795 cpb ORF. (A) RT-PCR was performed using two primer sets (each set containing a common reverse primer [R] to an internal cpb ORF sequence plus the forward primer F1 or F2), with the size shown of the product (if any) that would be amplified using each forward primer shown. (B) RNA of each sample was purified from 3-h or 5-h CN1795 cultures in TY broth before RT-PCR. Purity of those RNA samples was assessed by subjecting each RNA sample to 16S rRNA gene PCR without reverse transcriptase enzyme. The size of each PCR product is shown on the left. Results representative of three repetitions are shown.

FIG 6

Measurement of culture GUS activity levels. To evaluate the role of the VirR binding motif in regulating CPB production, two plasmid constructs were generated and transformed into wild-type CN1795 or CN1795::virS strains. The first plasmid (VirR boxes) contains ∼1.4 kb of sequences upstream of the cpb ORF fused upstream of the gusA ORF, including the VirR binding motif containing two VirR boxes. The second plasmid (No VirR boxes) contains the same sequences fused upstream of the gusA ORF, except for deletion of the VirR binding motif. Another plasmid carrying the gusA ORF unfused to any sequences upstream of the cpb gene was used as a background control in this experiment. A 1-ml aliquot of a 5-h TY culture of each strain was centrifuged, and the resulting cell pellet was resuspended in phosphate-buffered saline (PBS). After brief sonication, supernatants were mixed with GusA substrate (6 mM 4-nitrophenyl-β-d-glucuronide) and incubated at 37°C for 30 min. Absorbance then was read at 405 nm, and Miller unit values were calculated. Background from controls, either CN1795 (pJIR750-GusA) or CN1795::virS (pJIR750-GusA) strains, was subtracted from corresponding samples. Data presented are means ± SD of three independent experiments. Error bars indicate standard deviations. ns, P > 0.05; *, P ≤ 0.05; ****, P ≤ 0.0001.

FIG 7

Updated model for gene regulation by the VirS/VirR TCRS of Clostridium perfringens. The VirS/VirR TCRS consists of a histidine kinase (VirS) and a response regulator (VirR). Upon stimulation by the autoinducing peptide (AIP) produced by the Agr-like quorum sensing system, VirS undergoes autophosphorylation, which in turn phosphorylates the response regulator VirR (47, 48). Subsequently, phosphorylated VirR binds to a pair of VirR boxes located either near (within 70 to 500 bp) or distant (∼1.4 kb) from the target genes. Phosphorylated VirR binds to a pair of VirR boxes located near the promoters of regulated genes. This results in direct regulation of some virulence genes (e.g., the cpb and pfoA genes). Alternatively, phosphorylated VirR may bind to the promoter of the small RNA regulatory protein genes (e.g., vrr) to indirectly regulate the expression of some other target genes (e.g., cpa) via still incompletely understood mechanisms (20, 29).