The Agr-Like Quorum-Sensing System Is Important for Clostridium perfringens Type A Strain ATCC 3624 To Cause Gas Gangrene in a Mouse Model

Bacteria, media, and reagents.C. perfringens type A strain ATCC 3624 was purchased from the American Type Culture Collection (ATCC). The following broth media were used in this study: fluid thioglycolate medium (FTG; Difco Laboratories), TY broth (3% tryptic soy broth [Becton, Dickinson], 1% yeast extract [Becton, Dickinson], and 0.1% sodium thioglycolate [Sigma-Aldrich]), and TGY broth (TY broth supplemented with 2% glucose [Sigma-Aldrich]). The agrB-null mutant and complementing strains were screened using brain heart infusion (BHI) agar (Research Products International) plates containing 15 μg ml⁻¹ chloramphenicol (Sigma-Aldrich). Tryptose-sulfite-cycloserine (TSC) agar plates made of SFP agar base (Becton, Dickinson) with 0.04% d-cycloserine (Sigma-Aldrich) were used for isolation of C. perfringens from challenged skeletal muscle of mice. All other chemical reagents used in this study were purchased from Fisher Scientific, Sigma-Aldrich, or Bio-Rad.

Peptide synthesis.Based upon results of a previous report (31), this study used a synthetic 6-mer (6-R) peptide which consists of the sequence CLWFTH in a thiolactone ring, Synthesis of the 6-R peptide was carried out by the Peptide and Peptoid Synthesis Core Facility Division of the Health Sciences Core Research Facilities (HSCRF) at the University of Pittsburgh. Synthesis was performed using standard FMOC (9-fluorenylmethoxy carbonyl) chemistry cycles on a Liberty CEM microwave synthesizer using Oxyma/DIC [ethyl-(2Z)-2-cyano-2-hydroxyiminoacetate/N,N-diisopropylcarbodiimide] activation in dimethylformamide (DMF). Briefly, FMOC-Thr(tBu), FMOC-His(trt), FMOC-Cys(trt), FMOC-Leu, FMOC-Trp(Boc), and FMOC-Phe were purchased from Peptides International and used for stepwise assembly of the linear sequence on bis(2-sulfanylethyl) amino polystyrene (SEA-PS) resin (0.11 mmol g⁻¹; Millipore Sigma). Cleavage of the 6-R-bis(2-sulfanylethyl) amino intermediate from the SEA solid support was accomplished using a mixture of TFA/TIPS/DMS/H₂O/thioanisole (90/2.5/2.5/2.5/2.5) for 2 h at room temperature and then precipitated with diethyl ether, followed by three ether washes. The resulting pellet containing the crude 6-R-bis(2-sulfanylethyl) amino intermediate was allowed to air dry and then dissolved in 0.2 M sodium phosphate/tris(2-carboxyethyl)phosphine (TCEP) at pH 4.0, layered with N₂, and incubated overnight at 37°C with gentle stirring. The resulting crude reaction mixture was directly loaded onto a Waters Prep 4000 series chromatography system and purified on a Phenomenex Gemini (21.2 × 250 mm) 10-μm C₁₈ column using standard acetonitrile/0.1% TFA gradient conditions. Final analytical determination of peptide purity for the cyclic 6-R-thioester was performed on a Waters Alliance chromatography system using a Phenomenex Gemini (4.6 × 250 mm) 5-μm C₁₈ column along with standard acetonitrile/0.1% TFA gradient conditions. Matrix-assisted laser desorption ionization–time-of-flight analysis on an Applied Biosystems Voyager workstation was used for final confirmation of the expected target mass of each peptide. The purity of the final peptide was >95%. The purified synthetic peptide was resuspended in DMSO (Fisher Scientific) at 50 mM for use and then stored in a –80°C freezer for no longer than 2 weeks. In experiments, the final concentrations of 6-R peptide ranged from 25 to 100 μM, as specified.

Plasmids and primers.An agrB knockout plasmid named pJIR750agrBNi was constructed to prepare an ATCC 3624 agrB-null mutant using Clostridium-modified group II TargeTron Technology (32). The intron on this plasmid was sense orientation targeted to insert into the agrB ORF between nucleotides 342 and 343. The primers used for intron targeting the agrB gene were as follows: agrB-342|343s-IBS, 5′-AAAAAAGCTTATAATTATCCTTAGTGTTCATTGGAGTGCGCCCAGATAGGGTG-3′; agrB-342|343s-EBS1d, 5′-CAGATTGTACAAATGTGGTGATAACAGATAAGTCATTGGAATTAACTTACCTTTCTTTGT-3′; and agrB-342|343s-EBS2, 5′-TGAACGCAAGTTTCTAATTTCGATTAACACTCGATAGAGGAAAGTGTCT-3′. The 350-bp intron PCR product was then inserted into pJIR750ai (32) between the HindIII and BsrGI enzyme (New England Biolabs) sites to construct the pJIR750agrBNi vector. The screening primers used to verify the agrB-null mutant were NagrBKOF (5′-TGGAACTTATGCTCTAATACAAACA-3′) and NagrBKOR (5′-AATCTATAGTTTTTAACAATATATTT-3′). The same primer pair was also used for RT-PCR analysis of agrB gene expression. 16S RNA was used as a control housekeeping gene for RT-PCR. In this study, all primers were synthesized by Integrated DNA Technologies.

The agrB complementation vector (CPJVp3) was constructed as described previously (30). This plasmid was electroporated into the ATCC 3624 agrB-null mutant, and transformants were selected on BHI agar containing 15 μg ml⁻¹ of chloramphenicol. The mutant and complementing strains were further confirmed and characterized by PCR, RT-PCR, and Southern blot analyses, as described below.

C. perfringens DNA isolation, PCR, and intron Southern blot analyses.A MasterPure Gram-positive DNA purification kit was used for DNA extraction from all C. perfringens strains according to the manufacturer’s instructions (Epicenter). PCR for the agrB gene was performed using the NagrBOF and NagrBKOR primers. For the wild-type strain the PCR product amplified using these primers was 536 bp, while for the agrB-null mutant strain the same primer pair amplified a PCR product of about 1,400 bp due to the insertion of a 900-bp intron.

For Southern blotting, aliquots (3 μg each) of wild-type or agrB-null mutant DNA samples were first digested overnight with EcoRI at 37°C according to the manufacturer’s instructions (New England Biolabs). The overnight-digested DNA samples were then electrophoresed on a 1% agarose gel, followed by transfer onto a positively charged nylon membrane (Roche) for hybridization with an intron-specific probe (46). The intron-specific probe was prepared using the PCR DIG probe synthesis kit (Roche) and the intron primers (IBS and EBS2). After hybridization, Southern blots were developed using reagents from a DIG DNA labeling and detection kit (Roche), according to the manufacturer’s instructions.

C. perfringens RNA isolation and RT-PCR analyses.The wild-type parent ATCC 3624 and derivatives (the agrB-null mutant or complementing strain) were grown in TY medium for 5 h at 37°C. Cultures were then pelleted and RNA was extracted using saturated phenol and purified by TRIzol and chloroform (Life Technology and Sigma), as previously described (36). After the absence of DNA was confirmed (36) by subjecting samples to PCR without reverse transcriptase, RNA was quantified by measuring the sample absorbance at 260 nm. An aliquot of 1 μl of purified RNA (100 ng) was then used in a one-step RT-PCR containing 10 μl of 2× Taq master mix (New England Biolabs), 4 U of avian myeloblastosis virus reverse transcriptase (Promega), and agrB gene primers (described earlier), with ddH₂O added to reach a 20-μl total volume. Similarly, 16S RNA RT-PCR was performed as a loading control (36). Reaction mixtures were incubated for 45 min at 45°C to allow cDNA synthesis, and then regular PCR cycling was performed using the following conditions: (i) 95°C for 2 min; (ii) 30 cycles of 95°C for 15s, 50°C for 30 s, and 68°C for 30 s; and (iii) a final extension of 68°C for 5 min.

Measurement of C. perfringens growth in vitro.For analysis of C. perfringens in vitro vegetative growth, a 0.2-ml aliquot from overnight FTG cultures of the wild type, agrB-null mutant, or complementing strain was inoculated into 10 ml of TY medium. The cultures were then incubated at 37°C; at various culture times (0, 1, 3, 5, 8, and 24 h) thereafter, a 1-ml aliquot of culture was removed to measure the optical density at 600 nm (OD₆₀₀) by using a Bio-Rad Smart spectrophotometer.

Western blot analyses of CPA and PFO production.TY cultures of the wild-type, agrB mutant, or complementing strain were adjusted to equal OD₆₀₀s, and 30-μl portions of supernatants from those cultures were then mixed with 5× SDS-PAGE loading buffer and boiled for 5 min. Agr locus controls the expression of a number of secreted proteins (27), so loading equal amounts of protein in these experiments is not applicable for comparing the relative production of PFO or CPA by wild-type, the agrB mutant, or complementing strain. Instead, we adjusted the OD₆₀₀ of each culture to equivalence (since Agr does not affect growth) and then loaded the same volume of supernatant from that culture. Portions (30 μl) of each sample were then electrophoresed on a 10% acrylamide SDS gel, and the separated proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with TBS-Tween 20 (0.05% [vol/vol]) and nonfat dry milk (5% [wt/vol]) for 1 h at room temperature, followed by probing with a 1:1,000 dilution of rabbit polyclonal PFO antibody (36) or a 1:250 dilution of mouse monoclonal CPA antibody (36) overnight at 4°C. Finally, bound antibody was detected with a horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody (Sigma-Aldrich) and the addition of SuperSignal West Pico chemiluminescence substrate (Fisher Scientific).

Inhibition of toxin production by the 6-R peptide.Wild-type ATCC 3624 was cultured overnight at 37°C in FTG before reculture in fresh TY broth overnight at 37°C. After that second overnight growth, a 15-μl aliquot of the TY culture was inoculated into 1 ml of TY with or without the specified concentration of 6-R peptide. The culture without 6-R peptide received 2 μl of DMSO as a control since this amount of DMSO was present in cultures receiving the 6-R peptide. After 5 h of culture at 37°C, the supernatants were collected and used for Western blot detection of CPA or PFO production, as described above. ImageJ analysis was performed on three separate Western blots to determine the fold changes of CPA and PFO in the supernatants.

Mouse model of gas gangrene.A 40-μl aliquot from overnight FTG cultures of the wild-type, agrB-null mutant, or complementing strain was inoculated into 1 ml of TY medium, followed by incubation at 37°C for 5 h. These cultures were then washed in sterile DPBS. An aliquot of 50 μl, equivalent to ∼10⁶ CFU, was injected intramuscularly in the thighs of eight BALB/c mice (20 to 25 g) per group of treatment. Another group of mice was challenged with 50 μl of sterile DPBS (control). The animals were euthanized by cervical dislocation 4 h after infection, and samples of skeletal muscle were collected. All experiments involving mice were reviewed and approved by the University of California, Davis, Institutional Animal Care and Use Committee (protocol 20513).

Histopathology.Sections of challenged skeletal muscle were fixed by immersion in 10% buffered formalin (pH 7.2), for 24 to 72 h. Sections (4 μm thick) were then prepared routinely and stained with hematoxylin and eosin. The sections were examined microscopically by a pathologist in a blinded fashion. A semiquantitative score of lesion severity was assigned to each section using an ordinal scale from 0 (no lesions observed) to 4 (most severe). The following scoring system was used, based on examination of 5, 200× microscopic fields: 0, no lesions; 1, changes observed in 0 to 25% of the area examined; 2, changes observed in 25 to 50% of the area examined; 3, changes observed in 50 to 75% of the area examined; and 4, changes observed in 75 to 100% of the area examined. The following criteria were considered in this score: inflammation (edema, hemorrhage, and presence of inflammatory cells) and changes in muscle fibers (loss of striations, loss of cytoplasm, vacuolation, swelling, and hypercontraction bands).

C. perfringens immunohistochemistry.Sections of skeletal muscle were processed by an indirect immunoperoxidase technique for C. perfringens as previously described (44) using a Dako EnVision kit (Dako, Carpinteria, CA) according to the instructions of the manufacturer. The primary antibody was rabbit polyclonal C. perfringens antibody (GenWay Bio, San Diego, CA). Samples of skeletal muscle from mice receiving no C. perfringens inoculation were used as negative controls. Additional negative controls consisted of serial tissue sections of the test tissue incubated with normal rabbit serum instead of the specific antibodies. The colon of a goat from which C. perfringens had been isolated was used as a positive control for C. perfringens immunohistochemistry.

Recovery of C. perfringens from challenged tissues.Skeletal muscle was collected aseptically. Tissues were weighed, macerated, and resuspended in DPBS. Serial dilutions (10⁻¹ to 10⁻⁸) in DPBS were plated on selective TSC agar plates for C. perfringens and anaerobically incubated overnight at 37°C. After 24 h, black colonies, indicative of C. perfringens (45), were counted to calculate the number of CFU per gram of muscle. A representative number (n = 15) of those colonies was screened by PCR for the cpa gene to confirm that they were C. perfringens. For this, we used the primers CPAF (5′-GCTAATGTTACTGCCGTTGA-3′) and CPAR (5′-CCTCTGATACATCGTGTAAG-3′), which amplify a PCR product of about 324 bp. The PCR program was as follows: initial denaturation at 94°C for 5 min; 30 cycles at 94°C for 45 s, 56°C for 45 s, and 72°C for 60 s; and final extension at 72°C for 7 min.

Inhibition of gas gangrene by the 6-R peptide in a mouse model.Aliquots (40 μl) of an overnight FTG cultures of wild-type, agrB-null mutant, or complementing strains were inoculated into 1 ml of TY medium containing 2 μl of the 6-R peptide (100 μM) in DMSO or an equal concentration of DMSO alone and then incubated at 37°C for 5 h. The cultures were then washed in sterile DPBS, and 1 μl of the 6-R peptide (100 μM) in DMSO or an equal concentration of DMSO alone was again added. An aliquot of 50 μl—equivalent to ∼10⁶ CFU—was injected intramuscularly in the left thighs of eight BALB/c mice (20 to 25 g) per treatment group. Another group of mice was injected with 50 μl of sterile DPBS (control). The animals were euthanized 4 h after infection, and samples of skeletal muscle were collected.

Statistical analyses.All statistical analyses were performed using R (v3.3.1). Histopathological scores were compared by using a nonparametric Kruskal-Wallis test, followed by the Dunn test as post hoc analysis. For CPA and PFO production in culture supernatants, one-way analysis of variance was applied followed by a post hoc analysis using Tukey’s multiple-comparison test. Bacterial counts were compared by negative binomial regression analysis. Differences were considered significant when the P value was <0.05.