Article Figures & Data
- FIG 1
Deletion of the C. difficile accessory gene regulator autoinducing peptide generation loci (agrB1D1 and agrB2D2) by allelic exchange. DNA sequences upstream and downstream of the agrB1D1 locus of strain 630 (A) and the agrB1D1 and agrB2D2 loci of strain R20291 (B) were cloned into the pMTL82151 plasmid and transferred into the C. difficile strains by conjugation with E. coli CA434 bearing the assembled allelic-exchange cassette. The deletion mutants were confirmed by PCR and analyzed by 1% agarose gel electrophoresis. (C) Western blot analysis of 48-h culture supernatant fluids from the R20291 agrB1D1 and agrB2D2 mutants. Supernatants from 48-h cultures were concentrated with the Pierce 150-kDa concentrator (Thermo Fisher Scientific Inc., Rockford, IL) and subjected to 6% PAGE. The protein bands were transferred onto a 0.45-µm nitrocellulose membrane and probed with monoclonal antibodies specific for toxins A and B. The toxin bands on the transferred membrane were detected with the Protein Detector Western blot BCIP/NBT kit (KPL, Gaithersburg, MD). WT, wild type; MT, mutant; I, wild type; II, agrB1D1 mutant; III, complemented agrB1D1 mutant; IV, agrB2D2 mutant.
- FIG 2
Analysis of agr mutants for transcription of the toxin genes (tcdA and tcdB) in BHI and TY media. The 630 wild-type, agrB1D1 mutant, and complemented agrB1D1 mutant strains were cultured in either BHI (A) or TY (B) medium. The R20291 wild-type, agrB1D1 mutant, complemented agrB1D1 mutant, and agrB2D2 mutant strains were also cultured in either BHI (C) or TY (D) medium. The cultures were incubated for 16 h anaerobically at 37°C. Total RNA was isolated with the RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using the ProtoScript AMV First Strand cDNA synthesis kit (New England Biolabs, Ipswich, MA) with 1 µg of the isolated total RNA. Quantitative PCR was performed with primers specific for tcdA, tcdB, and the cDNA used as the template. Known quantities of tcdA and tcdB DNA were used as standards. The difference between the tcdA and tcdB transcript levels detected in the wild-type and agrB1B1 mutant strains was significant (P = 0.0001), but there was no significant difference (P = 0.235) between the two culture media. Error bars represent the standard deviations of three independent experiments.
- FIG 3
Plasmid-mediated and extracellular complementation restores toxin production in the hypervirulent NAP1/027 R20291 (A) and nonhypervirulent 630 (B) toxin-deficient agrB1D1 mutants. The R20291 and 630 agrB1D1 mutants were complemented with a plasmid bearing the wild-type agrB1D1 locus, and the complemented mutants were tested for toxin production. The mutants were also incubated in BHI medium anaerobically for 48 h in the presence or absence of the TI signal purified from wild-type strain 630. Toxin production was detected with the Cdifftox activity assay (30). agrB2D2 Mut, R20291 agrB2D2 deletion mutant; agrB1D1 Mut, agrB1D1 deletion mutant; agrB1D1 Mut+Comp, agrB1D1 deletion mutant complemented with a plasmid bearing the wild-type agrB1D1 locus. The difference in toxin activity between the wild type and the agrB1D1 mutant was significant (P = 0.0001 for both strains). Error bars represent the standard deviations of three independent experiments.
- FIG 4
Culture supernatant fluids collected from the agrB1D1 mutants are not cytotoxic to human foreskin fibroblast cells. Strain 630 and R20291 agrB1D1 and agrB2D2 mutants were cultured for 48 h in BHI medium anaerobically at 37°C. The culture supernatant fluids were collected, filter sterilized with a 0.45-µm filter, and examined for cytotoxicity with the Bartels Clostridium difficile cytotoxicity assay kit (Trinity Biotech, Jamestown, NY). The culture fluids were incubated with the fibroblast cells for 24 h and observed under a microscope for cytotoxic effects. Images were taken with an EVOS XL microscope (Advanced Microscopy Group) at ×20 magnification. Panels: A, a representative image of fibroblast cells cultured in growth medium only; B, wild-type 630; C, 630 agrB1D1 mutant; D, 630 complemented agrB1D1 mutant; E, wild-type R20291; F, R20291 agrB1D1 mutant; G, complemented R20291 agrB1D1 mutant; H, R20291 agrB2D2 mutant.
- FIG 5
Culture supernatants from C. difficile agrB1D1 mutants have no effect on cell viability. Strain 630 (A) and R20291 (B) agrB1D1 and agrB2D2 mutants were cultured for 48 h in BHI medium anaerobically at 37°C. The culture supernatant fluids were collected, filter sterilized with a 0.45-µm filter, and tested for their effect on cell viability. Briefly, ready-made fibroblast cells (Trinity Biotech, Jamestown, NY) were treated with the culture supernatant fluids and incubated for 48 h at 37°C aerobically. Following the incubation period, cell viability was examined with the CellTiter 96 nonradioactive cell proliferation assay (Promega, Madison, WI) in accordance with the instructions provided by the manufacturer. agrB1D1 Mut, agrB1D1 deletion mutant; agrB1D1 Mut+Comp, agrB1D1 mutant complemented with a plasmid bearing the wild-type agrB1D1 locus; agrB2D2 Mut, R20291 agrB2D2 deletion mutant. The differences between the amounts of formazan produced by fibroblast cells treated with supernatants from the agrB1D1 mutants and wild-type strains, the complemented agrB1D1 mutants, and the R20291 agrB2D2 mutant were significant (P = 0.0012). Error bars represent the standard deviations of three independent experiments.
- FIG 6
Hypervirulent R20291 agrB1D1 mutant is unable to cause C. difficile-associated disease in mice. Seven-week-old (both male and female) C57BL/6 mice (n = 12 in each group) were first treated with a cocktail of antibiotics; infected with the R20291 wild type, agrB1D1 mutant, agrB2D2 mutant, or complemented agrB1D1 mutant; and then monitored for 14 days. The animals were scored on the basis of the following endpoint symptoms: (i) diarrhea, hunched posture, and physical appearance (0, normal [good hair coat, normal posture, frequent grooming, no discharge from the eyes or nose]; 1, hunched posture and infrequent grooming; 2, hunched posture, no grooming [ruffled hair], distended stomach, and diarrhea [noticeable dehydration]); (ii) movement and response to external stimuli (0, normal [normal activity, alertness, response to stimuli, regular eating and drinking habits]; 1, reduced movement, reduced alertness, reduced response to stimuli, or uncoordinated movement; 2, no movement, no activity, no eating, no drinking or alertness); and (iii) body weight changes (0, normal [normal food consumption with normal urine and fecal output]; 1, 5 to 15% loss of body weight; 2, 15 to 20% loss of body weight). Animals that were assessed an endpoint score of 2 for any of the above three parameters during the 14-day observation period were euthanized because they were always moribund. Percent survival is the proportion of animals assessed an endpoint score of <2. (B) colonization of mice by the agrB1D1 mutant and the complemented mutant. (C) No toxins were detected in pellets of mice infected with the R20291 agrB1D1 mutant. Pellets collected from the mice on day 5 were tested for the presence of C. difficile cells by counting the CFU on selective plates and for toxins A and B with the Wampole C. difficile TOX A/B II assay (Technologies Lab, Blacksburg, VA). Data presented are the average readings from the 12 mice evaluated per group. The difference between the survival rates of mice treated with the agrB1D1 mutant and the wild type was statistically significant (P = 0.0001). There was no significant difference (P = 0.439) between the CFU counts of mice infected with the wild-type strain, the agrB1D1 mutant, or the complemented agrB1D1 mutant. Error bars represent the standard deviations of the 12 samples tested per group.
Supplemental Material
Figure S1
The C. difficile accessory gene regulator loci showing the location of the region deleted by allelic exchange. The agr1 locus in strains 630 (A) and R20291 (B) and the agr2 locus in strain R20291 (C) are shown. Download Figure S1, TIF file, 0.4 MB.
Copyright © 2016 Darkoh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S2
(A) Western blot analysis of 48-h culture supernatant fluid from the 630 agrB1D1 mutant. Supernatant from a 48-h culture was concentrated with the Pierce 150-kDa concentrator (Thermo Fisher Scientific Inc., Rockford, IL) and subjected to 6% PAGE. The protein bands were transferred onto a 0.45-µm nitrocellulose membrane and probed with monoclonal antibodies specific for toxins A and B. The toxin bands on the transferred membrane were detected with the Protein Detector Western blot BCIP/NBT kit (KPL, Gaithersburg, MD). I, wild type; II, agrB1D1 mutant. (B) The agr1 deletion abolishes toxin production in both the 630 (B) and R20291 (C) strains. The agrB1D1 mutants of both strains and a agrB2D2 mutant of the R20291 strain were incubated in BHI medium for 48 h anaerobically at 37°C. Toxin production was tested by ELISA with the Wampole C. difficile TOX A/B II assay (Technologies Lab, Blacksburg, VA). agrB1D1 Mut, agrB1D1 deletion mutant; agrB1D1 Mut+Comp, agrB1D1 mutant complemented with a plasmid bearing the wild-type agrB1D1 locus; agrB2D2 Mut, agrB2D2 deletion mutant. There were significant differences (P = 0.0023 for 630 and 0.0001 for R20291) between the amounts of toxins produced by the wild-type and agrB1D1 mutant strains. Error bars represent the standard deviations of three independent experiments. Download Figure S2, TIF file, 0.5 MB.
Copyright © 2016 Darkoh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S3
The agr1 mutants do not produce toxin in TY medium. agrB1D1 mutants of both strains and the R20291 agrB2D2 mutant strain were incubated in TY medium for 48 h anaerobically at 37°C. Toxin production was detected with the Cdifftox activity assay. agrB2D2 Mut, R20291 agrB2D2 mutant; agrB1D1 Mut, agrB1D1 mutant. There was a significant difference (P = 0.003 for 630 and 0.0001 for R20291) between the levels of toxin activity produced by the wild-type and agrB1D1 mutant strains. Error bars represent the standard deviations of three independent experiments. Download Figure S3, TIF file, 0.3 MB.
Copyright © 2016 Darkoh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S4
The R20291 agrB2D2 mutant produces an active TI signal that restores toxin production in 630 and R20291 agrB1D1 mutants unable to make toxins. 630 and R20291 agrB1D1 mutant strains were incubated in BHI medium anaerobically for 24 h in the presence of the TI signal purified from the R20291 agrB2D2 mutant. Toxin activity was detected with the Cdifftox activity assay (A), and toxin production was tested by nonquantitative ELISA (B) with the Wampole C. difficile TOX A/B II assay (Technologies Lab, Blacksburg, VA). agrB1D1 Mut, agrB1D1 deletion mutant; TI, TI signal. There were significant differences (P = 0.0041 for 630 and 0.0001 for R20291) between the amounts of toxins produced by the wild-type and agrB1D1 mutant strains in the absence of the TI signal. Error bars represent the standard deviations of three independent experiments. Download Figure S4, TIF file, 0.5 MB.
Copyright © 2016 Darkoh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Table S1
Primers used in this study. Table S1, TIF file, 0.4 MB.
Copyright © 2016 Darkoh et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Additional Files
Supplementary Data
Supplementary Data
- Figure sf1, TIF - Figure sf1, TIF
- Figure sf2, TIF - Figure sf2, TIF
- Figure sf3, TIF - Figure sf3, TIF
- Figure sf4, TIF - Figure sf4, TIF
- Table st1, TIF - Table st1, TIF
