Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile

Abstract

Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.

Figure 1. Confirmation of spo0A mutant construction in C. difficile strains by PCR and Southern blot.

Diagram of spo0A and surrounding genes in the wild type strain R20291 (A) and mutant derivative in which the targetron has inserted (B), showing primers and restriction sites used to confirm disruption of spo0A in C. difficile. PCR using the EBS-universal primer and the spo0A-specific primer JRP2528 resulted in a product of approximately 0.6 kb for the mutant strains, while no product was generated for the wild type parental strains (C). PCR with primers flanking spo0A showed that the mutant generated a product of approximately 3 kb, confirming an insertion of approximately 1.8 kb was present in these strains when compared to wild type which produced a product of 1.2 kb (D). Southern blot analysis using probes specific for spo0A (E) and the intron (F) confirmed the disruption of spo0A with the targetron. The probe for spo0A hybridised to a band of 3 kb for the wild type strains M7404, R20291 and 630Δerm and 2.7 kb for JGS6133, while for each mutant the probe hybridised to a band 1.8 kb greater. The intron-specific probe hybridised to a band of the corresponding size in the mutants only and did not bind to wild type strain DNA.

Figure 2. Characterisation of sporulation over time in each of the spo0A mutants.

The wild type, mutant and complemented derivatives in the M7404 (A), R20291 (B), 630Δerm (C) and JGS6133 (D) backgrounds were assayed for total viable count (black bars) and heat-shocked spore count (white bars) after incubation for 12, 24, 48 and 72 hours. Three biological replicates were tested for each strain; the mean values of these replicates are shown together with the standard errors of the means. The dashed line represents the limit of detection for this assay.

Figure 3. Analysis of toxin A and toxin B production by M7404 spo0A mutant.

The wild type, mutant and complemented derivatives were examined for toxin production by Western immunoblotting using (A) TcdA-specific and (B) TcdB-specific antibodies and precipitated supernatant proteins from the strains indicated. Toxin production by these strains was also quantified. Serial doubling dilutions of culture supernatants were made in MEM alpha medium supplemented with 1% HI FCS and used in Vero cell cytotoxicity assays (C). Morphological changes of the Vero cells were observed and scored by microscopy after 24 hrs. The toxin titre is the reciprocal of the endpoint dilution. Vero cell viability (D) was also measured using MTT reagent. •, M7404 (vector control); ▪, M7404 spo0A mutant (vector control); ▴, complemented derivative. All assays were performed in duplicate on at least three independent culture supernatants; the mean values of these assays are shown together with the standard errors of the means. ***, p ≤ 0.001. For Western immunoblot analysis, three independent culture supernatants were tested; the image shown is representative.

Figure 4. Analysis of toxin A and toxin B production by R20291 spo0A mutant.

The wild type, mutant and complemented derivatives were examined for toxin production by Western immunoblotting using (A) TcdA-specific and (B) TcdB-specific antibodies and precipitated supernatant proteins from the strains indicated. Toxin production by these strains was also quantified. Serial doubling dilutions of culture supernatants were made in MEM alpha medium supplemented with 1% HI FCS and used in Vero cell cytotoxicity assays (C). Morphological changes of the Vero cells were observed and scored by microscopy after 24 hrs. The toxin titre is the reciprocal of the endpoint dilution. Vero cell viability (D) was also measured using MTT reagent. •, R20291 (vector control); ▪, R20291 spo0A mutant (vector control); ▴, complemented derivative. All assays were performed in duplicate on at least three independent culture supernatants; the mean values of these assays are shown together with the standard errors of the means. **, p ≤ 0.01. For Western immunoblot analysis, three independent culture supernatants were tested; the image shown is representative.

Figure 5. Analysis of toxin A and toxin B production by 630Δerm spo0A mutant.

The wild type, mutant and complemented derivatives were examined for toxin production by Western immunoblotting using (A) TcdA-specific and (B) TcdB-specific antibodies and precipitated supernatant proteins from the strains indicated. Toxin production by these strains was also quantified. Serial doubling dilutions of culture supernatants were made in MEM alpha medium supplemented with 1% HI FCS and used in Vero cell cytotoxicity assays (C). Morphological changes of the Vero cells were observed and scored by microscopy after 24 hrs. The toxin titre is the reciprocal of the endpoint dilution. Vero cell viability (D) was also measured using MTT reagent. •, 630▵erm (vector control); ▪, 630▵erm spo0A mutant (vector control); ▴, complemented derivative. All assays were performed in duplicate on at least three independent culture supernatants; the mean values of these assays are shown together with the standard errors of the means. For Western immunoblot analysis, three independent culture supernatants were tested; the image shown is representative.

Figure 6. Analysis of toxin A and toxin B production by JGS6133 spo0A mutant.

The wild type, mutant and complemented derivatives were examined for toxin production by Western immunoblotting using (A) TcdA-specific and (B) TcdB-specific antibodies and precipitated supernatant proteins from the strains indicated. Toxin production by these strains was also quantified. Serial doubling dilutions of culture supernatants were made in MEM alpha medium supplemented with 1% HI FCS and used in Vero cell cytotoxicity assays (C). Morphological changes of the Vero cells were observed and scored by microscopy after 24 hrs. The toxin titre is the reciprocal of the endpoint dilution. Vero cell viability (D) was also measured using MTT reagent. •, JGS6133 (vector control); ▪, JGS6133 spo0A mutant (vector control); ▴, complemented derivative. All assays were performed in duplicate on at least three independent culture supernatants; the mean values of these assays are shown together with the standard errors of the means. For Western immunoblot analysis, three independent culture supernatants were tested; the image shown is representative.

Figure 7. Time course analysis of cytotoxic activity of C. difficile spo0A mutants.

The wild type, mutant and complemented derivatives of M7404 (A), R20291 (B), 630Δerm (C), and JGS6133 (D) were examined for toxin production over 12, 24, 48 and 72 hours. Serial doubling dilutions of culture supernatants were made in MEM alpha medium supplemented with 1% HI FCS and used in Vero cell cytotoxicity assays. Morphological changes of the Vero cells were observed and scored by microscopy after 24 hrs. The toxin titre is the reciprocal of the endpoint dilution. All assays were performed in duplicate on at least three independent culture supernatants; the mean values of these assays are shown together with the standard errors of the means. **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 8. Disruption of spo0A in C. difficile does not affect in vitro swimming motility.

Overnight cultures of the isogenic panels of wild type, mutant and complemented derivatives were used to inoculate semi-solid (0.0175% (w/v)) HIS agar. After 24 hours incubation, motility was observed for the M7404 derivatives (A), the R20291 derivatives (B) and the 630▵erm derivatives (C); JGS6133 and derivatives are non-motile under these conditions (D). No clear observable difference was noted between wild type and spo0A mutant for any strain background tested. The assay was performed in technical duplicates and repeated three times for each strain. Images are representative of each assay.

Figure 9. Disruption of spo0A in C. difficile leads to altered motility on solid medium.

For each strain a 15 µl spot of overnight culture was placed onto a 1% (w/v) HIS agar plate which was then incubated for four days. Following incubation, the plates were observed for any changes in growth across the agar surface in the mutant compared to wild type and the complemented derivative for strains M7404, R20291, 630▵erm and JGS6133 (A-D, respectively). WT  =  wild type; M  =  spo0A mutant; C  =  complemented. The white scale bar represents 1 cm. Images are representative of three independent experiments.

Figure 10. Colony morphology is altered in C. difficile spo0A mutants.

For each strain a 10 µl aliquot of glycerol stock was spread across a 1% (w/v) HIS agar plate. After incubation, colonies were examined microscopically for any morphological changes in the mutant compared to wild type and the complemented derivatives for strains M7404, R20291, 630▵erm and JGS6133 (A-D, respectively). The yellow scale bar in each image is 10 µm. Images are representative of three independent experiments.