Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production

Abstract

Toxigenic Clostridium difficile strains produce two toxins (TcdA and TcdB) during the stationary phase of growth and are the leading cause of antibiotic-associated diarrhea. C. difficile isolates of the molecular type NAP1/027/BI have been associated with severe disease and hospital outbreaks worldwide. It has been suggested that these "hypervirulent" strains produce larger amounts of toxin and that a mutation in a putative negative regulator (TcdC) allows toxin production at all growth phases. To rigorously explore this possibility, we conducted a quantitative examination of the toxin production of multiple hypervirulent and nonhypervirulent C. difficile strains. Toxin gene (tcdA and tcdB) and toxin gene regulator (tcdR and tcdC) expression was also monitored. To obtain additional correlates for the hypervirulence phenotype, sporulation kinetics and efficiency were measured. In the exponential phase, low basal levels of tcdA, tcdB, and tcdR expression were evident in both hypervirulent and nonhypervirulent strains, but contrary to previous assumptions, toxin levels were below the detectable thresholds. While hypervirulent strains displayed robust toxin production during the stationary phase of growth, the amounts were not significantly different from those of the nonhypervirulent strains tested; further, total toxin amounts were directly proportional to tcdA, tcdB, and tcdR gene expression. Interestingly, tcdC expression did not diminish in stationary phase, suggesting that TcdC may have a modulatory rather than a strictly repressive role. Comparative genomic analyses of the closely related nonhypervirulent strains VPI 10463 (the highest toxin producer) and 630 (the lowest toxin producer) revealed polymorphisms in the tcdR ribosome binding site and the tcdR-tcdB intergenic region, suggesting that a mechanistic basis for increased toxin production in VPI 10463 could be increased TcdR translation and read-through transcription of the tcdA and tcdB genes. Hypervirulent isolates produced significantly more spores, and did so earlier, than all other isolates. Increased sporulation, potentially in synergy with robust toxin production, may therefore contribute to the widespread disease now associated with hypervirulent C. difficile strains.

FIG. 1.

(A) Growth curves of the eight strains used in this study. Absorbance at 600-nm wavelength was measured over 72 h of growth. (B) Total toxin levels over time. The toxin levels represent combined TcdA and TcdB levels by ELISA (see Materials and Methods). Exponential-phase samples were taken when each individual culture reached an OD₆₀₀ of 0.5. Means and standard errors from three biological replicates are shown. The sensitivity of the ELISA was approximately 0.8 ng/ml for TcdA and 2.5 ng/ml for TcdB.

FIG. 2.

(A) Transcription of tcdA during exponential phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 replicates are shown. (B) Transcription of tcdA during stationary phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 biological replicates are shown.

FIG. 3.

(A) Transcription of tcdB during exponential phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 replicates are shown. (B) Transcription of tcdB during stationary phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 biological replicates are shown.

FIG. 4.

(A) Transcription of tcdR during exponential phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 replicates are shown. (B) Transcription of tcdR during stationary phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 biological replicates are shown.

FIG. 5.

(A) Transcription of tcdC during exponential phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 replicates are shown. (B) Transcription of tcdC during stationary phase relative to transcription of the housekeeping gene rpoA. Means and standard errors of 3 biological replicates are shown.

FIG. 6.

Schematic of the C. difficile PaLoc region. Coding regions and predicted regulatory elements are depicted. The cdu1 gene has three predicted Rho-independent terminators (indicated by hairpins). The formation of any given hairpin usually prevents the formation of others downstream. Further, if ter2 forms, transcription from tcdRp is blocked. ter, terminator; p, promoter; RBS, ribosome binding site. Not to scale.

FIG. 7.

(A) Spores as heat-resistant CFU over 48 h of growth. Means and standard errors of three biological replicates are shown. (B) Mean spores counted by microscopy per field.