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. 2023 Sep 28;12(10):1202.
doi: 10.3390/pathogens12101202.

Potent Bile Acid Microbial Metabolites Modulate Clostridium perfringens Virulence

Tahrir Alenezi  1   2   3 , Ying Fu  1 , Bilal Alrubaye  1 , Thamer Alanazi  1   2 , Ayidh Almansour  1   2 , Hong Wang  1   2 , Xiaolun Sun  1   2
Affiliations

Potent Bile Acid Microbial Metabolites Modulate Clostridium perfringens Virulence

Tahrir Alenezi et al. Pathogens. .

Abstract

Clostridium perfringens is a versatile pathogen, inducing diseases in the skin, intestine (such as chicken necrotic enteritis (NE)), and other organs. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduced NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H2S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H2S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H2S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids. In conclusion, DCA and isoalloLCA reduced C. perfringens virulence by transcriptionally modulating the pathogen signaling pathways. The findings could be used to design new strategies to prevent and treat C. perfringens-induced diseases.

Keywords: C. perfringens; bile acid; necrotic enteritis; toxic gas.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
DCA reduces C. perfringens strain CP1 growth and H2S production. C. perfringens was cultured with DCA for 24 h under anaerobic conditions. (A) C. perfringens enumeration by serial dilution and plating. (B) Detection of H2S production via the lead acetate (Pb(OAc)₂) discs. Results are representative of 3 independent experiments. All graphs show mean + SEM. Different letters of a–c mean p < 0.05.
Figure 2
Figure 2
IsoalloLCA reduces C. perfringens strain CP1 growth and H2S production. C. perfringens was cultured with CA, CDCA, LCA, or isoalloLCA for 24 h under anaerobic conditions. (A) C. perfringens enumeration by serial dilution and plating. (B) Detection of H2S production via the lead acetate discs. Results are representative of 3 independent experiments. All graphs show mean + SEM. Different letters of a–f mean p < 0.05.
Figure 3
Figure 3
C. perfringens CP1 virulence gene expression is reduced by DCA and isoalloLCA. C. perfringens CP1 was cultured with CA, CDCA, DCA, LCA, and isoalloLCA for 2 h. After RNA extraction and cDNA reverse transcription, the virulence gene accumulation was quantified by real-time PCR (qPCR). Results are representative of 3 independent experiments. All graphs show mean + SEM. Different letters of a–e mean p < 0.05.
Figure 4
Figure 4
The effects of DCA on C. perfringens HN13 and its mutant growth and H2S production. C. perfringens strain HN13 and its three mutants were cultured with DCA for 24 h under anaerobic conditions. (A) C. perfringens enumeration by serial dilution and plating. (B) Detection of H2S production via the lead acetate discs. # means No. All graphs show mean + SEM. ****, p < 0.0001. ns, not significant.
Figure 5
Figure 5
The effect of DCA on C. perfringens HN13 mutant virulence gene expression. C. perfringens HN13 and its three mutants were cultured with DCA for 2 h. After RNA extraction and cDNA reverse transcription, the virulence gene accumulation was quantified by real-time PCR (qPCR). Results are representative of 3 independent experiments. # means No. All graphs show mean + SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, not significant.
Figure 6
Figure 6
The effects of isoalloLCA on C. perfringens HN13 mutant growth, H2S production, and the virulence gene expression. (A) C. perfringens strain HN13 and its three mutants were cultured with isoalloLCA for 24 h under anaerobic conditions. C. perfringens enumeration by serial dilution and plating. (B) C. perfringens strain HN13 and its three mutants were cultured with isoalloLCA for 24 h under anaerobic conditions. Detection of H2S production via the lead acetate discs. (C) C. perfringens HN13 and its three mutants were cultured with isoalloLCA for 2 h. After RNA extraction and cDNA reverse transcription, the virulence gene accumulation was quantified by real-time PCR (qPCR). Results are representative of 3 independent experiments. # means No. All graphs show mean + SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns, not significant.
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