CD630_27900 gene deletion significantly reduces autolysis rate and virulence of Clostridioides difficile and the tolerance to acids and antibiotics

Abstract:

The function of CD630_27900 gene at the slpA-cwp66 locus of Clostridioides difficile is still unclear. The encoded enzyme is putatively regarded as a member of Lmbe family. [Objective] To construct CD630_27900 gene-deleted mutant, compare the phenotypes of the wild-type strain (CD630) and the mutant, and discuss the effect of CD630_27900 gene on the infection of C. difficile. [Methods] The CD630_27900-deleted mutant and the complementary strain were constructed by allele-coupled exchange (ACE). The growth curves, expression of autolysin genes (cwp19, Acd), cytotoxicity, expression of major toxin genes, and sensitivity to antibiotics and pH were compared among the three strains. [Results] The mutant strain ∆CD630_27900 and the complementary strain ::CD630_27900 were successfully constructed. At the decline phase, the autolysis rate of ∆CD630_27900 was significantly lower than that of CD630, and the autolysis rate of ::CD630_27900 was restored. The real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) results showed that the expression of cwp19 and Acdwas decreased in ∆CD630_27900 and increased in ::CD630_27900 compared with that in the wild type. The cytotoxicity assay showed that the cytotoxicity of ∆CD630_27900 was significantly reduced compared with that of CD630 and the complementary strain, which is consistent with reduced expression levels of thetoxin genes tcdA and tcdB. Furthermore, ∆CD630_27900 showed significantly stronger sensitivity to ampicillin, metronidazole, amoxicillin, vancomycin, norfloxacin, cefoxitin, and kanamycin than CD630 and ::CD630_27900. Strain ∆CD630_27900 was more sensitive to acid than strain CD630 and ::CD630_27900. However, the sensitivity of ∆CD630_27900 to the alkaline environment was comparable to that of CD630. [Conclusion] Upon the deletion of CD630_27900 gene, C. difficile demonstrated slow autolysis, low expression of autolys in genes cwp19 and Acd, low cytotoxicity, and low expression of toxin genes tcdA and tcdB. Thus, CD630_27900 may influence the autolysis and virulence of C. difficile. ∆CD630_27900 was more sensitive to the antibiotics commonly used in clinical settings than the wild type. These changes were reversed after the complementation of the gene. Thus, CD630_27900 can be a potential target in the treatment of C. difficile infection (CDI) with antibiotics.

Key words:Clostridioes difficile;CD630_27900 gene;peptidoglycan deacetylation;allele-coupled exchange (ACE);phenotypes of mutants