CD630＿27900基因缺失显著降低艰难拟梭菌自溶速率、毒力及对酸和抗生素的耐受性

艰难拟梭菌(Clostridioidesdifficile)CD630＿27900基因位于slpA-cwp66基因座上，CD630＿27900基因属于假定的Lmbe家族的酶，但基因功能尚未明确。【目的】本研究通过构建艰难拟梭菌CD630＿27900基因敲除菌株，比较野生型菌株(CD630)与突变株表型差异，探讨CD630＿27900基因对艰难拟梭菌感染的影响。【方法】用非等长同源臂偶联等位交换(allele-coupled exchange, ACE)构建CD630＿27900基因缺失菌株与回补菌株。比较它们在生长曲线、自溶素(cwp19,Acd)基因表达、细胞毒力、主要毒素基因表达、抗生素及pH敏感性差异，以研究CD630＿27900基因的功能。【结果】成功构建△CD630＿27900突变菌株和::CD630＿27900回补菌株。菌株△CD630＿27900在衰亡期自溶速率显著低于菌株CD630,::CD630＿27900自溶速率恢复。实时荧光定量聚合酶链反应(real-timefluorescencequantitative polymerasechainreaction,RT-q...

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

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.