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

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

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-qPCR)结果显示,缺失CD630_27900基因,自溶素cwp19、Acd基因表达量降低,::CD630_27900自溶素基因表达增强。相较于CD630,△CD630_27900菌株细胞毒力、毒素基因tcdA、tcdB表达量降低。相较于CD630,△CD630_27900对氨苄青霉素、甲硝唑、阿莫西林、万古霉素、诺氟沙星、头孢西丁、卡那霉素更加敏感,::CD630_27900对以上抗生素敏感性恢复。此外,△CD630_27900对酸比CD630敏感,对碱敏感性未发生变化。::CD630_27900对酸敏感性恢复至野生型水平。【结论】敲除CD630_27900基因,艰难拟梭菌自溶速率变慢、自溶素cwp19、Acd基因表达量降低、细胞毒力、毒素基因tcd A和tcd B降低,说明CD630_27900基因影响菌株自溶以及毒力释放。菌株△CD630_27900对临床上常见的抗生素及酸性环境更加敏感,且这些变化均可通过基因回补恢复。提示该基因可作为联合抗生素治疗艰难梭菌感染(Clostridioides difficile infection, CDI)的潜在靶点。

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.