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

摘要:

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-time fluorescence quantitative polymerase chain reaction,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tcdA和tcdB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。

关键词:艰难拟梭菌;CD630_27900基因;肽聚糖脱乙酰化;非等长同源臂偶联等位交换;突变表型

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