克雷白氏肺炎桿菌CG43抗氧化逆境反應的研究 -超氧化歧化酶SodA的表現調控

Abstract

在感染的過程中，細菌的抗氧化防禦機制對於其毒性扮演著重要的角色。本研究中，我們探討克雷白氏肺炎桿菌CG43的氧化逆境反應。首先，我們建構ΔsodA、ΔsodB、ΔsodC、ΔkatE、ΔkatG和ΔrpoS等突變株，並分析這些基因缺損後對克雷白氏肺炎桿菌抗氧化逆境反應造成的影響，結果發現ΔkatG的生長速度變快，而ΔsodA菌落大小不一。利用生存率測試與紙錠分析的結果發現，相較於CG43S3野生株以及ΔsodB、ΔsodC、ΔkatE和ΔrpoS突變株，ΔsodA突變株對paraquat的處理最為敏感，而ΔkatG則對H2O2處理最為敏感，這些結果暗示SodA或KatG可能是克雷白氏肺炎桿菌在抗氧化壓力主要的反應因子。接著，我們利用LacZ報導系統和即時聚合酶連鎖反應來分析，發現在fur基因缺損的情況下，sodA基因啟動子的活性及mRNA的表現量增加，而在fur和ryhB雙基因缺損菌株，Fur的抑制作用消失，這些結果暗示著Fur是sodA基因表現的抑制蛋白，而RyhB則會促進sodA基因表現。最後，藉由細胞膜間隙蛋白質的分析，我們發現SodA或SodB雖然缺乏N端訊號胜肽，卻和膜間隙蛋白SodC一樣在經過paraquat處理後會出現在細胞膜間隙。

During infection, defenses against oxidative stress play an important role in determining the bacterial virulence. Here, the antioxidative response in Klebsiella pneumoniae CG43 was investigated. Specific gene-deletion mutants including ΔsodA, ΔsodB, ΔsodC, ΔkatE, ΔkatG and ΔrpoS were generated and the deletion effects were analyzed. Each of the deletion had no apparent effect on the cell growth and phenotypic presentation except that ΔkatG showed faster growth and ΔsodA exerted varied colony forms. Compared to the parental strain K. pneumoniae CG43S3 or the deletion mutant ΔsodB, ΔsodC, ΔkatE and ΔrpoS, the survival analysis or the disc diffusion assay revealed that ΔsodA mutant was most sensitive to the treatment of paraquat (PQ) and ΔkatG was most sensitive to H2O2 treatment. These results indicated that SodA and KatG likely play a major role in the oxidative stress response. Using LacZ reporter analysis and qRT-PCR measurement, the sodA promoter activity and mRNA level were found to be increased in the fur deletion strain but the deleting effect was suppressed by further deleting the ryhB gene. This suggests a negative role of Fur but a positive role of RyhB on the expression of sodA. Finally, the periplasmic fractionation analysis indicated that although SodA and SodB carrying no N-terminal signal peptide, they as well as SodC were found in the periplasmic fraction after PQ treatment.