克雷白氏肺炎桿菌CG43中二價鐵螯合系統FeoABC、SitABCD和EfeUOB之特性探討與比較

Comparative characterization of the ferrous iron transport systems FeoABC, SitABCD and EfeUOB in Klebsiella pneumoniae CG43

郭俐君(Li-Ching Kok)

指導教授 ： 彭慧玲

國立交通大學/生物科技學院/生物科技系所/碩士(2014年)

https://doi.org/10.6842/NCTU.2014.00773

摘要

如同其他致病細菌，克雷白氏肺炎桿菌在宿主環境中需要克服缺鐵的考驗。克雷白氏肺炎桿菌的二價鐵的螯合系統基因組feoABC、sitABCD和efeUOB在含鐵量豐富的培養條件中，會受轉錄因子Fur抑制其表現。本研究以克雷白氏肺炎桿菌CG43S3為親本株分別建構了∆feoA、∆feoC、∆feoA∆fur、∆feoB∆fur、∆feoC∆fur、∆sitCD、∆sitCD∆fur、∆efeUOB、∆efeUOB∆fur、∆feoB∆sitCD、∆feoB∆efeUOB、∆sitCD∆efeUOB、∆efeUOB∆sitCD∆feoB和∆efeUOB∆sitCD∆feoB∆fur等基因缺損突變株，並比較分析這些基因缺損後對生長及致病性相關的表現型之影響；同時，這三套螯鐵系統預測的啟動子片段也被轉殖至啟動子報導質體placZ15中，再藉由β-半乳糖苷酶（β-galactosidase）活性評估其基因表現。結果顯示這三套螯鐵系統的啟動子活性皆可因fur基因缺損或缺鐵環境所誘導；在缺鐵、微氧條件下，feo的啟動子活性會被FeoC負向調控；而sitABCD活性最高。SitABCD系統能幫助細菌對抗氧化壓力；而sit及feo基因都剔除結果會使細菌的抗氧化能力明顯下降；EfeUOB表現可受酸性誘導，並被雙分子訊息系統的反應蛋白CpxR負向調控，此結果顯示EfeUOB可能在有氧、缺鐵及弱酸的特定環境中扮演重要角色。莢膜多醣體的生成與鐵的多寡有關，然而FeoABC、SitABCD或EfeUOB基因剔除並不影響克雷白氏肺炎桿菌CG43的莢膜多醣體產生；而除了∆sitCD∆efeU和∆feoC之外，其他的基因缺損株都會降低生物膜的生成，在添加了三價鐵螯合劑Deferoxamine後，這些缺損株的生物膜減少更為顯著；有趣的是，受鐵濃度影響表現、而且是決定生物膜生成的主要因子第三型線毛，其主要單位蛋白MrkA並沒有受到這些基因缺損的影響，這結果暗示這些螯鐵系統並不是透過影響MrkA表現來影響生物膜的生成。

關鍵字

二價鐵螯合系統 ； 克雷白氏肺炎桿菌

並列摘要

As the other pathogenic bacteria, Klebsiella pneumoniae often encounters the challenge of iron depletion surroundings in the host cells. The iron transport system encoding operon feoABC, sitABCD, and efeUOB are present in the genome of K. pneumoniae CG43. Under iron rich cultured condition such as LB medium, the expression of feoABC, efeUOB and sitABCD are repressed by the global regulator Fur (ferric uptake regulator). In the study, ∆feoA∆fur, ∆feoB∆fur, ∆feoC∆fur, ∆sitCD, ∆sitCD∆fur, ∆efeUOB, ∆efeUOB∆fur, ∆feoB∆sitCD, ∆feoB∆efeUOB, ∆sitCD∆efeUOB, ∆efeoUOB∆sitCD∆feoB and ∆efeoUOB∆sitCD∆feoB∆fur derived from K. pneumoniae CG43S3 have been generated. The mutation effects on the bacterial growth and virulence-related properties such as capsular polysaccharide (CPS) production, oxidative stress responses, type 3 fimbriae expression, and biofilm formation were analyzed and compared. The putative promoter of the three iron transport systems were isolated and individually cloned in front of the promoterless lacZ on the reporter plasmid pLacZ15 for the optimal expression and condition analysis using β-galactosidase activity. All three iron transport systems could be induced by the absence of Fur repressor and depleting iron from the culture medium. The feo promoter was negatively influenced under iron-depleted and microaerobic condition by FeoC, a predicted Fe-S sensor regulator. The microaerobically-induced sitABCD showed the highest promoter activity among these three systems. SitABCD plays important role in protecting bacteria from oxidative stress, with combined loss of sit and feo further decreased bacterial oxidative-resistance. EfeUOB is induced by weak acid and negatively regulated by the two-component regulator CpxR. This indicates its specific role in aerobic, iron-depleted and low pH environment. Although iron-availability affects CPS biosynthesis, FeoABC, SitABCD and EfeUOB transport systems do not involved in regulation of CPS biosynthesis. Except ∆sitCD∆efeUOB and ∆feoC, deletion of genes from these acquisition systems decreased biofilm formation of K. pneumoniae CG43S3, wherein iron chelators deferoxamine exerted more decreasing effects of biofilm formation. Interestingly, these deletion mutants did not decrease the expression of MrkA, the major pilin of type3 fimbriae which is iron dependent and the major determinant of biofilm formation. These results imply that these ferrous iron transport systems regulate biofilm formation independently of the expression of type 3 fimbriae.

並列關鍵字

ferrous iron uptake system ； Klebsiella pneumoniae ； FeoABC ； SItABCD ； EfeUOB

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