Characterization of the Ferric uptake regulator Fur-dependent regulation on type 3 fimbriae expression in Klebsiella pneumoniae CG43

• Main Authors: Hsu, Chi-Hsiang, 徐啟翔
• Other Authors: Peng, Hwei-Ling
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/skwy82

碩士 === 國立交通大學 === 生物科技學系 === 104 === In Escherichia coli, Fur, RyhB, and the RNA chaperone Hfq are interacting for the control of iron acquisition and storage. We have previously demonstrated in Klebsiella pneumoniae CG43S3 that the expression of type 3 fimbriae is iron dependent and regulated by Fur. Fur indirectly increases type 3 fimbriae expression through direct activation of the expression of mrkH.Here we investigate if the Fur-dependent expression of type 3 fimbriae is mediated by ryhB and Hfq. Western blot analysis revealed that the abolished production of the major pilin MrkA in ∆fur could be restored by deleting ryhB from ∆fur. The promoter activity and biofilm formation measurements also showed a similar results which suggesting ryhB directly binds to the mRNA of mrkA for the MrkA expression inhibition. Hence Fur activation of MrkA production is probably through repression of the ryhB expression. In addition, overexpression of ryhB blocked the MrkA production and the repression was not observed by deleting the Hfq gene indicating the RyhB-mediated regulation is Hfq dependent. The repression on MrkA production was no longer detected when RyhB was replaced by the site-directed mutant RyhB* of which the Hfq binding sequences were altered. This further supported that the repression of RyhB on MrkA production is Hfq dependent. Secondly, we investigated whether RcsB or Cpx interacts with Fur to influence the Fur-mediated regulation. Comparative analysis of the MrkA production in ∆fur, ∆rcsB, ∆fur∆rcsB, ∆fur∆rcsB[pRK415], ∆fur∆rcsB[pRK415-fur], and ∆fur∆rcsB[pRK415-rcsB]. Western blot analysis revealed that increased expression of fur had no complementation effect for MrkA production in ∆fur∆rcsB. On the other hand, the analysis for ∆fur∆cpxAR suggested that Fur may play an upatream negative regulatory role on the CpxAR-dependent MrkA expression. Type 1 fimbriae are crucial factors involved in the urinary tract infection by K. pneumoniae. Finally, we investigated if the expression of the type 1 fimbriae is iron-dependent.Western blot analysis revealed that addition of iron chelator Dip decreased the type 1 fimbriae major pilin FimA production in ∆mrkA or ∆mrkI. Compared to that of CG43S3, FimA production in ∆iscR was slightly increased. These suggested that the expression of type 1 fimbriae is iron-dependent and IscR may participate in the regulatory pathway.