探討克雷白氏肺炎桿菌 CG43 攝鐵調控蛋白 Fur 對第三型纖毛表現的調控

Abstract

在大腸桿菌中，攝鐵蛋白 Fur、小 RNA RyhB 及小 RNA 伴護蛋白 Hfq 可分別調控鐵離子的攝取及儲存，先前的研究指出，克雷白氏肺炎桿菌 CG43S3 在缺鐵環境中，無法偵測到第三型纖毛主要單位蛋白 MrkA 的生成；已知 Fur 可提升 MrkH 蛋白表現再間接影響第三型纖毛的表現，本論文在探討 Fur 是否經由 RyhB 來調控第三型纖毛的表現，並且確認 Hfq 是否參與 RyhB 的調控作用。首先，以西方墨點法分析 MrkA 的生成，我們發現 ∆fur 中被抑制表現的 MrkA，會因 RyhB 基因剔除而回復 MrkA 的生成量，進一步以啟動子活性和生物膜生成測試也發現類似結果，此暗示 RyhB 經由直接結合 mrkA 的 mRNA 來抑制 MrkA 表現，Fur可能是經由抑制 RyhB 而活化 MrkA 的表現；另外，在 CG43S3 中大量表現 ryhB 時可阻斷 MrkA 的生成，再次顯示 RyhB 對 MrkA 表現的抑制作用，而此抑制現象在剔除 hfq 基因後則觀察不到，此結果顯示 RyhB 抑制 MrkA 表現的作用需要 Hfq 的參與，為了進一步確認 Hfq 與 RyhB 的協同調控作用，我在 RyhB 被預測與 Hfq 結合的序列上做了定點突變，而在 CG43S3 中大量表現此突變過的 ryhB 時，MrkA的生成不受影響，此結果證實 RyhB 抑制 MrkA 表現的作用需要 Hfq 的參與。第二部份我更進一步探討 Fur、RcsB 或 Cpx 系統間是否有交互作用，首先建構 fur、rcsB 或 cpxAR 雙基因缺損株，以回補試驗與西方墨點法分析顯示在增加 fur 基因表現後仍無法回復 fur、rcsB 雙基因缺損對於 MrkA 生成影響，相對的， fur、cpxAR 雙基因缺損株的 MrkA 表現增加，此暗示 Fur 在 Cpx 系統上游扮演負向調控角色。第一型纖毛是克雷白氏肺炎桿菌造成泌尿道感染的重要因子，本論文最後探討鐵離子是否調控第一型纖毛的表現，在添加螯鐵劑 Dip 培養下，以西方墨點法在 ∆mrkA 或 ∆mrkI 菌株中，均可偵測第一型纖毛主要單位蛋白 FimA 表現量降低，初步分析發現相較於 CG43S3，FimA 在 ∆iscR 中表現會略為提升，此暗示第一型纖毛表現與鐵濃度有關，而 IscR 可能參與其表現調控。

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.