研究RcsB、Hfq、HNS對克雷白氏肺炎桿菌 CG43 酸壓力反應之調控

Abstract

腸類屬細菌的抗酸作用是保護自身免受胃酸損害的常見反應。我們在先前的研究發現剔除調控莢膜生合成的雙分子系統 Rcs 中的反應調控蛋白基因 rcsB 會使克雷白氏肺炎桿菌 CG43 在酸環境的生存率大幅下降，其基因體含有兩套同源的酸適應島嶼 AFI-I 和 AFI-II，將 AFI-I 上的基因 yfdX、 hdeB、 hdeD 或 hdeB1，或AFI-II的hdeA、hdeD1及hdeB2 基因剔除，皆會對抵抗酸壓力有負向影響。本篇研究中，我們先利用電泳遲滯試驗證明重組的 RcsB 蛋白能夠與帶有生物素標記的 yfdX 啟動子片段結合，而此結合會被未帶有生物素標記、不同濃度的 yfdX、 hdeB1、 hdeA 及 hdeD1B2 等任一啟動子片段所抑制，此暗示著 RcsB 能直接調控 yfdX,、 hdeB1、 hdeA 與 hdeD1B2 的轉錄表現。除外，我們也探討在別的細菌中被報導與其毒性或壓力反應有顯著影響的類核相關蛋白 HNS 與 RNA伴隨蛋白 Hfq 在克雷白氏肺炎桿菌 CG43抗酸壓力反應所扮演的角色： 在克雷白氏肺炎桿菌 CG43基因體可找到兩個與大腸桿菌中 HNS 類似的同源基因，分別命名為 hns-1 與 hns-2；我們發現在 hns-1 缺失下會使細菌抗酸能力下降，而 hns-2 的缺失對其於酸性環境的生存率沒有明顯影響，以啟動子活性分析發現，當細菌靜置培養於 pH 5 的 LB 時，hns-1 缺失可降低 yfdX 的啟動子活性，卻提升 hdeA 及 hdeD1B2 的啟動子活性；此外，剔除 hfq 或 fur 會顯著降低其酸壓力下的生存率，而同時剔除 fur 及 ryhB 基因時，其酸生存率則較剔除 fur之突變株高，此結果暗示 Hfq 與Fur對克雷白氏肺炎桿菌 CG43 的抗酸能力具有正向調控的功能，RyhB 則扮演負向調控的角色；而以西方墨點法分析發現 hfq 基因缺失會阻斷受酸誘導的 YfdX 表現，fur基因缺失沒有明顯影響，只有 fur及 ryhB 雙基因缺失才有明顯增加 YfdX 表現的現象。最後，利用即時定量聚合酶連鎖反應分析結果確認 RcsB 與 Hfq 正向影響 yfdX、 hdeA、 hdeB 與的表現，Fur 及 RyhB 則對 yfdX 與 hde 基因有不同的調控功能。

Acid resistance is a general response for enterobacteria to protect themselves from the damages by gastric acid. We have previously reported that deletion of the rcsB gene which coding for the response regulator of the two-component system Rcs (regulation of capsule synthesis) decreased the survivals of Klebsiella pneumoniae CG43 upon acid treatment. Two sets of acid fitness island (AFI) homologue were discovered in the genome of K. pneumoniae CG43, and the deletion of yfdX gene as well as of the AFI-I genes hdeB, hdeD and hdeB1, or of the AFI-II genes hdeA, hdeD1, and hdeB2, also have negative effects on the acid survivals. Here, we report using electrophoresis mobility shift assay (EMSA) to demonstrate that the recombinant RcsB could bind the biotin-labeled yfdX promoter, and either the non-labeled promoter DNA of yfdX, hdeB1, hdeA and hdeD1B2 inhibited the binding phenomenon, with different affinities. This suggested a direct regulation at the transcriptional level of RcsB on the expression of yfdX, hdeB1, hdeA and hdeD1B2. Besides RcsB, we also investigated the regulatory role of histone-like nucleoid protein H-NS and the RNA chaperone Hfq, both shown significant impacts on virulence and stress responses of other bacteria, in the acid stress response. In K. pneumoniae CG43, two homologous HNS coding genes were identified and namely hns-1 and hns-2. Loss of hns-1 decreased the acid survival while hns-2 deletion showed no apparent effect. Promotor activity analysis revealed that the hns-1 deletion decreased the promoter activity of yfdX but increased the expression of hdeA and hdeD1B2 when the bacteria cultured at pH 5 statically. Furthermore, deletion of hfq or fur significantly reduced the acid survival and furryhB had increased the acid survival when compared to fur. This suggested a positive regulatory role of Hfq and Fur but a negative role of the small RNA RyhB on the acid stress response. Western blot analysis revealed that the deletion of hfq blocked the acid-inducible expression of YfdX, while deletion of fur had no apparent effect but fur and ryhB double deletion significantly enhanced the YfdX expression at weak acid environment (pH 5). Deletion of rcsB had no effect on the expression of Hfq at either pH 5, pH 6 or pH 7, suggesting the RcsB regulation on the YfdX expression is independent of Hfq. Finally, qRT-PCR analysis further confirmed that RcsB and Hfq positively influenced the expression of yfdX, hdeB and hdeA, however, Fur and RyhB exerted different regulations on the expression of yfdX and the hde genes.