YfdX在克雷白氏肺炎桿菌CG43抗酸反應中所扮演的角色

Abstract

已知雙分子調控系統RcsB正向調控克雷白氏肺炎桿菌的抗酸反應。rcsB基因的剔除使細菌於低酸鹼值(pH3)之生存率顯著的降低。蛋白質體的研究發現，經過弱酸的適應後rcsB基因剔除使特定蛋白質表現量顯著降低，而後此蛋白質被鑑定為YfdX。位於克雷白氏肺炎桿菌酸適應島嶼的yfdX基因經剔除後，細菌於低酸鹼值的生存率顯著降低。在靜態培養以及弱酸的條件下，剔除rcsB基因或kvhA基因的菌株觀察到PyfdX表現量的降低。本研究中，我們製作ΔrcsBΔyfdX 以及 ΔkvhAΔyfdX之雙突變菌株。抗酸存活率試驗指出ΔkvhAΔyfdX 以及 ΔyfdX 反應相似，然而ΔrcsBΔyfdX 的存活率則相似於ΔrcsB 突變株。顯示酸適應島嶼之雙分子調控系統KvhA在靜態培養條件下的抗酸反應主要透過YfdX表現。目前尚未有研究指出yfdX基因的功能，而使用生物資訊的工具(BLAST, pfam)搜尋指出YfdX具有訊息胜肽但並無其他保守性的序列。酸適應島嶼的伴隨蛋白基因hdeB亦具有訊號序列，且hdeB-hdeD相鄰於yfdX而同樣受到KvhA以及RcsB的調控，於是假設yfdX與hdeB-hdeD在抗酸功能上具有關聯性。我們再進一步製作ΔhdeBΔhdeDΔyfdX以及ΔyfdXΔhdeB1突變菌株，結果顯示ΔhdeBΔhdeD與ΔhdeBΔhdeDΔyfdX有近似的酸存活率;ΔhdeB1基因不具備訊息胜肽ΔyfdX而在抗酸功能未具相關性。接下來，我們建構YfdX表現載體pET30a-yfdX，測試YfdX蛋白質之功能或用於YfdX抗體之製作。體外的伴隨蛋白活性試驗指出His6-YfdX降低ADH的沉澱，His6-YfdX相較於His6-HdeB具有較高的體外伴隨蛋白質活性，但YfdX與HdeB體外的伴隨蛋白活性並沒有明顯的加乘效果。抗體製作完成後，西方點墨法的測試顯示YfdX表現於膜間質，並受靜置培養及弱酸的影響增加表現量。最後，免疫共沉澱的實驗指出在弱酸環境下YfdX能與多數膜間質蛋白質作用，而在中性時與膜間質蛋白質的結合能力較低。以上體外的試驗指出，YfdX在酸性環境下具備伴隨蛋白的活性，保護蛋白質發生沉澱。

The two component system (2CS) response regulator (RR) RscB has been previously shown to play a positive role in the acid-resistance response in Klebsiella pneumoniae CG43. Deletion of rcsB apparently reduced the bacterial survival under pH 3 culture condition. After mild acid adaptation, the proteome study revealed a missing protein spot in the rcsB deletion mutant which later identified as YfdX. Deletion of the yfdX, located on a putative acid fitness island (AFI), appeared to reduce the bacterial survival at pH 3. In statically or mild acidic cultured condition, a reduced level of PyfdX promoter activity in the rcsB deletion and kvhA deletion mutant was also observed. Here, ΔrcsBΔyfdX and ΔkvhAΔyfdX mutants were generated. Acid survival analysis revealed that ΔkvhAΔyfdX and ΔyfdX had similar response to pH 3 treatment, while ΔrcsBΔyfdX revealed same survival level as ΔrcsB mutant. These suggested that KvhA, a 2CS RR encoding gene on the AFI, also plays a regulatory role of acid stress primarily on YfdX expression under statically cultured condition. Furthermore, A signal peptide has been found on YfdX by BLAST but no conserved domain on YfdX protein. We proposed that there are some functional relationships between yfdX and the nearby gene hdeB-hdeD of acid resistance. Therefore, ΔhdeBΔhdeDΔyfdX and ΔyfdXΔhdeB1 mutants were generated and confirmed the correlation between yfdX and hdeB-hdeD under acid stress. Thus, Recombinant YfdX protein has been generated for testing chaperone activity and raising antibodies for YfdX localization detection. Western blotting analysis further confirmed rcsB and kvhA controlling the periplasmic expression of YfdX under acidic and static culture condition. in vitro acid chaperone activity assay revealed the potent protective effects on ADH of YfdX then HdeB. Finally, co-immunoprecipitation test indicated that YfdX can interact with periplasmic proteins under acidic condition.