探討克雷伯氏肺炎桿菌NarX-NarL雙分子調控系統之功能

Abstract

本實驗室先前研究發現在分枝結核桿菌中，過度表現雙分子調控系統（TCS）的缺氧反應基因devS及devR會抑制NLRP3的活化，而克雷伯氏肺炎桿菌的narX及narL和前兩者皆有高達32%的序列相似度。已有研究指出NarX-NarL 雙分子調控系統在E. coli中可以調控硝酸鹽之呼吸作用，但此系統與細菌致病力的關係尚未受到討論。本研究目的為瞭解NarX-NarL 雙分子調控系統在克雷伯氏肺炎桿菌感染時的重要性，並探討其相關下游傳導路徑。我們以酵素結合免疫吸附分析法測量無標記基因剔除株∆narX 感染THP-1巨噬細胞時IL-1β的釋放量，結果顯示野生株及∆narX之間並無顯著差異，表示narX不太參與活化發炎小體的路徑。並且，剔除narX也不會影響生物膜的生成。然而，我們觀察到在厭氧環境的minimal agar上， ∆narX生長不論有無硝酸鹽的刺激，生長情形都相似，但視野生株的生長情形在添加硝酸鹽後有明顯好轉。基因微陣列分析結果發現narX在有氧環境下似乎對整體細胞的total RNA表現影響不大。接著，in vitro的實驗顯示∆narX對THP-1巨噬細胞的胞噬作用具更高的抵抗力，而從比色法再加上轉錄活性稍微較高的莢膜多醣基因群來推知，此現象可能是由較厚的莢膜多醣所造成。綜合上述，此研究再次證明narX能夠調節硝酸鹽呼吸作用的相關基因，且可能參與莢膜多醣的合成，影響感染時宿主胞噬作用的效力。

Klebsiella pneumoniae genes: narX and narL exhibit 32% sequence homology with hypoxia-induced two component system genes: devS and devR in Mycobacterium tuberculosis, whose over-expressions were shown to surpress NLRP3 inflammasome activation from our previous study. The molecular function of NarX-NarL two component system was known as the nitrate respiration modulator in E. coli. However, the role it plays in bacterial pathogenicity remains unknown. This study aims to understand the importance of NarX-NarL system during Klebsiella pneumoniae infection. ELISA was performed to record IL-1β release upon THP-1 macrophage infection by narX deletion mutant, ∆narX, which was constructed through unmarked deletion technique. The result showed no significant change in IL-1β release compared to the wild type strain, indicating narX played a minor role in inflammasome activation. Insignificant change in biofilm formation was also observed upon deletion of narX. However, although cell growth assay revealed that the growth of ∆narX remained the same with or without nitrate induction anaerobically, wild type strain’s growth was improved upon nitrate supplementation. DNA array analysis demonstrated that narX had less impact on the overall RNA expression profile while growing aerobically. Further in vitro experiment showed that ∆narX displayed increased resistance to phagocytosis by THP-1 macrophage. This might be explained by higher capsule polysaccharide (CPS) production as was determined with a colormeteric assay and with a mild elevation in transcriptional activity within the CPS loci. Collectively, these data confirmed the role of narX in regulating nitrate respiration genes and capsule polysaccharide production, and demonstrated its relevance to phagocytic escape during infection.