克雷伯氏肺炎桿菌 celB 之功能及對生物膜生成與致病力之影響

Abstract

克雷伯氏肺炎桿菌（Klebsiella pneumoniae）為革蘭氏陰性桿菌，屬於腸內菌科（Enterobacteriacea），常引起泌尿道感染、呼吸道感染及菌血症等院內感染，也會對免疫機能不全的患者造成肺炎和敗血性休克，為一重要的伺機性感染病原菌（opportunistic pathogen）。生物膜（biofilm）在許多細菌的致病過程扮演重要角色，克雷伯氏肺炎桿菌亦可形成生物膜以增加對抗生素、抗菌劑及宿主免疫系統反應的抵抗力。本實驗室先前已利用跳躍基因（transposon）建構二千五百株的克雷伯氏肺炎桿菌NTUH-K2044菌株之突變株庫（mutant library），因此我們藉由生物膜微量培養盤試驗（biofilm microtiter plate assay）來篩選生物膜生成量有明顯下降（down-regulation）的突變株。過程中篩選出20株生物膜生成量有明顯減少的突變菌株，並挑選NTUH-K2044染色體DNA上之醣類磷酸轉移酶系統（carbohydrate phosphortransferase system, PTS system cellobiose-specific IIC component） celB 基因被嵌入跳躍基因之突變株NTUH-K12-16進行探討。本實驗採用無抗生素基因標記的方式剔除基因，將上述基因的剔除株（unmarked deletion mutant）與其染色體互補株（chromosome complementation）進行比較，NTUH-K2044ΔcelB基因剔除菌株證實影響生物膜生成量下降是由於喪失 celB基因功能，而非染色體上其他自發性突變（spontaneous mutation）所造成。在LB培養液中，基因剔除菌株的生長速率與野生株無差異，但形成生物膜的能力下降；在50ppm纖維雙糖M9最基本培養液（cellobiose M9 minimal medium）中，基因剔除菌株的生長速率落後於野生菌株。在LB培養液與50ppm纖維雙糖M9最基本培養液的纖維雙糖的醣類磷酸轉移系統活性分析（cellobiose-specific PTS activity assay），NTUH-K2044ΔcelB基因剔除株的活性均較野生株降低。基因剔除株在小鼠模式的致病力也較野生株降低。顯示克雷伯氏肺炎桿菌的 celB 基因可利用纖維雙糖為碳源進行生長代謝，促進生物膜的形成，並提高致病力。

Klebsiella pneumoniae is a Gram-negative bacillus belonging to Enterobacteriace, which has become the predominant pathogen causing community acquired pyogenic liver abscess (PLA). The formation of biofilm benefits bacteria to colonize, and has been known to involve in the increasing resistance to antibiotic, antibacterial, and host immune responses. In order to explore the genes related to the biofilm formation in K. pneumoniae, biofilm down-regulation mutants were screened by microtiter plate assay using a NTUH-K2044 transposon mutant library. Twenty mutants revealed decreased biofilm formation compared with wild-type. One of the mutants was disrupted in celB, the putative cellobiose-specific IIC component of carbohydrate phosphotransferase system (PTS system). Unmarked deletion and chromosomal complementation of celB confirmed that celB was responsible for the biofilm formation by microtiter plate assay and slide culture. In 50ppm cellobiose minimal medium, the growth rate of NTUH-K2044ΔcelB deletion mutant showed a significant delay compared with that of wild-type. Cellobiose-specific PTS activity of deletion mutant grown in LB broth and 50ppm cellobiose minimal medium were markedly lower than that of the wild-type strain grown under the same conditions, indicating that celB is involved in cellobiose transport. Virulence of deletion mutant was also lower than wild-type strain in the murine model of intragastrical infection. Therefore, celB of K. pneumoniae contributes to biofilm formation and virulence may through the metabolism of cellobiose.