從突變基因庫找出克雷伯氏肺炎桿菌基因體中與生物膜生成相關的基因

克雷伯氏肺炎桿菌（Klebsiella pneumoniae）為革蘭氏陰性的桿菌，屬於腸內菌科（Enterobacteriacea），是一重要的伺機性感染致病菌（opportunistic pathogen），常引起泌尿道感染、菌血症及肺炎等院內感染，也會對免疫機能不全的患者造成肺炎和敗血性休克。目前已知生物膜（biofilm）的形成與發展在許多種細菌的致病過程中扮演著重要的角色，克雷伯氏肺炎桿菌會藉由形成生物膜，以增加對抗生素、抗菌劑及宿主免疫系統反應的抵抗力。之前本實驗室利用跳躍基因 (transposon) 建構成包含2500株的克雷伯氏肺炎桿菌NTUH-K2044菌株之突變株庫（mutant library），因此我們想藉由此突變株庫篩選出與生物膜形成有關的基因。挑選的方式採用生物膜微量培養盤試驗（biofilm microtiter plate assay），在篩選突變株的過程中，觀察到其中有3株突變菌株其生物膜量有明顯增加（up-regulation）：NTUH-K10-19、NTUH-K10-23、NTUH-K10-30，有3株突變菌株其生物膜量有明顯減少（down-regulation）：NTUH-K10-33、NTUH-K10-43、NTUH-K10-49。其中NTUH-K10-23為sugE基因突變株，NTUH-K10-33為clpX基因突變株，其餘突變株所破壞的基因仍未知。因為ClpX蛋白質已知在金黃色葡萄球菌（Staphylococcus aureus）與生物膜形成相關，所以本篇實驗選擇被跳躍子破壞sugE基因造成生物膜量上升的NTUH-K10-23突變株作為研究目標。用無抗生素基因標記的方式再次將剔除得NTUH-K2044ΔsugE突變菌株（unmarker deletion mutant）與其染色體互補株（chromosome complementation）證實影響生物膜生成是由於喪失sugE基因的功能，而非因為在細菌染色體上其他地方的自發性突變（spontaneous mutation）。為了進一步研究sugE基因的功能，我們選取NTUH-K2044野生菌株與NTUH-K2044ΔsugE突變菌株來進行微陣列實驗。利用兩者相較RNA表現量≧5倍的條件篩選所挑出12個RNA表現量上升的點中，大部分可歸類成與麥芽糖調節子（maltose regulon）相關的基因（malP、malF、lamB、treB）以及與碳水化合物磷酸轉移酶系統（carbohydrate phosphotransferase system, PTS）相關的基因（treB、licC、casA、celA），這些基因均會影響細菌生物膜的形成。微陣列實驗的結果，提供了ΔsugE突變菌株對於調控生物膜形成量上升的線索，但這之間的調控機制仍待進一步實驗。

關鍵字

克雷伯氏肺炎桿菌；生物膜微量培養盤試驗；生物膜量上升； sugE 基因；麥芽糖調節子；碳水化合物磷酸轉移酶系統

並列摘要

Klebsiella pneumoniae is an opportunistic pathogen associated with nosocomial infections such as urinary tract infection, bacteremia and pneumonia. The biofilm formation of bacteria has been known to involve in the increasing resistance to antibiotic, antibacterial, and host immune responses. In order to explore the genes responsible for the biofilm formation in K. pneumoniae, a NTUH-K2044 mutant transposon library was screened by biofilm microtiter plate assay. Three mutants revealed decreased biofilm formation, and three mutants revealed increased biofilm formation compared with wild-type. The interrupted gene of one mutant with increased biofilm formation was similar with sugE in Escherichia coli. Unmarked deletion and chromosomal complementation of sugE demonstrated that sugE was responsible for the biofilm formation. In order to sutudy the role of gene regulation in sugE in biofilm formation, the RNA expression profiles of ΔsugE mutant was compared with those of wild type by microarray. The expressions of twelve clones were up-regulated in ΔsugE mutant. These twelve clones contained genes involved in maltose catabolism and carbohydrate phosphotransferase system (PTS) that had been proven to associate with biofilm formation. These results suggested that sugE might affect the biofilm formation through the regulation of genes in maltose regulon and carbohydrate phosphotransferase system.

並列關鍵字

Klebsiella pneumoniae ； biofilm microtiter plate assay ； up-regulated biofilm formation ； sugE gene ； maltose regulon ； carbohydrate phosphotransferase system (PTS)

參考文獻

Barton, E. N., H. Daisley, et al. (1991). Diabetes mellitus and Klebsiella pneumoniae liver abscess in adults. Trop Geogr Med 43(1-2): 100-4.

Abranches, J., M. M. Candella, et al. (2006). Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence in Streptococcus mutans. J Bacteriol 188(11): 3748-56.

Aguilar, J., A. Cruz, et al. (1994). Multiple hepatic and pulmonary abscesses caused by Klebsiella pneumoniae. Enferm Infecc Microbiol Clin 12(5): 270-1.

Anderl, J. N., M. J. Franklin, et al. (2000). Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother 44(7): 1818-24.

Ayinala, S. R., M. Vulpe, et al. (2001). Pyogenic liver abscesses due to Klebsiella pneumoniae in a diabetic patient. J Miss State Med Assoc 42(3): 67-70.

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從突變基因庫找出克雷伯氏肺炎桿菌基因體中與生物膜生成相關的基因

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