病原菌大腸桿菌O157的biofilm形成及抗生素抗性之機制

Mechanism of biofilm formation and antibiotics resistance in pathogenic E. coli O157

劉學鴻(Hsua-Hon Liu)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02411

摘要

之前我們已發現大腸桿菌DH5a以某些抗生素處理會形成biofilm，由蛋白體學二維膠片的分析發現，某些蛋白質會有消長。 在此論文我們使用大腸桿菌的重要病原菌O157從事實驗。 在枯草桿菌素及kanamycin的處理都可見到biofilm的形成。 我們又改進二維膠片的分析技術，明確的從膠片上發現有表現量不同的蛋白質並施以質譜分析，蛋白包括TnaA, PspA, MenB, TnaA, Aco2, EFG,及PspA在被枯草桿菌素處理的細胞表現較多，而CH10, EftS, TalB, UidR, PhojH, PtnA, AroB在被kanamycin處理的細胞表現較多。 其中TnaA: Tryptophanase可分解Tryptophan成Indole，而大量(>250 uM)分泌到細胞表面可刺激細菌biofilm形成。 其調控的分子機制也被研究。
除了Indole，在枯草桿菌素處理過的大腸桿菌可分泌一些lipid態費洛蒙到胞外造成biofilm形成。 為了發現費洛蒙刺激細胞形成biofilm的機制，我收集及純化這些少量費洛蒙，並使用純化的費洛蒙來刺激經跳躍子基因破壞的突變株，視其對費洛蒙的反應，發現B3355, YdgR, YaaA, BglG, B0359。這一群跳躍子基因破壞的突變株不能形成biofilm，我們認為這些突變株被改變之基因與lipid態費洛蒙胞外造成biofilm形成的機制有關，這可為抑制biofilm形成有用的藥物標的。

關鍵字

病原菌大腸桿菌 ； 生物膜 ； 蛋白質體學

並列摘要

In previous studies, we found that adding some antibiotics to E. coli DH5a induced its biofilm formation. The 2-D gel electrophoresis showed that some proteins were expressed more and some less upon the treatment. In the present study, bacitracin and kanamycin were used to treat the pathogenic E. coli O157. We first improved the technology of running 2-D gel and clearly identified the proteins with different expression level. The proteins including TnaA, PspA, MenB, TnaA, Aco2, EFG, and PspA were expressed more in the cytoplasm of E. coli O157 upon incubation with bacitracin. Proteins CH10, EftS, TalB, UidR, PhojH, PtnA, and AroB were expressed more upon kanamycin treatment. Among them, TnaA (tryptophanase) catalyzes the formation of indole from Tryptophane and indole can secret into the medium to reach a critical concentration (250 uM), thereby triggering the biofilm formation. Its regulatory mechanism was then investigated.
Besides indole, a small quantity of an unknown autoinducer was released to the medium while the E. coli O157 was treated with bacitracin. In the present study, we treated the mutants of transposon gene knockout with the partially purified autoinducer to examine the response of the mutant bacteria. We discovered B3355, YdgR, YaaA, BglG, and B0359 that could not form biofilm. This allowed the identification of important proteins involved in the autoinducer signaling and biofilm formation, which may serve as anti-biofilm drug targets.

並列關鍵字

pathogenic E. coli ； biofilm ； proteomics

參考文獻

[1] Walsh, C. (2000) Molecular mechanisms that confer antibacterial drug resistance. Nature 406, 775-781.

[2] Spratt, B. G., and Cromic, K. D. (1988) Penicillin-binding proteins of gram-negative bacteria. Rev. Infect. Dis. 10, 699-711.

[3] Williams, D. H. (1996) The glycopeptide story-how to kill the deadly “superbugs”. Natl. Prod. Rep. 13, 469-477.

[4] Brisson-Noel, A., Tricu, Cuot,P., and Courvalin, P. (1988) Mechanism and action of spiramycin and other macrolides. J. Abntimicrob. Chemother. 22 (Suppl. B), 13-23.

[5] Chopra, I. (1985) in the tetracyclins, Handbook of Experimental Pharmacology 78, (eds Hlavaka, J. J. & Boothe, J. H.) 317-392.