十字花科黑腐病菌中類纖毛蛋白XpsG、H、I、J 和 K間交互作用關係的探討

Study of the interactions between XpsG, H, I, J and K pseudopilins in Xanthomonas campestris pv. campestris

官孝勳(Hsiao-Hsun Kuan)

指導教授 ： 陳凌雲

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00059

摘要

十字花科黑腐病菌為格蘭氏陰性菌的一種，會利用第二型分泌系統分泌蛋白而感染宿主。分泌的蛋白會先藉由Sec-dependent系統到達膜間質，再由一群12~14個蛋白所組成的分泌機器將分泌蛋白運送出細胞外。其中XpsG、H、I、J和K蛋白NH2-端與第四型纖毛生成系統中的纖毛組成蛋白有同源性，推測可在內外膜之間組裝成類纖毛構造，並推動分泌蛋白進入外膜通道。過去的研究發現XpsG蛋白是類纖毛的主要組成，且橫跨於內外膜之間，另外，XpsH可調節XpsG與XpsJ之間的交互作用，並推測類纖毛蛋白的排列順序為XpsG-XpsI-XpsH-XpsJ。由於XpsK抗體已製備完成，因此本實驗藉由鎳離子親和性管柱探討五個類纖毛蛋白XpsG、H、I、J和K在各個缺損株中的交互作用情形。結果發現XpsI可以抑制XpsH與XpsJ彼此間的交互作用，說明了XpsI可能扮演一個會與XpsH競爭XpsJ的負調控角色。另外，發現XpsK可促進XpsI與XpsJ彼此間的交互作用，說明XpsK可能位於XpsI與XpsJ之間而扮演一個正調控的角色。同時也發現XpsH、XpsI與XpsK可能以兩兩結合(XpsH-XpsI、XpsH-XpsK和XpsI-XpsK)的形式出現。綜合以上的結果推測類纖毛的生合成遠比想像中還要複雜，需要更多實驗加以釐清。

關鍵字

十字花科黑腐病菌 ； 第二型分泌系統 ； 類纖毛蛋白

並列摘要

Xanthomonas campestris pv. Campestris is one of the Gram-negtive bacteria which secretes protein to infect plants by typeⅡ secretion system. The secreted protein can reach to periplasm by the Sec-dependent system, and to export from cellular matrix by a machine composed of 12~14 proteins. The N terminal of XpsG, H, I , J and K protein are homologous with the pilin of typeⅣ secretion system, those are predicted to be assembled into a pilus-like structure between the cytoplasmic and outer membrane. The pseudopilus is presumed to push exproteins into the outer membrane. On previous studys showed that XpsG was a mainly composed of pseudopilus to span between inner and outer membrane, and could regulate the interaction between XpsG and XpsJ, and thought that XpsG, I, H and J should act sequentially in such order. We have prepared specific antibody against XpsK that be used to analyze the interaction between XpsG、H、I、J and K in various bacterial strains with different xps gene knockout after Ni2+ affinity chromatography. Result revealed that XpsI could inhibit the interaction between XpsH and XpsJ, and act as a negative regulator. XpsK could promote the interaction between XpsI and XpsJ, act as a positive regulator. Take together, we believed that XpsG and XpsJ are located at a extremity of pseudopilin complex , and there are three interaction forms between XpsH, I and K , including XpsH-XpsI, XpsH-XpsK and XpsI-XpsK. We believe that the copmposed of the pseudopilus is a complicated mechanism, and need more experiments to explain any possibility.

並列關鍵字

Xanthomonas campestris pv. Campestris ； typeⅡ secretion system ； pseudopilus

參考文獻

Bally, M., Filloux, A., Akrim, M., Ball, G., Lazdunski, A., and Tommassen, J. (1992). Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol 6, 1121-1131.

Bitter, W. (2003). Secretins of Pseudomonas aeruginosa: large holes in the outer membrane. Arch Microbiol 179, 307-314.

Bitter, W., Koster, M., Latijnhouwers, M., de Cock, H., and Tommassen, J. (1998). Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 27, 209-219.

Bleves, S., Lazdunski, A., and Filloux, A. (1996). Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa. J Bacteriol 178, 4297-4300.

Bleves, S., Voulhoux, R., Michel, G., Lazdunski, A., Tommassen, J., and Filloux, A. (1998). The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family). Mol Microbiol 27, 31-40.