以共純化方式分析內膜蛋白 XpsF 與 XpsE ATPase 或與另一內膜蛋白 XpsL 間的相互作用關係

Abstract

十字花科黑腐病菌 ( Xanthomonas campestris ) 屬於格蘭氏陰性菌，為農作物上常見的病原菌，藉由其第二型分泌系統可將各種水解酵素分泌至胞外，進而破壞並感染宿主細胞。在第二型分泌系統中，XpsE 為一具有 ATPase 活性的可溶性蛋白，推測 XpsE 可藉由與內胞膜蛋白 XpsL 相互作用將水解 ATP 的能量轉換為分泌所需的動能。XpsF 也是一個內胞膜蛋白，但具有三段穿膜序列，以 N-端起始於胞內，C-端結束於胞質週緣區的形式，在胞內形成兩個較大的區域。過去發現當菌株缺少 XpsF 蛋白即無法行使分泌功能，顯示 XpsF 為第二型分泌系統必要的組成蛋白。先前利用免疫共沉澱的分析方式，發現在同時大量表現 XpsF、XpsE、XpsL、XpsM、XpsC 的 xps 缺損株中，可以觀察到 XpsF、XpsE、XpsL 三者的相互作用關係 然而在具有分泌能力的互補菌株 (將 xpsL-strep 送入 xpsL 缺。損株中) 所進行的共純化實驗中， XpsF、XpsE、XpsL 三者的相互作用關係則減弱許多，需添加 DSP (crosslinker) 才可被觀察到，推測具備正常分泌功能的菌株中，XpsF、XpsE、XpsL 三者為動態的短暫性結合。為了進一步探討 XpsF 與 XpsE或 XpsL 之間的相互作用 本文利用共純化實驗進行分析 將 C-端具有 His-tag 的，全長 XpsFh 蛋白送入 XC1722 (xpsF 基因刪除株)，或是將共表現 XpsE 突變蛋白與 XpsFh 的質體送入 XC1736 (xpsE, xpsF 基因刪除株)，發現在野生型 XpsE或是 XpsEK331M、XpsEK331M, R504A、XpsER286A、XpsEV11A, L25A, L39A 參與下皆無法觀察到 XpsF 與 XpsL 或 XpsE 及其突變蛋白的相互作用關係，暗示這些由 XpsE突變蛋白所模擬的 XpsE 不同運作步驟，並非 XpsF 與 XpsL 或 XpsE 相互作用的最佳狀態。本研究採用的 XpsE 與 XpsFh 共表現質體，有助於未來測試其它XpsE 突變蛋白是否促進 XpsF 與第二型分泌系統中其它組成蛋白的相互作用關係。

Xanthomonas campestris is a common agricultural pathogen. Being a Gram-negative bacterium, X. campestris utilizes the type II secretion system to secret a number of hydrolytic enzymes outside the cell in its attack and infection of plant cells.Of all components in the X. campestris type II secretion system, XpsE is the only soluble protein exhibiting ATPase activity. It was hypothesized that the energy generated from ATP hydrolysis by XpsE is transmitted for secretion by its interaction with a cytoplasmic membrane protein XpsL. In addition to XpsL, XpsF is also a cytoplasmic membrane protein , however with three transmembrane sequences. It is constituted of two main cytoplasmic domains with its N-terminus facing cytoplasm and C-terminus facing periplasm. It was previously shown that the xpsF gene deficiency causes protein secretion defective, suggesting that the XpsF protein is an essential component of the type II secretion system. As shown in previous coimmune precipitation experiment, interaction between XpsF with XpsE and XpsL was observed by simultaneous overexpression of XpsF with XpsE, XpsL, XpsM and XpsC in a mutant stain XC17433 that is deleted in the xps gene cluster. However, when assayed in a secretion-proficient strain, association between XpsL with XpsF or XpsE appeared very weak, as suggested by the observation that a crosslinker DSP is required for copurification of XpsF or XpsE with XpsL-Strep. It was presumed that the association among XpsF, XpsE and XpsL may be transient in a secretion-proficient strain. In order to improve our understanding of interactive relationship among XpsF, XpsE and XpsL, copurification of XpsE or XpsL with the xpsFh gene was introduced into the xpsF-deficient strain XC1722. Alternatively, a plasmid co-expressing XpsFh and XpsE or its variants was introduced into the mutant strain XC1736 that is doubly deficient in xpsE and xpsF. The results indicated that none of the XpsE mutants included here, i.e. XpsEK331M, XpsEK331M ,R504A,XpsER286A and XpsEV11A, L25A,L39A,enhanced copurification of XpsE or XpsL with XpsFh. The implication is that none of the secretion steps blocked by these mutations in the xpsE gene is favorable for detecting stable association of XpsE or XpsL with XpsF. However, the coexpression plasmid employed in this study may be useful for future test of other xpsE gene mutations in enhancing of XpsF with other protein components of the type II secretion system.