十字花科黑腐病菌第二型分泌機制：XpsE 蛋白N端區域的特性分析

Abstract

十字花科黑腐病菌 (Xanthomonas campestris pv. campestris) 利用第二型分泌機制，分泌各種水解酵素藉以破壞植物細胞的表面構造。此分泌路徑中參與成員共有12個蛋白組成，負責將各種水解酵素自胞質週緣區 (periplasm) 送至胞外。XpsE 蛋白是成員中唯一的非膜蛋白，此外序列中含有Walk A box，因此被推論具有ATP水解酵素 (ATPase) 或激活酵素 (kinase) 活性，可能在分泌過程中扮演能源提供者或訊號發送者的角色。Vibrio cholerae 的EpsE蛋白研究結果顯示此蛋白可能藉其N-端區域和嵌在內膜的EpsL蛋白交互作用。利用胰蛋白酶 (trypsin) 有限地切割全長XpsE蛋白，先前的研究發現全長XpsE位於第153個胺基酸附近有一個對胰蛋白酶敏感的位置，暗示全長XpsE 蛋白可能以此位置被分割為兩個獨立區域 (domains)。爲檢驗全長XpsE以第153個胺基酸為界分割為兩個獨立區域的推論，本研究首先利用一廣寄主載體pBBRMCS-5殖入全長 XpsE蛋白N端1-155胺基酸 (XpsEN) 與全長XpsE蛋白C端153-567胺基酸 (XpsEC) 的基因片段，送入X. campestris pv. campestris野生型菌株，分析此XpsEN或XpsEC蛋白是否呈現dominant negative 的現象，結果顯示只有XpsEN蛋白會明顯抑制α-amylase的分泌，而此抑制現象可藉額外送入正常的全長XpsE蛋白而恢復正常。進一步分析全長XpsE和XpsEN在分泌受阻細胞中的分佈，發現兩個蛋白主要都位於細胞質中。利用鎳離子親和性管柱分析結果證實全長XpsE與具有N端His6-tag的XpsEN (hXpsEN) 間有直接結合的關係。純化的hXpsEN蛋白經過交叉連結劑處裡會形成雙聚體以及更大的分子，而XpsEN蛋白晶體結構也呈現雙聚體。為檢驗XpsEN形成雙聚體的生理意義，將其相互作用區域中保留性極高的Gly63突變為alanine或valine，並分析它們在野生菌株中抑制分泌的能力，結果發現以valine取代glycine的突變蛋白抑制能力稍有減弱，暗示XpsE的N端區域間相互作用可能具有功能上的重要性。

There are at least 12 Xps protein components constituting the type II secretion apparatus, which is utilized by Xanthomonas campestris pv. campestris for secreting extracellular proteins across the outer membrane. Among them, the XpsE is the only protein without any membrane spanning sequence. It was postulated to provide energy or signal for the secretion process by acting as an ATPase or a kinase for its possession of the indispensable nucleotide-binding motifs. The N-terminal domain of the Vibrio cholerae EpsE protein was demonstrated to mediate its association with the cytoplasmic membrane protein EpsL. Limited trypsin treatment of XpsE produced a 50 kDa fragment beginning at the 153rd residue, implicating a two-domain model for XpsE. In contrast to the C-terminal domain, the sequence of the N-terminal domain of XpsE does not show significant similarity with those of its homologues. To find out if indeed the XpsE is composed of two domains, I analyzed in this study the in vivo effect caused by overexpressing the N-terminal or the C-terminal domain of XpsE as XpsEN or XpsEC, respectively, in the wild type strain of Xanthomonas campestris pv. campestris. Severe interference in α-amylase secretion was caused only by XpsEN. Moreover, the interference was restored by simultaneous overexpression of the full-lengh XpsE. Subcellular fractionation revealed that the overexpressed XpsEN was present largely in the cytoplasm. Direct interaction between N-terminally His6-tagged XpsEN (hXpsEN) and XpsE was demonstrated by their co-elution upon nickel affinity chromatography. Crosslinking treatment of purified hXpsEN caused appearance of dimer and oligomers of larger sizes. Its crystal structure also appears as dimer. In order to examine the physiological significance of XpsEN dimerization, I took genetic approach by analyzing mutated XpsEN for the loss of interfering ability. The Gly63, a highly conserved residue located at the dimer interface, was mutated to alanine or valine. The latter produced a mutant causing slightly less severe interference in the wild type strain than the unaltered XpsEN, suggesting that the interaction between the N-terminal domain of the full-length XpsE probably has some functional significance.