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標題: 利用X-ray繞射研究黑腐病菌 Xanthomonas campestris 與廣泛抗藥性病菌 Stenotrophomonas maltophilia 的多重抗藥操作子所表達的蛋白結構
Structural Studies of Proteins Implicated in the Multiple Antibiotic Resistance Regulons in the Xanthomonas Campestris and Stenotrophomonas Maltophilia Pathogens Using X-Ray Diffraction Methodology
作者: 周三和
關鍵字: 生物技術, 藥學;基礎研究;Stenotrophomonas maltophilia;抗藥性病菌;蛋白結構;黑腐病菌;X-ray繞射;Xanthomonas campestris;Multiple Antibiotic Resistance;X-ray Diffraction
植物病原菌十字花科黑腐病菌Xanthomonas campestris為一格蘭式陰性病原菌,為一頗俱學術性及應用性的菌種。本實驗室在過去三年已積極從事此一病原菌的結構基因體研究,以期對此一病原菌做一更全面性的瞭解。在研究此一病原菌的過程中,本實驗室發現了一與目前極重要的廣泛細菌抗藥性有關的操作子(mar operon)。它包含MarR (Multiple Antibiotic Resistance Repressor),MarA (transcription activator),及MarC蛋白 (可能為一運送抗生素到胞外的內膜蛋白) 。我們已得到解析度良好 (1.7 Å) 的MarR蛋白結晶及其與抗生素Salicylate的共結晶 (見圖一) 。在此一計畫中我們將利用X-ray繞射法決定單一MarR蛋白,其與抗生素Salicylate複合体,及其與它的啟動子(promoter)複合体的結構。同時也將表達MarA蛋白及尋找其結晶條件。由於MarA蛋白為活化此一操作子的重要蛋白,所以將是一個極重要的藥物標的。MarC蛋白則因其為膜蛋白的緣故,結晶條件較難掌握,但也值得一試,因為它也將是一個重要的新型藥物標的。它的抑制物將可避免細菌將各類型抗生物排出胞外,達到殺菌的目的。而另一與Xanthomonas campestris基因序列極為相近的Stenotrophomonas maltophilia (之前稱為 Xanthomonas maltophilia) 則為一廣泛存在之格蘭氏陰性菌。在醫學上,此菌卻可造成嚴重的院內感染。由於臨床上分離到的 Stenotrophomonas maltophilia (S. maltophilia) 菌株大多具有多重抗藥性,對於免疫抑制或身体抵抗力差的病人常造成致命的嚴重後果,所以在治療上相當棘手。有趣的是Stenotrophomonas maltophilia也可作用於植物上,抑制植物黴菌的繁殖,並促進植物的生長。它也可去除土壤之污染,用來復育受污染的土地,所以是一多功能的超級菌種。它的命名1983年以前為Xanthomonas maltophila,直到1997年時才被改為Stenotrophomonas maltophilia。它的基因體序列與植物病原菌十字花科黑腐病菌Xanthomonas campestris極為相近,兩者的16S rRNA有96.9%的相似度,16S-23S spacer片段有83.8%的相似度。本團隊先前已建立Xcc的基因定序及有效的蛋白載体体系,供蛋白質高度表達、純化、功能及結構分析的工作。鑑於目前細菌抗藥性的嚴重性,本計劃除了解析目前十字花科黑腐病菌的mar regulon有關蛋白複合体的結構外,將進一步拓展到此一人體病原菌的研究。X-ray晶体繞射技術將是主要的技術,此乃因它沒有分子量大小的限制,同時也非常適合複合体結構的解析。希望藉由Xcc所建立的研究体系,進一步研究S. maltophila與mar有關的多重抗藥性蛋白結構,並比較其與Xcc蛋白結構的異同。目前S. maltophilia的基因序列也已由英國Sanger Institute基因体中心完成定序,總共4,851,126 bp ( S_maltophilia/),與Xcc相當。我們將利用生物資訊的方法,建立S. maltophilia的基因庫資料 (此一部份將由國家基因体生物資訊核心設施主持人清大生科系呂平江教授負責),並找出類似Xcc mar regulon的基因,以解析其結構。此一計畫相信對嚴重的細菌多重抗藥性問題有所助益,並希望能藉此建立一堅強的結構生物學基礎以進一步探討解決細菌抗藥性的新方法。

The plant pathogen Xanthomonas campestris is a Gram-negative bacterium that is of considerable importance both in academic and in applied science. Due to its great impact in these areas, we have in the past three years engaged in studying its structural genomics using NMR technology to gain a better understanding of this phytopathogen. During these studies, we have found an important mar regulon in Xcc, which includes MarR (multiple antibiotics resistance repressor), MarA (a transcription activator), and MarC (a putative inner membrane protein for transporting antibiotics outside). We have successfully expressed and purified the MarR protein in an amount sufficient for its crystallization studies. High quality crystals for this protein and its salicylate complex have been obtained. We will try to determine the structures of free MarR, MarR/salicylate complex, and MarR/DNA complex using X-ray crystallography in this project. We will also express and crystallize the MarA protein, which will be an important drug target due to its important role in activating this operon. We will also try to express and crystallize the MarC protein, which is harder to do due to its membrane-bound feature but is worthy of trying. Furthermore, we are also going to investigate a similar MarR system in Stenotrophomonas maltophili (named Xanthomonas maltophilia before), a ubiquitous, aerobic, non-fermentative, Gram-negative bacterium closely related to the Xanthomonas species. It has been reported as an opportunistic pathogen, responsible for serious infectious complications in immunocompromised patients due to its multiple antibiotics resistance. It is therefore rather difficult to deal with such an infection. Interestingly, all Stenotrophomonas strains can inhibit the growth of fungus rooted in plants and therefore influence the plant growth. It can also be used to remove soil contamination and to regenerate contaminated ground. Its genome is quite similar with Xanthomonas campestris, bearing a 97% similarity in 16S rRNA or 84% similarity in 16S-23S spacer fragments. Since we have already set up a high-throughput protein expression and purification facility for Xanthomonas campestris, we therefore plan to extend the mar regulon study to Stenotrophomonas maltophili due to the contemporary importance of drug resistance issue. These studies would allow us to gain a better understanding of this important issue. X-ray crystallography will be the prime technology used for these studies since it has no molecular weight limit and is very suitable for complex structural studies. The whole genome sequence of Stenotrophomonas maltophili has been decoded by the Sanger Institute in England successfully and is freely available for downloading ( Projects/S_maltophilia/). It consists of 4,851,126 bp and is comparable in size to that of Xanthomonas campestris. The director of the Bioinformatics core facility of the National Genomics Medicine Program, Dr. Lyu will use bioinformatics method to set up a Stenotrophomonas maltophili database first to locate the mar regulon genes, which will then be cloned, expressed, purified, crystallized, and its structure solved using X-ray crystallography. Its complex with salicylate and with promoter DNA will also be investigated. We believe such studies will be of considerable value toward the multiple-antibiotic-resistance problem occurring contemporarily
其他識別: DOH94-TD-G-111-041
Appears in Collections:生物化學研究所

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