利用大腸桿菌蛋白體微陣列晶片系統性探討抗菌?的胞內作用目標; Systematic Analysis of Intracellular Targeting Antimicrobial Peptides Using E. coli Proteome Microarray

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Title:	利用大腸桿菌蛋白體微陣列晶片系統性探討抗菌?的胞內作用目標;Systematic Analysis of Intracellular Targeting Antimicrobial Peptides Using E. coli Proteome Microarray
Authors:	何宇軒;Ho,Yu-Hsuan
Contributors:	系統生物與生物資訊研究所
Keywords:	蛋白質體晶片;抗菌?;生物資訊;proteome microarray;antimicrobial peptide;bioinformatics
Date:	2013-07-01
Issue Date:	2013-08-22 11:45:51 (UTC+8)
Publisher:	國立中央大學
Abstract:	抗藥性在近年來已受到越來越多的關注，因為缺乏新穎的抗生素，一些平常的感染在未來或許都會對人類造成致命的威脅。因此，對新型抗生素的需求是極為迫切的。天然的抗菌?是一種演化上非常重要的防禦系統，他們保護了多細胞生物免於微生物的侵襲。目前比較為人所知的抗菌?抗菌機制主要是透過破壞微生物細胞膜的完整性以達到殺死微生物的目的，然而，有一部分的抗菌?已被證實會穿過微生物的細胞膜，並且進一步影響微生物的胞內生理作用。而這些有胞內作用的抗菌?一旦進入細胞內就會影響不只一個細菌蛋白質的作用，因此，他們可能有非常複雜的胞內抗菌機制。而這項複雜的特性也使得這群抗菌?成為一個新的發展新型抗生素的來源，因為對細菌來說，要對這樣複雜的機制產生抗藥性也是相對的比較困難。目前，已經有一些具有胞內活性的抗菌?已經被辨識出來，但是他們的胞內作用機制仍然是未知的，像是Bac 7, Lactoferricin B (Lfcin B), P-Der及PR-39。為了瞭解這些抗菌?的胞內作用機制，我們先使用大腸桿菌蛋白體晶片去找出Lfcin B可能的作用目標蛋白。晶片結果顯示，Lfcin B對於兩個蛋白質有很強的親和力(basR and creB)，而這兩個蛋白質皆是屬於雙因子調控系統的反應子(response regulator of two component system)。為了進一步的分析，我們使用了一些實驗及生物資訊的分析去釐清Lfcin B與這兩個蛋白質間的關係。蛋白質凝膠電泳與激?實驗顯示Lfcin B影響了這兩個蛋白質的磷酸化能力。抗菌實驗顯示Lfcin B減低了大腸桿菌對特定環境的反應能力，像是在充滿鐵離子與微酸的環境以及探來源受到限制的環境下。為了更系統性的分析有胞內活性的抗菌?，我們也針對Bac 7, P-Der 及PR-39與晶片進行反應，同時，也加入之前已分析過的Lfcin B的晶片資料。首先，我們先分析各個抗菌?獨特的蛋白質目標，此結果顯示。Bac 7主要影響DNA的合成藉由攻擊purine代謝的功能。Lfcin B主要攻擊轉錄以及碳水化合物代謝的相關的功能。P-Der 主要影響細胞內的小分子代謝作用。PR-39攻擊許多跟RNA相關的細胞功能及合成。此外，細胞途徑的分析也發現Bac 7 及Lfcin B共同攻擊purine的代謝路徑，Lfcin B及 PR-39共同攻擊細胞內的脂多糖的合成路徑。更進一步，我們分析了四個抗菌?共同的目標蛋白，發現他們共同的作用目標為精氨酸脫梭酵素，因此，我們用抗菌實驗進一步去驗證，發現這四種抗菌?的確都有非常好的抑菌效果當細菌需要使用精氨酸脫梭酵素來當作存活的手段時。我們的發現使得對於這些胞內作用的抗菌?有了更多更深入的了解，也提供了後續多研究的方向。 It is a wide acceptance that antibiotics resistance now has become a global issue among the world. According to a recent report conducted by World Health Organization (WHO), the multidrug-resistant tuberculosis alone causes more than 150,000 deaths each year. Because of the lack of conventional antibiotics, normal infections could also become lethal. It’s an urgent need to develop novel strategies against the antibiotics resistance strains. The natural antimicrobial peptide (AMP) is an evolutionary important defense system for multicellular organisms to protect them from the invasions of the microbes. Although the well-known mechanism for AMP to kill the microbes is via the membrane permeabilization, some AMPs still have the ability to interrupt the intracellular cellular functions of microbes. The intracellular targeting AMPs may influence more than one protein inside the cells thus, the intracellular targeting AMPs have multiple modes of actions to inhibit the microbes. The complex mechanisms of intracellular targeting AMPs makes them become attractive resource for developing novel strategies against microbes because for microorganisms, it’s hard for them to exhibit the antimicrobial resistance to such complicated process. Yet several intracellular targeting AMPs have been identified, some of them still have the unknown target and function such as Bactenecin 7, Lactoferricin B, a hybrid of Pleurocidin and Dermaseptin (P-Der) and a proline-arginine-rich antibacterial peptide (PR-39). In addition, there is no systematic analysis to study the mechanisms of intracellular targeting AMPs. To elucidate the intracellular behavior of Lactoferricin B (Lfcin B), we first used E. coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system (TCS). For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli’s tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of TCS directly. To identify the protein targets of 3 intracellular active antimicrobial peptides, Bac 7, P-Der and PR-39, we used the E. coli proteome microarray to identify the hits for each AMP. In addition, to provide systematic level analysis, we also included the data of Lactoferricin B (Lfcin B) from our previous studies to give a more comprehensive analysis of 4 intracellular targeting antimicrobial peptides using several bioinformatics methods. First, we analyzed the unique protein hits of each AMP. Our results indicate that Bac 7 mainly target in the DNA synthesis via influencing the purine metabolism. Lfcin B mainly attacks the transcriptional and cellular carbohydrate metabolism related functions. P-Der affects several catabolic processes of small molecules. PR-39 shows strong preference to recognize the proteins that involved in RNA related cellular processes. In addition, the KEGG analysis of unique hits of each AMP indicates that the synergistic effects may appear among these 4 peptides. Bac 7 and Lfcin B may target on the same pathway, purine metabolism while, Lfcin B and PR-39 both target on Lipopolysaccharide biosynthesis. Furthermore, we analyzed the common hits of 4 AMPs and this result indicates that these 4 AMPs all target on the arginine decarboxylase. To validate this finding, the antimicrobial assay was conducted. The 4 AMPs all leaded to a significant growth inhibition of bacteria under the extreme acidic environment (pH < 3) compared to a membrane active peptide, Cecropin P1.
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