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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/80668


    Title: 應用大腸桿菌與酵母菌蛋白質體晶片系統性分析抗菌肽及抗生素作用之目標蛋白質;Systematically analyzing the protein targets of antimicrobial peptides and antibiotics by using Escherichia coli and Saccharomyces cerevisiae proteome microarrays
    Authors: 巴拉莫;SHAH, PRAMOD
    Contributors: 系統生物與生物資訊研究所
    Keywords: 蛋白質組微陣列;抗菌肽;抗生素;大腸桿菌;酵母菌;手術;Proteome microarray;Antimicrobial peptides (AMPs);Antibiotics;Escherichia coli;Saccharomyces cerevisiae;synergy
    Date: 2019-08-24
    Issue Date: 2019-09-03 14:54:13 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 抗藥性病原菌的增加淡化了抗生素的使用潛力,因此,替代性療法的發展刻不容緩。抗菌肽是先天性免疫系統重要的分子並存在於所有生物體中,近期因其抗菌活性而受到矚目,且有成為抗生素替代品的潛力。抗菌肽有著多元的效用,包括對於病原菌的選擇性、低毒性、低抗藥性和多靶向作用機制。儘管已知其影響細胞膜和細胞內分子多靶點的活性,但目前僅少數靶點被解析出來。在此研究中,高通量蛋白質體微列陣平台被用於系統性分析並廣泛地鑑定抗菌肽的蛋白質靶點,透過平行分析大腸桿菌與酵母菌蛋白質體中所受影響的生物模式。將具有抗細菌和抗真菌活性的抗菌肽,分別使用大腸桿菌蛋白質體和酵母菌蛋白質體微列陣辨識其靶點的蛋白質。其中抗菌肽(Polyphemusin-I、Sub-5、Penetratin、Histatin-5和TWF)將各別應用大腸桿菌蛋白質體微列陣做系統性地辨識其細菌的標靶蛋白質。其中分別找出109個Polyphemusin-I、92個Sub-5、118個Penetratin、93個Histatin-5和48個TWF標靶蛋白質。此外抗菌肽(Lfcin B、Sub-5、Penetratin 和Histatin-5)將各別應用酵母菌蛋白質體微列陣做系統性地辨識其真菌的標靶蛋白質。其中分別找出140個Lfcin B、137個Sub-5、128個Penetratin和123個Histatin-5標靶蛋白質。而這些被抗菌肽(Polyphemusin-I、Sub-5、Penetratin、Histatin-5和TWF)所辨識的細菌標靶蛋白質,透過生物資訊合併分析以了解其抗細菌的活性。同樣地;抗菌肽(Lfcin B、Sub-5、Penetratin 和Histatin-5)所辨識的真菌標靶蛋白質,也透過生物資訊合併分析以了解其抗真菌的活性。在上述實驗中,使用生物素-鏈親和素檢測系統作為訊號檢測的方式,透過標記Dylight(螢光物質)的鏈親和素,辨識生物素化抗菌肽所結合到蛋白質體微列陣上的標靶蛋白質。此外將標記Dylight的鏈親和素直接在蛋白質體微列陣上探測並作為陰性對照組。有趣的是當標記Dylight的鏈親和素直接在大腸桿菌蛋白質體以及酵母菌蛋白質體微列陣上探測時,分別辨識出大腸桿菌與酵母菌生物素化的蛋白質。到目前為止在大腸桿菌與酵母菌分別僅有一種與六種生物素化蛋白質被鑑定出來,而單一生物素修飾蛋白質在生物體的重要性並不清楚。因此透過標記DyLight的鏈親和素在大腸桿菌蛋白質體和酵母菌蛋白質體微陣列上探測,分別鑑定出12種生物素化大腸桿菌蛋白質和44種酵母菌生物素化蛋白質。在鏈親和素的44個標靶蛋白質中,共有30個標靶蛋白可透過標記Dylight抗生物素抗體在酵母菌蛋白質體微列陣中共同被辨識到,這也證明多種生物素化蛋白質存在於酵母菌當中。關於生物肽的靶點以及臨床使用的抗生素靶點也是所知之甚少,而在此研究中也使用一種新穎的方法,分別應用大腸桿菌蛋白質體和酵母菌蛋白質體微陣列來全面性的辨識抗生素的蛋白質靶標。在大腸桿菌蛋白體微列陣辨識已商業化的抗生素靶點,其中鑑定出93個Sulfamethoxazole、81個Trimethoprim、87個 Minocycline、65個 Streptomycin和88個Vancomycin的蛋白質靶點。將這些被鑑定出的抗生素蛋白質靶點進行生物資訊分析一併了解其抗菌活性。而酵母菌蛋白質體微列陣解析出Sulfamethoxazole總共有33個真菌蛋白質靶點。透過生物資訊學探究Sulfamethoxazole的抗真菌活性,也同時比較其抗菌和抗真菌靶點。此外從大腸桿菌蛋白質體和酵母菌蛋白質體微列陣所發現的抗菌肽標靶蛋白質,進一步探究其抗細菌和抗真菌病原體活性的機制差異,比較的結果指出相同抗菌肽或抗生素在細菌與真菌中所採用的機制完全不同。此外透過蛋白質體微列陣的分析方法,鑑定抗菌肽和抗生素的蛋白質靶點不僅提供了對抗菌肽和抗生素機制的理解,也被證明可作為探討組合協同作用機制的工具。相較於單獨使用的抑制效果總和,合併治療中觀察到更顯著優異的抑制效果被稱為組合協同作用。組合協同作用具有多種優點,如增強抗生素治療的潛力,減少單一抗生素使用的劑量從而降低其毒性、延長抗藥性的產生以及對於抗藥性病原體產生的強勁影響。關於抗生素靶點的知識有限,以至於組合協同作用的機制尚不清楚。因此鑑定抗菌肽和抗生素的整體蛋白質靶點,將更清楚地解釋組合協同作用的機制。在相同途徑中的共同富集導致在抗菌肽-抗菌肽,抗菌肽-抗生素以及抗生素-抗生素之間發現新的協同組合預測。另外合成致死方法用於研究抗菌肽真菌蛋白質靶點之間的合成致死組合。基於已發現的合成致死組合及其參與相同的蛋白質複合物和可逆功能,預測了Lfcin B和Histatin-5的組合協同作用,並且在體內實驗中透過單獨使用或組合的Lfcin B和Histatin-5,驗證了其對酵母菌的生長抑制曲線。;Abstract
    Increase in resistance pathogens have fade the potential of antibiotics. Thus, alternatives therapies are investigated to conquer the battle of resistance. Antimicrobial peptides (AMPs), the key molecules of innate immunity, are present in all organism and recently gaining attention for its antimicrobial activities as well as antibiotics’ alternative. AMPs exert wide range of activity, have selective nature for pathogenic, lower toxicity, minimal resistance development properties and multiple-targeting mechanism of actions. Despite the knowledge of its multi-targeting activities on cellular membrane and intracellular molecules, only few targets have been identified. In this study, the high-throughput platform of proteome microarrays was utilized for systematical and comprehensive identification of the entire protein targets of AMPs in parallel analysis to the entire proteome of the model organisms: Escherichia coli and Saccharomyces cerevisiae. Intracellular targeting AMPs with antibacterial and antifungal activities were probed on Escherichia coli proteome microarrays and Saccharomyces cerevisiae proteome microarrays to identify their antibacterial and antifungal protein targets, respectively. The bacterial protein targets of AMPs with antibacterial activities (Polyphemusin-I, Sub-5, Penetratin, Histatin-5 and TWF) were systematically identified by individually probing the AMPs on Escherichia coli proteome microarrays. In total of 109, 92, 118, 93 and 48 protein targets were identified for Polyphemusin-I, Sub-5, Penetratin, Histatin-5 and TWF, respectively. The fungal protein targets of AMPs with antifungal activities (Lfcin B, Sub-5, Penetratin and Histatin-5) were systematically identified by individually probing the AMPs on Saccharomyces cerevisiae proteome microarrays. In total of 140, 137, 128 and 123 protein targets were identified for Lfcin B, Sub-5, Penetratin and Histatin-5,respectively. These identified bacterial protein targets of AMPs (Polyphemusin-I, Sub-5, Penetratin, Histatin-5 and TWF) were bioinformatically analyzed together to understand their antibacterial activities. Also, the identified fungal protein targets of AMPs (Lfcin B, Sub-5, Penetratin and Histatin-5) were bioinformatically analyzed together to understand their antifungal activities. In above assays, biotin-streptavidin detection system was used for the signal detection of the biotinylated AMPs bound to the protein targets on proteome microarrays with streptavidin labeled DyLight (fluorescence). As a negative control, only streptavidin labeled DyLight was probing on proteome microarrays. Interesting by probing only streptavidin labeled DyLight on the Escherichia coli proteome microarrays as well as Saccharomyces cerevisiae proteome microarrays, the biotinylated proteins (proteins modification by biotin) of the Escherichia coli and Saccharomyces cerevisiae were identified, respectively. So far, only one biotinylated protein has been identified in Escherichia coli and six biotinylated proteins in Saccharomyces cerevisiae. The essentiality of biotin in living organism cannot be understood by single biotin modified protein. Thus, by probing streptavidin label DyLight on Escherichia coli proteome microarrays and Saccharomyces cerevisiae proteome microarrays, in total of 12 biotinylated proteins for Escherichia coli and 44 biotinylated proteins for Saccharomyces cerevisiae were identified, respectively. Among 44 protein targets of Streptavidin, 30 protein targets overlapped with the protein targets of Anti-biotin labeled Dylight on Saccharomyces cerevisiae proteome microarrays. This anti-biotin probing confirmed the presence of several biotinylated proteins in Saccharomyces cerevisiae. In regard to the targets of AMPs, the entire targets of clinically used antibiotics are also poorly understood. Thus, a novel approach was also used in this study to identify the entire protein targets of antibiotics by utilizing Escherichia coli proteome microarrays and Saccharomyces cerevisiae proteome microarrays, respectively. In total of 93, 81, 87, 65 and 88 protein targets of Sulfamethoxazole, Trimethoprim, Minocycline, Streptomycin and Vancomycin, the commercial antibiotics, were identified from Escherichia coli proteome microarrays. These identified protein targets of antibiotics were bioinformatically analyzed together to understand their antibacterial activities. Whereas, the fungal protein targets of Sulfamethoxazole obtained from Saccharomyces cerevisiae proteome microarrays probing was 33 protein targets in total. Bioinformatics were performed to explore the antifungal activities of Sulfamethoxazole as well as compared the antibacterial and antifungal targets of Sulfamethoxazole. Moreover, the identified protein targets of AMPs from Escherichia coli proteome microarrays as well as Saccharomyces cerevisiae proteome microarrays were further compared to identify the mechanistic difference in their activities against bacterial and fungal pathogens. The comparison results showed completely different mechanism of action of same AMPs and antibiotic in case of bacteria and fungi. Furthermore, the identified protein targets of AMPs and antibiotics from the proteome microarrays approach not only provided the understanding for the mechanism of AMPs and antibiotics but also proved to be a useful tool to study the mechanism of synergistic combinations. The significant higher inhibition effect observed in combination than the sum of individual inhibition is termed as synergistic combination. Synergistic combination has multiple advantages, like enhancing the potential of antibiotics, reducing the doses of individual antibiotics thus lowering their toxicity, prolong in the resistance development as well as exert powerful effect on resistance pathogens. In regard to the limited knowledge of antibiotic target, the mechanism of synergistic combination is unclear. Hence, the entire protein targets identified for AMPs and antibiotics will more clearly explain the mechanism of synergistic combination. The common enrichment in same pathways resulted in the prediction of new synergistic combinations were discovered between AMP and AMP, AMP and antibiotic as well as antibiotic and antibiotic. As well as synthetic lethality approach was used to identify the synthetic lethal pairs between the identified fungal protein targets of AMPs. Based on identified synthetic lethal pairs and their involvement in same protein complex and reversible functions, the synergistic combination was predicted between Lfcin B and Histatin-5 and was experimentally validate in vivo by inhibition growth curve of Saccharomyces cerevisiae in the presence of individual and combination of Lfcin B and Histatin-5.
    Appears in Collections:[系統生物與生物資訊研究所] 博碩士論文

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