本文選題：木糖葡萄球菌 + 生物被膜　； 參考：《東北農(nóng)業(yè)大學》2017年博士論文

【摘要】：奶牛乳房炎不僅對奶牛業(yè)危害十分嚴重,而且也是導致奶牛淘汰的疾病之一,引起奶牛乳房炎的病原菌除了常見的金黃色葡萄球菌、無乳鏈球菌、大腸桿菌等以外,凝固酶陰性葡萄球菌如木糖葡萄球菌(Staphylococcus xylosus,S.xylosus)引起的感染呈上升趨勢,已成為引起奶牛乳房炎發(fā)病的新特點。當木糖葡萄球菌產(chǎn)生的多聚復合物基質(zhì)將自身包繞,黏附于無活性物體或活體表面,形成了有一定結(jié)構(gòu)的細菌群體即生物被膜(biofilm,BF)后,生物被膜內(nèi)細菌可以抵抗宿主的免疫,同時對抗菌藥物產(chǎn)生耐藥性,造成乳房炎感染難以治愈,反復發(fā)作,由此引發(fā)奶牛乳房炎的持續(xù)性感染已引起部分學者的關(guān)注,成為奶牛乳房炎防治領域研究的新熱點。因此,對木糖葡萄球菌生物被膜調(diào)控機制的研究,尋找新型藥物作用靶標,提高奶牛乳房炎的預防和治療效果,已成為國內(nèi)外科研工作者關(guān)注的焦點之一。紅霉素(erythromycin)屬于大環(huán)內(nèi)酯類藥物,不僅對革蘭氏陽性菌,如葡萄球菌、化膿性鏈球菌等有較強的抑制作用,而且減少豬鏈球菌、銅綠假單胞菌和某些表皮葡萄球菌菌株的生物被膜形成,增加白喉棒狀桿菌和某些表皮葡萄球菌菌株生物被膜形成,但不影響牙齦卟啉單胞菌和解脲棒桿菌生物被膜的形成,這提示我們,紅霉素對生物被膜的影響因細菌種屬不同而不同,而目前尚未見紅霉素對木糖葡萄球菌生物被膜形成的影響及其作用機制的研究,故本研究通過蛋白質(zhì)組學i TRAQ技術(shù),在亞抑菌濃度紅霉素干預下篩選木糖葡萄球菌生物被膜形成的差異表達蛋白,為確定紅霉素的作用蛋白靶標提供理論支撐。在分析差異表達蛋白的基礎上,進一步研究谷氨酰胺合成酶在生物被膜形成中的作用及其是否是紅霉素的作用靶標,故采用基因敲除技術(shù)獲得glnA基因缺失株,證明其不僅在生物被膜形成中發(fā)揮作用,而且是紅霉素的作用靶標,研究的主要結(jié)果如下:(1)基于結(jié)晶紫染色法、掃描電子顯微鏡和激光共聚焦顯微鏡的方法,揭示亞抑菌濃度的紅霉素對生物被膜的干預效果。1/2 MIC(0.8μg/m L)、1/4 MIC(0.4μg/mL)、1/8 MIC(0.2μg/mL)和1/16 MIC(0.1μg/mL)的紅霉素顯著減少了木糖葡萄球菌生物被膜形成和活菌數(shù),使生物被膜立體結(jié)構(gòu)完全消失。(2)基于蛋白質(zhì)組學i TRAQ技術(shù),對1/2 MIC(0.8μg/m L)紅霉素干預木糖葡萄球菌生物被膜差異蛋白質(zhì)組定量分析,揭示紅霉素可能作用的蛋白靶標。根據(jù)差異蛋白選擇標準即ratio2.0 or0.5(p-value0.05),篩選并鑒定出24個差異表達蛋白,其中4個(16.7%)蛋白上調(diào)表達,20個(83.3%)蛋白下調(diào)表達。在上述差異表達蛋白中,如Preprotein translocase subunit Sec G(A0A060MLT7),Fmt B protein(A0A068E3T6),Gluconate permease(A0A068E1W8),Poly-gamma-glutamate synthase subunit Pgs B/Cap B(A0A068E9C1),Sodium/proline symporter(A0A068E9F7),Oligopeptide transport system permease protein Opp B(A0A060MF11)and Sodium/alanine symporter family protein(A0A068E6R7)均屬于膜蛋白和細胞表面蛋白,影響細菌黏附作用,因此,這些蛋白在生物被膜形成過程中發(fā)揮著重要作用。在上述差異表達蛋白中,Poly-gamma-glutamate synthase subunit Pgs B/Cap B(A0A068E9C1)參與莢膜多糖的生物合成過程,莢膜多糖的生物合成影響生物被膜的形成,故紅霉素通過影響莢膜多糖的生物合成過程從而干預木糖葡萄球菌生物被膜的形成是其發(fā)揮作用的機制之一。在上述差異表達蛋白中,Glutamine synthetase(A0A068E8L1)參與谷氨酰胺的生物合成,亞抑菌濃度的紅霉素減弱谷氨酰胺合成酶的活性,進而導致谷氨酰胺生物合成的減少。總之,紅霉素影響細菌的黏附作用和莢膜多糖的生物合成,減弱谷氨酰胺合成酶的活性導致谷氨酰胺生物合成減少。(3)基于基因敲除技術(shù)和分子對接方法,揭示谷氨酰胺合成酶不僅在生物被膜形成中發(fā)揮作用,而且是紅霉素的作用靶標。glnA基因缺失株形成生物被膜能力明顯降低,而當glnA基因缺失株添加谷氨酰胺后,生物被膜形成能力顯著性增加。紅霉素減少野生菌株生物被膜形成,減弱谷氨酰胺合成酶活性導致谷氨酰胺生物合成減少,然而紅霉素對glnA基因缺失株生物被膜形成無影響,也未能改變谷氨酰胺合成酶活性和谷氨酰胺生物合成量。分子對接結(jié)果顯示紅霉素和谷氨酰胺合成酶活性中心直接結(jié)合,導致谷氨酰胺合成酶活性下降,260位的組氨酸(His),261位的亮氨酸(Leu),346位的精氨酸(ARG),347位的苯丙氨酸(PHE)為主要結(jié)合位點。總之,谷氨酰胺合成酶在生物被膜形成中發(fā)揮重要作用,能夠與紅霉素直接結(jié)合是其作用靶標。綜上所述,紅霉素對木糖葡萄球菌生物被膜的形成具有干預作用,其對膜蛋白、細胞表面蛋白和莢膜多糖的生物合成有影響。谷氨酰胺合成酶參與谷氨酰胺的生物合成,其在生物被膜形成中發(fā)揮重要作用。紅霉素通過減弱谷氨酰胺合成酶活性,導致谷氨酰胺生物合成減少,干預生物被膜的形成,谷氨酰胺合成酶是其作用靶標。
[Abstract]:Dairy cow mastitis is not only a serious damage to the dairy industry, but also one of the diseases that lead to the elimination of dairy cows. The pathogenic bacteria causing cow mastitis are caused by coagulase negative Staphylococcus like Staphylococcus aureus (Staphylococcus xylosus, S.xylosus) in addition to common Staphylococcus aureus, Streptococcus free Streptococcus and Escherichia coli. The infection shows a rising trend and has become a new feature of the pathogenesis of dairy cow mastitis. When the polypolymer matrix produced by Staphylococcus aureus is wrapped around itself and adhered to the inactive or living surface, the bacteria in the membrane (biofilm, BF) formed a certain structure, and the bacteria in the biofilm can resist the host immunity. It is difficult to cure the infection of the mastitis, which causes the persistent infection of the dairy cow mastitis, which has aroused the attention of some scholars and has become a new hot spot in the field of dairy cow mastitis prevention and control. Therefore, the study of the regulation mechanism of the biofilm of the raw material of Staphylococcus xyus is to search for the new drug effect. Target, improving the prevention and treatment effect of Dairy Mastitis, has become one of the focus of researchers at home and abroad. Erythromycin (erythromycin) belongs to macrolide drugs. It not only has strong inhibition effect on Gram-positive bacteria, such as Staphylococcus and Streptococcus pyogenes, but also reduces Streptococcus suis, Pseudomonas aeruginosa and some The biofilm formation of some Staphylococcus epidermidis strains, increasing the formation of Corynebacterium diphtheria and some Staphylococcus epidermidis biofilms, but does not affect the formation of Porphyromonas gingivalis and bacilli biofilm, which suggests that the effect of erythromycin to the biofilm varies with the species of bacteria and is not yet red. The effect of mycophentin on the biofilm formation of Staphylococcus xyus and its mechanism of action, therefore, this study screened the differential expression proteins formed by the I TRAQ in the subantimicrobial concentration erythromycin, and provided theoretical support for the determination of the action protein target of erythromycin. The effect of Glutamine Synthetase on the formation of biofilm and whether it is the target of erythromycin is further studied on the basis of the expression of the protein. Therefore, the gene knockout technique is used to obtain the glnA gene deletion strain, which proves that it not only plays a role in the biofilm formation but also is the target of the action of erythromycin, the main result of the study. As follows: (1) based on the method of crystal violet staining, scanning electron microscopy and laser confocal microscopy, the interference effect of erythromycin on the biofilm of subinhibitory concentration.1/2 MIC (0.8 mu g/m L), 1/4 MIC (0.4 u g/mL), 1/8 MIC (0.2 mu g/mL) and 1/16 MIC (0.1 micron) significantly reduced the formation of biofilm formation of Staphylococcus aureus The three-dimensional structure of the biofilm completely disappeared. (2) based on the proteomics I TRAQ technology, the quantitative analysis of 1/2 MIC (0.8 g/m L) erythromycin interfered with the differential proteome of Staphylococcus xylose biofilm, and revealed the possible protein target of erythromycin. According to the differential protein selection criterion, ratio2.0 or0.5 (p-value0.05), screening 24 differentially expressed proteins were identified, of which 4 (16.7%) proteins were up-regulated and 20 (83.3%) proteins were down regulated. In the above differential expression proteins, such as Preprotein translocase subunit Sec G (A0A060MLT7), Fmt B protein (A0A068E3T6), Gluconate permease E9C1), Sodium/proline symporter (A0A068E9F7), Oligopeptide transport system permease protein Opp B (A0A060MF11) belong to membrane protein and cell surface protein, which affect the adhesion of bacteria. Therefore, these proteins play an important role in the process of biofilm formation. In the above differential expression protein, Poly-gamma-glutamate synthase subunit Pgs B/Cap B (A0A068E9C1) participates in the biosynthesis process of capsular polysaccharide, and the biosynthesis of capsular polysaccharide affects the formation of biofilm, so erythromycin interferes with the formation of the biofilm formation by affecting the biosynthesis process of capsule polysaccharide. Glutamine synthetase (A0A068E8L1) participates in the biosynthesis of glutamine in the above differential expression proteins. Erythromycin at subinhibitory concentration weakens the activity of glutamine synthetase and leads to the reduction of glutamine biosynthesis. In a word, erythromycin affects the adhesion of bacteria and the polysaccharide of the capsule. Biosynthesis, reducing the activity of glutamine synthetase leads to the decrease of glutamine biosynthesis. (3) based on gene knockout technique and molecular docking method, it is revealed that glutamine synthetase not only plays a role in the formation of biofilm, but also the ability of erythromycin target.GlnA gene deletion strain to form biological membrane. When the glnA gene deletion plant added glutamine, the biofilm formation ability increased significantly. Erythromycin reduced the biofilm formation of the wild strain, weakened the activity of glutamine synthetase and reduced the glutamine biosynthesis. However, erythromycin had no effect on the formation of the biofilm of the glnA gene deletion strain and failed to change the glutamine synthesis. Enzyme activity and glutamine biosynthesis. Molecular docking results show that the activity of erythromycin and glutamine synthetase is directly combined, resulting in a decrease in the activity of glutamine synthetase, 260 - bit histidine (His), 261 - bit leucine (Leu), 346 - bit arginine (ARG), and 347 - phenylalanine (PHE) as the main binding site. Amine synthetase plays an important role in the formation of biofilm, which is the target of direct binding with erythromycin. To sum up, erythromycin has an effect on the formation of biofilm of Staphylococcus xyleus, which affects the biosynthesis of membrane protein, cell surface protein and capsule polysaccharide. Glutamine synthetase is involved in glutamyl The biosynthesis of amines plays an important role in the formation of biofilm. By reducing the activity of glutamine synthetase, it leads to the reduction of the biosynthesis of glutamine and interfering with the formation of the biofilm, and the glutamine synthetase is the target of its action.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.61

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