基于谷氨酰胺合成酶靶标探讨红霉素干预木糖葡萄球菌生物被膜形成的机制

发布时间：2018-07-04 07:51

本文选题：木糖葡萄球菌 + 生物被膜　；参考：《东北农业大学》2017年博士论文

【摘要】：奶牛乳房炎不仅对奶牛业危害十分严重,而且也是导致奶牛淘汰的疾病之一,引起奶牛乳房炎的病原菌除了常见的金黄色葡萄球菌、无乳链球菌、大肠杆菌等以外,凝固酶阴性葡萄球菌如木糖葡萄球菌(Staphylococcus xylosus,S.xylosus)引起的感染呈上升趋势,已成为引起奶牛乳房炎发病的新特点。当木糖葡萄球菌产生的多聚复合物基质将自身包绕,黏附于无活性物体或活体表面,形成了有一定结构的细菌群体即生物被膜(biofilm,BF)后,生物被膜内细菌可以抵抗宿主的免疫,同时对抗菌药物产生耐药性,造成乳房炎感染难以治愈,反复发作,由此引发奶牛乳房炎的持续性感染已引起部分学者的关注,成为奶牛乳房炎防治领域研究的新热点。因此,对木糖葡萄球菌生物被膜调控机制的研究,寻找新型药物作用靶标,提高奶牛乳房炎的预防和治疗效果,已成为国内外科研工作者关注的焦点之一。红霉素(erythromycin)属于大环内酯类药物,不仅对革兰氏阳性菌,如葡萄球菌、化脓性链球菌等有较强的抑制作用,而且减少猪链球菌、铜绿假单胞菌和某些表皮葡萄球菌菌株的生物被膜形成,增加白喉棒状杆菌和某些表皮葡萄球菌菌株生物被膜形成,但不影响牙龈卟啉单胞菌和解脲棒杆菌生物被膜的形成,这提示我们,红霉素对生物被膜的影响因细菌种属不同而不同,而目前尚未见红霉素对木糖葡萄球菌生物被膜形成的影响及其作用机制的研究,故本研究通过蛋白质组学i TRAQ技术,在亚抑菌浓度红霉素干预下筛选木糖葡萄球菌生物被膜形成的差异表达蛋白,为确定红霉素的作用蛋白靶标提供理论支撑。在分析差异表达蛋白的基础上,进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红霉素的作用靶标,故采用基因敲除技术获得glnA基因缺失株,证明其不仅在生物被膜形成中发挥作用,而且是红霉素的作用靶标,研究的主要结果如下:(1)基于结晶紫染色法、扫描电子显微镜和激光共聚焦显微镜的方法,揭示亚抑菌浓度的红霉素对生物被膜的干预效果。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)的红霉素显著减少了木糖葡萄球菌生物被膜形成和活菌数,使生物被膜立体结构完全消失。(2)基于蛋白质组学i TRAQ技术,对1/2 MIC(0.8μg/m L)红霉素干预木糖葡萄球菌生物被膜差异蛋白质组定量分析,揭示红霉素可能作用的蛋白靶标。根据差异蛋白选择标准即ratio2.0 or0.5(p-value0.05),筛选并鉴定出24个差异表达蛋白,其中4个(16.7%)蛋白上调表达,20个(83.3%)蛋白下调表达。在上述差异表达蛋白中,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)均属于膜蛋白和细胞表面蛋白,影响细菌黏附作用,因此,这些蛋白在生物被膜形成过程中发挥着重要作用。在上述差异表达蛋白中,Poly-gamma-glutamate synthase subunit Pgs B/Cap B(A0A068E9C1)参与荚膜多糖的生物合成过程,荚膜多糖的生物合成影响生物被膜的形成,故红霉素通过影响荚膜多糖的生物合成过程从而干预木糖葡萄球菌生物被膜的形成是其发挥作用的机制之一。在上述差异表达蛋白中,Glutamine synthetase(A0A068E8L1)参与谷氨酰胺的生物合成,亚抑菌浓度的红霉素减弱谷氨酰胺合成酶的活性,进而导致谷氨酰胺生物合成的减少。总之,红霉素影响细菌的黏附作用和荚膜多糖的生物合成,减弱谷氨酰胺合成酶的活性导致谷氨酰胺生物合成减少。(3)基于基因敲除技术和分子对接方法,揭示谷氨酰胺合成酶不仅在生物被膜形成中发挥作用,而且是红霉素的作用靶标。glnA基因缺失株形成生物被膜能力明显降低,而当glnA基因缺失株添加谷氨酰胺后,生物被膜形成能力显著性增加。红霉素减少野生菌株生物被膜形成,减弱谷氨酰胺合成酶活性导致谷氨酰胺生物合成减少,然而红霉素对glnA基因缺失株生物被膜形成无影响,也未能改变谷氨酰胺合成酶活性和谷氨酰胺生物合成量。分子对接结果显示红霉素和谷氨酰胺合成酶活性中心直接结合,导致谷氨酰胺合成酶活性下降,260位的组氨酸(His),261位的亮氨酸(Leu),346位的精氨酸(ARG),347位的苯丙氨酸(PHE)为主要结合位点。总之,谷氨酰胺合成酶在生物被膜形成中发挥重要作用,能够与红霉素直接结合是其作用靶标。综上所述,红霉素对木糖葡萄球菌生物被膜的形成具有干预作用,其对膜蛋白、细胞表面蛋白和荚膜多糖的生物合成有影响。谷氨酰胺合成酶参与谷氨酰胺的生物合成,其在生物被膜形成中发挥重要作用。红霉素通过减弱谷氨酰胺合成酶活性,导致谷氨酰胺生物合成减少,干预生物被膜的形成,谷氨酰胺合成酶是其作用靶标。
[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.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.61

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