金黄色葡萄球菌eLtaS蛋白的生物学功能研究

发布时间：2018-03-18 18:38

本文选题：金黄色葡萄球菌　切入点：eLtaS　出处：《广西医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：金黄色葡萄球菌(Staphylococcus aureus)是一种重要的革兰氏阳性致病菌,其能够引起广泛的感染并引发多种疾病如中毒性休克、肺炎、皮肤病到严重的心内膜炎、败血症等,此外还可以引起骨髓炎、尿路感染等疾病。目前临床上抗金葡菌感染主要通过联合应用抗生素的方法,但是由于金葡菌抗药性问题不能解决,致使许多抗生素变得无能为力。脂磷壁酸(Lipoteichoic acid, LTA)是革兰氏阳性细菌细胞壁所特有的、具有高度免疫原性的聚合物,由核糖醇磷酸盐和/或甘油磷酸盐与细胞膜脂质共价结合形成,其为两性分子,一端可以与细胞膜脂质共价结合,另一端可以穿过细胞壁延伸在细胞外。在金葡菌中,脂磷壁酸的缺失会导致细菌停止生长,细胞形态发生严重变化,因此降低了金葡菌的感染能力以及致病性。目前已知脂磷壁酸合成酶LtaS (Lipoteichoic acid Synthase)蛋白在LTA的合成中起主要作用。LtaS蛋白是金葡菌在生长过程中表达的跨膜蛋白,全长646个氨基酸,相对分子质量大小为74.4×103Da,在金葡菌的各菌株中高度保守。已有研究表明,在金葡菌生长过程中,LtaS蛋白胞外217～218位的丙氨酸在细胞外多肽水解酶spsB作用下水解并形成细胞外蛋白eLtaS (Extracellular Lipoteichoic acid Synthase)。同时,人们发现虽然LtaS蛋白合成LTA的活性结构域主要位于其胞外eLtaS的氨基酸残基中,但是体外研究表明,eLtaS蛋白没有合成LTA的生物学功能。因此,受到水解游离在细胞外的eLtaS蛋白其生物学功能目前尚不明确。本研究的第一部分为金葡菌中一种新的补体相互作用蛋白的发现。我们前期研究中构建了金葡菌RNaseⅢ突变菌株(△RNaseⅢ-8325),并发现△RNaseⅢ-8325生长过程中的分泌上清对补体激活有明显的抑制作用。在此基础上,我们将突变株上清与野生型菌株8525-4上清中的总蛋白进行双向电泳分析比较,并将△RNaseⅢ-8325突变株上清中高表达的蛋白进行了质谱鉴定,结果发现有两种蛋白在△RNaseⅢ-8325表达水平明显升高,分别为脂磷壁酸合成酶LtaS蛋白的细胞外部分eLtaS蛋白和糖肽水解酶LtyM。通过基因工程重组表达,我们发现只有eLtaS能够有效的抑制补体介导的红细胞裂解。我们利用基因同源重组方法构建了ltaS的缺失突变菌株(△ltaS-8325),发现ltaS突变株生长较野生型菌株缓慢,将eLtaS蛋白与突变株共培养后对突变株的生长缓慢现象没有回复作用,这也表明eLtaS蛋白在培养上清中不具有合成金葡菌脂磷壁酸(LTA)的功能,同时我们发现突变株在外毒素水平、全血存活率方面都与野生型发生了明显变化。本文的第二部分内容为eLtaS抑制补体激活的机制研究。具体内容如下： 1. eLtaS抑制补体激活的机制。在这部分实验中,我们利用CH50和ELISA实验以及进一步的C5a释放检测实验证明了eLtaS是通过与补体C3片段C3d的结合导致C5转化酶失活,进而抑制补体的激活。 2. eLtaS与补体C3d相互作用的功能位点的确定。我们利用生物信息学方法模拟了eLtaS与补体C3d的相互作用并预测了其结合位点,然后体外重组表达了相应的突变体蛋白,并在此基础上通过实验学方法确认了二者相互作用的位点。 3. eLtaS蛋白与金葡菌致病性的关联性研究。通过构建的肺炎、腹膜炎动物模型,我们发现eLtaS蛋白可以加剧金葡菌8325-4对野生型小鼠肺部的感染以及提高腹膜炎对动物的致死率,但是在C3缺陷性的小鼠模型上,eLtaS则没有明显的加重金葡菌引发的感染。同时我们发现eLtaS蛋白可以抑制中性粒细胞对细菌的吞噬杀伤作用。综上所述,我们的实验结果首先发现LtaS蛋白细胞外部分eLtaS蛋白能够特异结合补体系统的C3d,抑制三条补体通路。进一步的实验结果表明其能够加重金葡菌引发的感染。我们的研究结果表明LtaS蛋白的双功能性,即：其全长形式的LtaS蛋白通过发挥酶学作用,促进LTA的合成。在多肽水解酶作用下生成的eLtaS蛋白不能够促进LTA的合成,但是其能够通过与补体系统相互作用,参与金葡菌的致病过程。我们的研究为抗金葡菌感染研究提供了新的理论基础,同时eLtaS也将有可能成为新抗金葡菌感染的药物靶点。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is a kind of important gram positive pathogens, which can cause widespread infection and cause a variety of diseases such as septic shock, pneumonia, severe skin disease, septicemia, endocarditis, also can cause osteomyelitis, urinary tract infections and other diseases. The current clinical anti Staphylococcus aureus methods through the combined application of antibiotics due to infection, but not Staphylococcus aureus resistant problem solving, causing many antibiotics become incapable of action.
Lipoteichoic acid (Lipoteichoic, acid, LTA) is unique to gram positive bacterial cell wall, a polymer having a highly immunogenic, formed by ribitol phosphate and / or glycerol phosphate and cell membrane lipid covalent binding of the amphiphilic molecules, end can be combined with cell membrane lipid covalent, the other end can be extended in outside the cell through the cell wall. In Staphylococcus aureus, lack of lipoteichoic acid will cause bacteria to stop the growth of cell morphogenesis serious changes, so as to reduce the infection of Staphylococcus aureus and pathogenicity. Currently known lipoteichoic acid synthase LtaS (Lipoteichoic acid Synthase) protein in the synthesis of LTA major the role of.LtaS protein expression of Staphylococcus aureus in the growth process of the transmembrane protein of 646 amino acids, the molecular weight is 74.4 * 103Da, highly conserved in all strains of Staphylococcus aureus. Studies have shown that, In the process of growth of Staphylococcus aureus, 217~218 alanine LtaS protein extracellular polypeptide in the extracellular hydrolase spsB hydrolyzed and the formation of extracellular protein eLtaS (Extracellular Lipoteichoic acid Synthase). At the same time, it was found that although amino acid residues in the active domain of LtaS protein synthesis of LTA is mainly located in the extracellular eLtaS, but in vitro studies showed that eLtaS protein had no biological function of synthesis of LTA. Therefore, by the hydrolysis of free extracellular eLtaS protein in its biological function remains unclear.
The first part of this study is a new complement interacting protein of Staphylococcus aureus found in our previous studies. Construct S.aureus RNase III mutant (RNase III -8325), and found that the supernatant from RNase III -8325 in the process of growth has obvious inhibiting effect on the activation of complement. Based on the comparative analysis of two-dimensional electrophoresis of total protein we mutant and wild type strain 8525-4 was in the supernatant, and RNase III -8325 mutation strains showed high expression in the supernatant proteins were identified by mass spectrometry, the results found that two kinds of protein increased significantly in RNase III -8325 expression levels were Lipoteinchoic acid synthase LtaS protein extracellular eLtaS protein and peptide hydrolase LtyM. by recombinant expression, we found that only eLtaS can inhibit the red blood cell lysis effective at complement mediated gene. We use the same source The method of constructing ltaS recombinant mutant strain (ltaS-8325), found that the ltaS mutant growth than the wild type strain will slow, eLtaS protein and mutant after co culture of mutant slow growth phenomenon did not respond, it also shows that the eLtaS protein in culture supernatant with synthetic staphylococcal lipoteichoic acid (LTA) function, at the same time we found that the mutant in exotoxin level, the survival rate of all aspects of the whole blood and the wild type has changed significantly.
The second part of this paper is the study of the mechanism of eLtaS inhibition of complement activation. The following is the following:
1., eLtaS inhibits the mechanism of complement activation. In this experiment, we used CH50 and ELISA experiments and further C5a release test to prove that eLtaS is inactivated by C5 binding enzyme combined with complement C3 fragment C3d, thereby inhibiting the activation of complement.
To determine the functional sites of 2. eLtaS and complement C3d interaction. We use biological information to simulate the interaction of eLtaS and complement C3d methodology and the binding sites, then the corresponding recombinant mutant protein, and on the basis of the experimental study confirmed the interaction between the two sites.
Association study of pathogenicity of 3. eLtaS protein and Staphylococcus aureus. By constructing pneumonia, peritonitis animal model, we found that eLtaS protein can aggravate the infection of Staphylococcus aureus in wild type mice lungs of 8325-4 and increase the mortality rate for animal peritonitis, but in a mouse model of C3 deficiency on eLtaS is not obvious exacerbation of Staphylococcus aureus causing infections. We also found that eLtaS protein can inhibit neutrophil phagocytosis in vitro.
In summary, our results first discovered LtaS protein extracellular eLtaS protein could bind specifically to the complement system C3d, inhibition of three complement pathway. Further experimental results show that it can increase the gold aureus infection. Our results show that the double function of LtaS protein, namely: the full-length form of LtaS protein by playing the role of enzyme, promote the synthesis of LTA. Under the action of the peptide hydrolase generated in eLtaS protein can promote the synthesis of LTA, but it can interact with the complement system, in the process of pathogenic Staphylococcus aureus. Our study provides a new theoretical foundation for the study of anti Staphylococcus aureus infection at the same time, eLtaS it will be possible to become a new target of drug resistant Staphylococcus aureus infection.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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