本文選題：金黃色葡萄球菌　切入點(diǎn)：eLtaS　出處：《廣西醫(yī)科大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：金黃色葡萄球菌(Staphylococcus aureus)是一種重要的革蘭氏陽(yáng)性致病菌,其能夠引起廣泛的感染并引發(fā)多種疾病如中毒性休克、肺炎、皮膚病到嚴(yán)重的心內(nèi)膜炎、敗血癥等,此外還可以引起骨髓炎、尿路感染等疾病。目前臨床上抗金葡菌感染主要通過(guò)聯(lián)合應(yīng)用抗生素的方法,但是由于金葡菌抗藥性問(wèn)題不能解決,致使許多抗生素變得無(wú)能為力。 脂磷壁酸(Lipoteichoic acid, LTA)是革蘭氏陽(yáng)性細(xì)菌細(xì)胞壁所特有的、具有高度免疫原性的聚合物,由核糖醇磷酸鹽和/或甘油磷酸鹽與細(xì)胞膜脂質(zhì)共價(jià)結(jié)合形成,其為兩性分子,一端可以與細(xì)胞膜脂質(zhì)共價(jià)結(jié)合,另一端可以穿過(guò)細(xì)胞壁延伸在細(xì)胞外。在金葡菌中,脂磷壁酸的缺失會(huì)導(dǎo)致細(xì)菌停止生長(zhǎng),細(xì)胞形態(tài)發(fā)生嚴(yán)重變化,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蛋白是金葡菌在生長(zhǎng)過(guò)程中表達(dá)的跨膜蛋白,全長(zhǎng)646個(gè)氨基酸,相對(duì)分子質(zhì)量大小為74.4×103Da,在金葡菌的各菌株中高度保守。已有研究表明,在金葡菌生長(zhǎng)過(guò)程中,LtaS蛋白胞外217～218位的丙氨酸在細(xì)胞外多肽水解酶spsB作用下水解并形成細(xì)胞外蛋白eLtaS (Extracellular Lipoteichoic acid Synthase)。同時(shí),人們發(fā)現(xiàn)雖然LtaS蛋白合成LTA的活性結(jié)構(gòu)域主要位于其胞外eLtaS的氨基酸殘基中,但是體外研究表明,eLtaS蛋白沒(méi)有合成LTA的生物學(xué)功能。因此,受到水解游離在細(xì)胞外的eLtaS蛋白其生物學(xué)功能目前尚不明確。 本研究的第一部分為金葡菌中一種新的補(bǔ)體相互作用蛋白的發(fā)現(xiàn)。我們前期研究中構(gòu)建了金葡菌RNaseⅢ突變菌株(△RNaseⅢ-8325),并發(fā)現(xiàn)△RNaseⅢ-8325生長(zhǎng)過(guò)程中的分泌上清對(duì)補(bǔ)體激活有明顯的抑制作用。在此基礎(chǔ)上,我們將突變株上清與野生型菌株8525-4上清中的總蛋白進(jìn)行雙向電泳分析比較,并將△RNaseⅢ-8325突變株上清中高表達(dá)的蛋白進(jìn)行了質(zhì)譜鑒定,結(jié)果發(fā)現(xiàn)有兩種蛋白在△RNaseⅢ-8325表達(dá)水平明顯升高,分別為脂磷壁酸合成酶LtaS蛋白的細(xì)胞外部分eLtaS蛋白和糖肽水解酶LtyM。通過(guò)基因工程重組表達(dá),我們發(fā)現(xiàn)只有eLtaS能夠有效的抑制補(bǔ)體介導(dǎo)的紅細(xì)胞裂解。我們利用基因同源重組方法構(gòu)建了ltaS的缺失突變菌株(△ltaS-8325),發(fā)現(xiàn)ltaS突變株生長(zhǎng)較野生型菌株緩慢,將eLtaS蛋白與突變株共培養(yǎng)后對(duì)突變株的生長(zhǎng)緩慢現(xiàn)象沒(méi)有回復(fù)作用,這也表明eLtaS蛋白在培養(yǎng)上清中不具有合成金葡菌脂磷壁酸(LTA)的功能,同時(shí)我們發(fā)現(xiàn)突變株在外毒素水平、全血存活率方面都與野生型發(fā)生了明顯變化。 本文的第二部分內(nèi)容為eLtaS抑制補(bǔ)體激活的機(jī)制研究。具體內(nèi)容如下： 1. eLtaS抑制補(bǔ)體激活的機(jī)制。在這部分實(shí)驗(yàn)中,我們利用CH50和ELISA實(shí)驗(yàn)以及進(jìn)一步的C5a釋放檢測(cè)實(shí)驗(yàn)證明了eLtaS是通過(guò)與補(bǔ)體C3片段C3d的結(jié)合導(dǎo)致C5轉(zhuǎn)化酶失活,進(jìn)而抑制補(bǔ)體的激活。 2. eLtaS與補(bǔ)體C3d相互作用的功能位點(diǎn)的確定。我們利用生物信息學(xué)方法模擬了eLtaS與補(bǔ)體C3d的相互作用并預(yù)測(cè)了其結(jié)合位點(diǎn),然后體外重組表達(dá)了相應(yīng)的突變體蛋白,并在此基礎(chǔ)上通過(guò)實(shí)驗(yàn)學(xué)方法確認(rèn)了二者相互作用的位點(diǎn)。 3. eLtaS蛋白與金葡菌致病性的關(guān)聯(lián)性研究。通過(guò)構(gòu)建的肺炎、腹膜炎動(dòng)物模型,我們發(fā)現(xiàn)eLtaS蛋白可以加劇金葡菌8325-4對(duì)野生型小鼠肺部的感染以及提高腹膜炎對(duì)動(dòng)物的致死率,但是在C3缺陷性的小鼠模型上,eLtaS則沒(méi)有明顯的加重金葡菌引發(fā)的感染。同時(shí)我們發(fā)現(xiàn)eLtaS蛋白可以抑制中性粒細(xì)胞對(duì)細(xì)菌的吞噬殺傷作用。 綜上所述,我們的實(shí)驗(yàn)結(jié)果首先發(fā)現(xiàn)LtaS蛋白細(xì)胞外部分eLtaS蛋白能夠特異結(jié)合補(bǔ)體系統(tǒng)的C3d,抑制三條補(bǔ)體通路。進(jìn)一步的實(shí)驗(yàn)結(jié)果表明其能夠加重金葡菌引發(fā)的感染。我們的研究結(jié)果表明LtaS蛋白的雙功能性,即：其全長(zhǎng)形式的LtaS蛋白通過(guò)發(fā)揮酶學(xué)作用,促進(jìn)LTA的合成。在多肽水解酶作用下生成的eLtaS蛋白不能夠促進(jìn)LTA的合成,但是其能夠通過(guò)與補(bǔ)體系統(tǒng)相互作用,參與金葡菌的致病過(guò)程。我們的研究為抗金葡菌感染研究提供了新的理論基礎(chǔ),同時(shí)eLtaS也將有可能成為新抗金葡菌感染的藥物靶點(diǎn)。
[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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R378

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本文編號(hào)：1630816