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ClpP蛋白酶在表皮葡萄球菌生物膜形成中的作用

發(fā)布時間:2018-06-06 04:50

  本文選題:表皮葡萄球菌 + 生物膜; 參考:《復(fù)旦大學(xué)》2008年博士論文


【摘要】: 表皮葡萄球菌是重要的醫(yī)院內(nèi)感染病原菌,它引起的感染主要是在一些植入人體的醫(yī)療器械表面形成生物膜,并造成持續(xù)性感染。以往報道ClpP蛋白酶在細菌耐受應(yīng)激環(huán)境、致病和形成生物膜等方面發(fā)揮作用,為了研究ClpP蛋白酶在表皮葡萄球菌中的功能,我們構(gòu)建了一株表皮葡萄球菌clpP突變菌株。與野生菌株相比,clpP突變菌株生物膜形成能力顯著下降。我們將表皮葡萄球菌clpP基因克隆到質(zhì)粒上,然后轉(zhuǎn)化入clpP突變菌株,得到的基因互補菌株生物膜形成能力回復(fù)到野生菌株的水平。我們將表皮葡萄球菌clpP基因點突變,使它編碼的蛋白失去肽酶的功能,然后將突變的clpP基因同樣克隆到質(zhì)粒上并轉(zhuǎn)化入clpP突變菌株,得到的菌株生物膜形成能力不能回復(fù)。這些結(jié)果表明表皮葡萄球菌生物膜形成需要ClpP蛋白酶的功能。與野生菌株相比,clpP突變菌株對高分子材料的粘附能力顯著下降,并且細胞間粘附因子PIA的合成量減少,這兩個表型變化可能是突變菌株生物膜形成能力下降的原因。通過比較表皮葡萄球菌agr突變菌株與野生菌株的clpP基因轉(zhuǎn)錄水平,我們發(fā)現(xiàn)ClpP受agr細菌數(shù)量感應(yīng)系統(tǒng)負調(diào)控。以往發(fā)現(xiàn)agr細菌數(shù)量感應(yīng)系統(tǒng)對表皮葡萄球菌粘附高分子材料的能力具有負調(diào)控作用,我們的研究結(jié)果提示agr系統(tǒng)有可能通過調(diào)控ClpP而調(diào)控表皮葡萄球菌對高分子材料的粘附。與野生菌株相比,clpP突變菌株生長減慢,并且對氧化應(yīng)激的耐受能力下降,這些研究結(jié)果表明表皮葡萄球菌正常生長和耐受氧化應(yīng)激均需要ClpP蛋白酶的功能。我們用大鼠中央靜脈插管感染模型比較clpP突變菌株與野生菌株引起插管相關(guān)感染的致病能力,結(jié)果顯示clpP突變菌株較不容易引起插管相關(guān)感染,這表明ClpP蛋白酶的功能有助表皮葡萄球菌引起插管相關(guān)感染。(第一部分) 已知細菌Spx蛋白是一種功能多樣的基因轉(zhuǎn)錄調(diào)控蛋白,在一般生理條件下受到ClpP蛋白酶的降解而維持相對較低的蛋白質(zhì)水平。在金黃色葡萄球菌中,Spx蛋白對生物膜形成有負調(diào)控作用。我們推測在表皮葡萄球菌中Spx蛋白同樣對生物膜形成有調(diào)控作用,ClpP蛋白酶可能通過降解Spx蛋白而影響表皮葡萄球菌生物膜形成。為了研究表皮葡萄球菌中Spx蛋白是否受ClpP蛋白酶降解,我們比較表皮葡萄球菌clpP突變菌株與野生菌株Spx蛋白的水平,結(jié)果發(fā)現(xiàn)Spx蛋白在clpP突變菌株中顯著增加,這驗證了表皮葡萄球菌Spx蛋白是ClpP蛋白酶的降解底物。為了研究Spx蛋白水平提高是否影響表皮葡萄球菌生物膜形成,我們通過構(gòu)建和轉(zhuǎn)化表達質(zhì)粒,在表皮葡萄球菌中過量表達Spx蛋白,然后檢測生物膜形成能力是否發(fā)生變化。與對照菌株相比,Spx過量表達的菌株生物膜形成能力顯著下降,這表明Spx蛋白負調(diào)控表皮葡萄球菌生物膜形成。深入研究發(fā)現(xiàn),Spx過量表達的菌株對高分子材料的粘附能力顯著下降、PIA合成相關(guān)基因ica操縱子轉(zhuǎn)錄水平降低、PIA的合成量減少,這些可能是Spx過量表達的菌株生物膜形成能力下降的原因。由于相比表皮葡萄球菌野生菌株,clpP突變菌株中Spx蛋白顯著增加,并且我們發(fā)現(xiàn)在表皮葡萄球菌野生菌株中過量表達Spx會抑制生物膜形成,因此論文第一部分中發(fā)現(xiàn)的clpP突變菌株生物膜形成能力下降至少部分原因是Spx蛋白堆積進而抑制生物膜的形成。我們的研究結(jié)果表明,降解Spx蛋白是ClpP影響表皮葡萄球菌生物膜形成的途徑之一。(第二部分)
[Abstract]:Staphylococcus epidermidis (Staphylococcus epidermidis) is an important pathogen of nosocomial infection. It causes infection mainly on the surface of some medical devices implanted in human body and causes persistent infection. ClpP protease has been reported to play a role in the bacteria tolerance stress environment, pathogenic and biofilm formation, in order to study the ClpP protease in the epidermis. We constructed a strain of Staphylococcus epidermidis clpP mutant strain. Compared with the wild strain, the biofilm formation ability of clpP mutant strain decreased significantly. We cloned the clpP gene of Staphylococcus epidermidis to the plasmid, then transformed into the clpP mutant strain, and the biofilm formation ability of the gene complementary strain was recovered to the strain. The level of the wild strain. We mutated the clpP gene point of Staphylococcus epidermidis, so that its encoded protein lost the function of peptidase, then the mutant clpP gene was cloned into the plasmid and transformed into the clpP mutant strain. The biofilm formation ability of the strain could not be recovered. These results suggest that the biofilm formation of Staphylococcus epidermidis needs to be formed. The function of ClpP protease. Compared with the wild strain, the adhesion ability of clpP mutant to polymer material decreased significantly, and the synthesis of intercellular adhesion factor PIA decreased. The two phenotypic changes may be the cause of the decline of the mutant strain's biofilm formation ability. By comparing the C of the agr mutant strain of Staphylococcus epidermidis and the wild strain of C LpP gene transcription level, we found that ClpP was negatively regulated by the agr bacterial quantitative induction system. It was found that the agr bacterial quantitative induction system has a negative regulatory effect on the ability of Staphylococcus epidermidis to adhere to polymer materials. Our results suggest that the agr system may regulate Staphylococcus epidermidis by regulating ClpP. Adhesion. Compared with the wild strain, the growth of clpP mutant strain slowed and the tolerance to oxidative stress decreased. These results showed that the normal growth of Staphylococcus epidermidis and tolerance to oxidative stress needed the function of ClpP protease. We used the rat central venous catheterization model to compare the clpP mutant strain with the wild strain. The results showed that the clpP mutant was less likely to cause intubation related infection, which indicates that the function of ClpP protease contributes to Staphylococcus epidermidis caused by intubation related infection. (Part I)
The known bacterial Spx protein is a functional gene transcriptional regulation protein that maintains relatively low protein levels by the degradation of ClpP protease under general physiological conditions. In Staphylococcus aureus, Spx protein has a negative regulatory effect on biofilm formation. We speculate that the Spx protein in Staphylococcus epidermidis is also a biofilm. ClpP protease may affect the formation of Staphylococcus epidermidis biofilm by degradation of Spx protein. In order to study whether Spx protein in Staphylococcus epidermidis is degraded by ClpP protease, we compare the level of Spx protein of Staphylococcus epidermidis clpP mutant strain and wild strain, and the result found that Spx protein is in clpP mutant strain. This demonstrated that the Spx protein of Staphylococcus epidermidis was the substrate for the degradation of ClpP protease. In order to study whether the level of Spx protein could affect the formation of Staphylococcus epidermidis biofilm, we overexpressed Spx protein in Staphylococcus epidermidis by constructing and transforming the expression plasmids, and then detected whether the biofilm formation ability changed. Compared with the control strain, the biofilm formation ability of Spx overexpressed strains decreased significantly, which indicated that Spx protein negatively regulates the formation of Staphylococcus epidermidis biofilm. In depth study, the adhesion ability of Spx overexpressed strains to polymer material decreased significantly, the PIA synthesis phase gene ica operon transcriptional level decreased and PIA synthesis. This may be the cause of the decline in the biofilm formation ability of Spx overexpressed strains. As compared to the wild strains of Staphylococcus epidermidis, the Spx protein in the clpP mutant strain increased significantly, and we found that excessive expression of Spx in the Wild Staphylococcus epidermidis could inhibit the formation of biofilm. Therefore, the first part of the paper found that the strain of Staphylococcus epidermidis was found. The decrease of the biofilm formation ability of clpP mutant strain is at least partly due to the accumulation of Spx protein to inhibit the formation of biofilm. Our results show that the degradation of Spx protein is one of the ways that ClpP affects the formation of Staphylococcus epidermidis biofilm. (second)
【學(xué)位授予單位】:復(fù)旦大學(xué)
【學(xué)位級別】:博士
【學(xué)位授予年份】:2008
【分類號】:R378

【共引文獻】

相關(guān)期刊論文 前5條

1 唐俊妮;周銳;王紅寧;史賢明;陳煥春;;葡萄球菌生物膜形成機制與ica之間的關(guān)系[J];微生物學(xué)通報;2008年08期

2 尚瑋;楊雅瓊;王學(xué)民;朱洪濤;;創(chuàng)傷患者凝固酶陰性葡萄球菌的分布及耐藥性分析[J];中華醫(yī)院感染學(xué)雜志;2011年05期

3 婁強;王艷歌;瞿滌;;表皮葡萄球菌雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)saeRS對相關(guān)蛋白調(diào)控的研究[J];西安交通大學(xué)學(xué)報(醫(yī)學(xué)版);2012年06期

4 王凌峰;李俊亮;;難愈性創(chuàng)面與細菌的探討[J];中華損傷與修復(fù)雜志(電子版);2012年04期

5 徐杰;莊萬強;斯海波;陳世榮;;低頻超聲聯(lián)合抗生素對表皮葡萄球菌生物膜滲透性的影響[J];中國抗生素雜志;2014年05期

相關(guān)博士學(xué)位論文 前4條

1 婁強;表皮葡萄球菌雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)SaeRS調(diào)控功能的研究[D];復(fù)旦大學(xué);2010年

2 劉倩;絲氨酸/蘇氨酸蛋白激酶Stk在表皮葡萄球菌生物膜形成中的作用及其機制研究[D];復(fù)旦大學(xué);2011年

3 陶亮;金黃色葡萄球應(yīng)答免疫球蛋白刺激并觸發(fā)聚集狀態(tài)對抗巨噬細胞吞噬作用的研究[D];中國科學(xué)技術(shù)大學(xué);2010年

4 王星;表皮葡萄球菌和分枝桿菌生物膜相關(guān)基因的鑒定和功能研究[D];復(fù)旦大學(xué);2012年

相關(guān)碩士學(xué)位論文 前4條

1 郜向娜;氨溴索對表皮葡萄球菌生物膜結(jié)構(gòu)破壞和殺菌作用的研究[D];重慶醫(yī)科大學(xué);2011年

2 董麗芬;兒童表皮葡萄球菌腦膜炎腦脊液蛋白質(zhì)組學(xué)的初步研究[D];中南大學(xué);2008年

3 范佳佳;表皮葡萄球菌新的生物膜調(diào)控因子絲氨酸、蘇氨酸蛋白激酶stk基因的功能研究[D];復(fù)旦大學(xué);2010年

4 廖欣;氨溴索對表皮葡萄球菌生物膜胞間多糖粘附素及其調(diào)控基因作用的體外研究[D];重慶醫(yī)科大學(xué);2013年

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