本文選題：表皮葡萄球菌 + 生物膜　； 參考：《復旦大學》2008年博士論文

【摘要】： 表皮葡萄球菌是重要的醫(yī)院內感染病原菌,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基因克隆到質粒上,然后轉化入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基因同樣克隆到質粒上并轉化入clpP突變菌株,得到的菌株生物膜形成能力不能回復。這些結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基因轉錄水平,我們發(fā)現(xiàn)ClpP受agr細菌數(shù)量感應系統(tǒng)負調控。以往發(fā)現(xiàn)agr細菌數(shù)量感應系統(tǒng)對表皮葡萄球菌粘附高分子材料的能力具有負調控作用,我們的研究結果提示agr系統(tǒng)有可能通過調控ClpP而調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,并且對氧化應激的耐受能力下降,這些研究結果表明表皮葡萄球菌正常生長和耐受氧化應激均需要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感染的致病能力,結果顯示clpP突變菌株較不容易引起插管相關感染,這表明ClpP蛋白酶的功能有助表皮葡萄球菌引起插管相關感染。(第一部分) 已知細菌Spx蛋白是一種功能多樣的基因轉錄調控蛋白,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水平。在金黃色葡萄球菌中,Spx蛋白對生物膜形成有負調控作用。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,我們通過構建和轉化表達質粒,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合成相關基因ica操縱子轉錄水平降低、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)
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R378

【共引文獻】

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本文編號：1985189