ClpP蛋白酶在表皮葡萄球菌生物膜形成中的作用

发布时间：2018-06-06 04:50

本文选题：表皮葡萄球菌 + 生物膜　；参考：《复旦大学》2008年博士论文

【摘要】： 表皮葡萄球菌是重要的医院内感染病原菌,它引起的感染主要是在一些植入人体的医疗器械表面形成生物膜,并造成持续性感染。以往报道ClpP蛋白酶在细菌耐受应激环境、致病和形成生物膜等方面发挥作用,为了研究ClpP蛋白酶在表皮葡萄球菌中的功能,我们构建了一株表皮葡萄球菌clpP突变菌株。与野生菌株相比,clpP突变菌株生物膜形成能力显著下降。我们将表皮葡萄球菌clpP基因克隆到质粒上,然后转化入clpP突变菌株,得到的基因互补菌株生物膜形成能力回复到野生菌株的水平。我们将表皮葡萄球菌clpP基因点突变,使它编码的蛋白失去肽酶的功能,然后将突变的clpP基因同样克隆到质粒上并转化入clpP突变菌株,得到的菌株生物膜形成能力不能回复。这些结果表明表皮葡萄球菌生物膜形成需要ClpP蛋白酶的功能。与野生菌株相比,clpP突变菌株对高分子材料的粘附能力显著下降,并且细胞间粘附因子PIA的合成量减少,这两个表型变化可能是突变菌株生物膜形成能力下降的原因。通过比较表皮葡萄球菌agr突变菌株与野生菌株的clpP基因转录水平,我们发现ClpP受agr细菌数量感应系统负调控。以往发现agr细菌数量感应系统对表皮葡萄球菌粘附高分子材料的能力具有负调控作用,我们的研究结果提示agr系统有可能通过调控ClpP而调控表皮葡萄球菌对高分子材料的粘附。与野生菌株相比,clpP突变菌株生长减慢,并且对氧化应激的耐受能力下降,这些研究结果表明表皮葡萄球菌正常生长和耐受氧化应激均需要ClpP蛋白酶的功能。我们用大鼠中央静脉插管感染模型比较clpP突变菌株与野生菌株引起插管相关感染的致病能力,结果显示clpP突变菌株较不容易引起插管相关感染,这表明ClpP蛋白酶的功能有助表皮葡萄球菌引起插管相关感染。(第一部分) 已知细菌Spx蛋白是一种功能多样的基因转录调控蛋白,在一般生理条件下受到ClpP蛋白酶的降解而维持相对较低的蛋白质水平。在金黄色葡萄球菌中,Spx蛋白对生物膜形成有负调控作用。我们推测在表皮葡萄球菌中Spx蛋白同样对生物膜形成有调控作用,ClpP蛋白酶可能通过降解Spx蛋白而影响表皮葡萄球菌生物膜形成。为了研究表皮葡萄球菌中Spx蛋白是否受ClpP蛋白酶降解,我们比较表皮葡萄球菌clpP突变菌株与野生菌株Spx蛋白的水平,结果发现Spx蛋白在clpP突变菌株中显著增加,这验证了表皮葡萄球菌Spx蛋白是ClpP蛋白酶的降解底物。为了研究Spx蛋白水平提高是否影响表皮葡萄球菌生物膜形成,我们通过构建和转化表达质粒,在表皮葡萄球菌中过量表达Spx蛋白,然后检测生物膜形成能力是否发生变化。与对照菌株相比,Spx过量表达的菌株生物膜形成能力显著下降,这表明Spx蛋白负调控表皮葡萄球菌生物膜形成。深入研究发现,Spx过量表达的菌株对高分子材料的粘附能力显著下降、PIA合成相关基因ica操纵子转录水平降低、PIA的合成量减少,这些可能是Spx过量表达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突变菌株中Spx蛋白显著增加,并且我们发现在表皮葡萄球菌野生菌株中过量表达Spx会抑制生物膜形成,因此论文第一部分中发现的clpP突变菌株生物膜形成能力下降至少部分原因是Spx蛋白堆积进而抑制生物膜的形成。我们的研究结果表明,降解Spx蛋白是ClpP影响表皮葡萄球菌生物膜形成的途径之一。(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)
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R378

【共引文献】