表皮葡萄球菌sarZ基因及hln基因对其生物膜形成及毒力的影响

发布时间：2018-03-21 05:14

本文选题：表皮葡萄球菌　切入点：生物膜　出处：《复旦大学》2008年硕士论文　论文类型：学位论文

【摘要】： 表皮葡萄球菌是引起院内感染的主要致病菌之一,其致病性主要为在植入性医疗器材表面形成生物膜。生物膜是细菌黏附于有机或无机材料表面,合成分泌细胞外基质包被细菌所形成的细菌群体结构。生物膜的形成是细菌逃逸宿主免疫清除的主要机制,也是细菌体内播散感染的病灶。因此,对于表皮葡萄球菌生物膜形成分子机制的深入研究,将有助于提供防治表皮葡萄球菌感染的有效措施。我们通过观察生物膜表型的改变从生物膜阳性的表皮葡萄球菌标准菌株S.epidermidis1457的Tn917转座子随机插入突变库中筛选出三株生物膜阴性的突变菌株,经鉴定Tn917在各突变株中插入位点均为同一基因,sarZ。比较sarZ突变株与野生株S.epidermidis1457,发现sarZ突变株在体外生物膜形成能力降低,在生物膜形成的初始黏附阶段形成生物膜减少,在第二阶段合成细胞间黏附素(PIA)减少,在大鼠中央静脉感染模型和小鼠皮下置管感染模型中的致病性均降低。基因互补使突变株恢复野生株生物膜表型,说明在生物膜形成的两个阶段,sarZ基因促进生物膜的形成。利用基因芯片方法研究sarZ突变株基因表达谱,发现sarZ突变株中79个基因的表达水平较野生株S.epidermidis1457发生改变,包括转录水平下调的催化合成PIA的icaADBC和ica调控基因icaR及转录水平上调的毒力相关基因—溶血素基因hln和有机过氧化物抗性基因ohr,并且该结果经定量Real Time PCR证实。经表型分析,我们也的确观察到sarZ突变株耐受有机过氧化物的能力较野生株降低和较野生株明显的溶血。sarZ突变株及过表达hln基因菌株溶血活性较野生株S.epidermidis1457升高,说明Hln在表皮葡萄球菌中具有溶血功能并且其表达水平受SarZ调控。有趣的是,我们还观察到过表达hln基因菌株形成生物膜减少,初始黏附能力降低,说明hln基因在表皮葡萄球菌中还具有抑制生物膜形成的功能。因此,我们利用家蚕感染模型比较过表达hln菌株与野生株,但是尚未观察到两者有明显的毒力差异。本研究揭示表皮葡萄球菌sarZ基因是其生物膜形成及毒力的重要的调控基因,并且可能通过不依赖IcaR的方式控制ica基因的表达,提示表皮葡萄球菌生物膜形成中存在新的以SarZ为调控蛋白的调控通路。我们也发现了表皮葡萄球菌一个新的溶血素Hln,菌株过表达hln抑制其生物膜形成,并且在对其研究中第一次在表皮葡萄球菌中使用了家蚕感染模型比较细菌毒力。另外,本文中对sarZ和hln基因的研究提示它们可能作为潜在的药物靶标用于防治表皮葡萄球菌感染。
[Abstract]:Staphylococcus epidermidis is one of the main pathogens causing nosocomial infection. The pathogenicity of Staphylococcus epidermidis is the formation of biofilm on the surface of implanted medical equipment. The formation of biofilm is the main mechanism of immune clearance of bacterial escape host, and is also the focus of bacterial disseminated infection in vivo. Further study on the molecular mechanism of biofilm formation in Staphylococcus epidermidis. By observing the phenotypic changes of biofilm, we screened the Tn917 transposon of S. epidermidis1457, a standard biofilm positive strain, into a random mutation library. Three biofilm-negative mutant strains, The insertion site of Tn917 in each mutant was identified as the same gene sarZ.Compared with the wild strain S.epidermidis1457, it was found that the ability of sarZ mutant to form biofilm in vitro was decreased, and the biofilm formation decreased during the initial adhesion stage of biofilm formation. In the second stage, the synthesis of intercellular adhesion (PIAA) was reduced, and the pathogenicity of PIAs was decreased in the model of central vein infection in rats and in the model of subcutaneous tube infection in mice. Gene complementation restored the biofilm phenotype of wild strain. The gene expression profile of sarZ mutant strain was studied by gene chip method. It was found that the expression level of 79 genes in sarZ mutant was different from that in wild strain S. epidermidis1457. The results were confirmed by quantitative Real Time PCR, including the down-regulated icaADBC and ica regulatory genes of PIA, icaR, and the virulence related genes hln and ohrl, which were up-regulated at transcription level, and the results were confirmed by quantitative Real Time PCR. We also observed that the ability of sarZ mutant to tolerate organic peroxide was lower than that of wild strain, and that the hemolytic activity of S.epidermidis1457 was higher than that of S.epidermidis1457, and the hemolytic activity of S.epidermidis1457 was higher than that of S.epidermidis1457. The results indicate that Hln has hemolytic function in Staphylococcus epidermidis and its expression level is regulated by SarZ. Interestingly, we also observed that the formation of biofilm and the decrease of initial adhesion ability of the strain with overexpression of hln gene were also observed. The results indicated that hln gene also inhibited biofilm formation in Staphylococcus epidermidis. Therefore, we compared the overexpression of hln with wild strains by using silkworm infection model, but no obvious virulence difference between them was observed. This study revealed that the sarZ gene of Staphylococcus epidermidis is an important regulatory gene for biofilm formation and virulence, and may control the expression of ica gene in a manner independent of IcaR. We also found a new hemolysin Hlnin in Staphylococcus epidermidis, which overexpressed hln to inhibit the biofilm formation of Staphylococcus epidermidis. For the first time in the study of Staphylococcus epidermidis, the silkworm infection model was used to compare the bacterial virulence. The study of sarZ and hln genes suggests that they may be used as potential drug targets for the prevention and treatment of Staphylococcus epidermidis infection.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378

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