一氧化氮调节金黄色葡萄球菌生物被膜形成的作用机制探讨

发布时间：2018-06-30 06:35

本文选题：一氧化氮 + 金黄色葡萄球菌　；参考：《中国海洋大学》2010年硕士论文

【摘要】：一氧化氮(nitric oxide, NO)是生物体内重要的活性分子,参与了生物体内许多重要的生理过程,例如,动物体内的血管松弛、免疫防御、神经传递以及植物体内的种子萌发、铁代谢、抗逆调节以及抗衰老调节等,被认为是多功能的第二信使。NO在动物以及高等植物体内的研究已受到广泛的重视并取得重要进展,然而其在细菌中,尤其是细菌形成的多细胞结构—细菌生物被膜(biofilm)中的作用却极少有人研究。细菌生物被膜是由细菌和其分泌的胞外基质在物体表面形成的高度组织化的多细胞结构,是细菌产生抗生素耐药和逃避机体免疫系统攻击的主要原因,但其形成的分子机制比较复杂,涉及到许多的信号通路和调节蛋白,阐明细菌生物被膜形成机理对于细菌生物被膜相关感染的治疗具有重要意义。实验室前期研究发现,NO能促进金黄色葡萄球菌生物被膜形成,本论文主要是在实验室前期研究的基础上,对NO调节金黄色葡萄球菌生物被膜形成的作用机制进行初步探讨。金黄色葡萄球菌致密的细胞壁结构和大量的胞外多糖(PIA/PNAG)是影响其体内遗传操作的主要原因,首先利用实验室分离得到的糖粘附素降解酶多聚β-1,6-N-乙酰葡聚糖胺裂解酶(DSPB)对金黄色葡萄球菌的电击转化条件进行进一步的优化。PIA是β-1-6-N-乙酰胺基葡萄糖多聚物,DSPB能专一降解PIA。实验结果表明在从限制缺陷菌株RN4220中提取质粒过程中,加入DSPB对细胞进行预处理可以明显提高质粒的纯度和数量。利用0.1U的DSPB制备感受态细胞,其转化效率相比于未处理组提高了1000倍,而且其效果优于溶菌酶和溶葡萄球菌酶。为了进一步确证内源NOS对金黄色葡萄球菌生物被膜的调节作用,利用表达载体对Saureus RN6390△nos中nos基因进行回补,结果表明nos基因回补后回复了其生物被膜形成能力,说明内源性NOS在金黄色葡萄球菌生物被膜形成中具有重要作用。同时,采用96孔板和Flow-cell两种不同的生物被膜模型,通过外源加入NO供体SNP进一步验证了外源性NO同样能够促进金黄色葡萄球菌RN6390的生物被膜形成。根据文献报道葡萄球菌生物被膜的形成方式分PIA依赖型和PIA非依赖型,PIA合成增加能促进生物被膜的形成。那么外源NO是否是通过调节PIA的合成促进生物被膜的形成呢?实时荧光定量PCR和胞外多糖检测结果表明加入NO供体SNP后ica的表达量和PIA的产生量均降低,证明NO对S.aureus生物被膜形成的调节作用不是通过促进PIA的产生而实现的。 S. aureusRN6390的双组分调节系统srrAB与枯草杆菌ResDE双组分系统高度同源,而ResDE能通过NsrR蛋白感受NO或者其衍生信号从而调节基因的表达。那么srrAB是否也能感受NO呢?定量PCR检测显示,生物被膜中srrAB表达量显著提高,表明srrAB与生物被膜形成密切相关。通过同源重组构建了S. aureusRN6390的双组份调节系统srrAB的缺失突变株,检测NO对其生物被膜形成的促进作用是否依赖于srrAB。结果表明srrAB缺失后生物被膜形成能力降低,但NO对其生物被膜形成的促进效应仍然存在,因此NO对金黄色葡萄生物被膜形成的促进与srrAB无关。综上,本论文在前期研究的基础上初步探讨了NO调节金黄色葡萄球菌生物被膜形成的作用机制,证实了这种调节作用并不依赖于能够编码多糖胞间粘附素的ica和双组份系统srrAB。本论文揭示了NO在细菌生物被膜形成过程中的作用,并对作用机制进行了初步探讨,为进一步的实验提供了理论依据,同时,为细菌生物被膜形成的调节机制和细菌生物被膜相关感染的预防和治疗提供了新的思路。
[Abstract]:Nitric oxide (NO) is an important active molecule in the organism. It participates in many important physiological processes in the organism, such as vascular relaxation, immune defense, neural transmission, seed germination in plants, iron metabolism, anti inverse regulation and anti aging regulation in the animal body. It is considered to be the second messenger of multifunction.NO in the body. Research in animals and higher plants has received extensive attention and made significant progress. However, it is rarely studied in bacteria, especially the multicellular structure bacteria biofilm (biofilm), which is formed by bacteria. The bacterial biofilm is the height of the bacteria and its extracellular matrix on the surface of the body. The organised multicellular structure is the main reason for bacteria to produce antibiotic resistance and escape from the attack of the body's immune system. But the molecular mechanism of its formation is complex, involving many signal pathways and regulatory proteins. It is important to clarify the mechanism of bacterial biofilm formation for the treatment of bacterial biofilm related infection. The previous study found that NO could promote the formation of biofilm of Staphylococcus aureus. This paper mainly discussed the mechanism of NO regulating the formation of Staphylococcus aureus biofilm on the basis of early laboratory studies.
The dense cell wall structure of Staphylococcus aureus and a large number of extracellular polysaccharides (PIA/PNAG) are the main factors affecting the genetic manipulation in the body. First, the electrical shock conversion conditions of Staphylococcus aureus were further optimized by using the glucose adhesion degrading enzyme poly beta -1,6-N- acetylglucan lyzase (DSPB) isolated from the laboratory. .PIA is a beta -1-6-N- acetylamine based glucose polymer. DSPB can degrade PIA. experiment results show that in the process of extracting plasmids from the restricted strain RN4220, adding DSPB to the cell can obviously improve the purity and quantity of the plasmids. Using DSPB of 0.1U to prepare the receptive cells, the conversion efficiency is compared to the untreated group. It is 1000 times higher than that of lysozyme and lysostinase.
In order to further confirm the regulatory effect of endogenous NOS on the biofilm of Staphylococcus aureus, the expression vector was used to supplement the NOS gene in Saureus RN6390 delta Nos. The results showed that the NOS gene returned to its biofilm formation ability, which indicated that endogenous NOS plays an important role in the formation of Staphylococcus aureus biofilm. At the same time, using two different biofilm models of 96 orifice and Flow-cell, and adding NO donor SNP to further verify that exogenous NO can also promote the formation of biofilm of Staphylococcus aureus RN6390.
It is reported that the formation of staphylococcal biofilm is divided into PIA dependent and PIA non dependent forms. The increase of PIA synthesis can promote the formation of biofilm. Then, does exogenous NO promote the formation of biofilm by regulating the synthesis of PIA? Real time fluorescence quantitative PCR and extracellular polysaccharide detection results show ICA's table after NO donor SNP Both the amounts of PIA and the amount of NO decreased, demonstrating that the regulation of S.aureus on biofilm formation by S.aureus was not achieved by promoting the production of PIA.
The dual component system of S. aureusRN6390 is highly homologous to the two component system of Bacillus subtilis ResDE, and ResDE can regulate the expression of genes through the NO or its derived signal through the NsrR protein. Then can srrAB also feel NO? Quantitative PCR detection shows that srrAB expression in the biofilm shows that srrAB and organisms are used. The membrane formation is closely related. Through homologous recombination, the deletion mutant strain of the S. aureusRN6390 dual component regulation system srrAB is constructed, and whether the promotion of NO to its biofilm formation depends on the srrAB. result indicates that the biofilm formation ability of the srrAB is reduced after the deletion of the biofilm, but the effect of NO on the formation of its biofilm still exists, because of the existence of the effect of NO on the formation of its biofilm. The promotion of NO to the biofilm formation of Golden Grape has nothing to do with srrAB.
On the basis of previous studies, this paper preliminarily discussed the mechanism of NO regulation of the biofilm formation of Staphylococcus aureus, and confirmed that this regulation does not depend on the ICA and the dual component system srrAB. that can encode polysaccharide intercellular adhesion. This paper reveals the role of NO in the formation of bacterial biofilm. The mechanism of action is preliminarily discussed, which provides a theoretical basis for further experiments. At the same time, it provides a new way of thinking for the regulation mechanism of bacterial biofilm formation and the prevention and treatment of bacterial biofilm related infection.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【引证文献】