【作者】 商飞；

【导师】 孙宝林；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 金黄色葡萄球菌是一种非常重要的人体致病菌,可以引发一系列疾病,从普通的皮肤类感染疾病,如丘疹、脓疱病、疔疮、烫伤样皮肤综合症,到可以致死的系统性感染疾病如肺炎、脑膜炎、骨髓炎、中毒性休克综合症以及败血症等。金黄色葡萄球菌之所以能成为重要致病菌并引发如此众多的疾病感染,主要原因在于可以表达和分泌大量的毒性因子。这些毒性因子主要包括分泌型蛋白:丝氨酸/半胱氨酸蛋白酶、核酸酶、溶血素、肠毒素、脂肪酶、凝固酵素,和细胞表面蛋白:蛋白质A(protein A),纤维蛋白原/胶原质-连接蛋白等。近年来,3’,5’-环二鸟苷酸(c-di-GMP)信号分子调控系统受到越来越多的关注。c-di-GMP最早作为木葡糖醋酸杆菌纤维素合成酶的别构激活子被发现,目前已被认定为广泛存在于细菌当中的第二信使信号分子,参与多种复杂的生理活动的调控。c-di-GMP是由一系列含有GGDEF结构域的蛋白合成并由含有EAL或者HD-GYP结构域的蛋白降解。尽管在很多细菌当中c-di-GMP做为第二信使行使功能已经被深入的研究,但是在一些低GC含量的革兰氏阳性细菌当中,c-di-GMP究竟扮演什么样的角色需要更多的研究来揭示。全基因组测序信息显示金黄色葡萄球菌的基因组只编码了一个GGDEF结构域蛋白(GdpS),而没有EAL或者HD-GYP结构域的蛋白。本研究采用生物基因芯片、实时荧光定量PCR等技术比较野生型金黄色葡萄球标准菌株NCTC8325与其gdpS基因敲除株的基因表达差异。结果显示金黄色葡萄球菌中唯一的GGDEF结构域蛋白(GdpS)影响了一些毒性基因的表达。在gdpS基因敲除株中,一系列毒性因子的转录水平发生了变化,例如丝氨酸/半胱氨酸蛋白酶、纤维蛋白原连接蛋白,尤其是蛋白A(protein A,Spa)。蛋白A是金黄色葡萄球菌中的一种重要的胞壁蛋白,可以和多种哺乳动物的免疫球蛋白IgG的Fc端结合并被认为是金黄色葡萄球菌免疫逃避机制的重要组成元件,蛋白A的致病性已通过多种动物模型得到证实。基于蛋白A的重要性,本研究对GdpS影响其表达的机制进行了更深入的探求。在gdpS敲除株中,编码蛋白A的基因spa的转录水平降低了约8倍。有趣的是,spa的一个正调控因子sarS的转录水平在gdpS敲除株中也有所下降,表明gdpS影响spa的表达可能通过sarS。为了证明这一点,我们在gdpS敲除株中转入含有编码全长gdpS的序列的互补质粒,结果发现spa和sarS的转录水平被都回补到了野生型的水平。而另一方面,同样作为spa表达的调节因子的agr、sarA、sarT和rot的转录水平在gdpS敲除株中则没有变化。因此,可以得出结论:gdpS影响spa的表达通过sarS而不依赖于agr、sarA、sarT、和rot。此外,互补实验的结果表明GdpS影响spa的表达依赖于其N-端结构域,而不依赖其C-端的GGDEF结构域,即GdpS影响spa的表达不依赖于c-di-GMP信号通路。更多还原

【Abstract】 Staphylococcus aureus is an important human pathogen that constitutes the principal causes of a variety of diseases, ranging from minor skin infections, such as pimples, impetigo, furuncles, and scalded skin syndrome, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, Toxic shock syndrome (TSS), and septicemia. The success of the organism as a pathogen and its ability to cause such a wide range of infections are due to its extensive virulence factors. These factors include secreted proteins, such as serine and cysteine proteases, nuclease, hemolysins, enterotoxins, lipase, coagulase, and proteins exposed on the cell surface, such as protein A (Spa) and fibrinogen- and collagen-binding proteins.In recent years, 3’,5’-cyclic diguanylic acid (c-di-GMP) signaling system has drawn much attention. The c-di-GMP was initially described as an allosteric activator of cellulose synthase in Gluconacetobacter Xylinus, and is now recognized as a second messenger ubiquitous in bacteria and is involved in the regulation of a number of complex physiological processes. This novel second messenger is synthesized by a class of enzymes containing GGDEF domains and hydrolyzed by EAL or HD-GYP domain-containing proteins. Although the role of c-di-GMP as a second messenger has been extensively studied in diverse bacteria, its role in some low GC gram-positive bacteria still remains obscure. The genome of S. aureus NCTC8325 encodes only one GGDEF domain-containing protein and another protein with a modified GGDEF domain, whereas no EAL domain-containing proteins are encoded.In this study, we identified the role of the only GGDEF domain protein (GdpS, GGDEF domain protein from Staphylococcus) in the virulence of S. aureus NCTC8325 by comparing the transcriptional profiling between the wild type and the gdpS mutant strain. Inactivation of gdpS results in an alteration in the production of a range of virulence factors, such as serine and cysteine proteases, fibrinogen-binding proteins, and specifically, protein A (Spa), a major surface protein of S. aureus that binds to the Fc region of immunoglobulin G (IgG) of diverse mammalian species, and is thought to be an important component of the immune evasion machinery of this pathogen. The role of Spa in the pathogenesis of staphylococcal infections has been investigated in animal models. We carried out a more detailed study on protein A, as it is a major determinant of the virulence of S. aureus. The transcript level of spa decreases eightfold in the gdpS mutant when compared with the parental NCTC8325 strain. Furthermore, the transcript level of sarS, which encodes a direct positive regulator of spa, also decreases in the gdpS mutant when compared with the wild type, indicating that GdpS might affect the expression of spa via SarS. To further determinate it, we attempted to complement the gdpS mutant with a plasmid containing the full-length gdpS and the transcript levels of spa and sarS in the gdpS mutant complemented almost to the same extent to those in the wild type. In addition, the transcript levels of agr, sarA, sarT, and rot display no apparent changes in the gdpS mutant, suggesting that GdpS affects the expression of spa through interaction with SarS by unknown mechanisms. Furthermore, the complementation assays show that the influences of GdpS on spa and sarS depend on its N-terminal domain, which is predicted to be the sensor of a two-component system, rather than its C-terminal GGDEF domain with conserved GGDEF, suggesting that GdpS functions in S. aureus by an unknown mechanism independent of 3’,5’-cyclic diguanylic acid signaling.更多还原