【作者】 陶亮；

【导师】 孙宝林；

【作者基本信息】 中国科学技术大学 ， 分子和生化生物学， 2010， 博士

【摘要】 金黄色葡萄菌是一种常见的条件致病菌,它可以引起包括慢性耳炎、鼻窦炎、败血症、心内膜炎、骨髓炎等在内的一系列人体感染。该病原菌表达一系列致病相关因子包括细胞毒素、荚膜、基质表面粘附蛋白、葡萄球菌激酶、超抗原毒素、凝固酶、脂酶、趋化因子抑制蛋白、补体抑制蛋白等。许多毒力因子编码基因位于可移动基因元件上如原噬菌体、致病岛、转座子等,金黄色葡萄球菌菌株的致病能力和毒力强弱主要取决于其携带的毒力因子种类和表达强度。在某些条件下,金黄色葡萄球菌可以在体内形成生物膜,它被认为是造成慢性感染的主要原因。通常认为,生物膜的形成有利于细菌抵抗外界环境中的不良因子。金黄色葡萄球菌的生物膜周期主要分三个阶段：第一阶段为与介质表面的粘附,表面粘附蛋白MSCRAMMs起了主要的粘附作用；第二阶段为聚集和生物膜的成熟,在该阶段中除了MSCRAMMs外,细胞壁多聚糖粘附素PIA在将细菌细胞相互粘着聚团的过程中起了重要作用,聚集蛋白Aap也参与了该过程；第三阶段为生物膜的解聚,当生物膜发展到一定阶段或营养受限时,细菌解聚生物膜以达到转移的目的,在葡萄球菌中,群体密度感受系统agr调控这一过程。在研究中我们发现,哺乳动物的血清可以强烈诱导金黄色葡萄球菌产生类似生物膜的聚集现象,这种聚团并不是依赖细菌体与粘连蛋白如纤维蛋白原的简单物理吸附来完成的。通过实时定量PCR检验,在血清诱导刺激下,许多金黄色葡萄球菌细胞壁粘附因子相关的编码基因如clfA、clfB、ica、spa、sasG、fnbA、fnbB的转录水平都得到了不同程度的提高,证明血清刺激通过信号传导触发了细菌的生理状态的改变。在对血清组分作进一步分离研究后发现,免疫球蛋白是血清中诱导金黄色葡萄球菌发生聚集的有效成分。免疫球蛋白的诱导也同样可以造成clfA、clfB、ica、spa、sasG、fnbA、fnbB的mRNA水平升高。金黄色葡萄球菌细胞壁粘附因子的表达受到一系列调控因子的协调控制。我们的研究发现在金黄色葡萄球菌MW2中,通常用于细菌感知环境信号的二元信号系统都没有在免疫球蛋白诱导金黄色葡萄球菌聚集的过程起主要作用,包括全局调控大量毒力因子的agr系统。同时,金黄色葡萄球菌生物膜形成过程中另一个重要的调控因子SarA显著响应了免疫球蛋白的诱导刺激,其转录水平升高了3-5倍左右。Northern印迹实验显示sarA从σB依赖的启动子处起始的一条转录产物在免疫球蛋白诱导后显著加强,而从另两个σA依赖的启动子处起始的转录产物水平则没有发生明显变化。该结果说明金黄色葡萄球菌可选择性sigma因子σB在免疫球蛋白诱导后被激活,从而触发细菌生理状态的改变。敲除σB将导致金黄色葡萄球菌MW2对免疫球蛋白诱导后聚集能力的削弱。我们通过巨噬细胞吞噬模型发现,免疫球蛋白诱导后的聚集细菌可以有效地躲避巨噬细胞的吞噬,此外被吞噬后在巨噬细胞内的存活能力也有少许加强。在本研究中,我们展示了哺乳动物免疫球蛋白可以强烈诱导金黄色葡萄球菌发生聚集。在诱导发生后,可选择性sigma因子σB被激活,上调重要的全局毒力调控蛋白SarA,进而加强各种细胞壁粘附因子相关基因的转录。这种免疫球蛋白诱导的细菌聚集可以有效地抵抗巨噬细胞的吞噬,从而达到免疫逃逸。我们认为,免疫球蛋白可能为金黄色葡萄球菌提供了特异性识别有机体入侵的环境因子信号。细菌特异性识别免疫球蛋白并触发聚集状态以躲避细胞吞噬是一种金黄色葡萄球菌与宿主长期相互作用进化的结果。我们的研究有助于阐释金黄色葡萄球菌在机体入侵过程中的环境信号感知机制,以及该信号在细菌细胞内的传导过程。更多还原

【Abstract】 Staphylococcus aureus is a widely distributed opportunistic human pathogen that is responsible for several organic infections such as chronic otitis, naso sinusitis, septicemia, endocarditis, and osteomyelitis. The human pathogen expresses a series virulence factors like cytotoxins, capsule, MSCRAMMs, staphylokinase, superantigen toxins, coagulase, lipase, CHIP, and Efb. Many of these virulence factor coding genes are located in the mobile genetic elements such as prophages, pathogenicity islands, and transposons. The virulence of Staphylococcus aureus mainly depends on the number of the virulence factors it carries and their expression level.S. aureus may form highly organized bacterial community, termed biofilm, in certain occasions. Its biofilm-forming ability is widely accepted as an important virulence that causes chronic infections. It is believed that a formed biofilm would help to cope with the variable microenvironment. S. aureus biofilm formation proceeds in three stages:primary attachment, biofilm maturation, and dispersal of the cells from the biofilm. The cell-wall associated MSCRAMMs play an important role in matrix adhesion during the primary attachment, while PIA mainly contributes to the maturation of a biofilm, and accumulation protein Aap is also involved. The detachment stage is crucial for bacteria dissemination to other colonization site, for instance when the nutrient is limited. In staphylococci, this mechanism is controlled by the quorum-sensing system agr.We have observed severe aggregation of S. aureus cells in the cultivation induced by mammalian serum. The clumps were somehow similar to biofilms. The phenomenon of aggregation was not cause by direct interaction between bacterial cells and adhesive proteins such as fibrinogen. Later we have found that protein A sefinose purified immunoglobulin would also induce S. aureus cells to form clumps during the cultivation. Real-time quantitative polymerase chain reaction showed that the mRNA levels of several cell-wall associated genes including ica, spa, clfA, clfB, fnbA,fnbB, and sasG were significantly increased in the Ig induced cells, similar to that in the serum induced group. These results indicated that Ig would efficiently induce the aggregation of S. aureus during the growth, while this procedure probably involved a switch in cell physiological conditions.The expression of S. aureus cell-wall associated adhesins is precisely regulated by several global regulators. Two component systems are usually responsible for bacterial perception of environmental changes. However, we have found that 15 tested two component systems, including the impotant global agr system, did not contributed to the clumping process induced by immunoglobulin. SarA is another important global regulator that modulates the biofilm formation. The real-time quantitative PCR showed that the transcriptional level of sarA in S. aureus MW2 was significantly increased after the induction of immunoglobulin. Previous study showed that sarA gene can be transcribed by three promoters. One of them isσB-dependent and the others areσA-dependent. Our Northern blot assay showed that the transcript produced from theσB-dependent promoter was gradually increased during the induction of immunoglobulin while the amount of transcripts produced from the other twoσA-dependent promoters remained unchanged. These results indicate that the alternative sigma factorσB was activated by the stimulation of immunoglobulin. The deletion mutation of the sigB gene weakened the aggregation ability induced by immunoglobulin. Besides, the phagocytosis assays showed that immunoglobulin induction would help the S. aureus cells to evade the phagocytosis by macrophage and slightly enhance the bacterial survival ability in macrophage.Here we have showed that the induction of mammalian immunoglobulin would cause severe aggregation of S. aureus cells during the bacterial growth. When stimulated by immunoglobulin, S. aureus alternative sigma factorσB was activated to elevate the transcription of several genes including the global regulator SarA, and further enhanced the transcription of various genes encoding cell-wall associated adhesins. The aggregation of the bacterial cells results in the efficient evasion of phagocytosis by macrophage. We postulated that the existence of mammalian immunoglobulin may provide S. aureus with specific environmental signals for organic invasion. The strategy is probably a result of the long time interactions between the S. aureus and its host during the evolution. Our study may help to understand how S. aureus receives specific environmental signals for organic invasion, and how this signal is transducted into the bacterial cell.更多还原