【作者】 赵丽萍；

【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， 2010， 博士

【摘要】 近年来的研究发现,细胞之间的信息交流不单单存在于多细胞生物中,细菌的细胞之间也存在着相互交流来协调行为方式。细菌在增殖的过程中,会产生一些次级代谢产物,有些次级代谢产物可以作为化学信号在细菌细胞间传递信息。这种信号分子的浓度会随着细菌的增殖而在胞外积累,即信号分子的浓度反映了细菌细胞的密度。当信号分子积累到一定的阈值浓度时,可被细菌感知,与相应的受体结合后在群体范围内引发一系列的基因调控,使细菌能在多细胞水平上采取协调一致的行动来完成一些重要的生理学功能,如生物发光、生物膜的形成、细胞分化、运动性、致病菌的毒力因子的诱导、抗生素与细菌素的合成等。这些重要的生理功能单凭细菌个体是无法完成的。这种信号分子称为自诱导物,这一过程称为群体感应或密度依赖的基因调控,这一调控系统称为群体感应系统。大多数细菌都有群体感应系统,在革兰氏阴性细菌中的种内信号通讯使用的语言是酰基高丝氨酸内酯(AHLs),革兰氏阳性细菌利用寡肽作为信号分子实现种内细胞通讯。不同种的细菌之间也能进行交流,交流所用的语言是Autoinducer-2 (AI-2),这种信号分子存在于大多数细菌中,是目前所知的唯一能进行种内和种间交流的通用语言。AI-2由LuxS催化合成,luxS基因广泛存在于革兰氏阴性细菌和革兰氏阳性细菌中,且具有很高的保守性。LuxS是细菌硫代谢中甲基循环中一个代谢酶,在代谢过程中AI-2作为副产物被合成。所以LuxS肩负着代谢和信号传递的双重功能,因此成为近年来研究的一个热点。但是针对于某一种细菌来说,AI-2信号系统通常不是起到主导地位的群体感应系统,和主要的群体感应一起共同参与基因的调控,所以研究起来相对比较困难。目前有很多研究证明LuxS/AI-2在多种细菌中能发挥信号调控功能,参与调控这些细菌一些重要的生物学途径,如在哈氏弧菌Vibrio harveyi中参与调控生物发光现象,在大肠杆菌Escherichia coli中参与调控生物膜的形成,在霍乱弧菌Vibrio cholerae中参与调控致病性,在链球菌Streptococcus anginosus中参与调控其对抗生素的敏感性等。然而在可以广泛引起人畜疾病的革兰氏阳性菌金黄色葡萄球菌中,LuxS/AI-2系统是否具有信号调控的功能,如果有信号调控功能,AI-2群体感应系统参与哪些生理功能的调控,调控的方式如何等等问题还没有详细的研究报道。金黄色葡萄球菌在环境中无处不在,能引起医院或社区获得感染,是一种非常重要的条件致病菌。金黄色葡萄球菌的感染能引起人畜多种疾病,从皮肤的浅表感染如丘疹、脓包、疥疮、烫伤样皮肤综合症,到可以致死的深层感染如肺炎、脑膜炎、骨髓炎、心内膜炎、中毒性休克综合症以及败血症等。金黄色葡萄球菌之所以有很强的致病性,主要归结于其能产生大量的毒性因子。这些毒性因子包括在金黄色葡萄球菌在生长的延滞期和对数前期表达的细胞表面毒力因子,如表面蛋白、超抗原、一些抗吞噬因子等,这些毒素因子能促进细菌在宿主表面的粘附。在对数后期和稳定期生长阶段,金黄色葡萄球菌表达的毒性因子主要是一些分泌到胞外的酶和外毒素等,能够促进对宿主的入侵和感染。这些毒性因子的产生与生长周期相关,大多都受金黄色葡萄球菌中已知的群体感应系统Agr系统的调控,同时也受一些重要的调控子的调控。金黄色葡萄球菌中也存在LuxS,而且具有合成AI-2的活性,因此我们感兴趣的是它是否能作为信号分子参与基因的调控。本课题通过基因芯片和实时荧光定量PCR等技术分析比较了金黄色葡萄球菌标准菌株NCTC8325的野生型、luxS基因敲除菌株以及在敲除菌株中添加外源AI-2时,这些菌株基因表达转录谱的差异。结果显示在luxS基因敲除菌株中荚膜多糖(Capsular Polysaccharide, CP)基因cap和二元信号系统kdpDE的转录水平都发生了变化,并且改变的水平能被AI-2回补。CP是金黄色葡萄球菌所产生的毒性因子之一,属于细胞表面成分,在侵染过程中能够起到抗吞噬的作用使细菌免受宿主免疫细胞杀伤。有趣的是被报道的能调控cap操纵子的调控子如yabJ-spoVG, arlRS, agr, sbcDC, ccpA, mgr, sae,以及sarA等的转录水平在luxS敲除菌株中都没有变化。因此我们推测,金黄色葡萄球菌中AI-2群体感应系统应该是通过一个未知的途径调控CP的转录。KdpDE由感应蛋白KdpD和效应蛋白KdpE组成一个二元信号系统,最先在大肠杆菌中被阐述与环境中K+缺少或高渗透压有关。在致病菌结核分枝杆菌Mycobacterium tuberculosis中kdpDE基因敲出后能导致其毒性增强,金黄色葡萄球菌中也存在KdpDE系统,虽然有报道它受环境压力的调控但是目前还没有具体功能的研究报道。为了研究KdpDE和CP之间关系,我们获得了kdpDE敲除菌株,发现kdpDE敲除后,CP的转录水平下降,DNA凝胶阻滞实验证明了KdpE可以结合到cap基因的启动子上直接调控其表达。kdpE基因敲除和互补以及KdpE磷酸化位点突变实验证明了KdpDE系统是通过磷酸化传递途径实现其对荚膜多糖表达的调控。细胞吞噬实验显示AI-2作为信号分子能调控生物膜的形成和细菌的抗吞噬能力,此结果和荚膜多糖的转录水平一致进一步验证了证明了我们的推测,即AI-2群体感应系统通过二元信号系统KdpDE调控荚膜多糖的表达进而影响一些表型的变化。此研究为LuxS/AI-2系统在金黄色葡萄球菌中的信号调控功能提供了直接的证据,首次阐述了二元信号系统KdpDE在金葡菌中与致病性相关的具体功能,为金葡菌感染的治疗提供了新的启示。更多还原

【Abstract】 Recently people found cell-cell communication is not just in multi-cellular organism, bacteria can also communicate with one another to coordinate their behavior. In the process of proliferation, bacteria secrete small diffusible molecules called autoinducers (AIs) continuously. While bacteria proliferated, AIs accumulated in the around environment, once reached a threshold concentration AIs can be detected and enter the cell, bind to its receptors and regulate transcription of lots of target genes. Thus bacteria can coordinate social behavior at multi-cellular level, such as bioluminescence, biofilm formation, swarming behavior, antibiotic production, and virulence factor secretion. This process is referred to as quorum sensing (QS), or density-dependent gene regulation, and this system is called QS system. Many QS mechanisms have evolved among bacteria. In general, gram-negative bacteria use acylated homoserine lactones (AHLs) as AIs, and gram-positive bacteria use oligopeptide AIs, which act through two-component phosphorelay cascades. There is one mechanism that is shared by both gram-positive and gram-negative bacteria, involving the production of autoinducer 2 (AI-2). In contrast to other autoinducers that are species specific, AI-2 is widely present in bacteria, leading to the suggestion that it is a universal language for interspecies communication. AI-2 is byproduct synthesized by LuxS enzyme in a metabolic pathway known as the activated methyl cycle. LuxS is conserved and widely present in both gram-negative and gram-positive bacteria. Due to its dual roles in metabolism and in QS, has been a hot topic to study, however, it is difficulty to find out its real rule in QS since it is not the main QS system and it is regulated by other regulators. The LuxS/AI-2 system is known to be functional as a QS system involved in the regulation of a range of behaviours in diverse bacteria, such as bioluminescence in Vibrio harveyi, biofilm formation in Escherichia coli, virulence-associated traits in Vibrio cholerae, and antibiotic susceptibility in Streptococcus anginosu. However, AI-2 QS role in gram-positive bacteria, especially in S. aureus, has not been studied in detail until now.S. aureus is a major nosocomial pathogen with the ability to cause a variety of infectious diseases, from relatively benign skin infections such as pimples, impetigo, furuncles, and scalded skin syndrome, to potentially fatal systemic disorders such as pneumonia, meningitis, osteomyelitis and septicemia. The strong pathogenesis of S. aureus is essentially determined by its virulence factors, including surface-associated adhesins, superantigens, exoenzymes and exotoxin, which are regulated by a wide range of regulatory systems. Among these regulatory elements, the Agr system (the accessory gene regulator) is the only characterized QS system in S. aureus. Interestingly, S. aureus also possesses a functional luxS gene and has the capability to produce AI-2. It will be of great importance to explore whether AI-2 can function as a QS signal to regulate physiological functions in S. aureus.In this study, we analyzed the transcript differences between the standard strains of S. aureus NCTC8325 wild type, the luxS mutant and the luxS mutant added exogenous AI-2 using microarray and real time RT-PCR. Our data show that Inactivation of luxS in S. aureus NCTC8325 resulted in higher transcription of capsular polysaccharide (CP) synthesis genes and the two component system kdpDE genes. The survival rate of the luxS mutant was higher than that of the wild type both in human blood and macrophage U937. Addition of exogenous AI-2 restored all the parental phenotypes by a concentration-dependent mechanism. CP is an important cell wall component that can interact with the host immune system during the invasive process, allowing the organism to resist uptake and killing by phagocytes. It is interesting that the transcript levels of various regulatory elements known to modulate CP synthesis in S. aureus such as agrA, sarA, sbcDC, rnaⅢarlRS, ccpA, mgrA, saeRS, and spoVG displayed no apparent changes in luxS mutant. So we suspected that LuxS/AI-2 modulated cap gene transcription through another mechanism. Sensor protein KdpD together with reaction protein KdpE constitutes a two-component signal transduction system, which was first characterized in E. coli. In this organism, KdpDE regulate severe K+ limitation or osmotic upshift. In Mycobacterium tuberculosis, deletion of kdpDE resulted in increased virulence. Although several reports have shown that, in S. aureus, the transcript level of kdpDE changes under certain environmental stresses, information about the role of KdpDE in S. aureus and how it functions remains incomplete.To study the relationship between KdpDE and CP, we constructed kdpDE mutant strain. Our data show that the transcript levels of cap genes were decreased in kdpDE mutant and gel-shift assay show that KdpE can bind to the promoter region of the cap operon, suggesting that the KdpDE system could regulate cap gene transcription. kdpE mutant and KdpE phosphorylation site mutant assays indicated that KdpE regulates the transcription of cap through a phosphorylation pathway. The alterated survival of S. aureus in human blood and monocytic cells correlated with the changes in transcript levels of CP. The data further validated our hypothesis that S. aureus AI-2 quorum sensing regulates CP synthesis and virulence through the two-component regulatory system KdpDE. Our study provided a direct proof that the LuxS/AI-2 system played a signaling role in S. aureus and new clues to the functional analysis of the KdpDE system in S. aureus. Our findings add new understanding to AI-2 quorum sensing regulation and mechanisms of innate immune evasion used by S. aureus and provide novel clues for antimicrobial chemotherapy of Staphylococal infection.更多还原