金黄色葡萄球菌非编码RNA-L8以及TRAP蛋白生物学功能研究
摘要
金黄色葡萄球菌(Staphylococcus aureus)是一种重要的人类致病菌,它能够引起广泛地感染,包括从皮肤炎症、中毒性休克、肺炎到威胁生命的骨髓炎、心内膜炎、败血症等一系列疾病。其通过表达各种表面粘附蛋白、分泌蛋白和毒力因子如蛋白A、蛋白酶、溶血素、肠毒素等引发各种疾病。目前临床上治疗金黄色葡萄球菌感染主要是采用联合使用抗生素的方法,但耐药性金黄色葡萄球菌的出现致使许多抗生素变的无能为力。
非编码RNA,指的是不被翻译成蛋白质的RNA,如tRNA, rRNA,miRNA,snRNA,sRNA等。它们广泛存在于真核和原核细胞中,操纵着细胞许多生理功能。过去对于sRNA(small non-coding RNA)的研究主要集中在真核生物,发现了许多具有功能的sRNA分子如miRNA,它可以与靶基因互补调节特定基因的表达。近年来,随着对原核生物研究的深入,发现在细菌体内也存在着类似的非编码sRNA,这些sRNA是一类长度一般在20至500个核苷酸左右,执行多种功能的非编码RNA分子。不同的sRNA具有不同的功能,不同的sRNA发挥功能的机制也不相同。50%—60%的sRNA是通过与靶mRNA的配对来发挥其调控功能。它们通过配对影响翻译的水平或影响mRNA的稳定性从而在转录水平对基因表达进行调控。有研究报道表明,细菌中的许多sRNA与其生长代谢和毒力调控过程密切相关。原核生物中sRNA的研究目前主要集中于大肠杆菌(Escherichia coli),已发现近百种sRNA。新近的研究结果表明在革兰氏阳性菌金黄色葡萄球菌中也存在新的sRNA,其中部分位于金黄色葡萄球菌基因组的病原岛或只存在于致病菌株中,提示这些sRNA可能参与了致病菌毒素的表达调控。金黄色葡萄球菌RNAIII(一种sRNA)已经被证实作用于多个毒力相关基因,参与调控金黄色葡萄球菌的致病性。
Hfq (a host factor for RNA phage Qβ)蛋白最早在大肠杆菌中发现,具有RNA伴侣分子活性,其主要的生物学功能是通过形成六聚体与RNA结合来影响RNA的稳定性或者是通过辅助sRNA与mRNA结合来调节靶基因的表达,它对于sRNA通过配对发挥功能至关重要。在研究基因表达调控时发现一些在转录后水平调控靶基因表达的sRNA分子通常需要Hfq蛋白的辅助。这表明这些sRNA分子可能是一个sRNA家族,这个家族的重要特征是能有效地与Hfq蛋白结合并利用碱基配对与靶mRNA分子相互作用,从而调节了靶mRNA的表达。目前在大肠杆菌中,超过30%的非编码sRNA能够与Hfq蛋白结合。
本研究的第一部分为金黄色葡萄球菌中新的sRNA-L8生物学功能的研究。我们结合生物信息学的预测,进一步利用RACE的方法获得一条新的全长sRNA,将其命名为L8。进一步构建L8缺失突变菌株,通过一系列表型和应激反应实验以及靶基因的寻找和鉴定,阐明了L8在金黄色葡萄球菌中的重要生物学功能。本部分的主要研究内容如下:
1. L8的发现、鉴定及相关检测。结合生物信息学,我们在金黄色葡萄球菌中预测到一些新的sRNA分子,并通过RACE的方法找到一条新的全长sRNA,命名为L8,进一步通过Northern Blot的方法验证了其在金黄色葡萄球菌中的存在。提取重金属离子和高渗应激条件下的总RNA,反转录成cDNA,进行荧光定量PCR检测L8的表达状况,结果发现L8的表达水平下降。L8可以与RNA伴侣分子Hfq蛋白特异性结合,并在hfq突变菌株中表达水平下降。L8在RNaseIII突变菌株中表达水平上升,在RNaseHII和RNaseZ突变菌株中表达水平下降。
2.金黄色葡萄球菌L8突变株制备及相关检测。利用基因同源重组的原理制备L8的缺失突变菌株,一系列表型和应激反应实验结果显示L8缺失后,菌株在生长、生物膜的形成、自溶、全血中存活、中性粒细胞中存活以及H2O2中的存活均发生明显变化,并且突变株对动物的致病性也明显降低,表明sRNA-L8在金黄色葡萄球菌中有重要的生物学功能。
3. L8靶基因的寻找和鉴定。通过生物信息学预测的L8靶基因为arlR,首先利用荧光定量PCR检测其在突变株中的表达状况,发现在L8突变株中,arlR的表达水平明显降低,提示L8可以正调控arlR的表达。Hfq蛋白能够辅助L8和arlR的相互作用。然后我们又通过构建LacZ报告载体进一步确定了arlR是L8的靶基因。
总之,本部分研究结果表明我们新发现的sRNA-L8能够正调控arlR的表达,从而影响金黄色葡萄球菌的致病性。
早期的研究结果认为TRAP是金黄色葡萄球菌内与其致病性相关的蛋白分子,其通过调控agr系统来影响金黄色葡萄球菌的致病性。但是后期的研究结果显示TRAP蛋白与agr系统无关,其是否影响金黄色葡萄球菌的致病性还存在争议。我们已有的结果和已发表的研究工作都表明TRAP蛋白的抗体能够抑制金黄色葡萄球菌对机体的感染。
本研究的第二部分为TRAP蛋白的生物学功能研究。我们通过构建突变菌株并对其表型和应激反应进行检测,鉴定其与agr系统的相关性,研究TRAP蛋白在金黄色葡萄球菌中的生物学功能。本部分的主要研究内容如下:
1.金黄色葡萄球菌traP突变株制备及相关检测。利用基因同源重组的原理制备traP的缺失突变菌株,突变株中agr系统没有发生突变,agr系统表达水平没有明显改变,这表明TRAP蛋白的确与agr系统无关。但是我们发现突变株对动物的致病性明显降低,一系列表型和应激反应实验结果也显示traP缺失后,菌株在生长、表面电荷、疏水性、中性粒细胞中存活以及H2O2中的存活均发生明显变化,证明TRAP蛋白在金黄色葡萄球菌中有重要的生物学功能。
2. TRAP蛋白影响中性粒细胞对金黄色葡萄球菌的吞噬。中性粒细胞吞噬实验显示traP缺失菌株更容易被吞噬,但重组表达的TRAP蛋白不能够影响中性粒细胞对金黄色葡萄球菌的吞噬。我们进一步通过分析突变菌株和野生型菌株细胞壁蛋白的表达谱,发现了一个明显升高的蛋白条带,质谱分析表明该蛋白为功能未知的蛋白,我们将其命名为SAP。实时定量PCR结果表明编码该蛋白的基因在突变株中的表达水平明显升高。SAP蛋白是否参与中性粒细胞对金黄色葡萄球菌吞噬过程有待于进一步鉴定。
总之,我们的实验结果初步表明TRAP蛋白的确与agr系统无关,但是其可能通过影响中性粒细胞对金黄色葡萄球菌吞噬,从而调控金黄色葡萄球菌对机体的感染。
Staphylococcus aureus (S.aureus) is an important pathogen capable of causing a variety of diseases in humans through virulence gene expression, ranging from localized infections of skin and soft tissue to life-threatening systemic infections, such as skin infections, toxic shock syndrome, pneumonia, endocarditis and meningitis etc.
Non-coding RNAs (ncRNAs) including tRNA, rRNA,miRNA,snRNA etc, exist extensively in the eukaryote and prokaryocyte, and carry out lots of physiological functions. The most well studied ncRNAs is miRNA in the past ten years, which can regulate sepecific gene by complementation with target gene. Recently, it is reported that there are some regulational ncRNAs play the import roles in prokaryocyte, which is named as small RNAs (sRNA). The length of sRNAs in bacteria ranges from about 20 to 500 neucletides. sRNAs perform their function with different mechanisms. About 50-60% of sRNAs play their function through base pairing with target mRNAs. They can regulate the target gene expression by influencing the translation process or the stability of mRNAs. A sRNA can target more than one genes. At the same time, more than one sRNA can regulate the same target. The present study have shown that sRNA play an important role in the bacteria growth metabolism and verulence regulation. There are about one hundred sRNAs have been indentified in Escherichia coli (E.coli). It is also reported that there are some functional sRNAs identified in S.aureus. These sRNAs are predicted to regulate the pathogenesis because they are located in the pathogenicity islands or only expressed in the pathogenic strains. RNAIII is a well studied sRNA, which is identified as a key regulator of toxin expression in S. aureus.
The Hfq protein is a RNA chaperone, which is firstly discovered in E.coli. It can form a homohexameric ring to bind RNA and change the stability of RNA or help sRNA pairing with the target mRNA. In E.coli, Hfq is vital for sRNAs functions. At present more than 30% noncoding RNA could bind with Hfq in E.coli. These sRNAs regulate the expession of target mRNA with the aid of Hfq.
Chapter I: Identification of a novel functional sRNA L8 in S.aureus. A novel sRNA was identified with prediction of bioinformatics and named as L8. The L8 deletion strain was constructed. The further study suggested that L8 could regulate the expression of arlR and play an important role in the pathogenicicy of S.aureus. This chapter is divided into three parts.
1, Identification of the novel small RNA. On the basis of the predication of bioinformatics, the transcript of a novel sRNA was identified. And the full lenth of this sRNA were obtained RACE in S.aureus and named L8. The length of L8 was confirmed by Northern blot. Total RNA under stress condition and cDNA was prepared. The results of qRT-PCR showed that the expression of L8 was significantly altered when S.aureus was grown under some different stress conditions.
2, Construction of L8 deletion mutant from S.aureus 8325-4. L8 deletion mutant strain was prepared with homologous recombination. It was found that the pathogenicity of mutant strain decreased significantly. Similarly, whole blood survival assay in vitro showed that the survival rate of L8 mutant strain remarkbly decreased compared with its parent strain. A serial of stress-response experiments results displayed that L8 was involved in growth, biofilm formation, autolysis of S.arueus. These data indicated that L8 play a pivotal role in S.aureus.
3, Idenfication of the target genes regulated by L8. The gene arlR was predicted as one target of L8 by bioibformatics. The results of qPCR showed that the expression of arlR was decreased in L8 mutant strain. It was found that L8 could specifically bind arlR with the aid of Hfq. In the further investigation, arlR was indentified as one target of L8 by construction of reporter vector.
In summary, a novel functional sRNA-L8 in S.aureus was identified, which could regulate the expression of alrR and play an important role in the pathogenicity of S.arueus.
TRAP was thought as an important protein to influence the pathogenicity of S.aureu by regulating the expression of agr, but recent papers showed that TRAP could not influence the expression of agr. The biological function of TRAP is controversial. Our previous report suggested that the antibodies of TRAP could inhibit the infenciton cause by S.aureus. We tried to investigate the biological function of TRAP in the pathogenicity of S.aureus in the following study.
Chapter II: Study of the biological function of TRAP in S.aureu. In this chapter, we construced a trap deletion mutant from S.arueus 8325-4. It was investigated whether TRAP could regulate the expression of agr. In the further study, it was demonstrated that the trap deletion mutant was more sensitive to the phagocytosis of neutrophil. This part was divided into two parts.
1, Construction of the traP deletion mutant from S.aureus 8325-4. traP deletion mutant strain was prepared by gene reconbination. The agr system was not changed in the mutant, which suggested that TRAP could not influence the expression of agr. While, the infection caused by the mutant strain was decreased significantly, which indicated that TRAP was involved in the pathogenicity of S.aureus. Compared with its parent strain, a serial of phenotypes alternation were observed in the traP deletion strain. These data suggested that TRAP play an important role in S.aureus.
2, The TRAP protein was involved in the neutrophil phagocytosis of S.arueus. It was found that traP mutant was sensitive to the neutrophil phagocytosis compared with its parent strain. But the recombinant TRAP protein could not influence this process. In the further study, it was found the level of a protein on the cell wall of S.aureus was increased. This protein was indentified by MS. It was a protein of unknown function and named as SAP. The results of qPCR showed that the expression of sap was higher in the trap mutant strain than that in the wild type. Whether SAP is involved in the neutrophil phagocytosis and killing will be investigated in the future.
In a word, The TRAP protein could not influence the expression of agr, but it might play an important role in the pathogenicity of S.arueus by regulationg the interaction between neutrophil and S.aureus.
非编码RNA,指的是不被翻译成蛋白质的RNA,如tRNA, rRNA,miRNA,snRNA,sRNA等。它们广泛存在于真核和原核细胞中,操纵着细胞许多生理功能。过去对于sRNA(small non-coding RNA)的研究主要集中在真核生物,发现了许多具有功能的sRNA分子如miRNA,它可以与靶基因互补调节特定基因的表达。近年来,随着对原核生物研究的深入,发现在细菌体内也存在着类似的非编码sRNA,这些sRNA是一类长度一般在20至500个核苷酸左右,执行多种功能的非编码RNA分子。不同的sRNA具有不同的功能,不同的sRNA发挥功能的机制也不相同。50%—60%的sRNA是通过与靶mRNA的配对来发挥其调控功能。它们通过配对影响翻译的水平或影响mRNA的稳定性从而在转录水平对基因表达进行调控。有研究报道表明,细菌中的许多sRNA与其生长代谢和毒力调控过程密切相关。原核生物中sRNA的研究目前主要集中于大肠杆菌(Escherichia coli),已发现近百种sRNA。新近的研究结果表明在革兰氏阳性菌金黄色葡萄球菌中也存在新的sRNA,其中部分位于金黄色葡萄球菌基因组的病原岛或只存在于致病菌株中,提示这些sRNA可能参与了致病菌毒素的表达调控。金黄色葡萄球菌RNAIII(一种sRNA)已经被证实作用于多个毒力相关基因,参与调控金黄色葡萄球菌的致病性。
Hfq (a host factor for RNA phage Qβ)蛋白最早在大肠杆菌中发现,具有RNA伴侣分子活性,其主要的生物学功能是通过形成六聚体与RNA结合来影响RNA的稳定性或者是通过辅助sRNA与mRNA结合来调节靶基因的表达,它对于sRNA通过配对发挥功能至关重要。在研究基因表达调控时发现一些在转录后水平调控靶基因表达的sRNA分子通常需要Hfq蛋白的辅助。这表明这些sRNA分子可能是一个sRNA家族,这个家族的重要特征是能有效地与Hfq蛋白结合并利用碱基配对与靶mRNA分子相互作用,从而调节了靶mRNA的表达。目前在大肠杆菌中,超过30%的非编码sRNA能够与Hfq蛋白结合。
本研究的第一部分为金黄色葡萄球菌中新的sRNA-L8生物学功能的研究。我们结合生物信息学的预测,进一步利用RACE的方法获得一条新的全长sRNA,将其命名为L8。进一步构建L8缺失突变菌株,通过一系列表型和应激反应实验以及靶基因的寻找和鉴定,阐明了L8在金黄色葡萄球菌中的重要生物学功能。本部分的主要研究内容如下:
1. L8的发现、鉴定及相关检测。结合生物信息学,我们在金黄色葡萄球菌中预测到一些新的sRNA分子,并通过RACE的方法找到一条新的全长sRNA,命名为L8,进一步通过Northern Blot的方法验证了其在金黄色葡萄球菌中的存在。提取重金属离子和高渗应激条件下的总RNA,反转录成cDNA,进行荧光定量PCR检测L8的表达状况,结果发现L8的表达水平下降。L8可以与RNA伴侣分子Hfq蛋白特异性结合,并在hfq突变菌株中表达水平下降。L8在RNaseIII突变菌株中表达水平上升,在RNaseHII和RNaseZ突变菌株中表达水平下降。
2.金黄色葡萄球菌L8突变株制备及相关检测。利用基因同源重组的原理制备L8的缺失突变菌株,一系列表型和应激反应实验结果显示L8缺失后,菌株在生长、生物膜的形成、自溶、全血中存活、中性粒细胞中存活以及H2O2中的存活均发生明显变化,并且突变株对动物的致病性也明显降低,表明sRNA-L8在金黄色葡萄球菌中有重要的生物学功能。
3. L8靶基因的寻找和鉴定。通过生物信息学预测的L8靶基因为arlR,首先利用荧光定量PCR检测其在突变株中的表达状况,发现在L8突变株中,arlR的表达水平明显降低,提示L8可以正调控arlR的表达。Hfq蛋白能够辅助L8和arlR的相互作用。然后我们又通过构建LacZ报告载体进一步确定了arlR是L8的靶基因。
总之,本部分研究结果表明我们新发现的sRNA-L8能够正调控arlR的表达,从而影响金黄色葡萄球菌的致病性。
早期的研究结果认为TRAP是金黄色葡萄球菌内与其致病性相关的蛋白分子,其通过调控agr系统来影响金黄色葡萄球菌的致病性。但是后期的研究结果显示TRAP蛋白与agr系统无关,其是否影响金黄色葡萄球菌的致病性还存在争议。我们已有的结果和已发表的研究工作都表明TRAP蛋白的抗体能够抑制金黄色葡萄球菌对机体的感染。
本研究的第二部分为TRAP蛋白的生物学功能研究。我们通过构建突变菌株并对其表型和应激反应进行检测,鉴定其与agr系统的相关性,研究TRAP蛋白在金黄色葡萄球菌中的生物学功能。本部分的主要研究内容如下:
1.金黄色葡萄球菌traP突变株制备及相关检测。利用基因同源重组的原理制备traP的缺失突变菌株,突变株中agr系统没有发生突变,agr系统表达水平没有明显改变,这表明TRAP蛋白的确与agr系统无关。但是我们发现突变株对动物的致病性明显降低,一系列表型和应激反应实验结果也显示traP缺失后,菌株在生长、表面电荷、疏水性、中性粒细胞中存活以及H2O2中的存活均发生明显变化,证明TRAP蛋白在金黄色葡萄球菌中有重要的生物学功能。
2. TRAP蛋白影响中性粒细胞对金黄色葡萄球菌的吞噬。中性粒细胞吞噬实验显示traP缺失菌株更容易被吞噬,但重组表达的TRAP蛋白不能够影响中性粒细胞对金黄色葡萄球菌的吞噬。我们进一步通过分析突变菌株和野生型菌株细胞壁蛋白的表达谱,发现了一个明显升高的蛋白条带,质谱分析表明该蛋白为功能未知的蛋白,我们将其命名为SAP。实时定量PCR结果表明编码该蛋白的基因在突变株中的表达水平明显升高。SAP蛋白是否参与中性粒细胞对金黄色葡萄球菌吞噬过程有待于进一步鉴定。
总之,我们的实验结果初步表明TRAP蛋白的确与agr系统无关,但是其可能通过影响中性粒细胞对金黄色葡萄球菌吞噬,从而调控金黄色葡萄球菌对机体的感染。
Staphylococcus aureus (S.aureus) is an important pathogen capable of causing a variety of diseases in humans through virulence gene expression, ranging from localized infections of skin and soft tissue to life-threatening systemic infections, such as skin infections, toxic shock syndrome, pneumonia, endocarditis and meningitis etc.
Non-coding RNAs (ncRNAs) including tRNA, rRNA,miRNA,snRNA etc, exist extensively in the eukaryote and prokaryocyte, and carry out lots of physiological functions. The most well studied ncRNAs is miRNA in the past ten years, which can regulate sepecific gene by complementation with target gene. Recently, it is reported that there are some regulational ncRNAs play the import roles in prokaryocyte, which is named as small RNAs (sRNA). The length of sRNAs in bacteria ranges from about 20 to 500 neucletides. sRNAs perform their function with different mechanisms. About 50-60% of sRNAs play their function through base pairing with target mRNAs. They can regulate the target gene expression by influencing the translation process or the stability of mRNAs. A sRNA can target more than one genes. At the same time, more than one sRNA can regulate the same target. The present study have shown that sRNA play an important role in the bacteria growth metabolism and verulence regulation. There are about one hundred sRNAs have been indentified in Escherichia coli (E.coli). It is also reported that there are some functional sRNAs identified in S.aureus. These sRNAs are predicted to regulate the pathogenesis because they are located in the pathogenicity islands or only expressed in the pathogenic strains. RNAIII is a well studied sRNA, which is identified as a key regulator of toxin expression in S. aureus.
The Hfq protein is a RNA chaperone, which is firstly discovered in E.coli. It can form a homohexameric ring to bind RNA and change the stability of RNA or help sRNA pairing with the target mRNA. In E.coli, Hfq is vital for sRNAs functions. At present more than 30% noncoding RNA could bind with Hfq in E.coli. These sRNAs regulate the expession of target mRNA with the aid of Hfq.
Chapter I: Identification of a novel functional sRNA L8 in S.aureus. A novel sRNA was identified with prediction of bioinformatics and named as L8. The L8 deletion strain was constructed. The further study suggested that L8 could regulate the expression of arlR and play an important role in the pathogenicicy of S.aureus. This chapter is divided into three parts.
1, Identification of the novel small RNA. On the basis of the predication of bioinformatics, the transcript of a novel sRNA was identified. And the full lenth of this sRNA were obtained RACE in S.aureus and named L8. The length of L8 was confirmed by Northern blot. Total RNA under stress condition and cDNA was prepared. The results of qRT-PCR showed that the expression of L8 was significantly altered when S.aureus was grown under some different stress conditions.
2, Construction of L8 deletion mutant from S.aureus 8325-4. L8 deletion mutant strain was prepared with homologous recombination. It was found that the pathogenicity of mutant strain decreased significantly. Similarly, whole blood survival assay in vitro showed that the survival rate of L8 mutant strain remarkbly decreased compared with its parent strain. A serial of stress-response experiments results displayed that L8 was involved in growth, biofilm formation, autolysis of S.arueus. These data indicated that L8 play a pivotal role in S.aureus.
3, Idenfication of the target genes regulated by L8. The gene arlR was predicted as one target of L8 by bioibformatics. The results of qPCR showed that the expression of arlR was decreased in L8 mutant strain. It was found that L8 could specifically bind arlR with the aid of Hfq. In the further investigation, arlR was indentified as one target of L8 by construction of reporter vector.
In summary, a novel functional sRNA-L8 in S.aureus was identified, which could regulate the expression of alrR and play an important role in the pathogenicity of S.arueus.
TRAP was thought as an important protein to influence the pathogenicity of S.aureu by regulating the expression of agr, but recent papers showed that TRAP could not influence the expression of agr. The biological function of TRAP is controversial. Our previous report suggested that the antibodies of TRAP could inhibit the infenciton cause by S.aureus. We tried to investigate the biological function of TRAP in the pathogenicity of S.aureus in the following study.
Chapter II: Study of the biological function of TRAP in S.aureu. In this chapter, we construced a trap deletion mutant from S.arueus 8325-4. It was investigated whether TRAP could regulate the expression of agr. In the further study, it was demonstrated that the trap deletion mutant was more sensitive to the phagocytosis of neutrophil. This part was divided into two parts.
1, Construction of the traP deletion mutant from S.aureus 8325-4. traP deletion mutant strain was prepared by gene reconbination. The agr system was not changed in the mutant, which suggested that TRAP could not influence the expression of agr. While, the infection caused by the mutant strain was decreased significantly, which indicated that TRAP was involved in the pathogenicity of S.aureus. Compared with its parent strain, a serial of phenotypes alternation were observed in the traP deletion strain. These data suggested that TRAP play an important role in S.aureus.
2, The TRAP protein was involved in the neutrophil phagocytosis of S.arueus. It was found that traP mutant was sensitive to the neutrophil phagocytosis compared with its parent strain. But the recombinant TRAP protein could not influence this process. In the further study, it was found the level of a protein on the cell wall of S.aureus was increased. This protein was indentified by MS. It was a protein of unknown function and named as SAP. The results of qPCR showed that the expression of sap was higher in the trap mutant strain than that in the wild type. Whether SAP is involved in the neutrophil phagocytosis and killing will be investigated in the future.
In a word, The TRAP protein could not influence the expression of agr, but it might play an important role in the pathogenicity of S.arueus by regulationg the interaction between neutrophil and S.aureus.
引文
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[2] Benton BM, Zhang JP, Bond S,et al. Large-scale identification of genes required for full virulence of Staphylococcus aureus.J Bacteriol,2004, 186:8478–8489.
[3] Chan PF, Foster SJ, Ingham E,et al. The Staphylococcus aureus alternative sigma factorσB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J Bacteriol,1998, 180:6082–6089.
[4] Lowy FD.Staphylococcus aureus infections. N Engl J Med,1998,339:520-532.
[5] Balaban N, Collins LV, Cullor JS,et al. Prevention of diseases caused by Staphylococcus aureus using the peptide RIP. Peptides ,2000, 21:1301–1311.
[6] McManus MT, Sharp PA. Gene Silencing in mammals by small interfering RNAs. Nat Rev Genet,2002, 3:737-747.
[7] Majdalani N, Vanderpool CK, Gottesman S. Bacterial Small RNA Regulators. Crit Rev Biochem Mol Biol, 2005,40:93–113.
[8] Wassarman KM.6S RNA: a regulator of transcription. Mol Microbiol 2007,65:1425–1431.
[9] Barrick JE, Sudarsan N, Weinberg Z, et al. 6S RNA is a widespread regulator ofeubacterial RNA polymerase that resembles an open promoter. RNA 2005,11: 774–784.
[10] Gildehaus N, Neusser T, Wurm R, et al. Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitrotranscription and synthesis of RNA-directed de novotranscripts. Nucleic Acids Res ,2007,35: 1885–1896.
[11] Gottesman S. Micros for microbes: Non-coding regulatory RNAs in bacteria. Trends Genet, 2005,21: 399–404.
[12] Storz G, Altuvia S, Wassarman KM. An abundance of RNA regulators. Annu Rev Biochem, 2005,74: 199–217.
[13] Pichon C, Felden B. Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains. Proc Natl Acad Sci, 2005, 102: 14249–14254.
[14] Roberts C, Anderson KL, Murphy E, et al. Characterizing the effect of the Staphylococcus aureus virulence factor regulator, SarA, on logphase mRNA half-lives. J Bacteriol, 2006, 188: 2593–2603.
[15] Abdelnour A, Arvidson S, Bremell T, et al. The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect. Immun, 1993,61:3879–3885.
[16] Chien Y and Cheung AL. Molecular interactions between two global regulators,sar and agr,in Staphylococcus aureus. J. Biol. Chem,1998, 273:2645–2652.
[17] Peng HL, Novick RP, Kreiswirth B, et al. Cloning, characterization, and sequencing of an accessory gene regu-lator (agr)in Staphylococcus aureus. J. Bacteriol. 1988,170:4365–4372.
[18] Novick RP, Ross HF, Projan SJ, et al. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J, 1993,12: 3967–3975.
[19] Novick RP. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol, 2003,48: 1429–1449.
[20] Morfeldt E, Taylor D, von Gabain A, et al. Activation ofα-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J, 1995,14:4569–4577.
[21] Huntzinger E, Boisset S, Saveanu C, et al. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J, 2005, 24: 824–835.
[22] Cheung AL, Eberhardt KJ, Chung E, et al. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis. J Clin Invest, 1994, 94: 1815–1822.
[23] Gillaspy AF, Hickmon SG, Skinner RA, et al. Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect Immun, 1995, 63: 3373–3380.
[24] Dunman PM, Murphy E, Haney S, et al. Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J Bacteriol, 2001, 183: 7341–7353.
[25] Gov Y, Borovok I, Korem M, et al. Quorum Sensing in Staphylococci Is Regulated via Phosphorylation of Three Conserved Histidine Residues. J. Biol. Chem,2004, 279:14665–14672.
[26] Vieira-da-Motta O, Ribeiro PD, Dias da Silva W, et al. RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production. Peptides, 2001, 22:1621-1627.
[27] Yang G, Gao Y, Dong J, et al. A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections. J Biol Chem,2005,280:27431–27435.
[28] Balaban N, Goldkorn T, Gov Y, et al. Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating protein(TRAP). J Biol Chem, 2001, 276:2658– 26.
[29] Shaw LN, Jonsson IM, Singh VK, et al. Inactivation of traP has no effect on the Agr quorum sensing system or virulence of Staphylococcus aureus. Infect and Immun, 2007,75(9): 4519-4527.
[30] Tsang LH, Daily ST, Weiss EC, et al. Mutation of traP in Staphylococcus aureushas no impact on expression of agr or biofilm formation. Infect and Immun, 2007,75:4528-4533.
[31] Adhikari RP, Arvidson S, Novick RP. A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP-. Infect and Immun, 2007,75:4534-4540.
[32] Singh VK, Jayaswal RK, Wilkinson BJ. Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiol Lett,2001,199:79-84.
[33] Valentin-Hansen P, Eriksen M and Udesen C. The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol,2004,51:1525–1533.
[34] Zhang A, Wassarman KM, Ortega J, et al. The Sm-like Hfq protein increases OxyS RNA interactionwith target mRNAs. Mol Cell, 2002,9:11–22。
[35] Brescia CC, Mikulecky PJ, Feig AL, et al. Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure. RNA,2003. 9: 33–43.
[36] Christiansen JK, Larsen MH, Ingmer H, et al. The RNA-binding protein Hfq of Listeria monocytogenes: Role in stress tolerance and virulence. J Bacteriol. 2004,186: 3355–3362.
[37] Fournier B and Hooper DC. A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus. J Bacteriol, 2000,182:3955–3964.
[38] Fournier B, Klier A and Rapoport G. The two-component system ArlS-ArlR is a regulator of virulence gene expression in Staphylococcus aureus. Mol Microbiol,2001,41:247–261.
[39] Liang X, Zheng L, Landwehr C, et al. Global Regulation of Gene Expression by ArlRS, a Two-Component Signal Transduction Regulatory System of Staphylococcus aureus. J Bacteriol,2005,8:5486–5492
[2] Benton BM, Zhang JP, Bond S,et al. Large-scale identification of genes required for full virulence of Staphylococcus aureus.J Bacteriol,2004, 186:8478–8489.
[3] Chan PF, Foster SJ, Ingham E,et al. The Staphylococcus aureus alternative sigma factorσB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J Bacteriol,1998, 180:6082–6089.
[4] Lowy FD.Staphylococcus aureus infections. N Engl J Med,1998,339:520-532.
[5] Balaban N, Collins LV, Cullor JS,et al. Prevention of diseases caused by Staphylococcus aureus using the peptide RIP. Peptides ,2000, 21:1301–1311.
[6] McManus MT, Sharp PA. Gene Silencing in mammals by small interfering RNAs. Nat Rev Genet,2002, 3:737-747.
[7] Majdalani N, Vanderpool CK, Gottesman S. Bacterial Small RNA Regulators. Crit Rev Biochem Mol Biol, 2005,40:93–113.
[8] Wassarman KM.6S RNA: a regulator of transcription. Mol Microbiol 2007,65:1425–1431.
[9] Barrick JE, Sudarsan N, Weinberg Z, et al. 6S RNA is a widespread regulator ofeubacterial RNA polymerase that resembles an open promoter. RNA 2005,11: 774–784.
[10] Gildehaus N, Neusser T, Wurm R, et al. Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitrotranscription and synthesis of RNA-directed de novotranscripts. Nucleic Acids Res ,2007,35: 1885–1896.
[11] Gottesman S. Micros for microbes: Non-coding regulatory RNAs in bacteria. Trends Genet, 2005,21: 399–404.
[12] Storz G, Altuvia S, Wassarman KM. An abundance of RNA regulators. Annu Rev Biochem, 2005,74: 199–217.
[13] Pichon C, Felden B. Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains. Proc Natl Acad Sci, 2005, 102: 14249–14254.
[14] Roberts C, Anderson KL, Murphy E, et al. Characterizing the effect of the Staphylococcus aureus virulence factor regulator, SarA, on logphase mRNA half-lives. J Bacteriol, 2006, 188: 2593–2603.
[15] Abdelnour A, Arvidson S, Bremell T, et al. The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect. Immun, 1993,61:3879–3885.
[16] Chien Y and Cheung AL. Molecular interactions between two global regulators,sar and agr,in Staphylococcus aureus. J. Biol. Chem,1998, 273:2645–2652.
[17] Peng HL, Novick RP, Kreiswirth B, et al. Cloning, characterization, and sequencing of an accessory gene regu-lator (agr)in Staphylococcus aureus. J. Bacteriol. 1988,170:4365–4372.
[18] Novick RP, Ross HF, Projan SJ, et al. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J, 1993,12: 3967–3975.
[19] Novick RP. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol, 2003,48: 1429–1449.
[20] Morfeldt E, Taylor D, von Gabain A, et al. Activation ofα-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J, 1995,14:4569–4577.
[21] Huntzinger E, Boisset S, Saveanu C, et al. Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J, 2005, 24: 824–835.
[22] Cheung AL, Eberhardt KJ, Chung E, et al. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis. J Clin Invest, 1994, 94: 1815–1822.
[23] Gillaspy AF, Hickmon SG, Skinner RA, et al. Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis. Infect Immun, 1995, 63: 3373–3380.
[24] Dunman PM, Murphy E, Haney S, et al. Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci. J Bacteriol, 2001, 183: 7341–7353.
[25] Gov Y, Borovok I, Korem M, et al. Quorum Sensing in Staphylococci Is Regulated via Phosphorylation of Three Conserved Histidine Residues. J. Biol. Chem,2004, 279:14665–14672.
[26] Vieira-da-Motta O, Ribeiro PD, Dias da Silva W, et al. RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production. Peptides, 2001, 22:1621-1627.
[27] Yang G, Gao Y, Dong J, et al. A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections. J Biol Chem,2005,280:27431–27435.
[28] Balaban N, Goldkorn T, Gov Y, et al. Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating protein(TRAP). J Biol Chem, 2001, 276:2658– 26.
[29] Shaw LN, Jonsson IM, Singh VK, et al. Inactivation of traP has no effect on the Agr quorum sensing system or virulence of Staphylococcus aureus. Infect and Immun, 2007,75(9): 4519-4527.
[30] Tsang LH, Daily ST, Weiss EC, et al. Mutation of traP in Staphylococcus aureushas no impact on expression of agr or biofilm formation. Infect and Immun, 2007,75:4528-4533.
[31] Adhikari RP, Arvidson S, Novick RP. A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP-. Infect and Immun, 2007,75:4534-4540.
[32] Singh VK, Jayaswal RK, Wilkinson BJ. Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiol Lett,2001,199:79-84.
[33] Valentin-Hansen P, Eriksen M and Udesen C. The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol,2004,51:1525–1533.
[34] Zhang A, Wassarman KM, Ortega J, et al. The Sm-like Hfq protein increases OxyS RNA interactionwith target mRNAs. Mol Cell, 2002,9:11–22。
[35] Brescia CC, Mikulecky PJ, Feig AL, et al. Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure. RNA,2003. 9: 33–43.
[36] Christiansen JK, Larsen MH, Ingmer H, et al. The RNA-binding protein Hfq of Listeria monocytogenes: Role in stress tolerance and virulence. J Bacteriol. 2004,186: 3355–3362.
[37] Fournier B and Hooper DC. A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus. J Bacteriol, 2000,182:3955–3964.
[38] Fournier B, Klier A and Rapoport G. The two-component system ArlS-ArlR is a regulator of virulence gene expression in Staphylococcus aureus. Mol Microbiol,2001,41:247–261.
[39] Liang X, Zheng L, Landwehr C, et al. Global Regulation of Gene Expression by ArlRS, a Two-Component Signal Transduction Regulatory System of Staphylococcus aureus. J Bacteriol,2005,8:5486–5492