摘要
在现代社会中,细菌性痢疾仍然是全球公共卫生安全面临重大威胁之一。在发展中国家,细菌性痢疾的危害尤为严重。志贺氏菌属于革兰氏阴性杆菌,是导致细菌性痢疾的首要病原体。志贺氏菌可分为四个血清群,包括了A群：志贺氏痢疾杆菌(S.dysenteriae),B群：福氏痢疾杆菌(S.flexneri),C群：鲍氏痢疾杆菌(S.boydii)和D群：宋内氏痢疾杆菌(S. sonnei)。在我国,福氏痢疾杆菌为主要的流行菌株。在以往的研究工作中,我们于2001年完成了福氏痢疾杆菌2a301株的全基因组序列测定,是我国首个独立完成的微生物基因组项目,为志贺氏菌的致病机理研究提供了完整的遗传背景信息。
志贺氏菌的基因组序列与大肠杆菌高度同源,其致病性主要基于细菌中约230Kb的侵袭性大质粒。痢疾杆菌的毒力基因集中分布在侵袭性大质粒中约31Kb的”entry region"中。这些毒力基因编码了包括细菌毒素,三型分泌系统(T3SS)的结构蛋白及效应因子等大量蛋白质。因此,毒力基因的表达无疑为细菌造成巨大的代谢负担。因此,在进化的过程中,志贺氏菌形成了一个精巧的毒力基因转录和表达调控体系,由来自侵袭性大质粒的调控系统和来自细菌基因组编码的全局调控系统共同组成。
在不适宜的环境条件下,志贺氏菌的毒力基因的表达受到抑制。染色体编码的H-NS蛋白通过结合毒力基因启动子附近的AT富集区组装转录抑制复合物,阻止RNA聚合酶向启动子下游移动,从而在转录水平抑制毒力基因表达。在适宜环境因素的诱导下,即温度,pH值和渗透压等环境参数与小肠内环境接近时,志贺氏菌的毒力基因的转录开始激活。毒力基因的激活通过一系列级联调控步骤实现。首先包括了virF基因的转录激活,其产物VirF蛋白继而激活virB基因的转录,造成VirB蛋白的表达上调。VirB蛋白能够特异性结合位于毒力基因启动子上游的DNA顺式作用位点(cis-acting site),从而解除H-NS介导的转录抑制作用,最终导致位于侵袭性大质粒上的各种毒力基因的转录的全面激活。受到VirB蛋白调控的毒力基因还包括了VirB自身的编码基因及其上游调控基因virF。
VirB蛋白在志贺氏菌的毒力基因转录调控中具有核心作用,然而VirB如何解除H-NS的转录抑制作用的分子机理尚不清楚。氨基酸序列比对分析表明VirB蛋白与传统转录调控因子的同源性很低,却与质粒分离蛋白ParB, SopB, KorB同源性较高。VirB特异性结合位于启动子上游的顺式作用位点,该位点与ParB类蛋白识别的ParS序列具有高度的同源性,均包括了两个保守的反向重复序歹Box-1和Box-2。由此可见,VirB蛋白与质粒分离蛋白密切相关。然而,现有的实验证据表明VirB蛋白未参与质粒分配过程。由此,我们推测,VirB蛋白可能在进化的过程中逐渐失去了原有的质粒分配功能,扮演了基因转录调控的角色,然而其祖先分子的结构特征及与DNA相互作用的特性被保留下来。
为揭示VirB蛋白特异性识别DNA顺式作用位点的分子基础和VirB蛋白激活毒力基因转录的分子机制,我们结合了结构生物学,生物物理及生物化学和细菌学等多学科技术方法,针对VirB-DNA相互作用开展了系统的研究。
我们首先解析了VirB蛋白核心结构域(VirB core)与icsB毒力基因上游DNA顺式作用位点icsB-WT (26bp)复合物,VirB蛋白核心结构域与icsP毒力基因上游DNA顺式作用位点icsP-WT (31bp)复合物晶体结构,揭示了VirB蛋白与ParB类蛋白在多肽骨架结构上同源,具有典型的HTH motif,我们的晶体结构展示：VirB核心结构域直接结合顺式作用位点DNA双螺旋结构的“大沟”(major groove),是VirB蛋白识别序列特异性的决定因素。VirB核心结构域与DNA磷酸骨架及DNA碱基之间形成11个稳定的分子间盐桥和氢键,从而实现对DNA序列的“阅读”,其中最重要的DNA序列识别由精氨酸R167完成。我们发现：R167的侧链与DNA顺式作用位点icsB-WT(?)UicsP-WT中的Box-2第三位的鸟氨酸碱基形成两个重要的氢键,是唯一识别DNA碱基的氨基酸。与ParB类似蛋白结合DNA复合物的晶体结构比对发现,ParB类似蛋白结合反向重复序列中的两个Box,而VirB蛋白仅结合反向重复序列中的一个,即Box-2,从而揭示了VirB蛋白与ParB类蛋白在DNA结合方面最核心的区别。我们随后开展突变实验,验证了参与DNA识别的11个氨基酸中有10个氨基酸是VirB转录激活活性所必须的,从而建立了VirB识别DNA序列特异性的结构基础并验证了序列识别与其功能的关联。我们进而对结合于VirB蛋白的DNA icsB-WT(?)UicsP-WT的结构进行分析,发现DNA双螺旋结构发生扭曲,主要体现在DNA螺旋轴发生明显的弯曲及DNA“小沟”(minor groove)变窄等。DNA双螺旋构象的变化证明了VirB蛋白对DNA结构改变的能力。而这种能力是ParB类似蛋白所不具有的。我们接下来利用荧光光光谱分析的方法研究了VirB全长蛋白(包括了N-端结构域,核心结构域和C端结构域)与DNA顺式作用位点的相互作用。我们发现VirB的N-端和C-端结构域均具有DNA结合能力,然而这种DNA结合能力是不依赖DNA的碱基序列的。VirB与DNA的结合具有显著的协同性,其原因为VirB的C-端结构域可介导VirB蛋白的寡聚化,从而为DNA的结合提供多个位点。
综合以上研究成果,我们最终提出VirB解除H-NS抑制毒力基因转录的分子模型,即VirB蛋白通过C-端结构域发生寡聚化,形成VirB的多聚体。VirB通过核心结构域准确定位到启动子上游的顺式结合位点,并结合反向重复序列中的Box-2。VirB的结合引发DNA双螺旋的构象变化,主要体现在：造成DNA双链在顺式作用位点处发生弯曲；DNA的弯曲使进而引起下游DNA结合到VirB多聚体的其他DNA结合位点；最终造成DNA在VirB多聚体上的缠绕。VirB诱导的DNA构象变化可破坏H-NS-DNA转录抑制复合物的稳定性,释放RNA聚合酶复合物,激活毒力基因的转录。
志贺氏菌不但是重要的病原微生物,而且是细菌致病性研究中重要的模式生物之一。细菌毒力和侵袭力的调控机制显然细菌致病性研究的核心科学问题之一,也是药物设计的重要靶标。本研究阐明了VirB识别DNA顺式作用序列的结构基础,提出了VirB解除H-NS介导的转录抑制作用的分子模型,为阐明痢疾杆菌毒力基因调控机理提供了重要基础,为相关的抑制剂及药物设计提供了科学理论依据。
Bacillary dysentery remains the leading public health threat, especially in developing country. Shigella, a Gram-negative facultative intracellular pathogen of humans, is the primary causative agent of bacillary dysentery. Shigella can be subdivided into four species, S. flexineri, S dysenteriae, S. sonnei and S. boydii, in which most of the pathogenicity studies were carried out on the prevalent S. flexineri. We previously completed the genome sequencing of the Shigella flexneri2a301strain in2001, the most prevalent strain in China, providing the complete genetic background for further studies. This project presents the first microbial genome decoded by chinese scientists.
The genomic sequence of S. flexneri is highly homologous to the non-pathogenic E. coli. The virulence of S. flexneri depends on the virulence genes clustered within the so called "entry region", a31-Kb segment on the virulence plasmid (～230-Kb). These genes encode a variety of proteins, including invasins, structural proteins forming the sophisticated type Ⅲ secretion system and the effector proteins. The vast quantity of the virulence genes products obviously presents a great metabolic burden for the bacteria; therefore, the bacteria has developed a sophisticated regulatory system to control the expression of the virulence genes, comprising of the plasmid encoded regulatory system and the chromosome encoded global gene regulation.
When the growth conditions are not ideal for invasion, the transcription of the virulence genes is repressed by a chromosomally encoded heat-stable nucleoid structural protein (H-NS). It is believed that H-NS can bind the AT-rich DNA segment near the promoters, forming the repressive nucleoprotein structures that function as the obstacles to impede the movements of RNA polymerase, thereby silent the transcription. In response to proper environmental signals, i.e. pH value, temperature and osmolarity similar to the environment in the lower intestine of the host, the H-NS mediated transcriptional repression can be alleviated by the plasmid encoded regulatory system. A transcriptional cascade is then initiated with the activation of virF gene expressing an AraC-like protein VirF that in turn activates the transcription of the virB regulatory gene. The gene product VirB protein consequently relieves the H-NS mediated transcriptional repression, leading to the activation of the virulence genes on the plasmid. VirB also activates the transcription of its own gene and feeds back positively onto the transcription of the virF upstream regulatory gene.
VirB play an central role in transcriptional regulation of the virulence genes; however, the molecular basis for the alleviation of H-NS mediated promoter repression remained unclear. The amino acid sequence homology analysis reveals that VirB shares little homology with the conventional transcriptional regulators, but the protein is homologues to the ParB like proteins involving the plasmid partitioning. VirB can specifically bind the cis-acting site upstream the promoter region, which share the key elements with the ParS site recognized by the ParB-like proteins. Therefore, the mode of VirB-DNA interaction must be related to ParB-ParS interactions. We speculate that VirB was reassigned exclusively to the regulatory roles during the evolution, and many key structural features of its ancestor were well preserved.
To understand molecular basis underlying recognition of the cis-acting site by VirB and its role in transcriptional activation of the virulence genes, we employed the methods of structural biology, biophysics&biochemistry and bacteriology to investigate the mode of VirB-DNA interaction.
We first solved the crystal structure of VirB core complexed by the cis-acting site upstream of icsB promoter icsB-WT (26bp), VirB core complexed by the cis-acting site upstream of icsP promoter icsP-WT (31bp). The crystal structures reveal that the overall structure of VirB core is related to ParB-like proteins, containg a typical HTH domain. The crystal structures show that VirB Core domain binds directly to DNA major groove of the cis-acting site, presenting the structural determinant for DNA sequence specificity. VirB core provides11stable intermolecular hydrogen bonds or salt bridges to the DNA backbone phosphates and base, facilitating readout of DNA sequence. We found that the hydrogen bonds donated by the side chain of R167to the guanosine base at the3rd position of the Box2in the cis-acting sites present the only DNA base reading by VirB. The structural comparison of VirB-DNA complex to the ParB-ParS like complexes, We found VirB binds only one of the half sites in the inverted repeats,ParB binds two of the half sites simultaneously.which inform the most difference between VirB and ParB for DNA recognizion We next carried out the mutagenesis studies, confirming that10residues out of the11residues providing contacts to DNA are essential for the activity of VirB in transcriptional activation, thereby established the structure basis for DNA sequence specificity and the correlation of the sequence recognition and the function of VirB. We further analyzed the structure of DNA double helices of icsB-WT and icsP-WT, and discovered the conformation of the DNA bound by VirB deviates from the standard DNA. The distortion of DNA involves the bending of helical axis and the narrowing of the DNA minor groove. The conformational changes observed in DNA demonstrated the capability of VirB of introducing distortion of DNA double helix, a capability not shared by ParB-like proteins.We performed fluorescence anisotropy experiments to study the binding capability of VirB variants to a fluorescently labeled DNA cis-acting site.the results showed that both N-and C-terminal portion of VirB contribute significantly to the DNA binding affinity, which is independent of DNA sequence. The cooperative DNA binding capability of VirB owns to its C-terminal domain that mediates the oligomerization of the protein, suggesting that the high-order VriB oligomer is the functional assembly providing multiple sites for DNA binding.
Our findings suggest a model for VirB-mediated promoter activation.The C-terminal domain of VirB facilitates the oligomerization and assembles the functional high-order VirB oligomer. The HTH domain of VirB recognizes a specific cis-acting site upstream the promoter, thus anchors the VirB oligomer to DNA. The binding of VirB HTH domain induces the distortion at the AT-rich segment in VirB binding site, which may subsequently promote the adjacent DNA strand bending toward the VirB oligomer, therefore assists the nearby DNA strand to be loaded on the other DNA binding site on the oligomer. Thereby, the DNA wrapping around VirB oligomer initiates. Due to that the specific VirB binding site is limited, the subsequent DNA strand loading requires nonspecific DNA binding capability of VirB. The high-order VirB oligomer combines multiple DNA binding sites from the VirB monomers, therefore a considerable long DNA strand could be accommodated. The DNA wrapping around VirB oligomer subsequently remodels the DNA upstream the promoter and destabilizes the repressive H-NS-DNA complexes, such as the H-NS-DNA-H-NS bridges. Eventually, the trapped RNA polymerase is then released and the transcription resumes.
Shigella is not only an important pathogen for humans but also a model organism for the research of the bacteria pathogenicity. The molecular mechanism underlying the virulence gene regulation is one of the key question to be addressed in the field of pathogenic bacteria, severing a important target for drug design. Our studies have provided the structural basis for the recognition of the cis-acting site by VirB, thus, proposed a model for the relieving of H-NS mediated transcriptional repression. Our works provided the basis for the understanding of virulence gene regulation and a framework for the structure-based inhibitor design.
引文
[1]Von seidlein,L.,e, al A multicentre study of Shigella diarrhoea in six Asian countries; disease burden, clinical manifestations and microbiology.[J].PloS Med 2006,3:e353
[2]Sansonetti, P. J. Shigellosis:an old disease in new clothes? [J].PloS Med.2006 3:e354.
[3]World Health Organization.Initiative for vaccine research (IVR).New vaccines against infectious disesases:research and development status. World Health Organization, Geneva, Switzerland.2006.
[4]DuPont, H. L., M. M. Levine, R. B. Hornick, and S. B. Formal. Inoculum size in shigellosis and implications for expected mode of transmission. [J]. J. Infect. Dis.1989.159:1126-1128.
[5]Gorden, J., and P. L. Small. Acid resistance in enteric bacteria. [J]. Infect. Immun.1993.61:364-367.
[6]Islam, D., L. Bandholtz, J. Nilsson, H. Wigzell, B. Christensson, B. Agerberth, and G. Gudmundsson. Downregulation of bactericidal peptides in enteric infections:a novel immune escape mechanism with bacterial DNA as a potential regulator. [J]. Nat. Med.2001.7:180-185
[7]Sansonetti, P. J. War and peace at mucosal surfaces. [J]. Nat. Rev. Immunol. 2004.4:953-964
[8]Sansonetti, P. J., J. Arondel, R. Cantey, M. C. Prevost, and M. Huerre. Infection of rabbit Peyer's patches by Shigella flexneri:effect of adhesive or invasive bacterial phenotypes on follicle-associated epithelium. [J].Infect. Immun. 1996.64:2752-2764
[9]Man, A. L., M. E. Prieto-Garcia, and C. Nicoletti. Improving M cell-mediated transport across mucosal barriers:do certain bacteria hold the keys? [J].Immunology.2004.113:15-22
[10]Mounier, J., T. Vasselon, R. Hellio, M. Lesourd, and P. J. Sansonetti. Shigella flexneri enters human colonic Caco-2 epithelial cells through the basolateral pole. [J]. Infect. Immun.1992.60:237-248
[11]Islam, D., B. Veress, P. K. Bardhan, A. A. Lindberg, and B. Christensson. In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis. [J]. Infect. Immun.1997.65:739-749
[12]Zychlinsky, A., M. C. Pre'vost, and P. J. Sansonetti. Shigella flexneri induces apoptosis in infected macrophages. [J]. Nature 1992.358:167-168.
[13]Zychlinsky, A., K. Thirumalai, J. Arondel, J. R. Cantey, A. Aliprantis, and P. J. Sansonetti. In vivo apoptosis in Shigella flexneri infections. [J]. Infect. Immun. 1996.64:5357-5365.
[14]Zychlinsky, A., C. Fitting, J. M. Cavaillon, and P. J. Sansonetti. Interleukin-1 is released by murine macrophages during apoptosis induced by Shigella flexneri. [J].J. Clin. Investig.1994.94:1328-1332.
[15]Sansonetti, P. J., A. Ryter, P. Clerc, A. T. Maurelli, and J. Mounier. Multiplication of Shigella flexneri within HeLa cells:lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. [J]. Infect. Immun.1986. 51:461-469
[16]Perdomo, O. J., J. M. Cavaillon, M. Huerre, H. Ohayon, P. Gounon, and P. J. Sansonetti. Acute inflammation causes epithelial invasion and mucosal destruction in experimental shigellosis. [J]. J. Exp. Med.1994.180:1307-1319.
[17]Sakaguchi, T., H. Kohler, X. Gu, B. A. McCormick, and H. C. Reinecker. Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells. [J].Cell. Microbiol.2002.4:367-381
[18]Laohachai, K. N., R. Bahadi, M. B. Hardo, P. G. Hardo, and J. I. Kourie. The role of bacterial and non-bacterial toxins in the induction of changes in membrane transport:implications for diarrhea. [J].Toxicon 2003.42:687-707.
[19]Jennison,A.V.,andVerma,N.K. Shigella flexneri infection:pathogenesis and vaccine development. [J]. FEMS Microbiol Rev.2004.28:43-58.
[20]Sasakawa, C., K. Kamata, T. Sakai, S. Y. Murayama, S. Makino, and M. Yoshikawa. Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigellaflexneri. [J]. Infect. Immun.1986.54:470-475.
[21]Buchrieser, C., P. Glaser, C. Rusniok, H. Nedjari, H. D'Hauteville, F. Kunst, P. Sansonetti, and C. Parsot. The virulence plasmid pWR100 and the repertoire of proteins secreted by the type Ⅲ secretion apparatus of Shigella flexneri. [J]. Mol. Microbiol.2000.38:760-771.
[22]Sasakawa, C., K. Komatsu, T. Tobe, T. Suzuki, and M. Yoshikawa. Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a. [J].J. Bacteriol.1993.175:2334-2346.
[23]Sasakawa, C., S. Makino, K. Kamato, and M. Yoshikawa. Isolation, characterization, and mapping of Tn5 insertions into the 140-megadalton invasion plasmid defective in the mouse Sereny test in Shigella flexneri 2a. [J].Jnfect. Immun.1986.54:32-36.
[24]Buysse, J. M., C. K. Stover, E. V. Oaks, M. Venkatesan, and D. J. Kopecko. Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping. [J].J.Bacteriol.1987. 169:2561-2569
[25]Oaks, E. V., T. L. Hale, and S. B. Formal. Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp. [J]. Infect. Immun.1986.53:57-63
[26]Adler, B., C. Sasakawa, T. Tobe, S. Makino, K. Komatsu, and M. Yoshikawa. A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. [J]. Mol. Microbiol. 1989.3:627-635.
[27]Van Eerde, A., C. Hamiaux, J. Perez, C. Parsot, and B. W. Dijkstra. Structure of Spa15, a type Ⅲ secretion chaperone from Shigella flexneri with broad specificity. [J].EMBO Rep.2004.5:477-483.
[28]Zurawski, D. V., C. Mitsuhata, K. L. Mumy, B. A. McCormick, and A. T. Maurelli. OspF and OspC1 are Shigella flexneri type Ⅲ secretion system effectors that are required for postinvasion aspects of virulence. [J]. Infect. Immun. 2006.74:5964-5976.
[29]Venkatesan, M. M., J. M. Buysse, and A. B. Hartman. Sequence variation in two ipaH genes of Shigella flexneri and homology to the LRG-like family of proteins. [J]. Mol. Microbiol.1991.5:2435-2446.
[30]Bell, J. K., G. E. Mullen, C. A. Leifer, A. Mazzoni, D. R. Davies, and D. M. Segal. Leucine-rich repeats and pathogen recognition in Toll-like receptors. [J].Trends Immunol.2003.24:528-533.
[31]Fritz, J. H., R. L. Ferrero, D. J. Philpott, and S. E. Girardin.2006. Nod-like proteins in immunity, inflammation and disease. [J].Nat. Immunol.7:1250- 1257.
[32]Martinon, F., and J. Tschopp. NLRs join TLRs as innate sensors of pathogens. [J].Trends Immunol.2005.26:447-454.
[33]Yoshida, S., Y. Handa, T. Suzuki, M. Ogawa, M. Suzuki, A. Tamai, A. Abe, E. Katayama, and C. Sasakawa. Microtubule-severing activity of Shigella is pivotal for intercellular spreading. [J].Science.2006.314:985-989.
[34]Yoshida, S., E. Katayama, A. Kuwae, H. Mimuro, T. Suzuki, and C. Sasakawa. Shigella delivers effector proteins to trigger host microtubule destabilization, which promotes Racl activity and efficient bacterial internalization. [J]. EMBO J. 2002.21:2923-2935.
[35]Bernardini, M.L., Sanna, M.G., Fontaine, A., and Sansonetti, P.J. OmpC is involved in invasion of epithelial cells by Shigella flexneri. [J]. Infect Immun.1993.61:3625-3635.
[36]Nakayama, S., and Watanabe, H. Involvement of cpxA, a sensor of a two component regulatory system, in the pH-dependent regulation of expression of Shigella sonnei virF gene. [J]. J Bacteriol.1995.177:5062-5069.
[37]Falconi, M., Prosseda, G., Giangrossi, M., Beghetto, E., and Colonna, B. Involvement of FIS in the H-NS-mediated regulation of VirF gene of Shigella and enteroinvasive Escherichia coli. [J]. Mol Microbiol.2001.42:439-452.
[38]Tobe, T., Yoshikawa, M., Mizuno, T., and Sasakawa, C. Transcriptional control of the invasion regulatory gene virB of Shigella flexneri:activation by VirF and repression by H-NS. [J]. J Bacteriol.1993.175:6142-6149.
[39]Sakai, T., Sasakawa, C., and Yoshikawa, M. Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kilo Dalton VirF protein. [J]. Mol Microbiol.1988.2:589-597.
[40]Le Gall, T., Mavris, M., Martino, M.C., Bernardini, M.L., Denamur, E., and Parsot, C. Analysis of virulence plasmid gene expression defines three classes of effectors in the type Ⅲ secretion system of Shigella flexneri. [J]. Microbiology. 2005.151:951-962.
[41]Backert, S., and T. F. Meyer. Type IV secretion systems and their effectors in bacterial pathogenesis. [J].Curr. Opin. Microbiol.2006.9:207-217.
[42]Blocker, A., N. Jouihri, E. Larquet, P. Gounon, F. Ebel, C. Parsot, P. Sansonetti, and A. Allaoui. Structure and composition of the Shigella flexneri "needle complex," a part of its type Ⅲ secreton. [J] Mol. Microbiol.2001. 39:652-663.
[43]Sani, M., A. Allaoui, F. Fusetti, G. T. Oostergetel, W. Keegstra, and E. J. Boekema. Structural organization of the needle complex of the type Ⅲ secretion apparatus ofShigella flexneri. [J].Micron.2007.38:291-301.
[44]Tamano, K., S. Aizawa, E. Katayama, T. Nonaka, S. Imajoh-Ohmi, A. Kuwae, S. Nagai, and C. Sasakawa. Supramolecular structure of the Shigella type Ⅲ secretion machinery:the needle part is changeable in length and essential for delivery of effectors. [J].J SMBO J.2000.19:3876-3887.
[45]Schuch, R., and A. T. Maurelli. MxiM and MxiJ, base elements of the Mxi-Spa type Ⅲ secretion system of Shigella, interact with and stabilize the MxiD secretin in the cell envelope. [J].J. Bacteriol 2001.183:6991-6998
[46]Blocker, A., K. Komoriya, and S. Aizawa. Type Ⅲ secretion systems and bacterial flagella:insights into their function from structural similarities. [J].Proc. Natl. Acad. Sci. USA.2003.100:3027-3030.
[47]Cornelis, G. R. The type Ⅲ secretion injectisome. [J].J Nat. Rev. Microbiol. 2006.4:811-825.
[48]Schuch, R., and A. T. Maurelli. Spa33, a cell surface-associated subunit of the Mxi-Spa type Ⅲ secretory pathway of Shigella flexneri, regulates Ipa protein traffic. [J].Infect. Immun.2001.69:2180-2189
[49]Tamano, K., E. Katayama, T. Toyotome, and C. Sasakawa. Shigella Spa32 is an essential secretory protein for functional type Ⅲ secretion machinery and uniformity of its needle length. [J].J. Bacteriol.2002.184:1244-1252
[50]West, N. P., P. Sansonetti, J. Mounier, R. M. Exley, C. Parsot, S. Guadagnini, M. C. Prevost, A. Prochnicka-Chalufour, M. Delepierre, M. Tanguy, and C. M. Tang. Optimization of virulence functions through glucosylation of Shigella LPS. [J].Science.2005.307:1313-1317
[51]Magdalena, J., A. Hachani, M. Chamekh, N. Jouihri, P. Gounon, A. Blocker, and A. Allaoui. Spa32 regulates a switch in substrate specificity of the type Ⅲ secreton of Shigella flexneri from needle components to Ipa proteins. [J].J. Bacteriol.2002.184:3433-3441
[52]Sorg, J. A., B. Blaylock, and O. Schneewind. Secretion signal recognition by YscN, the Yersinia type Ⅲ secretion ATPase.[J].Proc. Natl. Acad. Sci. USA.2006.103:16490-16495.
[53]Veenendaal, A. K., J. L. Hodgkinson, L. Schwarzer, D. Stabat, S. F. Zenk, and A. J. Blocker. The type Ⅲ secretion system needle tip complex mediates host cell sensing and translocon insertion. [J].Mol. Microbiol.2007.63:1719-1730
[54]Johnson, S., P. Roversi, M. Espina, A. Olive, J. E. Deane, S. Birket, T. Field, W. D. Picking, A. J. Blocker, E. E. Galyov, W. L. Picking, and S. M. Lea. Self-chaperoning of the type Ⅲ secretion system needle tip proteins IpaD and BipD. [J].J. Biol. Chem.2007.282:4035-4044
[55]Blocker, A., P. Gounon, E. Larquet, K. Niebuhr, V. Cabiaux, C. Parsot, and P. Sansonetti. The tripartite type Ⅲ secreton of Shigella flexneri inserts IpaB and IpaC into host membranes. [J].J. Cell Biol.1999.147:683-693
[56]Espina, M., A. J. Olive, R. Kenjale, D. S. Moore, S. F. Ausar, R. W. Kaminski, E. V. Oaks, C. R. Middaugh, W. D. Picking, and W. L. Picking. IpaD localizes to the tip of the type Ⅲ secretion system needle of Shigella flexneri. [J].Infect. Immun.2006.74:4391-4400
[57]Garner, M. J., R. D. Hayward, and V. Koronakis. The Salmonella pathogenicity island 1 secretion system directs cellular cholesterol redistribution during mammalian cell entry and intracellular trafficking. [J]. Cell. Microbiol. 2002.4:153-165.
[58]Simons, K., and W. L. Vaz. Model systems, lipid rafts, and cell membranes. [J]. Annu. Rev. Biophys. Biomol. Struct.2004.33:269-295.
[59]Hayward, R. D., R. J. Cain, E. J. McGhie, N. Phillips, M. J. Garner, and V.Koronakis. Cholesterol binding by the bacterial type Ⅲ translocon is essential for virulence effector delivery into mammalian cells. [J]Mol. Microbiol. 2005. 56:590-603
[60]Skoudy, A., J. Mounier, A. Aruffo, H. Ohayon, P. Gounon, P. Sansonetti, and G. Tran Van Nhieu. CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells. [J]. Cell. Microbiol.2000.2:19-33.
[61]Watarai, M., S. Funato, and C. Sasakawa. Interaction of Ipa proteins of Shigella flexneri with integrin promotes entry of the bacteria into mammalian cells. [J].J. Exp. Med.1996.183:991-999.
[62]Boquet, P., and E. Lemichez. Bacterial virulence factors targeting Rho GTPases:parasitism or symbiosis? [J]. Trends Cell Biol.2003.13:238-246
[63]Tran Van Nhieu, G., E. Caron, A. Hall, and P. J. Sansonetti. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells. [J].EBO J.1999.18:3249-3262
[64]Yoshida, S., E. Katayama, A. Kuwae, H. Mimuro, T. Suzuki, and C. Sasakawa. Shigella delivers effector proteins to trigger host microtubule destabilization, which promotes Racl activity and efficient bacterial internalization.[J].EMBO J.2002.21:2923-2935
[65]Alto, N. M., F. Shao, C. S. Lazar, R. L. Brost, G. Chua, S. Mattoo, S. A. McMahon, P. Ghosh, T. R. Hughes, C. Boone, and J. E. Dixon. Identification of a bacterial type Ⅲ effector family with G protein mimicry functions. [J].Cell.2006. 124:133-145.
[66]Demali, K. A., A. L. Jue, and K. Burridge. IpaA targets beta 1 integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry. [J]. J. Biol. Chem.2006.281:39534-39541.
[67]Ramarao, N., C. Le Clainche, T. Izard, R. Bourdet-Sicard, E. Ageron, P. J. Sansonetti, M. F. Carlier, and G. Tran Van Nhieu. Capping of actin filaments by vinculin activated by the Shigella IpaA carboxyl-terminal domain. [J]. FEBS Lett. 2007.581:853-857.
[68]Stevens, J. M., E. E. Galyov, and M. P. Stevens. Actin-dependent movement of bacterial pathogens. [J]. Nat. Rev. Microbiol.2006.4:91-101.
[69]Santapaola, D., F. Del Chierico, A. Petrucca, S. Uzzau, M. Casalino, B. Colonna, R. Sessa, F. Berlutti, and M. Nicoletti. Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene of Shigella flexneri, is necessary for proper unipolar IcsA localization and for efficient intercellular spread. [J]. J. Bacteriol.2006.188:1620-162
[70]Suzuki, T., H. Miki, T. Takenawa, and C. Sasakawa. Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flexneri. [J]. EMBO J.1998.17:2767-2776.
[71]Suzuki, T., H. Mimuro, H. Miki, T. Takenawa, T. Sasaki, H. Nakanishi, Y. Takai, and C. Sasakawa.2000. Rho family GTPase Cdc42 is essential for the actin-based motility of Shigella in mammalian cells. [J]. J. Exp. Med.191: 1905-1920.
[72]Suzuki, T., H. Mimuro, S. Suetsugu, H. Miki, T. Takenawa, and C. Sasakawa. Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading. [J]. Cell. Microbiol.2002.4:223-233.
[73]Yoshida, S., Y. Handa, T. Suzuki, M. Ogawa, M. Suzuki, A. Tamai, A. Abe, E. Katayama, and C. Sasakawa. Microtubule-severing activity of Shigella is pivotal for intercellular spreading. [J]. Science 2006.314:985-989.
[74]Ogawa, M., T. Yoshimori, T. Suzuki, H. Sagara, N. Mizushima, and C. Sasakawa. Escape of intracellular Shigella from autophagy.[J]. Science.2005.307 :727-731.
[75]Picking, W. L., H. Nishioka, P. D. Hearn, M. A. Baxter, A. T. Harrington, A. Blocker, and W. D. Picking. IpaD of Shigella flexneri is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes. [J]. Infect. Immun.2005.73:1432-1440.
[76]Rathman, M., N. Jouirhi, A. Allaoui, P. Sansonetti, C. Parsot, and G. Tran Van Nhieu. The development of a FACS-based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread. [J]. Mol. Microbiol. 2000.35:974-990
[77]Schuch, R., R. C. Sandlin, and A. T. Maurelli. A system for identifying post-invasion functions of invasion genes:requirements for the Mxi-Spa type Ⅲ secretion pathway of Shigella flexneri in intercellular dissemination.[J]. Mol. Microbiol. 1999.34:675-689.
[78]Watanabe H., Arakawa E., Ito K., Kato J., Nakamura A. Genetic analysis of an invasion region by use of a Tn3-lac transposon and identification of a second positive regulator gene, invE, for cell invasion of Shigella sonnei: significant homology of invE with ParB of plasmid P1. [J].J. Bacteriol. 1990.172:619-629
[79]Radnedge L, Davis MA, Austin SJ. P1 and P7 plasmid partition:ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity. [J]. EMBO J.1996.5.1;15(5):1155-1162
[80]Surtees, J. A., and B. E. Funnell. The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex. [J]. J. Biol. Chem.2001. 276:12385-12394.
[81]Surtees, J.A. & Funnell, B.E. P1 ParB domain structure includes two independent multimerization domains. [J]. J Bacteriol.1999.181:5898-5908.
[82]Beloin, C., S. McKenna, and C. J. Dorman. Molecular dissection of VirB, a key regulator of the virulence cascade of Shigella flexneri. [J]. J. Biol. Chem. 2002.277:15333-15344
[83]Taniya, T., J. Mitobe, S. Nakayama, Q. Mingshan, K. Okuda, and H. Watanabe. Determination of the InvE binding site required for expression of IpaB of the Shigella sonnei virulence plasmid:involvement of a ParB BoxA-like sequence. [J].J. Bacteriol.2003.185:5158-5165
[84]Wing, H. J., Yan, A. W., Goldman, S. R. & Goldberg, M. B. Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB. [J].JBacteriol.2004.186,699-705.
[85]Badaut, C., Williams, R., Arluison, V., Bouffartigues, E., Robert, B., Buc, H. & Rimsky, S. The degree of oligomerization of the H-NS nucleoid structuring protein is related to specific binding to DNA. [J].J Biol Chem.2002.211, 41657-41666
[86]Noom, M. C., Navarre, W. W., Oshima, T., Wuite, G. J.& Dame, R. T. H-NS promotes looped domain formation in the bacterial chromosome. [J]. Curr Biol.2001.17,R913-R914
[87]Tolstorukov, M. Y., Virnik, K. M., Adhya, S. & Zhurkin, V. B. A-tract clusters may facilitate DNA packaging in bacterial nucleoid. [J].Nucleic Acids Res.2005. 33,3907-3918.
[88]Turner, E. C. & Dorman, C. J. H-NS antagonism in Shigella flexneri by VirB, a virulence gene transcription regulator that is closely related to plasmid partition factors. [J]. J Bacteriol.2001.189,3403-3413.
[89]Castellanos MI, et al. VirB alleviates H-NS repression of the icsP promoter in Shigella flexneri from sites more than one kilobase upstream of the transcription start site. [J]. J. Bacteriol.2009.191:4047-4050.
[90]Schumacher, M. A.& Funnell, B. E. Structures of ParB bound to DNA reveal mechanism of partition complex formation. [J]. Nature.2005.438,516-519
[91]Radnedge, L., Davis, M. A.& Austin, S. J. P1 and P7 plasmid partition:ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity. [J]. EMBO J..1996.15,1155-1162
[92]Surtees, J. A.& Funnell, B. E. The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex. [J]. J. Biol. Chem.2001.276, 12385-12394
[93]Lukaszewicz, M. et al. Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2. [J]. Nucleic Acids Res.2002.30,1046-1055
[94]Delbruck, H., Ziegelin, G, Lanka, E. & Heinemann, U. An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4.[J]. J.Biol Chem.2002.277, 4191-4198
[95]Khare, D., Ziegelin, G., Lanka, E. & Heinemann, U. Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB. [J]. Nature Struct. Mol. Biol 2004.11,656-663
[96]Leonard, T. A., Butler, P. J. G. & Lowe, J. Structural analysis of the chromosome segregation protein SpoOJ from Thermus thermophilus. [J]. Mol. Microbiol.2004.53,419-432
[97]Bouet JY, Ah-Seng Y, Benmeradi N, Lane D:Polymerization of SopA partition ATPase:regulation of DNA binding and SopB. [J]. Mol Microbiol.2007, 63:468-481.
[98]Castaing JP, Bouet JY, Lane D:F plasmid partition depends on interaction of SopA with non-specific DNA. [J]. Mol Microbiol.2008,70:1000-1011.
[99]Bouet JY, Lane D:Molecular basis of the supercoil deficit induced by the mini-F plasmid partition complex. [J]. JBiol Chem.2009,284:165-173.
[100]Schumacher MA, Piro KM, Xu W:Insight into F plasmid DNA segregation revealed by structures of SopB and SopB-DNA complexes. [J]. Nucl Acids Res 2010,38:4514-4526.
[101]Schumacher, M.A. Structural biology of plasmid partition:uncovering the molecular mechanisms of DNA segregation. [J]. Biochem J.2008.412,1-18.
[102]Wang YM, Tegenfeldt JO, Reisner W, Riehn R, Guan XJ, Guo L, Golding I, Cox EC, Sturm J & Austin RH. Single molecule studies of repressor-DNA interactions show longrange interactions. [J]. Pro Natl Acad Sci USA.2005.102: 9796-9801
[103]Pedersen AG, Jensen LJ, Brunak S, Staerfeldt HH & Ussery DW. A DNA structural atlas for Escherichia coli. [J]. J Mol Biol.2000.299:907-930.
[104]Bouffartigues E, Buckle M, Badaut C, Travers A & Rimsky S H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing. [J]. Nat Struct Mol Biol.2007.14:441-448.
[105]Doyle M, Fookes M, Ivens A, Mangan MW,Wain J & Dorman CJ.An H-NS-like stealth protein aids horizontal DNA transmission in bacteria. [J]. Science.2007.315:251-252.
[106]Prosseda G, Falconi M, Giangrossi M, Gualerzi CO, Micheli G & Colonna B The virF promoter in Shigella:more than just a curved DNA stretch. [J]. Mol Microbiol.2004.51:523-537
[107]Swinger KK & Rice PA IHF and HU:flexible architects of bent DNA. [J]. Curr Opin Struc Biol.2004.14:28-35.
[108]Stoebel DM, Free A, Dorman CJ:Anti-silencing:overcoming HNS-mediated repression of transcription in Gram-negative bacteria. [J]. Microbiology.2008, 154:2533-2545
[109]Dorman CJ, Ni Bhriain N, Higgins CF. DNA supercoiling and environmental regulation of virulence gene expression in Shigella flexneri. [J].Nature.1990.344: 789-792.
[110]Maurelli AT, Sansonetti PJ. Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. [J].Proc. Natl. Acad. Sci. U. S. A.1988.85:2820-2824.
[111]Nakayama S, Watanabe H. Involvement of cpxA, a sensor of a two-component regulatory system, in the pH-dependent regulation of expression of Shigella sonnei virF gene. [J].J. Bacteriol.1995.177:5062-5069.
[112]Nakayama S, Watanabe H. Identification of cpxR as a positive regulator essential for expression of the Shigella flexneri virF gene. [J].J. Bacteriol.1998. 180:3522-3528
[113]Porter ME, Dorman CJ. In vivo DNA-binding and oligomerization properties of the Shigella flexneri AraC-like transcriptional regulator VirF as identified by random and site-specific mutagenesis. [J] J. Bacteriol.2002.184:531-539.
[114]冯炜玮,陈志伟蛋白质结晶及其影响因素研究进展[J].安徽农业科学,201038(18)：9412-941
[115]梁毅.结构生物学.[M.北京：科学出版社,2010.
[116]Kabsch, W. Xds. [J].Acta Crystallogr D Biol Crystallogr.2010 66,125-32.
[117]Sheldrick, G.M. Experimental phasing with SHELXC/D/E:combining chain tracing with density modification. [J]Acta Crystallogr D Biol Crystallogr.2010, 479-85.
[118]Emsley, P. & Cowtan, K. Coot:model-building tools for molecular graphics. [J].Acta Crystallogr D Biol Crystallogr.2004.60,2126-32.
[119]McCoy, A.J. et al. Phaser crystallographic software. [J].J Appl Crystallogr .2007.40,658-674
[120]Adams, P.D. et al. PHENIX:a comprehensive Python-based system for macromolecular structure solution.[J].Acta Crystallogr D Biol Crystallogr.2010. 66,213-21
[121]Jeffrey H. Miller. Experiments in molecular genetics. [Cold Spring Harbor, N.Y.] Cold Spring Harbor Laboratory,1972.
[122]Holm, L.& Sander, C. Protein structure comparison by alignment of distance matrices. [J].JMol Bio.1993.233,123-38.
[123]Lavery, R.& Sklenar, H. The definition of generalized helicoidal parameters and of axis curvature for irregular nucleic acids. [J]J Biomol Struct Dyn.1988.6, 63-91.
[124]Lu, X.J.& Olson, W.K.3DNA:a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structures. [J].Nat Protoc.2008.3,1213-27.
[125]Radnedge, L., Davis, M.A., Youngren, B. & Austin, S.J. Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri. [J].J Bacteriol.1997. 179,3670-5
[126]Kane, K.A.& Dorman, C.J. VirB-mediated positive feedback control of the virulence gene regulatory cascade of Shigella flexneri. [J].J Bacteriol.2012.194, 5264-73
[127]Stella, S., Cascio, D. & Johnson, R.C. The shape of the DNA minor groove directs binding by the DNA-bending protein Fis. [J]. Genes Dev.201024,814-26