志贺氏菌Ⅲ型分泌系统效应子蛋白IpaH4.5功能研究
|
摘要
志贺氏菌是一类不形成芽孢的革兰氏阴性致病菌,其致病性依赖于Ⅲ型分泌系统(Type III secretion systems, T3SS)。T3SS是一种能分泌细菌毒力因子的多蛋白复合体,是细菌致病的重要毒力武器。许多动植物致病菌利用T3SS对宿主侵袭致病。细菌的T3SS由分泌器装置(apparatus)、转位子(translocators)、效应子(effectors)、分子伴侣(chaperones)等构成。T3SS分泌的效应子具有诱导侵袭、介导吞噬逃逸、促进细菌在宿主细胞内运动和细胞间播散、干扰宿主细胞信号通路等功能。
志贺氏菌在侵袭致病过程中,利用T3SS向宿主细胞释放超过25种毒力蛋白,这些毒力蛋白称为效应子(effectors),目前仅对少数效应子的功能有研究报道,大部分效应子功能未知。志贺氏菌IpaH家族蛋白由毒力大质粒和染色体上的基因共同编码,福氏志贺氏菌2a 301株的毒力大质粒上共有5个ipaH拷贝:ipaH1.4、ipaH2.5、ipaH4.5、ipaH7.8和ipaH9.8,染色体上有7个ipaH的拷贝。IpaH家族成员氨基端为6~8个富含亮氨酸蛋白(LRR)的结构域,羧基端为839个碱基构成的保守区。目前已知志贺氏菌IpaH家族蛋白由T3SS分泌,但对其功能研究并不多见。有报道IpaH7.8能够促进志贺氏菌吞噬逃逸、IpaH9.8能与哺乳动物剪接因子U2AF35结合干扰宿主的免疫反应、染色体IpaH能够抑制宿主炎症反应等,但对于毒力大质粒编码的IpaH4.5的功能,尚未见到详细的研究报道。为此,我们利用基因缺失突变技术、细胞侵袭实验、蛋白质相互作用等技术手段对IpaH4.5的功能进行了深入系统的研究。
研究IpaH4.5的功能,首先需要构建ipaH4.5缺失突变株和互补株。我们利用λ-red同源重组技术构建了ipaH4.5缺失突变株,利用能够在志贺氏菌中复制且携带卡那抗性基因的pAK载体,构建了ipaH4.5缺失突变株的互补株。经PCR证实突变株中ipaH4.5缺失,互补株中ipaH4.5回复。利用RT-PCR技术对突变株和互补株中ipaH4.5的转录进行验证,结果表明突变株中的ipaH4.5没有转录活性,互补株中ipaH4.5的转录活性得到恢复,表明突变株和互补株构建成功。
接下来我们对突变株、野生株和互补株的生存和侵袭相关表型进行了比较,分析ipaH4.5对志贺氏菌生长、代谢、侵袭力和毒力的影响。通过测定突变株、野生株、互补株的生长曲线和生化实验,我们发现ipaH4.5不影响志贺氏菌的生长和代谢。利用体外应激实验,我们又发现ipaH4.5也不影响志贺氏菌的对极端环境如热休克、低pH值、氧压力、渗透压等的抗力。HeLa细胞和鼠J774.A.1细胞侵袭实验结果表明,ipaH4.5缺失突变株、野生株和互补株入侵细胞的能力无明显差异。我们进一步测定了突变株、野生株和互补株感染鼠J774.A.1细胞后,培养上清中炎性因子TNF-α、IL-1β的分泌水平。结果表明,与突变株相比,野生株和互补株能够抑制鼠J774.A.1细胞分泌TNF-α和IL-1β等炎性因子,提示IpaH4.5具有抑制宿主炎症反应的功能。通过小鼠肺感染模型和豚鼠角膜实验,进一步证实IpaH4.5能够抑制宿主炎症反应,志贺氏菌缺失ipaH4.5后引起小鼠肺组织和豚鼠角膜炎症反应增强。
为深入探讨IpaH4.5抑制炎症反应的机制,我们利用双荧光素酶报告基因系统检测IpaH4.5对NF-κB信号途径的影响。构建带有Flag标签和GFP标签的IpaH4.5的真核表达载体。Flag-IpaH4.5和Flag空载体分别转染293T细胞后,用相同剂量TNF-α刺激细胞,与空载体相比,IpaH4.5能够抑制NF-κB的转录活性,并且这种抑制作用随着ipaH4.5转染剂量的增加而增强,呈现明显的剂量反应关系,说明IpaH4.5能够抑制宿主细胞的NF-κB信号途径。我们随后利用激光共聚焦技术观察GFP-IpaH4.5在真核细胞中的定位,结果表明,IpaH4.5分布于细胞质和细胞核,提示IpaH4.5在细胞内通过与多种蛋白相互作用来执行功能。
为阐明IpaH4.5抑制NF-κB信号途径的机制,我们利用酵母双杂交技术筛选了细胞内与IpaH4.5相互作用的蛋白。我们以ipaH4.5基因全长为诱饵,从人脾cDNA文库中筛选与其相互作用的蛋白质,共得到52个候选基因,通过回筛实验初步验证筛选结果的可靠性,通过测序、序列比对、功能分类,发现筛选到的蛋白基因主要涉及以下一些功能:凋亡、免疫调控、细胞黏附、转录调控等。其中包括NF-κB的p65亚基、PMSB9、HLA-C、UXT、POMP等蛋白基因,我们选择感兴趣的p65蛋白进行深入的研究。通过体外的GST pull-down实验和体内的免疫共沉淀实验(Co-IP)验证IpaH4.5与p65亚基确实存在相互作用,进一步的研究表明p65亚基上与IpaH4.5相互作用的结构域位于p65的第1~190个aa之间。随后我们又利用双荧光素酶报告基因系统验证IpaH4.5与NF-κB的p65亚基相互作用的生物学意义,证实IpaH4.5通过结合p65直接抑制NF-κB信号途径。
以上研究结果表明,志贺氏菌IpaH家族蛋白成员之一—IpaH4.5与其他一些已报道的志贺氏菌T3SS效应子蛋白一样,能够干扰宿主的天然免疫、抑制宿主的炎症反应。双荧光素酶报告系统和酵母双杂交实验证明IpaH4.5通过与NF-κB的p65亚基相互作用直接抑制NF-κB信号途径,进而抑制宿主的天然免疫。有关IpaH家族蛋白功能最新的研究表明,IpaH家族蛋白成员具有非常独特的E3泛素连接酶活性,生物化学实验证据表明IpaH选择性地利用宿主中一类特殊的泛素结合酶,通过泛素化途径导致靶蛋白降解,进而在志贺氏菌对宿主的免疫抑制中起重要作用。目前还不清楚IpaH作为一种特殊的E3泛素连接酶,能够泛素化哪些宿主靶蛋白。我们通过研究筛选到与IpaH4.5相互作用的宿主蛋白,这些蛋白很可能是IpaH型E3泛素连接酶作用的底物。因此有必要进一步通过泛素化实验来证实这一猜想。
通过本课题的研究,揭示了IpaH4.5抑制宿主炎症反应的分子机制,使我们对IpaH4.5的功能有了比较全面的认识,为阐明研究志贺氏菌致病的分子机制提供了重要线索,也为研制志贺氏菌疫苗提供新的思路。
Shigellosis (bacillary dysentery) is one of the most important intestine infection diseases which are epidemic wordwide. Shigella spp. is the causative agent of shigellosis which specifically infect and cause disease in the large intestine of human hosts. Shigella species which are Gram-negative enteric bacteria cause disease by a type III secretion system (T3SS). Type III secretion system (T3SS) is a family of multi-protein complex which serve to secrete macromolecules. Type III secretion (T3SS) systems are used by numerous Gram-negative pathogenic bacteria to deliver effector proteins into cells of their human, animal or plant hosts. The T3SS systems are comprised of the T3SS apparatus, translocators, effectors and specific chaperones.
Shigella ?exneri harbours a large virulence plasmid that encodes T3SS responsible for S. ?exneri pathogenesis, through which about 25 effectors are delivered. Some effectors target the actin cytoskeleton to promote entry or inhibit phagocytosis of the bacterium, others promote the spread of the bacterium into adjacent cells or interfere with the host’s innate immune responses. Only a small number of these effectors are known to us, most effectors remain to be elucidated. It is reported that IpaH proteins are encoded by plasmid and chromosome of shigella and secreted via the T3SS. Previous studies indicate that five copies of the ipaH genes exist on the large plasmid and seven copies of the ipaH genes exist on the chromosome of S. flexneri 2a 301. All ipaH copies have almost identical C-terminal halves. Although the N-terminal portions of ipaH differ in each gene, they all included a common leucine-rich repeat (LRR) motif. The biological significance of each IpaH protein still largely remains speculative. It has been reported that IpaH7.8 facilitated S. flexneri escape from the vacuole when mouse macrophages or human monocyte-derived macrophages was infected with S. flexneri, IpaH9.8 binded to a splicing factor U2AF35 to modulate host immune response, chromosomal IpaH proteins acted synergistically to modulate the host inflammatory responses. IpaH4.5, another member of IpaH family ecoded by large invasion plasmid, was never reported detailed. Therefore, we systematically explored the role of IpaH4.5 by gene deletion, invasion assay and yeast two-hybrid screening to discover the function of IpaH4.5.
To explore the function of ipaH4.5 gene of S. flexneri 2a 301, aλ-Red recombination system was used to knock out this gene first. Then, a pAK plasmid haboring a kanamycin gene was constructed and ipaH4.5gene was subcloned into it to construct a complementary plasmid. A complementary strain was obtained by introducing the complementary plasmid into the deletion mutant. Semi-quantitative RT-PCR results showed that transcription of the ipaH4.5 gene could be detected in the wild-type and complementary strain, but not ipaH4.5 deletion mutant, indicating that ipaH4.5 was successfully deleted in the mutant and restored in the complementary strain.
Subsequently, the related phenotypes of wild-type, mutant and complementary strain were compared by some experiments which were carried out as following: 1) the growth curves of wild-type strain, deletion mutant and complementary strain were measured respectively. 2) Some basic biochemical events were also investigated. 3) Survival ability of S. flexneri in extreme conditions such as heat, low pH, high oxygen, and so on. The results of above studies showed no significant differences between three strains, suggesting that the function of this protein might be unrelated to the surviaval ability and metabolism of S. flexneri. Then we performed invasion assays using HeLa cells and murine macrophage cell line J774.A.1, and no difference in invasion ability was observed between the three strains either. After that, quantities of cytokines in the culture supernatants of murine macrophages cell line J774.A.1 after being infected for two hours was measured by enzyme-linked immunosorbent assays (ELISA) for mouse IL-1β, tumor necrosis factor alpha (TNF-а). More IL-1βand TNF-аwere observed in murine macrophages infected with deletion mutant than wild type and complementary strain. So we concluded that IpaH4.5 could inhibit inflammation response of the host. The conjection was further conformed by the murine pneumonia model and the sereny test.
In order to explore why the inflammatory response of cell and animals was inhibited by IpaH4.5, we used a dual-luciferase reporter system to detect whether NF-κB signaling was affected by IpaH4.5. NF-κB signaling plays an important role in activating host innate immune responses and is a frequent target of pathogenic effectors. The results show that after transfecting Flag-IpaH4.5 and empty vector into 293T cells, NF-κB signaling was inhibited obviously by IpaH4.5 compared with the empty vector when 293T cells were stimulated by TNF-а. Then intracellular localization of IpaH4.5 was observed by a laser-scanning confocal microscopy, the results showed that ipaH4.5 localized in cytoplasm and nucleus.
We also performed yeast two-hybrid screening to indentify IpaH4.5-binding proteins to find out why IpaH4.5 inhibited NF-κB signaling pathway. Full length of IpaH4.5 was used as a bait to screen human spleen cDNA liberary. Finally 52 clones that interacted with IpaH4.5 were identified. Sequence analysis and functional classification showed that these clones were involved in the following functions: apoptosis, immunity regulation, cell adhesion and transcriptional regulation. The candidate protein in which we were most interested was the p65 subunit of NF-κB protein. Full-length cDNA for this protein was then cloned from the cDNA library. The interaction between IpaH4.5 and p65 were confirmed by in vitro GST pull-down assays and in vivo co-immumoprecitipation analysis. Further studies showed that the interacting domain of p65 with IpaH4.5 was located in section between 1~190 of p65. To determine the significance of the interaction between IpaH4.5 and p65, we used dual-luciferase reporter system to verify the interaction. Luciferase activity assays showed IpaH4.5 directly down-regulated the transcriptional activity of NF-κB by binding with p65 subunit.
The above results indicate that the function of IpaH4.5 is the same as other reported effectors like OspG, OspF and IpaH9.8 to modulate host innate immune system. Dual-luciferase reporter system and the yeast two-hybrid screening show that IpaH4.5 downregulate the host inflammatory via binding with p65 subunit of NF-κB. Latest researches on the function of the IpaH protein family show that all members of IpaH protein family have E3 ubiquitin ligase activity. But it is still unclear that what kind of host protein do these specific E3 ubiquitin ligase target. Our research has found out about 50 proteins binding with IpaH4.5 by yeast two-hybrid screening. These proteins may be substrates of IpaH-type E3 ubiquitin ligase. Therefore it is necessary to adoption of ubiquitination experiment to prove this conjecture.
In conclusion, our data demonstrated that IpaH4.5 directly inhibited NF-κB by interacting with p65 subunit. These findings expand our knowledge of the function of the IpaH family, and provide important clues for understanding the molecular pathogenesis of shihella and new ideas for vaccine development.
志贺氏菌在侵袭致病过程中,利用T3SS向宿主细胞释放超过25种毒力蛋白,这些毒力蛋白称为效应子(effectors),目前仅对少数效应子的功能有研究报道,大部分效应子功能未知。志贺氏菌IpaH家族蛋白由毒力大质粒和染色体上的基因共同编码,福氏志贺氏菌2a 301株的毒力大质粒上共有5个ipaH拷贝:ipaH1.4、ipaH2.5、ipaH4.5、ipaH7.8和ipaH9.8,染色体上有7个ipaH的拷贝。IpaH家族成员氨基端为6~8个富含亮氨酸蛋白(LRR)的结构域,羧基端为839个碱基构成的保守区。目前已知志贺氏菌IpaH家族蛋白由T3SS分泌,但对其功能研究并不多见。有报道IpaH7.8能够促进志贺氏菌吞噬逃逸、IpaH9.8能与哺乳动物剪接因子U2AF35结合干扰宿主的免疫反应、染色体IpaH能够抑制宿主炎症反应等,但对于毒力大质粒编码的IpaH4.5的功能,尚未见到详细的研究报道。为此,我们利用基因缺失突变技术、细胞侵袭实验、蛋白质相互作用等技术手段对IpaH4.5的功能进行了深入系统的研究。
研究IpaH4.5的功能,首先需要构建ipaH4.5缺失突变株和互补株。我们利用λ-red同源重组技术构建了ipaH4.5缺失突变株,利用能够在志贺氏菌中复制且携带卡那抗性基因的pAK载体,构建了ipaH4.5缺失突变株的互补株。经PCR证实突变株中ipaH4.5缺失,互补株中ipaH4.5回复。利用RT-PCR技术对突变株和互补株中ipaH4.5的转录进行验证,结果表明突变株中的ipaH4.5没有转录活性,互补株中ipaH4.5的转录活性得到恢复,表明突变株和互补株构建成功。
接下来我们对突变株、野生株和互补株的生存和侵袭相关表型进行了比较,分析ipaH4.5对志贺氏菌生长、代谢、侵袭力和毒力的影响。通过测定突变株、野生株、互补株的生长曲线和生化实验,我们发现ipaH4.5不影响志贺氏菌的生长和代谢。利用体外应激实验,我们又发现ipaH4.5也不影响志贺氏菌的对极端环境如热休克、低pH值、氧压力、渗透压等的抗力。HeLa细胞和鼠J774.A.1细胞侵袭实验结果表明,ipaH4.5缺失突变株、野生株和互补株入侵细胞的能力无明显差异。我们进一步测定了突变株、野生株和互补株感染鼠J774.A.1细胞后,培养上清中炎性因子TNF-α、IL-1β的分泌水平。结果表明,与突变株相比,野生株和互补株能够抑制鼠J774.A.1细胞分泌TNF-α和IL-1β等炎性因子,提示IpaH4.5具有抑制宿主炎症反应的功能。通过小鼠肺感染模型和豚鼠角膜实验,进一步证实IpaH4.5能够抑制宿主炎症反应,志贺氏菌缺失ipaH4.5后引起小鼠肺组织和豚鼠角膜炎症反应增强。
为深入探讨IpaH4.5抑制炎症反应的机制,我们利用双荧光素酶报告基因系统检测IpaH4.5对NF-κB信号途径的影响。构建带有Flag标签和GFP标签的IpaH4.5的真核表达载体。Flag-IpaH4.5和Flag空载体分别转染293T细胞后,用相同剂量TNF-α刺激细胞,与空载体相比,IpaH4.5能够抑制NF-κB的转录活性,并且这种抑制作用随着ipaH4.5转染剂量的增加而增强,呈现明显的剂量反应关系,说明IpaH4.5能够抑制宿主细胞的NF-κB信号途径。我们随后利用激光共聚焦技术观察GFP-IpaH4.5在真核细胞中的定位,结果表明,IpaH4.5分布于细胞质和细胞核,提示IpaH4.5在细胞内通过与多种蛋白相互作用来执行功能。
为阐明IpaH4.5抑制NF-κB信号途径的机制,我们利用酵母双杂交技术筛选了细胞内与IpaH4.5相互作用的蛋白。我们以ipaH4.5基因全长为诱饵,从人脾cDNA文库中筛选与其相互作用的蛋白质,共得到52个候选基因,通过回筛实验初步验证筛选结果的可靠性,通过测序、序列比对、功能分类,发现筛选到的蛋白基因主要涉及以下一些功能:凋亡、免疫调控、细胞黏附、转录调控等。其中包括NF-κB的p65亚基、PMSB9、HLA-C、UXT、POMP等蛋白基因,我们选择感兴趣的p65蛋白进行深入的研究。通过体外的GST pull-down实验和体内的免疫共沉淀实验(Co-IP)验证IpaH4.5与p65亚基确实存在相互作用,进一步的研究表明p65亚基上与IpaH4.5相互作用的结构域位于p65的第1~190个aa之间。随后我们又利用双荧光素酶报告基因系统验证IpaH4.5与NF-κB的p65亚基相互作用的生物学意义,证实IpaH4.5通过结合p65直接抑制NF-κB信号途径。
以上研究结果表明,志贺氏菌IpaH家族蛋白成员之一—IpaH4.5与其他一些已报道的志贺氏菌T3SS效应子蛋白一样,能够干扰宿主的天然免疫、抑制宿主的炎症反应。双荧光素酶报告系统和酵母双杂交实验证明IpaH4.5通过与NF-κB的p65亚基相互作用直接抑制NF-κB信号途径,进而抑制宿主的天然免疫。有关IpaH家族蛋白功能最新的研究表明,IpaH家族蛋白成员具有非常独特的E3泛素连接酶活性,生物化学实验证据表明IpaH选择性地利用宿主中一类特殊的泛素结合酶,通过泛素化途径导致靶蛋白降解,进而在志贺氏菌对宿主的免疫抑制中起重要作用。目前还不清楚IpaH作为一种特殊的E3泛素连接酶,能够泛素化哪些宿主靶蛋白。我们通过研究筛选到与IpaH4.5相互作用的宿主蛋白,这些蛋白很可能是IpaH型E3泛素连接酶作用的底物。因此有必要进一步通过泛素化实验来证实这一猜想。
通过本课题的研究,揭示了IpaH4.5抑制宿主炎症反应的分子机制,使我们对IpaH4.5的功能有了比较全面的认识,为阐明研究志贺氏菌致病的分子机制提供了重要线索,也为研制志贺氏菌疫苗提供新的思路。
Shigellosis (bacillary dysentery) is one of the most important intestine infection diseases which are epidemic wordwide. Shigella spp. is the causative agent of shigellosis which specifically infect and cause disease in the large intestine of human hosts. Shigella species which are Gram-negative enteric bacteria cause disease by a type III secretion system (T3SS). Type III secretion system (T3SS) is a family of multi-protein complex which serve to secrete macromolecules. Type III secretion (T3SS) systems are used by numerous Gram-negative pathogenic bacteria to deliver effector proteins into cells of their human, animal or plant hosts. The T3SS systems are comprised of the T3SS apparatus, translocators, effectors and specific chaperones.
Shigella ?exneri harbours a large virulence plasmid that encodes T3SS responsible for S. ?exneri pathogenesis, through which about 25 effectors are delivered. Some effectors target the actin cytoskeleton to promote entry or inhibit phagocytosis of the bacterium, others promote the spread of the bacterium into adjacent cells or interfere with the host’s innate immune responses. Only a small number of these effectors are known to us, most effectors remain to be elucidated. It is reported that IpaH proteins are encoded by plasmid and chromosome of shigella and secreted via the T3SS. Previous studies indicate that five copies of the ipaH genes exist on the large plasmid and seven copies of the ipaH genes exist on the chromosome of S. flexneri 2a 301. All ipaH copies have almost identical C-terminal halves. Although the N-terminal portions of ipaH differ in each gene, they all included a common leucine-rich repeat (LRR) motif. The biological significance of each IpaH protein still largely remains speculative. It has been reported that IpaH7.8 facilitated S. flexneri escape from the vacuole when mouse macrophages or human monocyte-derived macrophages was infected with S. flexneri, IpaH9.8 binded to a splicing factor U2AF35 to modulate host immune response, chromosomal IpaH proteins acted synergistically to modulate the host inflammatory responses. IpaH4.5, another member of IpaH family ecoded by large invasion plasmid, was never reported detailed. Therefore, we systematically explored the role of IpaH4.5 by gene deletion, invasion assay and yeast two-hybrid screening to discover the function of IpaH4.5.
To explore the function of ipaH4.5 gene of S. flexneri 2a 301, aλ-Red recombination system was used to knock out this gene first. Then, a pAK plasmid haboring a kanamycin gene was constructed and ipaH4.5gene was subcloned into it to construct a complementary plasmid. A complementary strain was obtained by introducing the complementary plasmid into the deletion mutant. Semi-quantitative RT-PCR results showed that transcription of the ipaH4.5 gene could be detected in the wild-type and complementary strain, but not ipaH4.5 deletion mutant, indicating that ipaH4.5 was successfully deleted in the mutant and restored in the complementary strain.
Subsequently, the related phenotypes of wild-type, mutant and complementary strain were compared by some experiments which were carried out as following: 1) the growth curves of wild-type strain, deletion mutant and complementary strain were measured respectively. 2) Some basic biochemical events were also investigated. 3) Survival ability of S. flexneri in extreme conditions such as heat, low pH, high oxygen, and so on. The results of above studies showed no significant differences between three strains, suggesting that the function of this protein might be unrelated to the surviaval ability and metabolism of S. flexneri. Then we performed invasion assays using HeLa cells and murine macrophage cell line J774.A.1, and no difference in invasion ability was observed between the three strains either. After that, quantities of cytokines in the culture supernatants of murine macrophages cell line J774.A.1 after being infected for two hours was measured by enzyme-linked immunosorbent assays (ELISA) for mouse IL-1β, tumor necrosis factor alpha (TNF-а). More IL-1βand TNF-аwere observed in murine macrophages infected with deletion mutant than wild type and complementary strain. So we concluded that IpaH4.5 could inhibit inflammation response of the host. The conjection was further conformed by the murine pneumonia model and the sereny test.
In order to explore why the inflammatory response of cell and animals was inhibited by IpaH4.5, we used a dual-luciferase reporter system to detect whether NF-κB signaling was affected by IpaH4.5. NF-κB signaling plays an important role in activating host innate immune responses and is a frequent target of pathogenic effectors. The results show that after transfecting Flag-IpaH4.5 and empty vector into 293T cells, NF-κB signaling was inhibited obviously by IpaH4.5 compared with the empty vector when 293T cells were stimulated by TNF-а. Then intracellular localization of IpaH4.5 was observed by a laser-scanning confocal microscopy, the results showed that ipaH4.5 localized in cytoplasm and nucleus.
We also performed yeast two-hybrid screening to indentify IpaH4.5-binding proteins to find out why IpaH4.5 inhibited NF-κB signaling pathway. Full length of IpaH4.5 was used as a bait to screen human spleen cDNA liberary. Finally 52 clones that interacted with IpaH4.5 were identified. Sequence analysis and functional classification showed that these clones were involved in the following functions: apoptosis, immunity regulation, cell adhesion and transcriptional regulation. The candidate protein in which we were most interested was the p65 subunit of NF-κB protein. Full-length cDNA for this protein was then cloned from the cDNA library. The interaction between IpaH4.5 and p65 were confirmed by in vitro GST pull-down assays and in vivo co-immumoprecitipation analysis. Further studies showed that the interacting domain of p65 with IpaH4.5 was located in section between 1~190 of p65. To determine the significance of the interaction between IpaH4.5 and p65, we used dual-luciferase reporter system to verify the interaction. Luciferase activity assays showed IpaH4.5 directly down-regulated the transcriptional activity of NF-κB by binding with p65 subunit.
The above results indicate that the function of IpaH4.5 is the same as other reported effectors like OspG, OspF and IpaH9.8 to modulate host innate immune system. Dual-luciferase reporter system and the yeast two-hybrid screening show that IpaH4.5 downregulate the host inflammatory via binding with p65 subunit of NF-κB. Latest researches on the function of the IpaH protein family show that all members of IpaH protein family have E3 ubiquitin ligase activity. But it is still unclear that what kind of host protein do these specific E3 ubiquitin ligase target. Our research has found out about 50 proteins binding with IpaH4.5 by yeast two-hybrid screening. These proteins may be substrates of IpaH-type E3 ubiquitin ligase. Therefore it is necessary to adoption of ubiquitination experiment to prove this conjecture.
In conclusion, our data demonstrated that IpaH4.5 directly inhibited NF-κB by interacting with p65 subunit. These findings expand our knowledge of the function of the IpaH family, and provide important clues for understanding the molecular pathogenesis of shihella and new ideas for vaccine development.
引文
[1]、World Health Organization, Global burden of disease (GBD) 2002 estimates 2004, World Health Organization, Geneva, Switzerland。
[2]、Kotloff KL, Winickoff JP, Ivanoff B, Levine MM, Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull. W. H. O. 1999, 77:651-666。
[3]、Canh, Chaicumpa W, Agtini MD, A multicentre study of Shigella diarrhoea in six Asian countries: disease burden, clinical manifestations and microbiology, PLoS Med, 2006, 3: e353。
[4]、Sansonetti PJ, Shigellosis: an old disease in new clothes? PLoS Med, 2006, 3: e354-368。
[5]、黄留玉,痢疾杆菌侵袭HeLa细胞基因表达谱的研究,第三军医大学博士学位论文,2007。
[6]、葛大放,陈道利,姜璐,安徽省马鞍山市细菌性痢疾监测与耐药性分析,疾病监测, 2007, 22(2): 96-97。
[7]、孙永祥,金以森,毛建勋,福氏志贺氏菌4C亚型生物学特性研究及ipaH基因检测,中国卫生检验杂志, 2005, 15( 5): 531-532。
[8]、陈伟鑫,上海嘉定区志贺氏菌新菌型的出现及菌型的变迁,中国卫生检验杂志, 2006, 16 (10): 1167~1169。
[9]、Jin Q, Yuan Z, Xu J, Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157, Nucleic Acids Res, 2002, 30(20): 4432-4441。
[10]、Wei J, Goldberg MB, Burland V, Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T, Infect Immun., 2003, 71(5): 2775-2786。
[11]、张继瑜,刘红,张笑兵,志贺氏菌福氏2a毒力大质粒DNA全序列测定与基因分析,中国科学, 2003 , 33(1) : 580-601。
[12]、Mavris M, Page AL, Tournebize R, Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus, Mol Microbiol, 2002, 43(6): 1543-1553。
[13]、Liao Xiang,Ying Tianyi, Wang Hengliang, Huang Liuyu, A two-dimentional proteome map of Shigella Flexneri, Electrophoresis, 2003, 24: 2864-2882。
[14]、Ying T, Wang H, Li M, Wang J, Shi Z, Feng E, Liu X, Su G, Wei K, Zhang X, Huang LY, Immunoproteomics of outer membrane proteins and extracellular proteins of Shigella flexneri 2a 2457T, Proteomics 2005, 5: 4777-93。
[15]、DuPont HL, Levine MM, Hornick RB, Formal SB, Inoculum size in shigellosis and implications for expected mode of transmission, J Infect Dis, 1989, 159: 1126-1128。
[16]、Gorden J, Small PL, Acid resistance in enteric bacteria, Infect Immun, 1993, 61: 364-367。
[17]、Islam D, Bandholtz L, Nilsson J, Wigzell H, Christensson B, Agerberth B, Gudmundsson G, Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator, Nat Med, 2001, 7: 180-185。
[18]、Schroeder GN, Hilbi H, Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion, Clin Microbiol Rev, 2008, 21: 134-156。
[19]、Sansonetti PJ, Arondel J, Cantey R, Prevost MC, Huerre M, Infection of rabbit Peyer’s patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on ollicle-associated Epithelium, Infect.Immun, 1996, 64: 2752-2764。
[20]、Wassef JS, Keren DF, Mailloux JL, Role of M cells in initial antigen uptake and in ulcer formation in rabbit intestinal loop model of shigellosis, Infect Immun, 1989, 57: 858-863。
[21]、Islam D, Veress B, Bardhan PK, Lindberg AA, Christensson B, In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis, Infect Immun, 1997,65:739-749。
[22]、Zychlinsky A, Pre′vost MC, Sansonetti PJ, Shigella flexneri induces apoptosis in infected Macrophages, Nature, 1992, 358: 167-168。
[23]、Zychlinsky A, Thirumalai K, Arondel J, Cantey JR, Aliprantis A, Sansonetti P J, In vivo apoptosis in Shigella flexneri infections, Infect Immun, 1996, 64: 5357-5365。
[24]、Sansonetti PJ, Phalipon A, Arondel J, Thirumalai K, Banerjee S, Akira S, Takeda K, Zychlinsky A, Caspase-1 activation of IL-1βand IL-18 are essential for Shigella flexneri-induced inflammation, Immunity, 2000, 12: 581-590。
[25]、Zychlinsky A, Fitting C, Cavaillon JM, Sansonetti PJ, Interleukin-1 is released by murine macrophages during apoptosis induced by Shigella flexneri, J Clin Investig,1994,94:1328-1332。
[26]、Bernardini ML, Mounier J, Hauteville H, Coquis-Rondon M, Sansonetti PJ, Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin, Proc Natl Acad Sci USA, 1989, 86: 3867-3871。
[27]、Fasano A, Noriega FR, Liao FM, Wang W, Levine MM, Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo, Gut, 1997, 40: 505-511。
[28]、Nataro JP, Seriwatana J, Fasano A, Maneval DR, Guers LD, Noriega F, Dubovsky F, Levine MM, Morris JG Jr, Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains, Infect Immun, 1995, 63: 4721-4728。
[29]、Cherla RP, Lee SY, Tesh VL, Shiga toxins and apoptosis, FEMS Microbiol Lett, 2003, 228: 159- 166。
[30]、Arbibe L, Kim DW, Batsche E, Pedron T, Mateescu B, Muchardt C, Parsot C, Sansonetti PJ, An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses, Nat Immunol, 2007, 8:47-56。
[31]、Ingersoll MA, Zychlinsky A, ShiA abrogates the innate T-cell response to Shigella flexneri Infection, Infect Immun, 2006, 74: 2317-2327。
[32]、Sansonetti PJ, Hale TL, Dammin GJ, Kapfer C, Collins HH, Formal SB, Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri, Infect, Immun, 1983, 39: 1392-1402。
[33]、Sansonetti PJ, Kopecko DJ, Formal SB, Involvement of a plasmid in the invasive ability of Shigella flexneri, Infect Immun, 1982, 35: 852-860。
[34]、Sasakawa C, Kamata K, Sakai T, Murayama SY, Makino S, Yoshikawa M, Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congored binding activity in Shigella flexneri, Infect Immun, 1986, 54: 470-475。
[35]、Buchrieser C, Glaser P, Rusniok C, Nedjari H, Hauteville HD’, Kunst F, Sansonetti P, Parsot C, The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri, Mol Microbiol, 2000, 38: 760-771。
[36]、JiangY, Yang F, Zhang X, Yang J, Chen L, Yan Y, Nie H, Xiong Z, Wang J, Dong J, Xue Y, Xu X, Zhu Y, Chen S, Jin Q, The complete sequence and analysis of the large virulence plasmid pSS of Shigella sonnei, Plasmid, 2005, 54: 149-159。
[37]、Venkatesan MM, Goldberg MB, Rose DJ, Grotbeck EJ, Burland V, Blattner FR, Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri, Infect Immun, 2001, 69: 3271-3285。
[38]、Yang F, Yang J, Zhang X, Chen L, Jiang Y, Yan Y, Tang X, Wang J, Xiong Z, Dong J, Xue Y, Zhu Y, Xu X, Sun L, Chen S, Nie H, Peng J, Xu J, Wang Y, Yuan Z, Wen Y, Yao Z, Shen Y, Qiang B, Hou Y, Yu J, Jin Q, Genome dynamics and diversity of Shigella species, the etiologic agents ofbacillary dysentery, Nucleic Acids Res, 2005, 33: 6445-6458。
[39]、Buysse JM, Stover CK, Oaks EV, Venkatesan M, Kopecko DJ, Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping, J Bacteriol, 1987, 169: 2561-2569。
[40]、Oaks EV, Hale TL, Formal SB, Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp., Infect Immun, 1986, 53: 57-63。
[41]、Blocker A, Gounon P, Larquet E, Niebuhr K, Cabiaux V, Parsot C, Sansonetti P, The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes, J Cell Biol, 1999,147: 683-693。
[42]、Me′nard R, Sansonetti P, Parsot C, The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD, EMBO J, 1994, 13: 5293-5302。
[43]、Adler B, Sasakawa C, Tobe T, Makino S, Komatsu K, Yoshikawa M, A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri, Mol Microbiol, 1989, 3: 627-635。
[44]、Dorman CJ, Porter ME, The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms, Mol Microbiol, 1998, 29: 677-684。
[45]、Kane CD, Schuch R, Day WA Jr, Maurelli AT, MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system, J Bacteriol, 2002, 184: 4409-4419。
[46]、Page AL, Ohayon H, Sansonetti PJ, Parsot C, The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri, Cell Microbiol, 1999, 1: 183-193。
[47]、Parsot C, Ageron E, Penno C, Mavris M, Jamoussi K, Hauteville Hd, Sansonetti P, Demers B, A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri, Mol Microbiol, 2005, 56: 1627-1635。
[48]、Parsot C, Hamiaux C, Page AL, The various and varying roles of specific chaperones in type III secretion systems, Curr Opin Microbiol, 2003, 6: 7-14。
[49]、van Eerde, Hamiaux AC, Perez J, Parsot C, Dijkstra BW, Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity, EMBO Rep, 2004, 5: 477-483。
[50]、Kim DW, Lenzen G, Page AL, Legrain P, Sansonetti PJ, Parsot C, The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes,Proc Natl Acad Sci USA, 2005,102: 14046-14051。
[51]、Zurawski DV, Mitsuhata C, Mumy KL, McCormick BA, Maurelli AT, OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence, Infect. Immun, 2006, 74: 5964-5976。
[52]、Okuda J, Toyotome T, Kataoka N, Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses, Biochem Biophys Res Commun, 2005, 333(2): 531-539。
[53]、Egile C, Hauteville Hd, Parsot C, Sansonetti PJ, SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri, Mol Microbiol, 1997, 23: 1063-1073。
[54]、Lett MC, Sasakawa C, Okada N, Sakai T, Makino S, Yamada M, Komatsu K, Yoshikawa M, virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the VirG protein and determination of the complete coding sequence, J Bacteriol,1989. 171:353-359。
[55]、Santapaola D, Chierico FD, Petrucca A, Uzzau S, Casalino M, Colonna B, Sessa R, Berlutti F, Nicoletti M, Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene ofShigella flexneri, is necessary for proper unipolar IcsA localization and for efficient Intercellular spread, J Bacteriol, 2006, 188: 1620-1627。
[56]、Yoshida S, Handa Y, Suzuki T, Ogawa M, Suzuki M, Tamai A, Abe A, Katayama E, Sasakawa C, Microtubule-severing activity of Shigella is pivotal for intercellular spreading, Science, 2006, 314: 985-989。
[57]、Benjelloun-Touimi Z, Sansonetti PJ, Parsot C, SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion, Mol Microbiol, 1995, 17: 123-135。
[58]、Newman CL, Stathopoulos C, Autotransporter and twopartner secretion: delivery of large-size virulence factors by gram-negative bacterial pathogens, Crit Rev Microbiol, 2004, 30: 275-286。
[59]、卜歆,福氏2a痢疾杆菌htpG基因功能的研究,西北农林科技大学硕士学位论文,2008
[60]、Guan S, Bastin DA, Verma NK, Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX, Microbiology, 1999, 145 ( Pt 5): 1263-1273。
[61]、Vokes SA, Reeves SA, Torres AG, The aerobactin iron transport system genes in Shigella flexneri are present within a pathogenicity island, Mol Microbiol, 1999, 33(1): 63-73。
[62]、Turner SA, Luck SN, Sakellaris H, Nested deletions of the SRL pathogenicity island of Shigella flexneri 2a, J Bacteriol, 2001, 183(19): 5535-5543。
[63]、王芳,袁静,何湘,黄留玉。志贺菌Ⅲ型分泌系统研究进展,中国病原生物学杂志,2009,4(3):215-218。
[64]、Purdy GE, Payne SM, The SHI-3 iron transport island of Shigella boydii 0-1392 carries the genes for aerobactin synthesis and transport. J Bacteriol, 2001, 183(14): 4176-4182。
[65]、Cornelis GR, The typeⅢsecretion injectisome, Nat Rev Microbio, 2006, 4(11):811-825。
[66]、Blocker A, Jouihri N, Larquet E, Structure and composition of the Shigella flexneri "needle complex", a part of its typeⅢsecretion, Mol Microbiol, 2001, 39(3): 652-663。
[67]、Schuch R, Maurelli AT, MxiM and MxiJ, base elements of the Mxi-Spa typeⅢsecretion system of Shigella, interact with and stabilize the MxiD secretion in the cell envelope, J Bacteriol, 2001, 183(24):6991-6998。
[68]、Morita-Ishihara T, Ogawa M, Sagara H, Shigella Spa33 is an essential C-ring component of typeⅢsecretion machinery, J Biol Chem, 2006, 281(1):599-607。
[69]、Tamano K, Katayama E, Toyotome T, Shigella Spa32 is an essential secretory protein for functional typeⅢsecretion machinery and uniformity of its needle length, J Bacteriol, 2002, 184(5): 1244-1252。
[70]、Akeda Y, Galan JE, Chaperone release and unfolding ofsubstrates in typeⅢsecretion, Nature, 2005, 437(7060):911-915。
[71]、Espina M, Olive AJ, Kenjale R, IpaD localizes to the tip of the typeⅢsecretion system needle of Shigella flexneri, Infect Immun, 2006, 74(8):4391-4400。
[72]、Kenjale R, Wilson J, Zenk SF, The needle component of the typeⅢsecreton of Shigella regulates the activity of the secretion apparatus, J Biol Chem, 2005, 280(52):42929-42937。
[73]、Demali, KA, Jue AL, Burridge K, IpaA targets beta 1integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry, J Biol Chem, 2006, 81(50):39534- 39541。
[74]、Hamiaux C, Eerde A, Parsot C, Structural mimicry for vinculin activation by IpaA, a virulence factor of Shigella flexneri, EMBO Rep, 2006, 7(8):794-799。
[75]、Blocker A, Gounon P, Larquet EK, The tripartite typeⅢsecreton of Shigella flexneriinserts IpaB and IpaC into host membranes, J Cell Biol, 1999, 147(3): 683-693。
[76]、Hayward RD, Cain RJ, McGhie EJ, Cholesterol binding by the bacterial typeⅢtranslocon is essential for virulence effector delivery into mammalian cells, Mol Microbiol, 2005, 56(3):590-603。
[77]、Hilbi H, Moss JE, Hersh D, Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB, J Biol Chem, 1998, 273(49): 32895-32900。
[78]、Harrington A, Darboe N, Kenjale R, Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes, Biochemistry, 2006, 45(2): 626-636。
[79]、Tran VN, Caron GE, Hall A, IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells, EMBO J, 1999, 18(12): 3249-3262。
[80]、Veenendaal AK, Hodgkinson JL, Schwarzer L, The typeⅢsecretion system needle tip complex mediates host cell sensing and translocon insertion, Mol Microbiol, 2007,63(6): 1719-1730。
[81]、Fernandez-Prada CM, Hoover DL, Tall BD, Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages, Infect Immun, 2000, 68(6) : 3608-3619。
[82]、Rhode JR, Breitkreutz A, Chenal A, et al. TypeⅢsecretion effectors of the IpaH family are E3 ubiquitin ligases, Cell Host Microbe, 2007, 1(1): 77-83。
[83]、Ogawa M, Yoshimori T, Suzuki T, Escape of intracellular Shigella from autophagy, Science, 2005,307(5710): 727-731。
[84]、Ohya K, Handa Y, Ogawa M, IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. Its activity to promote membrane ruffling via Rac1 and Cdc42 activation, J Biol Chem, 2005, 280(25): 24022-24034。
[85]、Alto NM, Shao F, Lazar CS, et al, Identification of a bacterial typeⅢeffector family with G protein mimicry functions, Cell, 2006, 124: 133-145。
[86]、Pendaries C, Tronchere H, Arbibe L, PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection, EMBO J, 2006, 25(5): 1024-1034。
[87]、Zurawski DV, Mitsuhata C, Mumy KL, OspF and OspC1 are Shigella flexneri typeⅢsecretion system effectors that are required for postinvasion aspects of virulence, Infect Immun, 2006, 74(10): 5964-5976。
[88]、Miura M, Terajima J, Izumiya H, OspE2 of Shigella sonnei is required for the maintenance of cell architecture of bacterium-infected cells, Infect Immun, 2006, 74(5): 2587-2595。
[89]、Li H, Xu H, Zhou Y, The phosphothreonine lyase activity of a bacterial typeⅢeffector Family, Science, 2007, 315(5814): 1000-1003。
[90]、Kim DW, Lenzen G, Page AL, The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes, Proc Natl Acad Sci, 2005, 102(39):14046-14051。
[91]、Yoshida S, Handa Y, Suzuki T, Microtubule-severing activity of Shigella is pivotal for intercellular spreading, Science, 2006, 314(5801): 985-989。
[92]、Johnson S, Roversi P, Espina M, Self-chaperoning of the typeⅢsecretion system needle tip proteins IpaD and BipD, J Biol Chem, 2007, 282(6): 4035-4044。
[93]、Boquet P, Lemichez E,. Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis? Trends Cell Biol, 2003,13: 238–246。
[94]、Goldberg MB, Theriot JA, Shigella flexneri surface protein IcsA is sufficient to direct actin-based Motility, Proc Natl Acad Sci, 1995, 92(14): 6572-6576。
[95]、Deretic V, Autophagy as an immune defense mechanism, Curr Opin Immunol, 2006, 8(4): 375- 382。
[96]、Clark CS, Maurelli AT, Shigella flexneri inhibits staurosporine-induced apoptosis in epithelial Cells, Infect Immun, 2007,75(5): 2531-2539。
[97]、Arbibe L, Kim DW, Batsche E, An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses, Nat Immunol, 2007,8(1): 47-56。
[98]、Ashida H, Toyotome T, Nagai T, Shigella chromosomal IpaH proteins are secreted via the typeⅢsecretion system and act as effectors, Mol Microbiol, 2007, 63(3):680–693。
[99]、DeLeo FR, Modulation of phagocyte apoptosis by bacterial pathogens, Apoptosis, 2004, 9(4): 399-413。
[100]、Luis JM, Isabel S, Guy R, Type III secretion: The bacteria-eukaryotic cell express, FEMS Microbiol Lett, 2005, 252(1): 1-10.
[101]、Hartman AB, Venkatesan M, Oaks EV, Sequence and Molecular Characterization of a Multicopy Invasion Plasmid Antigen Gene, IpaH, of Shigella flexneri, J Bacteri, 1990, 12(4): 1905-1915.
[102]、Passy SI, Yu X, Li Z, Radding CM, Egelman EH, Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. Proc Natl Acad Sci U S A, 1999, 96(8): 4279-84.
[103]、张影,朱力,袁静等,弗氏2a志贺氏菌2457T株yciD基因缺失突变株的构建,生物技术通讯,2005,16(5):488-490。
[104]、卜歆,朱力,刘先凯等,福氏2a志贺氏菌2457T HtpG蛋白诱导小鼠炎性反应,微生物学报,2008,48(7):1-6。
[105]、王玉飞,IV型分泌系统调控布鲁氏菌胞内生存的分子机制研究,中国人民解放军军事医学科学院博士学位论文,2008。
[106]、葛堂栋,冯尔玲,晏本菊等,痢疾杆菌酸抗性系统相关基因缺失突变体的构建,生物技术通讯, 2005, 16(5): 488-91。
[107]、Haraga Andrea, Miller SI, A Salmonnella enterica Serovar Typhimurium Translocated Leucine-Rich Repeat Effector Protein Inhibits NF-B-Dependent Gene Expression, Infect Immun, 2003,71(7): 4052-4058。
[108]、Kramer, R.W, Slagowski N L, Eze NA, Yeast Functional Genomic Screens Lead to Identification of a Role for a Bacterial Effector in Innate Immunity Regulation. PLos pathogens. 2007, 3(2): 179-184
[109]、Zhu Y, Li H, Hu L, Wang J, Zhou Y, Pang Z, Liu L, and Shao F. Structure of a Shigella effector reveals a new class of ubiquitin ligases, Nat Struct Mol Biol , 2008, 15: 1302-1308。
[110]、Singer AU, Rohde JR., Lam R., Skarina T, Kagan O, Dileo R., Chirgadze NY, Cuff ME, Joachimiak A, Tyers M, Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases, Nat Struct Mol Biol, 2008, 15: 1293-1301。
[1] Luis JM, Isabel S, Guy R, et al. TypeⅢsecretion: The bacteria-eukaryotic cell express[J]. FEMS Microbiol Lett, 2005, 252(1): 1-10.
[2] Mavris M, Page AL, Tournebize R, et al. Regulation of transcription by the activity of the Shigella flexneri typeⅢsecretion apparatus[J]. Mol Microbiol,2002, 43(6):1543-1553.
[3] Cornelis, GR. The typeⅢsecretion injectisome[J]. Nat Rev Microbio,2006,4(11):811-825.
[4] Blocker A, Jouihri N, Larquet E, et al. Structure and composition of the Shigella flexneri "needle complex", a part of its typeⅢsecretion[J]. Mol Microbiol, 2001, 39(3): 652-663.
[5] Schuch R, Maurelli AT. MxiM and MxiJ, base elements of the Mxi-Spa typeⅢsecretion system of Shigella, interact with and stabilize the MxiD secretion in the cell envelope[J]. J Bacteriol, 2001, 83(24):6991-6998.
[6] Morita-Ishihara T, Ogawa M, Sagara H, et al. Shigella Spa33 is an essential C-ring component of typeⅢsecretion machinery[J]. J Biol Chem, 2006, 281(1):599-607.
[7] Tamano K, Katayama E, Toyotome T, et al. Shigella Spa32 is an essential secretory protein for functional typeⅢsecretion machinery and uniformity of its needle length[J]. J Bacteriol, 2002, 184(5): 1244-1252.
[8] Akeda Y, Galan JE. Chaperone release and unfolding ofsubstrates in typeⅢsecretion[J]. Nature, 2005, 437(7060):911-915.
[9] Espina M, Olive AJ, Kenjale R, et al. IpaD localizes to the tip of the typeⅢsecretion system needle of Shigella flexneri[J]. Infect Immun, 2006, 74(8):4391-4400.
[10] Kenjale R, Wilson J, Zenk SF, et al. The needle component of the typeⅢsecreton of Shigella regulates the activity of the secretion apparatus[J]. J Biol Chem, 2005, 280(52):42929-42937.
[11] Demali, KA, Jue AL, Burridge K. IpaA targets beta 1integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry[J]. J Biol Chem, 2006, 281(50):39534-39541.
[12] Hamiaux C, Eerde A, Parsot C, et al. Structural mimicry for vinculin activation by IpaA, a virulence factor of Shigella flexneri[J]. EMBO Rep, 2006, 7(8):794-799.
[13] Blocker A, Gounon P, Larquet EK, et al. The tripartite typeⅢsecreton of Shigella flexneri insertsIpaB and IpaC into host membranes[J]. J Cell Biol, 1999, 147(3): 683-693.
[14] Hayward RD, Cain RJ, McGhie EJ, et al. Cholesterol binding by the bacterial typeⅢtranslocon is essential for virulence effector delivery into mammalian cells[J]. Mol Microbiol, 2005, 56(3):590-603.
[15] Hilbi H, Moss JE, Hersh D, et al. Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB[J]. J Biol Chem, 1998, 273(49):32895-32900.
[16] Harrington A, Darboe N, Kenjale R, et al. Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes[J]. Biochemistry, 2006, 45(2):626-636.
[17] Tran VN, Caron GE, Hall A, et al. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells[J]. EMBO J, 1999, 18(12):3249-3262.
[18] Veenendaal AK, Hodgkinson JL, Schwarzer L, et al. The typeⅢsecretion system needle tip complex mediates host cell sensing and translocon insertion[J]. Mol Microbiol, 2007, 63(6):1719-1730.
[19] Fernandez-Prada CM, Hoover DL, Tall BD, et al. Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages[J]. Infect Immun, 2000, 68(6) :3608-3619.
[20] Okuda J, Toyotome T, Kataoka N, et al. Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses[J]. Biochem Biophys Res Commun, 2005, 333(2):531-539.
[21] Rhode JR, Breitkreutz A, Chenal A, et al. TypeⅢsecretion effectors of the IpaH family are E3 ubiquitin ligases[J]. Cell Host Microbe, 2007, 1(1):77-83.
[22] Ogawa M, Yoshimori T, Suzuki T, et al. Escape of intracellular Shigella from autophagy[J]. Science, 2005,307(5710):727-731.
[23] Ohya K, Handa Y, Ogawa M, et al. IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. Its activity to promote membrane ruffling via Rac1 and Cdc42 activation[J]. J Biol Chem, 2005, 280(25):24022-24034.
[24] Alto NM, Shao F, Lazar CS, et al. Identification of a bacterial typeⅢeffector family with G protein mimicry functions[J]. Cell, 2006, 124:133-145.
[25] Pendaries C, Tronchere H, Arbibe L, et al. PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection[J]. EMBO J, 2006, 25(5):1024-1034.
[26] Zurawski DV, Mitsuhata C, Mumy KL, et al. OspF and OspC1 are Shigella flexneri typeⅢsecretion system effectors that are required for postinvasion aspects of virulence[J]. Infect Immun, 2006, 74(10):5964-5976.
[27] Miura M, Terajima J, Izumiya H, et al. OspE2 of Shigella sonnei is required for the maintenance ofcell architecture of bacterium-infected cells[J]. Infect Immun, 2006, 74(5):2587-2595.
[28] Li H, Xu H, Zhou Y, et al. The phosphothreonine lyase activity of a bacterial typeⅢeffector family[J]. Science, 2007, 315(5814):1000-1003.
[29] Kim DW, Lenzen G, Page AL, et al. The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes[J]. Proc Natl Acad Sci, 2005, 102(39):14046-14051.
[30] Yoshida S, Handa Y, Suzuki T, et al. Microtubule-severing activity of Shigella is pivotal for intercellular spreading[J]. Science, 2006, 314(5801):985-989.
[31] Johnson S, Roversi P, Espina M, et al. Self-chaperoning of the typeⅢsecretion system needle tip proteins IpaD and BipD[J]. J Biol Chem, 2007, 282(6):4035-4044.
[32] Boquet P, Lemichez E. Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis?[J].Trends Cell Biol, 2003, 13(5):238-246.
[33] Picking WL, Nishioka H, Hearn PD, et al. 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(3):1432-1440.
[34] Goldberg MB, Theriot JA. Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility[J]. Proc Natl Acad Sci, 1995, 92(14):6572-6576.
[35] Deretic V. Autophagy as an immune defense mechanism[J]. Curr Opin Immunol, 2006, 18(4):375-382.
[36] Clark CS, Maurelli AT. Shigella flexneri inhibits staurosporine-induced apoptosis in epithelial cells[J]. Infect Immun,2007,75(5):2531–2539.
[37] Arbibe L, Kim DW, Batsche E, et al. An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses[J]. Nat Immunol, 2007,8(1):47-56.
[38] Ashida H, Toyotome T, Nagai T, et al. Shigella chromosomal IpaH proteins are secreted via the typeⅢsecretion system and act as effectors[J]. Mol Microbiol, 2007, 63(3):680–693.
[39] DeLeo FR. Modulation of phagocyte apoptosis by bacterial pathogens[J]. Apoptosis, 2004, 9(4):399-413.
[1]王芳,袁静,黄留玉.志贺氏菌III型分泌系统研究进展.中国病原生物学杂志, 2009, 4(3):215-218.
[2] Me′nard R, Sansonetti P, Parsot C. The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J, 1994, 13: 5293?5302.
[3]葛堂栋,冯尔玲,晏本菊,等.痢疾杆菌酸抗性系统相关基因缺失突变体的构建.生物技术通讯, 2005, 16(5): 488?490.
[4]卜歆,朱力,刘先凯,等.福氏2a志贺氏菌2457T HtpG蛋白诱导小鼠炎性反应.微生物学报, 2008, 48(7): 1?6.
[5]张影,朱力,袁静,等.弗氏2a志贺氏菌2457T株ycid基因缺失突变株的构建.生物技术通讯, 2005, 16(5): 488?490.
[6] Zurawski DV, Mitsuhata C, Mumy KL. OspF and OspC1 are Shigella flexneri typeⅢsecretion system effectors that are required for postinvasion aspects of virulence. Infect Immun, 2006, 74(10): 5964?5976.
[7] Fernandez-Prada CM, Hoover DL, Tall BD. Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages, Infect Immun, 2000, 68(6): 3608?3619.
[8] Arbibe L, Kim DW, Batsche E, et al. An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses. Nat Immunol, 2007, 8(1): 47?56.
[9] Kim DW, Lenzen G, Page AL, et al. The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes. Proc Natl Acad Sci, 2005, 102(39): 14046?14051.
[10] Zurawski DV, Mitsuhata C, Mumy KL, et al. OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence. Infect. Immun, 2006, 74(10): 5964?5976.
[11] Okuda J, Toyotome T, Kataoka N. Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses. Biochem Biophys Res Commun, 2005, 333(2): 531?539.
[12]王芳,马红芳,何湘,等.福氏志贺氏菌III型分泌系统效应子IpaH4.5功能的初步研究.军事医学科学院院刊, 2009, 33(2): 120?123.
[2]、Kotloff KL, Winickoff JP, Ivanoff B, Levine MM, Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull. W. H. O. 1999, 77:651-666。
[3]、Canh, Chaicumpa W, Agtini MD, A multicentre study of Shigella diarrhoea in six Asian countries: disease burden, clinical manifestations and microbiology, PLoS Med, 2006, 3: e353。
[4]、Sansonetti PJ, Shigellosis: an old disease in new clothes? PLoS Med, 2006, 3: e354-368。
[5]、黄留玉,痢疾杆菌侵袭HeLa细胞基因表达谱的研究,第三军医大学博士学位论文,2007。
[6]、葛大放,陈道利,姜璐,安徽省马鞍山市细菌性痢疾监测与耐药性分析,疾病监测, 2007, 22(2): 96-97。
[7]、孙永祥,金以森,毛建勋,福氏志贺氏菌4C亚型生物学特性研究及ipaH基因检测,中国卫生检验杂志, 2005, 15( 5): 531-532。
[8]、陈伟鑫,上海嘉定区志贺氏菌新菌型的出现及菌型的变迁,中国卫生检验杂志, 2006, 16 (10): 1167~1169。
[9]、Jin Q, Yuan Z, Xu J, Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157, Nucleic Acids Res, 2002, 30(20): 4432-4441。
[10]、Wei J, Goldberg MB, Burland V, Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T, Infect Immun., 2003, 71(5): 2775-2786。
[11]、张继瑜,刘红,张笑兵,志贺氏菌福氏2a毒力大质粒DNA全序列测定与基因分析,中国科学, 2003 , 33(1) : 580-601。
[12]、Mavris M, Page AL, Tournebize R, Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus, Mol Microbiol, 2002, 43(6): 1543-1553。
[13]、Liao Xiang,Ying Tianyi, Wang Hengliang, Huang Liuyu, A two-dimentional proteome map of Shigella Flexneri, Electrophoresis, 2003, 24: 2864-2882。
[14]、Ying T, Wang H, Li M, Wang J, Shi Z, Feng E, Liu X, Su G, Wei K, Zhang X, Huang LY, Immunoproteomics of outer membrane proteins and extracellular proteins of Shigella flexneri 2a 2457T, Proteomics 2005, 5: 4777-93。
[15]、DuPont HL, Levine MM, Hornick RB, Formal SB, Inoculum size in shigellosis and implications for expected mode of transmission, J Infect Dis, 1989, 159: 1126-1128。
[16]、Gorden J, Small PL, Acid resistance in enteric bacteria, Infect Immun, 1993, 61: 364-367。
[17]、Islam D, Bandholtz L, Nilsson J, Wigzell H, Christensson B, Agerberth B, Gudmundsson G, Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator, Nat Med, 2001, 7: 180-185。
[18]、Schroeder GN, Hilbi H, Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion, Clin Microbiol Rev, 2008, 21: 134-156。
[19]、Sansonetti PJ, Arondel J, Cantey R, Prevost MC, Huerre M, Infection of rabbit Peyer’s patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on ollicle-associated Epithelium, Infect.Immun, 1996, 64: 2752-2764。
[20]、Wassef JS, Keren DF, Mailloux JL, Role of M cells in initial antigen uptake and in ulcer formation in rabbit intestinal loop model of shigellosis, Infect Immun, 1989, 57: 858-863。
[21]、Islam D, Veress B, Bardhan PK, Lindberg AA, Christensson B, In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis, Infect Immun, 1997,65:739-749。
[22]、Zychlinsky A, Pre′vost MC, Sansonetti PJ, Shigella flexneri induces apoptosis in infected Macrophages, Nature, 1992, 358: 167-168。
[23]、Zychlinsky A, Thirumalai K, Arondel J, Cantey JR, Aliprantis A, Sansonetti P J, In vivo apoptosis in Shigella flexneri infections, Infect Immun, 1996, 64: 5357-5365。
[24]、Sansonetti PJ, Phalipon A, Arondel J, Thirumalai K, Banerjee S, Akira S, Takeda K, Zychlinsky A, Caspase-1 activation of IL-1βand IL-18 are essential for Shigella flexneri-induced inflammation, Immunity, 2000, 12: 581-590。
[25]、Zychlinsky A, Fitting C, Cavaillon JM, Sansonetti PJ, Interleukin-1 is released by murine macrophages during apoptosis induced by Shigella flexneri, J Clin Investig,1994,94:1328-1332。
[26]、Bernardini ML, Mounier J, Hauteville H, Coquis-Rondon M, Sansonetti PJ, Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin, Proc Natl Acad Sci USA, 1989, 86: 3867-3871。
[27]、Fasano A, Noriega FR, Liao FM, Wang W, Levine MM, Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo, Gut, 1997, 40: 505-511。
[28]、Nataro JP, Seriwatana J, Fasano A, Maneval DR, Guers LD, Noriega F, Dubovsky F, Levine MM, Morris JG Jr, Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains, Infect Immun, 1995, 63: 4721-4728。
[29]、Cherla RP, Lee SY, Tesh VL, Shiga toxins and apoptosis, FEMS Microbiol Lett, 2003, 228: 159- 166。
[30]、Arbibe L, Kim DW, Batsche E, Pedron T, Mateescu B, Muchardt C, Parsot C, Sansonetti PJ, An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses, Nat Immunol, 2007, 8:47-56。
[31]、Ingersoll MA, Zychlinsky A, ShiA abrogates the innate T-cell response to Shigella flexneri Infection, Infect Immun, 2006, 74: 2317-2327。
[32]、Sansonetti PJ, Hale TL, Dammin GJ, Kapfer C, Collins HH, Formal SB, Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri, Infect, Immun, 1983, 39: 1392-1402。
[33]、Sansonetti PJ, Kopecko DJ, Formal SB, Involvement of a plasmid in the invasive ability of Shigella flexneri, Infect Immun, 1982, 35: 852-860。
[34]、Sasakawa C, Kamata K, Sakai T, Murayama SY, Makino S, Yoshikawa M, Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congored binding activity in Shigella flexneri, Infect Immun, 1986, 54: 470-475。
[35]、Buchrieser C, Glaser P, Rusniok C, Nedjari H, Hauteville HD’, Kunst F, Sansonetti P, Parsot C, The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri, Mol Microbiol, 2000, 38: 760-771。
[36]、JiangY, Yang F, Zhang X, Yang J, Chen L, Yan Y, Nie H, Xiong Z, Wang J, Dong J, Xue Y, Xu X, Zhu Y, Chen S, Jin Q, The complete sequence and analysis of the large virulence plasmid pSS of Shigella sonnei, Plasmid, 2005, 54: 149-159。
[37]、Venkatesan MM, Goldberg MB, Rose DJ, Grotbeck EJ, Burland V, Blattner FR, Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri, Infect Immun, 2001, 69: 3271-3285。
[38]、Yang F, Yang J, Zhang X, Chen L, Jiang Y, Yan Y, Tang X, Wang J, Xiong Z, Dong J, Xue Y, Zhu Y, Xu X, Sun L, Chen S, Nie H, Peng J, Xu J, Wang Y, Yuan Z, Wen Y, Yao Z, Shen Y, Qiang B, Hou Y, Yu J, Jin Q, Genome dynamics and diversity of Shigella species, the etiologic agents ofbacillary dysentery, Nucleic Acids Res, 2005, 33: 6445-6458。
[39]、Buysse JM, Stover CK, Oaks EV, Venkatesan M, Kopecko DJ, Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping, J Bacteriol, 1987, 169: 2561-2569。
[40]、Oaks EV, Hale TL, Formal SB, Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp., Infect Immun, 1986, 53: 57-63。
[41]、Blocker A, Gounon P, Larquet E, Niebuhr K, Cabiaux V, Parsot C, Sansonetti P, The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes, J Cell Biol, 1999,147: 683-693。
[42]、Me′nard R, Sansonetti P, Parsot C, The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD, EMBO J, 1994, 13: 5293-5302。
[43]、Adler B, Sasakawa C, Tobe T, Makino S, Komatsu K, Yoshikawa M, A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri, Mol Microbiol, 1989, 3: 627-635。
[44]、Dorman CJ, Porter ME, The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms, Mol Microbiol, 1998, 29: 677-684。
[45]、Kane CD, Schuch R, Day WA Jr, Maurelli AT, MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system, J Bacteriol, 2002, 184: 4409-4419。
[46]、Page AL, Ohayon H, Sansonetti PJ, Parsot C, The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri, Cell Microbiol, 1999, 1: 183-193。
[47]、Parsot C, Ageron E, Penno C, Mavris M, Jamoussi K, Hauteville Hd, Sansonetti P, Demers B, A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri, Mol Microbiol, 2005, 56: 1627-1635。
[48]、Parsot C, Hamiaux C, Page AL, The various and varying roles of specific chaperones in type III secretion systems, Curr Opin Microbiol, 2003, 6: 7-14。
[49]、van Eerde, Hamiaux AC, Perez J, Parsot C, Dijkstra BW, Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity, EMBO Rep, 2004, 5: 477-483。
[50]、Kim DW, Lenzen G, Page AL, Legrain P, Sansonetti PJ, Parsot C, The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes,Proc Natl Acad Sci USA, 2005,102: 14046-14051。
[51]、Zurawski DV, Mitsuhata C, Mumy KL, McCormick BA, Maurelli AT, OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence, Infect. Immun, 2006, 74: 5964-5976。
[52]、Okuda J, Toyotome T, Kataoka N, Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses, Biochem Biophys Res Commun, 2005, 333(2): 531-539。
[53]、Egile C, Hauteville Hd, Parsot C, Sansonetti PJ, SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri, Mol Microbiol, 1997, 23: 1063-1073。
[54]、Lett MC, Sasakawa C, Okada N, Sakai T, Makino S, Yamada M, Komatsu K, Yoshikawa M, virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the VirG protein and determination of the complete coding sequence, J Bacteriol,1989. 171:353-359。
[55]、Santapaola D, Chierico FD, Petrucca A, Uzzau S, Casalino M, Colonna B, Sessa R, Berlutti F, Nicoletti M, Apyrase, the product of the virulence plasmid-encoded phoN2 (apy) gene ofShigella flexneri, is necessary for proper unipolar IcsA localization and for efficient Intercellular spread, J Bacteriol, 2006, 188: 1620-1627。
[56]、Yoshida S, Handa Y, Suzuki T, Ogawa M, Suzuki M, Tamai A, Abe A, Katayama E, Sasakawa C, Microtubule-severing activity of Shigella is pivotal for intercellular spreading, Science, 2006, 314: 985-989。
[57]、Benjelloun-Touimi Z, Sansonetti PJ, Parsot C, SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion, Mol Microbiol, 1995, 17: 123-135。
[58]、Newman CL, Stathopoulos C, Autotransporter and twopartner secretion: delivery of large-size virulence factors by gram-negative bacterial pathogens, Crit Rev Microbiol, 2004, 30: 275-286。
[59]、卜歆,福氏2a痢疾杆菌htpG基因功能的研究,西北农林科技大学硕士学位论文,2008
[60]、Guan S, Bastin DA, Verma NK, Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX, Microbiology, 1999, 145 ( Pt 5): 1263-1273。
[61]、Vokes SA, Reeves SA, Torres AG, The aerobactin iron transport system genes in Shigella flexneri are present within a pathogenicity island, Mol Microbiol, 1999, 33(1): 63-73。
[62]、Turner SA, Luck SN, Sakellaris H, Nested deletions of the SRL pathogenicity island of Shigella flexneri 2a, J Bacteriol, 2001, 183(19): 5535-5543。
[63]、王芳,袁静,何湘,黄留玉。志贺菌Ⅲ型分泌系统研究进展,中国病原生物学杂志,2009,4(3):215-218。
[64]、Purdy GE, Payne SM, The SHI-3 iron transport island of Shigella boydii 0-1392 carries the genes for aerobactin synthesis and transport. J Bacteriol, 2001, 183(14): 4176-4182。
[65]、Cornelis GR, The typeⅢsecretion injectisome, Nat Rev Microbio, 2006, 4(11):811-825。
[66]、Blocker A, Jouihri N, Larquet E, Structure and composition of the Shigella flexneri "needle complex", a part of its typeⅢsecretion, Mol Microbiol, 2001, 39(3): 652-663。
[67]、Schuch R, Maurelli AT, MxiM and MxiJ, base elements of the Mxi-Spa typeⅢsecretion system of Shigella, interact with and stabilize the MxiD secretion in the cell envelope, J Bacteriol, 2001, 183(24):6991-6998。
[68]、Morita-Ishihara T, Ogawa M, Sagara H, Shigella Spa33 is an essential C-ring component of typeⅢsecretion machinery, J Biol Chem, 2006, 281(1):599-607。
[69]、Tamano K, Katayama E, Toyotome T, Shigella Spa32 is an essential secretory protein for functional typeⅢsecretion machinery and uniformity of its needle length, J Bacteriol, 2002, 184(5): 1244-1252。
[70]、Akeda Y, Galan JE, Chaperone release and unfolding ofsubstrates in typeⅢsecretion, Nature, 2005, 437(7060):911-915。
[71]、Espina M, Olive AJ, Kenjale R, IpaD localizes to the tip of the typeⅢsecretion system needle of Shigella flexneri, Infect Immun, 2006, 74(8):4391-4400。
[72]、Kenjale R, Wilson J, Zenk SF, The needle component of the typeⅢsecreton of Shigella regulates the activity of the secretion apparatus, J Biol Chem, 2005, 280(52):42929-42937。
[73]、Demali, KA, Jue AL, Burridge K, IpaA targets beta 1integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry, J Biol Chem, 2006, 81(50):39534- 39541。
[74]、Hamiaux C, Eerde A, Parsot C, Structural mimicry for vinculin activation by IpaA, a virulence factor of Shigella flexneri, EMBO Rep, 2006, 7(8):794-799。
[75]、Blocker A, Gounon P, Larquet EK, The tripartite typeⅢsecreton of Shigella flexneriinserts IpaB and IpaC into host membranes, J Cell Biol, 1999, 147(3): 683-693。
[76]、Hayward RD, Cain RJ, McGhie EJ, Cholesterol binding by the bacterial typeⅢtranslocon is essential for virulence effector delivery into mammalian cells, Mol Microbiol, 2005, 56(3):590-603。
[77]、Hilbi H, Moss JE, Hersh D, Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB, J Biol Chem, 1998, 273(49): 32895-32900。
[78]、Harrington A, Darboe N, Kenjale R, Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes, Biochemistry, 2006, 45(2): 626-636。
[79]、Tran VN, Caron GE, Hall A, IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells, EMBO J, 1999, 18(12): 3249-3262。
[80]、Veenendaal AK, Hodgkinson JL, Schwarzer L, The typeⅢsecretion system needle tip complex mediates host cell sensing and translocon insertion, Mol Microbiol, 2007,63(6): 1719-1730。
[81]、Fernandez-Prada CM, Hoover DL, Tall BD, Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages, Infect Immun, 2000, 68(6) : 3608-3619。
[82]、Rhode JR, Breitkreutz A, Chenal A, et al. TypeⅢsecretion effectors of the IpaH family are E3 ubiquitin ligases, Cell Host Microbe, 2007, 1(1): 77-83。
[83]、Ogawa M, Yoshimori T, Suzuki T, Escape of intracellular Shigella from autophagy, Science, 2005,307(5710): 727-731。
[84]、Ohya K, Handa Y, Ogawa M, IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. Its activity to promote membrane ruffling via Rac1 and Cdc42 activation, J Biol Chem, 2005, 280(25): 24022-24034。
[85]、Alto NM, Shao F, Lazar CS, et al, Identification of a bacterial typeⅢeffector family with G protein mimicry functions, Cell, 2006, 124: 133-145。
[86]、Pendaries C, Tronchere H, Arbibe L, PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection, EMBO J, 2006, 25(5): 1024-1034。
[87]、Zurawski DV, Mitsuhata C, Mumy KL, OspF and OspC1 are Shigella flexneri typeⅢsecretion system effectors that are required for postinvasion aspects of virulence, Infect Immun, 2006, 74(10): 5964-5976。
[88]、Miura M, Terajima J, Izumiya H, OspE2 of Shigella sonnei is required for the maintenance of cell architecture of bacterium-infected cells, Infect Immun, 2006, 74(5): 2587-2595。
[89]、Li H, Xu H, Zhou Y, The phosphothreonine lyase activity of a bacterial typeⅢeffector Family, Science, 2007, 315(5814): 1000-1003。
[90]、Kim DW, Lenzen G, Page AL, The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes, Proc Natl Acad Sci, 2005, 102(39):14046-14051。
[91]、Yoshida S, Handa Y, Suzuki T, Microtubule-severing activity of Shigella is pivotal for intercellular spreading, Science, 2006, 314(5801): 985-989。
[92]、Johnson S, Roversi P, Espina M, Self-chaperoning of the typeⅢsecretion system needle tip proteins IpaD and BipD, J Biol Chem, 2007, 282(6): 4035-4044。
[93]、Boquet P, Lemichez E,. Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis? Trends Cell Biol, 2003,13: 238–246。
[94]、Goldberg MB, Theriot JA, Shigella flexneri surface protein IcsA is sufficient to direct actin-based Motility, Proc Natl Acad Sci, 1995, 92(14): 6572-6576。
[95]、Deretic V, Autophagy as an immune defense mechanism, Curr Opin Immunol, 2006, 8(4): 375- 382。
[96]、Clark CS, Maurelli AT, Shigella flexneri inhibits staurosporine-induced apoptosis in epithelial Cells, Infect Immun, 2007,75(5): 2531-2539。
[97]、Arbibe L, Kim DW, Batsche E, An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses, Nat Immunol, 2007,8(1): 47-56。
[98]、Ashida H, Toyotome T, Nagai T, Shigella chromosomal IpaH proteins are secreted via the typeⅢsecretion system and act as effectors, Mol Microbiol, 2007, 63(3):680–693。
[99]、DeLeo FR, Modulation of phagocyte apoptosis by bacterial pathogens, Apoptosis, 2004, 9(4): 399-413。
[100]、Luis JM, Isabel S, Guy R, Type III secretion: The bacteria-eukaryotic cell express, FEMS Microbiol Lett, 2005, 252(1): 1-10.
[101]、Hartman AB, Venkatesan M, Oaks EV, Sequence and Molecular Characterization of a Multicopy Invasion Plasmid Antigen Gene, IpaH, of Shigella flexneri, J Bacteri, 1990, 12(4): 1905-1915.
[102]、Passy SI, Yu X, Li Z, Radding CM, Egelman EH, Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. Proc Natl Acad Sci U S A, 1999, 96(8): 4279-84.
[103]、张影,朱力,袁静等,弗氏2a志贺氏菌2457T株yciD基因缺失突变株的构建,生物技术通讯,2005,16(5):488-490。
[104]、卜歆,朱力,刘先凯等,福氏2a志贺氏菌2457T HtpG蛋白诱导小鼠炎性反应,微生物学报,2008,48(7):1-6。
[105]、王玉飞,IV型分泌系统调控布鲁氏菌胞内生存的分子机制研究,中国人民解放军军事医学科学院博士学位论文,2008。
[106]、葛堂栋,冯尔玲,晏本菊等,痢疾杆菌酸抗性系统相关基因缺失突变体的构建,生物技术通讯, 2005, 16(5): 488-91。
[107]、Haraga Andrea, Miller SI, A Salmonnella enterica Serovar Typhimurium Translocated Leucine-Rich Repeat Effector Protein Inhibits NF-B-Dependent Gene Expression, Infect Immun, 2003,71(7): 4052-4058。
[108]、Kramer, R.W, Slagowski N L, Eze NA, Yeast Functional Genomic Screens Lead to Identification of a Role for a Bacterial Effector in Innate Immunity Regulation. PLos pathogens. 2007, 3(2): 179-184
[109]、Zhu Y, Li H, Hu L, Wang J, Zhou Y, Pang Z, Liu L, and Shao F. Structure of a Shigella effector reveals a new class of ubiquitin ligases, Nat Struct Mol Biol , 2008, 15: 1302-1308。
[110]、Singer AU, Rohde JR., Lam R., Skarina T, Kagan O, Dileo R., Chirgadze NY, Cuff ME, Joachimiak A, Tyers M, Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases, Nat Struct Mol Biol, 2008, 15: 1293-1301。
[1] Luis JM, Isabel S, Guy R, et al. TypeⅢsecretion: The bacteria-eukaryotic cell express[J]. FEMS Microbiol Lett, 2005, 252(1): 1-10.
[2] Mavris M, Page AL, Tournebize R, et al. Regulation of transcription by the activity of the Shigella flexneri typeⅢsecretion apparatus[J]. Mol Microbiol,2002, 43(6):1543-1553.
[3] Cornelis, GR. The typeⅢsecretion injectisome[J]. Nat Rev Microbio,2006,4(11):811-825.
[4] Blocker A, Jouihri N, Larquet E, et al. Structure and composition of the Shigella flexneri "needle complex", a part of its typeⅢsecretion[J]. Mol Microbiol, 2001, 39(3): 652-663.
[5] Schuch R, Maurelli AT. MxiM and MxiJ, base elements of the Mxi-Spa typeⅢsecretion system of Shigella, interact with and stabilize the MxiD secretion in the cell envelope[J]. J Bacteriol, 2001, 83(24):6991-6998.
[6] Morita-Ishihara T, Ogawa M, Sagara H, et al. Shigella Spa33 is an essential C-ring component of typeⅢsecretion machinery[J]. J Biol Chem, 2006, 281(1):599-607.
[7] Tamano K, Katayama E, Toyotome T, et al. Shigella Spa32 is an essential secretory protein for functional typeⅢsecretion machinery and uniformity of its needle length[J]. J Bacteriol, 2002, 184(5): 1244-1252.
[8] Akeda Y, Galan JE. Chaperone release and unfolding ofsubstrates in typeⅢsecretion[J]. Nature, 2005, 437(7060):911-915.
[9] Espina M, Olive AJ, Kenjale R, et al. IpaD localizes to the tip of the typeⅢsecretion system needle of Shigella flexneri[J]. Infect Immun, 2006, 74(8):4391-4400.
[10] Kenjale R, Wilson J, Zenk SF, et al. The needle component of the typeⅢsecreton of Shigella regulates the activity of the secretion apparatus[J]. J Biol Chem, 2005, 280(52):42929-42937.
[11] Demali, KA, Jue AL, Burridge K. IpaA targets beta 1integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry[J]. J Biol Chem, 2006, 281(50):39534-39541.
[12] Hamiaux C, Eerde A, Parsot C, et al. Structural mimicry for vinculin activation by IpaA, a virulence factor of Shigella flexneri[J]. EMBO Rep, 2006, 7(8):794-799.
[13] Blocker A, Gounon P, Larquet EK, et al. The tripartite typeⅢsecreton of Shigella flexneri insertsIpaB and IpaC into host membranes[J]. J Cell Biol, 1999, 147(3): 683-693.
[14] Hayward RD, Cain RJ, McGhie EJ, et al. Cholesterol binding by the bacterial typeⅢtranslocon is essential for virulence effector delivery into mammalian cells[J]. Mol Microbiol, 2005, 56(3):590-603.
[15] Hilbi H, Moss JE, Hersh D, et al. Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB[J]. J Biol Chem, 1998, 273(49):32895-32900.
[16] Harrington A, Darboe N, Kenjale R, et al. Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes[J]. Biochemistry, 2006, 45(2):626-636.
[17] Tran VN, Caron GE, Hall A, et al. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells[J]. EMBO J, 1999, 18(12):3249-3262.
[18] Veenendaal AK, Hodgkinson JL, Schwarzer L, et al. The typeⅢsecretion system needle tip complex mediates host cell sensing and translocon insertion[J]. Mol Microbiol, 2007, 63(6):1719-1730.
[19] Fernandez-Prada CM, Hoover DL, Tall BD, et al. Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages[J]. Infect Immun, 2000, 68(6) :3608-3619.
[20] Okuda J, Toyotome T, Kataoka N, et al. Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses[J]. Biochem Biophys Res Commun, 2005, 333(2):531-539.
[21] Rhode JR, Breitkreutz A, Chenal A, et al. TypeⅢsecretion effectors of the IpaH family are E3 ubiquitin ligases[J]. Cell Host Microbe, 2007, 1(1):77-83.
[22] Ogawa M, Yoshimori T, Suzuki T, et al. Escape of intracellular Shigella from autophagy[J]. Science, 2005,307(5710):727-731.
[23] Ohya K, Handa Y, Ogawa M, et al. IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. Its activity to promote membrane ruffling via Rac1 and Cdc42 activation[J]. J Biol Chem, 2005, 280(25):24022-24034.
[24] Alto NM, Shao F, Lazar CS, et al. Identification of a bacterial typeⅢeffector family with G protein mimicry functions[J]. Cell, 2006, 124:133-145.
[25] Pendaries C, Tronchere H, Arbibe L, et al. PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection[J]. EMBO J, 2006, 25(5):1024-1034.
[26] Zurawski DV, Mitsuhata C, Mumy KL, et al. OspF and OspC1 are Shigella flexneri typeⅢsecretion system effectors that are required for postinvasion aspects of virulence[J]. Infect Immun, 2006, 74(10):5964-5976.
[27] Miura M, Terajima J, Izumiya H, et al. OspE2 of Shigella sonnei is required for the maintenance ofcell architecture of bacterium-infected cells[J]. Infect Immun, 2006, 74(5):2587-2595.
[28] Li H, Xu H, Zhou Y, et al. The phosphothreonine lyase activity of a bacterial typeⅢeffector family[J]. Science, 2007, 315(5814):1000-1003.
[29] Kim DW, Lenzen G, Page AL, et al. The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes[J]. Proc Natl Acad Sci, 2005, 102(39):14046-14051.
[30] Yoshida S, Handa Y, Suzuki T, et al. Microtubule-severing activity of Shigella is pivotal for intercellular spreading[J]. Science, 2006, 314(5801):985-989.
[31] Johnson S, Roversi P, Espina M, et al. Self-chaperoning of the typeⅢsecretion system needle tip proteins IpaD and BipD[J]. J Biol Chem, 2007, 282(6):4035-4044.
[32] Boquet P, Lemichez E. Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis?[J].Trends Cell Biol, 2003, 13(5):238-246.
[33] Picking WL, Nishioka H, Hearn PD, et al. 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(3):1432-1440.
[34] Goldberg MB, Theriot JA. Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility[J]. Proc Natl Acad Sci, 1995, 92(14):6572-6576.
[35] Deretic V. Autophagy as an immune defense mechanism[J]. Curr Opin Immunol, 2006, 18(4):375-382.
[36] Clark CS, Maurelli AT. Shigella flexneri inhibits staurosporine-induced apoptosis in epithelial cells[J]. Infect Immun,2007,75(5):2531–2539.
[37] Arbibe L, Kim DW, Batsche E, et al. An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses[J]. Nat Immunol, 2007,8(1):47-56.
[38] Ashida H, Toyotome T, Nagai T, et al. Shigella chromosomal IpaH proteins are secreted via the typeⅢsecretion system and act as effectors[J]. Mol Microbiol, 2007, 63(3):680–693.
[39] DeLeo FR. Modulation of phagocyte apoptosis by bacterial pathogens[J]. Apoptosis, 2004, 9(4):399-413.
[1]王芳,袁静,黄留玉.志贺氏菌III型分泌系统研究进展.中国病原生物学杂志, 2009, 4(3):215-218.
[2] Me′nard R, Sansonetti P, Parsot C. The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J, 1994, 13: 5293?5302.
[3]葛堂栋,冯尔玲,晏本菊,等.痢疾杆菌酸抗性系统相关基因缺失突变体的构建.生物技术通讯, 2005, 16(5): 488?490.
[4]卜歆,朱力,刘先凯,等.福氏2a志贺氏菌2457T HtpG蛋白诱导小鼠炎性反应.微生物学报, 2008, 48(7): 1?6.
[5]张影,朱力,袁静,等.弗氏2a志贺氏菌2457T株ycid基因缺失突变株的构建.生物技术通讯, 2005, 16(5): 488?490.
[6] Zurawski DV, Mitsuhata C, Mumy KL. OspF and OspC1 are Shigella flexneri typeⅢsecretion system effectors that are required for postinvasion aspects of virulence. Infect Immun, 2006, 74(10): 5964?5976.
[7] Fernandez-Prada CM, Hoover DL, Tall BD. Shigella flexneri IpaH7.8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages, Infect Immun, 2000, 68(6): 3608?3619.
[8] Arbibe L, Kim DW, Batsche E, et al. An injected bacterial effector targets chromatin access for transcription factor NF-kappaB to alter transcription of host genes involved in immune responses. Nat Immunol, 2007, 8(1): 47?56.
[9] Kim DW, Lenzen G, Page AL, et al. The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes. Proc Natl Acad Sci, 2005, 102(39): 14046?14051.
[10] Zurawski DV, Mitsuhata C, Mumy KL, et al. OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence. Infect. Immun, 2006, 74(10): 5964?5976.
[11] Okuda J, Toyotome T, Kataoka N. Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses. Biochem Biophys Res Commun, 2005, 333(2): 531?539.
[12]王芳,马红芳,何湘,等.福氏志贺氏菌III型分泌系统效应子IpaH4.5功能的初步研究.军事医学科学院院刊, 2009, 33(2): 120?123.