福氏2a志贺氏菌2457T HtpG蛋白诱导小鼠炎性反应

[目的]构建福氏2a志贺氏菌2457T株的htpG缺失突变株和回复株,对HtpG蛋白的功能进行初步研究.[方法]采用X-Red重组系统对htpG基因进行缺失突变,构建了福氏2a志贺氏菌2457T株的htpG缺失突变株,并利用低拷贝质粒构建了htpG突变株的回复株.在此基础上,对野生株、突变株和回复株的生长曲线、生化反应、豚鼠角膜试验进行了比较分析,并考察了野生株、突变株和回复株腹腔注射引起小鼠炎症反应的强弱.[结果]HtpG蛋白功能与福氏志贺氏菌的基本生化代谢无关,也不影响细菌穿透上皮细胞的能力,但腹腔注射后能够引起小鼠强烈的炎症反应.[结论]HtpG蛋白功能可能与细菌的免疫致病性相关.     

弗氏志贺菌毒力大质粒导入大肠杆菌MG1655

目的:将弗氏2a志贺菌2457T的毒力大质粒pSF导入大肠杆菌MG1655。方法:通过诱动转移技术,将弗氏2a志贺菌2457T的毒力大质粒导入大肠杆菌MG1655。结果:构建了MG1655/pSF:pXL275-virG的毒力大质粒导入突变株,双向电泳初步比较分析表明在重组MG1655中有志贺菌毒力的表达。结论:成功地将弗氏2a志贺菌2457T毒力大质粒pSF导入了大肠杆菌MG1655。     

福氏2a志贺氏菌2457T株argT基因突变体的构建

目的：构建福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体,以进行后续ArgT功能研究。方法：根据福氏2a志贺氏菌2457T株基因组全序列,采用λ-Red重组系统对argT基因进行缺失,并经PCR验证;采用定点突变的方法构建ArgT非降解株,并经SDS-PAGE验证;对野生株、argT缺失突变株和ArgT非降解突变株37℃时的生长曲线及生化反应进行比较研究。结果：构建了2457T的argT缺失突变株和ArgT非降解突变株;2种突变株初始生长均较慢,但最终和野生株状态一致;2种突变株利用甘露醇的能力都比野生株强,而利用葡萄糖的能力降低。结论：获得了福氏2a志贺氏菌2457T株argT基因缺失突变体和ArgT蛋白非降解突变体。     

驱除痢疾杆菌侵袭大质粒的新方法

用质粒不相容性原理驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒 ,先从福氏2a侵袭大质粒分别扩增ori和inc基因 ,将它们克隆至 pMD18 T载体 ,得重组质粒pMDori和 pMDinc ,然后转化 2 4 5 7T和S7,不管是pMDori还是 pMDinc都能竞争驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒。     

志贺菌福氏2a信号标签诱变突变体文库的构建

以合成的单链序列特异性标签为模板,通过PCR得到双链DNA标签并将其克隆到自杀质粒pUT-Tn5 Km2的转座子中,转化大肠杆菌S17-1λpir;然后用经转化的S17-1λpir与福氏志贺菌2a 2457T交配,挑出对氨苄青霉素敏感,对卡那霉素和萘啶酮酸抗性的菌落,结果表明构建了包含4376个福氏志贺菌突变体信号标签诱变库,为进一步鉴定该病原体的毒力基因打下了基础。     

弗氏2a志贺菌htrA基因突变体的构建

目的:构建弗氏2a志贺菌2457T的htrA基因缺失突变株及HtrA酶失活突变株,以便进一步研究HtrA蛋白的功能。方法:用PCR扩增htrA基因上下游同源臂,构建含有kan基因的打靶片段,采用λ-Red重组系统对htrA基因进行缺失,用PCR进行验证;通过定点突变的方法构建HtrA酶失活突变株,并测序验证。结果与结论:构建了2457T htrA缺失突变株和2457T/htrAFSA酶失活突变株。     

Shigella flexneri effectors OspE1 and OspE2 mediate induced adherence to the colonic epithelium following bile salts exposure

Shigella flexneri is a Gram-negative pathogen that invades the colonic epithelium. While invasion has been thoroughly investigated, it is unknown how Shigella first attaches to the epithelium. Previous literature suggests that Shigella utilizes adhesins that are induced by environmental signals, including bile salts, encountered in the small intestine prior to invasion. We hypothesized that bile would induce adherence factors to facilitate attachment to colonic epithelial cells. To test our hypothesis, S. flexneri strain 2457T was subcultured in media containing bile salts, and the ability of the bacteria to adhere to the apical surface of polarized T84 epithelial cells was measured. We observed a significant increase in adherence, which was absent in a virulence plasmid-cured strain and a type-III secretion system mutant. Microarray expression analysis indicated that the ospE1/ospE2 genes were induced in the presence of bile, and bile-induced adherence was lost in a ΔospE1/ΔospE2 mutant. Further studies demonstrated that the OspE1/OspE2 proteins were localized to the bacterial outer membrane following exposure to bile salts. The data presented are the first demonstration that the OspE1/OspE2 proteins promote initial adherence to the intestinal epithelium. The adhesins required for Shigella attachment to the colonic epithelium may serve as ideal targets for vaccine development.     

鉴定痢疾杆菌毒力基因的SW480细胞模型构建

信号标签诱变技术（STM）是一种在体内高通量筛选病原体毒力基因的新方法,在应用时的一个先决条件是要建立合适的体内筛选系统。为将该技术应用于福氏痢疾杆菌,我们使用三个福氏痢疾杆菌菌株进行了预试验：通过同源重组构建而成的带有氯霉素抗性且aroA和virG基因失活的突变株RC426;因在侵袭质粒上自发缺失3个基因座(ipaBCDA, invA 和 virG)的另一减毒突变株T32,其曾被用作福氏痢疾杆菌的口服疫苗;还有具侵袭宿主细胞能力的野生性菌株2457T。将RC426、T32和2457T混合后侵袭结肠细胞系SW480,不同时间回收经侵袭后细胞裂解液中的菌体并统计。结果显示在侵袭12h内回收到减毒突变株的量与野生有毒株存在显著性差异,表明SW480 细胞系可用于痢疾杆菌的STM研究。Abstract: Signature-tagged mutagenesis (STM) is a novel technology with high throughput screening ability to identify virulent genes of pathogen in vivo. An appropriate animal or cell line model is one of prerequisites by exploiting this technique. In order to apply STM to Shigella flexneri, RC426 was constructed as an attenuated mutant with chloramphenicol resistance and aroA and virG genes inactivated by homologous recombination; Another attenuated strain T32 was used as an oral S. flexneri 2a vaccine due to a spontaneous deletion in three loci (ipaBCDA, invA and virG) on the virulence plasmid. The wild type strain 2457T had the invasion ability into host cells. The three strains, RC426, T32 and 2457T, were mixed together to invade colon cancer cell line SW480, and the distinct strains were recovered and counted from cell lysates of invaded SW480 in different time. The results showed that there were statistically significant differences between the amounts of two attenuated strains recovered and that of virulent strain within 12h invasion, indicating SW480 was a suitable cell model for applying STM to screen virulent genes of Shigella flexneri.     

福氏2a志贺氏菌△aroA突变减毒株的构建

志贺氏菌芳香族氨基酸合成酶基因缺陷能够使菌体明显减毒,并有可能成为新一代痢疾疫苗．用ＰＣＲ技术从野生型福氏２ａ志贺氏菌２４５７Ｔ中克隆出ａｒｏＡ基因,在体外进行精确的缺失突变,并通过体内同源重组,构建成△ａｒｏＡ突变体ＲＳ４２６．实验结果表明,这种突变体仍保持了侵袭能力和保护性Ｏ抗原的表达,但其毒力已明显降低,不能产生豚鼠角结膜炎,小鼠半数致死量明显提高．免疫保护试验显示,ＲＳ４２６可在小鼠中产生对福氏２ａ野生菌１００％的保护作用．     

Multicopy icsA is able to suppress the virulence defect caused by the wzz(SF) mutation in Shigella flexneri

The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells. S. flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface. A S. flexneri 2457T wzz(SF) mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM. Introduction of a plasmid encoding IcsA into S. flexneri wzz(SF) showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzz(SF) mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM.     

弗氏2a志贺菌2457T株碱性蛋白质组图谱的建立

目的:建立弗氏2a志贺菌2457T株的碱性蛋白质组图谱。方法:首先采用双向电泳技术对弗氏2a志贺菌2457T株表达的全部碱性菌体蛋白及碱性膜蛋白进行分离,再通过基质辅助激光解析/电离串联飞行时间质谱进行鉴定。结果:共鉴定到46个蛋白点,对应于38种蛋白质。结论:首次完成了弗氏2a志贺菌2457T株的碱性蛋白质组图谱。     

New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies

It has been difficult to evaluate the protective efficacy of vaccine candidates against shigellosis, a major form of bacillary dysentery caused by Shigella spp. infection, because of the lack of suitable animal models. To develop a proper animal model representing human bacillary dysentery, guinea pigs were challenged with virulent Shigella flexneri serotype 2a (strains 2457T or YSH6000) or S. flexneri 5a (strain M90T) by the intrarectal (i.r.) route. Interestingly, all guinea pigs administered these Shigella strains developed severe and acute rectocolitis. They lost approximately 20% of their body weight and developed tenesmus by 24 h after Shigella infection. Shigella invasion and colonization of the distal colon were seen at 24 h but disappeared by 48 h following i.r. infection. Histopathological approaches demonstrated significant damage and destruction of mucosal and submucosal layers, thickened intestinal wall, edema, erosion, infiltration of neutrophils, and depletion of goblet cells in the distal colon. Furthermore, robust expression of IL-8, IL-1beta, and inducible NO synthase mRNA was detected in the colon from 6 to 24 h following Shigella infection. Most importantly, in our new shigellosis model, guinea pigs vaccinated with an attenuated S. flexneri 2a SC602 strain possessing high levels of mucosal IgA Abs showed milder symptoms of bacillary dysentery than did animals receiving PBS alone after Shigella infection. In the guinea pig, administration of Shigella by i.r. route induces acute inflammation, making this animal model useful for assessing the protective efficacy of Shigella vaccine candidates.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.     

Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors

Shigella possess 220 kb plasmid, and the major virulence determinants, called effectors, and the type III secretion system (TTSS) are exclusively encoded by the plasmid. The genome sequences of S. flexneri strains indicate that several ipaH family genes are located on both the plasmid and the chromosome, but whether their chromosomal IpaH cognates can be secreted from Shigella remains unknown. Here we report that S. flexneri strain, YSH6000 encodes seven ipaH cognate genes on the chromosome and that the IpaH proteins are secreted via the TTSS. The secretion kinetics of IpaH proteins by bacteria, however, showed delay compared with those of IpaB, IpaC and IpaD. Expression of the each mRNA of ipaH in Shigella was increased after bacterial entry into epithelial cells, and the IpaH proteins were secreted by intracellular bacteria. Although individual chromosomal ipaH deletion mutants showed no appreciable changes in the pathogenesis in a mouse pulmonary infection model, the DeltaipaH-null mutant, whose chromosome lacks all ipaH genes, was attenuated to mice lethality. Indeed, the histological examination for mouse lungs infected with the DeltaipaH-null showed a greater inflammatory response than induced by wild-type Shigella, suggesting that the chromosomal IpaH proteins act synergistically as effectors to modulate the host inflammatory responses.     

Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.     

A type III effector protease NleC from enteropathogenic Escherichia coli targets NF-κB for degradation

Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that enteropathogenic Escherichia coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IκB degradation and nuclear translocation of the p65 subunit of NF-κB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP-NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-κB components, c-Rel and p50. Recombinant His(6) -NleC induced p65 and p50 cleavage in HeLa cell lysates and mutation of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-κB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation.     

Binding affects the tertiary and quaternary structures of the Shigella translocator protein IpaB and its chaperone IpgC

Shigella flexneri uses its type III secretion system (T3SS) to promote invasion of human intestinal epithelial cells as the first step in causing shigellosis, a life-threatening form of dysentery. The Shigella type III secretion apparatus (T3SA) consists of a basal body that spans the bacterial envelope and an exposed needle that injects effector proteins into target cells. The nascent Shigella T3SA needle is topped with a pentamer of the needle tip protein invasion plasmid antigen D (IpaD). Bile salts trigger recruitment of the first hydrophobic translocator protein, IpaB, to the tip complex where it senses contact with a host membrane. In the bacterial cytoplasm, IpaB exists in a complex with its chaperone IpgC. Several structures of IpgC have been determined, and we recently reported the 2.1 ? crystal structure of the N-terminal domain (IpaB(74.224)) of IpaB. Like IpgC, the IpaB N-terminal domain exists as a homodimer in solution. We now report that when the two are mixed, these homodimers dissociate and form heterodimers having a nanomolar dissociation constant. This is consistent with the equivalent complexes copurified after they had been co-expressed in Escherichia coli. Fluorescence data presented here also indicate that the N-terminal domain of IpaB possesses two regions that appear to contribute additively to chaperone binding. It is also likely that the N-terminus of IpaB adopts an alternative conformation as a result of chaperone binding. The importance of these findings within the functional context of these proteins is discussed.     

Structural and functional studies on the N-terminal domain of the Shigella type III secretion protein MxiG

MxiG is a single-pass membrane protein that oligomerizes within the inner membrane ring of the Shigella flexneri type III secretion system (T3SS). The MxiG N-terminal domain (MxiG-N) is the predominant cytoplasmic structure; however, its role in T3SS assembly and secretion is largely uncharacterized. We have determined the solution structure of MxiG-N residues 6-112 (MxiG-N(6-112)), representing the first published structure of this T3SS domain. The structure shows strong structural homology to forkhead-associated (FHA) domains. Canonically, these cell-signaling modules bind phosphothreonine (Thr(P)) via highly conserved residues. However, the putative phosphate-binding pocket of MxiG-N(6-112) does not align with other FHA domain structures or interact with Thr(P). Furthermore, mutagenesis of potential phosphate-binding residues has no effect on S. flexneri T3SS assembly and function. Therefore, MxiG-N has a novel function for an FHA domain. Positioning of MxiG-N(6-112) within the EM density of the S. flexneri needle complex gives insight into the ambiguous stoichiometry of the T3SS, supporting models with 24 MxiG subunits in the inner membrane ring.