Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.     

LPS Unmasking of Shigella flexneri Reveals Preferential Localisation of Tagged Outer Membrane Protease IcsP to Septa and New Poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP^HA was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP^HA was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP^HA was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP^HA and IcsA showed that IcsP^HA preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP^HA in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP^HA was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP^HA detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.     

Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz

Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16-35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random-length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Actin-based motility is sufficient for bacterial membrane protrusion formation and host cell uptake

Shigella flexneri replicates in the cytoplasm of host cells, where it nucleates host cell actin filaments at one pole of the bacterial cell to form a 'comet tail' that propels the bacterium through the host's cytoplasm. To determine whether the ability to move by actin-based motility is sufficient for subsequent formation of membrane-bound protrusions and intercellular spread, we conferred the ability to nucleate actin on a heterologous bacterium, Escherichia coli . Previous work has shown that IcsA (VirG), the molecule that is necessary and sufficient for actin nucleation and actin-based motility, is distributed in a unipolar fashion on the surface of S. flexneri . Maintenance of the unipolar distribution of IcsA depends on both the S. flexneri outer membrane protease IcsP (SopA) and the structure of the lipopolysaccharide (LPS) in the outer membrane. We co-expressed IcsA and IcsP in two strains of E. coli that differed in their LPS structures. The E. coli were engineered to invade host cells by expression of invasin from Yersinia pseudotuberculosis and to escape the phagosome by incubation in purified listeriolysin O (LLO) from Listeria monocytogenes . All E. coli strains expressing IcsA replicated in host cell cytoplasm and moved by actin-based motility. Actin-based motility alone was sufficient for the formation of membrane protrusions and uptake by recipient host cells. The presence of IcsP and an elaborate LPS structure combined to enhance the ability of E. coli to form protrusions at the same frequency as S. flexneri , quantitatively reconstituting this step in pathogen intercellular spread in a heterologous organism. The frequency of membrane protrusion formation across all strains tested correlates with the efficiency of unidirectional actin-based movement, but not with bacterial speed.     

Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host neural Wiscott-Aldrich syndrome protein

The IcsA (VirG) protein of Shigella flexneri is a polarly localized, outer membrane protein that is essential for virulence. Within host cells, IcsA activates the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn recruits the Arp2/3 complex, which nucleates host actin to form F-actin comet tails and initiate bacterial motility. Linker insertion mutagenesis was undertaken to randomly introduce 5-amino-acid in-frame insertions within IcsA. Forty-seven linker insertion mutants were isolated and expressed in S. flexneri Delta icsA strains. Mutants were characterized for IcsA protein production, cell surface expression and localization, intercellular spreading, F-actin comet tail formation, and N-WASP recruitment. Using this approach, we have identified a putative autochaperone region required for IcsA biogenesis, and our data suggest an additional region, not previously identified, is required for N-WASP recruitment.     

Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement.

Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 3'(2')-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form.     

The making of a gradient: IcsA (VirG) polarity in Shigella flexneri

The generation and maintenance of subcellular organization in bacteria is critical for many cell processes and properties, including growth, structural integrity and, in pathogens, virulence. Here, we investigate the mechanisms by which the virulence protein IcsA (VirG) is distributed on the bacterial surface to promote efficient transmission of the bacterium Shigella flexneri from one host cell to another. The outer membrane protein IcsA recruits host factors that result in actin filament nucleation and, when concentrated at one bacterial pole, promote unidirectional actin-based motility of the pathogen. We show here that the focused polar gradient of IcsA is generated by its delivery exclusively to one pole followed by lateral diffusion through the outer membrane. The resulting gradient can be modified by altering the composition of the outer membrane either genetically or pharmacologically. The gradient can be reshaped further by the action of the protease IcsP (SopA), whose activity we show to be near uniform on the bacterial surface. Further, we report polar delivery of IcsA in Escherichia coli and Yersinia pseudotuberculosis, suggesting that the mechanism for polar delivery of some outer membrane proteins is conserved across species and that the virulence function of IcsA capitalizes on a more global mechanism for subcellular organization.     

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.     

IcsA surface presentation in Shigella flexneri requires the periplasmic chaperones DegP, Skp, and SurA

A Shigella flexneri degP mutant, which was defective for plaque formation in Henle cell monolayers, had a reduced amount of IcsA detectable on the bacterial surface with antibody. However, the mutant secreted IcsA to the outer membrane at wild-type levels. This suggests that IcsA adopts an altered conformation in the outer membrane of the degP mutant with reduced exposure on the cell surface. IcsA is, therefore, unlikely to be accessible to actin-nucleating proteins within the eukaryotic cell cytoplasm, which is required for bacterial movement within the host cell and cell-to-cell spread. The degP mutant was somewhat more sensitive to detergents, antibiotics, and the antimicrobial peptide magainin, indicating that the degP phenotype was not limited to IcsA surface presentation. The plaque defect of the degP mutant, which is independent of DegP protease activity, was suppressed by overexpression of the periplasmic chaperone Skp but not by SurA. S. flexneri skp and surA mutants failed to form plaques in Henle cell monolayers and were defective in cell surface presentation and polar localization of IcsA. Therefore, the three periplasmic folding factors DegP, Skp, and SurA were all required for IcsA localization and plaque formation by S. flexneri.     

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。     

Outer Membrane Protein A (OmpA): A New Player in Shigella flexneri Protrusion Formation and Inter-Cellular Spreading

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.     

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.     

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.     

Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin-based motility

Shigella pathogenesis involves bacterial invasion of colonic epithelial cells and movement of bacteria through the cytoplasm and into adjacent cells by means of actin-based motility. The Shigella protein IcsA (VirG) is unipolar on the bacterial surface and is both necessary and sufficient for actin-based motility. IcsA is inserted into the outer membrane as a 120-kDa polypeptide that is subsequently slowly cleaved, thereby releasing the 95-kDa amino-terminal portion into the culture supernatant. IcsP, the major Shigella protease that cleaves IcsA, was identified and cloned. It has significant sequence similarity to the E. coli serine proteases, OmpP and OmpT. Disruption of icsP in serotype 2a S. flexneri leads to a marked reduction in IcsA cleavage, increased amounts of IcsA associated with the bacterium and altered distribution of IcsA on the bacterial surface. The icsP mutant displays significantly increased rates of actin-based motility, with a mean speed 27% faster than the wild-type strain; moreover, a significantly greater percentage of the icsP mutant moves in the cytoplasm. Yet, plaque formation on epithelial monolayers by the mutant was not altered detectably. These data suggest that IcsA, and not a host protein, is limiting in the rate of actin-based motility of wild-type serotype 2a S. flexneri .     

Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that infects immunocompromised patients and trauma victims and causes fatal lung infections in people with cystic fibrosis. This microorganism produces a number of virulence factors, one of which is lipopolysaccharide (LPS), which has been shown to mediate many biological effects including resistance to serum killing and phagocytosis. These biological activities have been correlated to the length of the O-polysaccharide and its distribution on the outer membrane. Wzz is responsible for regulation of the size distribution of the O-antigen. Wzz has been found to participate solely in the Wzy-dependent pathway for LPS biosynthesis, which produces heteropolymeric O-polysaccharide such as the B-band LPS of P. aeruginosa. Our laboratory has previously reported characterization of a Wzz protein encoded in the B-band O-antigen biosynthesis cluster of PAO1. The availability of the genome sequence of P. aeruginosa PAO1 has made it possible to identify a second functional Wzz protein (PA0938, Wzz2). Gene replacement was used to generate an unmarked wzz2delta knock-out and a wzz2delta/wzz1::Gm double knock-out. As expected, the wzz2delta strain produced LPS with modal length imparted by Wzz1, and the wzz2delta/wzz1::Gm strain produced LPS O-antigen with a non-modal (random) length. Both wzz1 and wzz2 from P. aeruginosa PAO1 were cloned and expressed with an N-terminal His6 tag. His6-Wzz1 and His6-Wzz2 were purified to near homogeneity by immobilized metal affinity chromatography (IMAC). These preparations were used to develop specific polyclonal antibodies against each of the proteins. In vivo protein cross-linking followed by Western immunoblotting indicated that Wzz1 forms dimers whereas Wzz2 forms octamers. By generation of a wzz2delta/rmlC double mutant and analysis of the LPS, we have made the novel observation that polymerization of modal chain length-distributed O-antigen occurred before ligation to the lipid A core. We have shown an association between the Wzz proteins and O-antigen polymer chains using immunoprecipitation with anti-O5 O-antigen monoclonal antibody MF15-4. Both Wzz1 and Wzz2 could be co-precipitated with O5 polymer.     

与福氏2a志贺氏菌2457T株ArgT相互作用蛋白的筛选及鉴定

目的：筛选、鉴定与福氏2a志贺氏菌2457T株ArgT相互作用的蛋白,以进一步研究ArgT在福氏2a志贺氏菌致病过程中发挥的作用。方法：将ArgT与GST融合表达,通过体外GST沉降实验和MALDI-TOF MS技术,筛选并鉴定与福氏2a志贺氏菌2457T株ArgT相互作用的蛋白。结果：筛选并鉴定到与福氏2a志贺氏菌2457TArgT相互作用的蛋白OmpR。结论：OmpR与ArgT存在体外相互作用。     

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。     

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。