High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli.

Lipopolysaccharide from smooth strains of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli O111:B4, O55:B5, and O127:B8 was fractionated by gel filtration chromatography. All lipopolysaccharide samples separated into three major populations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions from S. typhimurium and S. minnesota indicated that the three peaks were made up of molecules with average O-antigen lengths of (i) 70 or more repeat units, (ii) 30 and 20 repeats units in the samples from S. typhimurium and S. minnesota, respectively, and (iii) 1 repeat unit. In contrast to the Salmonella samples, peak 1 from the E. coli samples was not detected on polyacrylamide gels and lacked detectable phosphate. This high-molecular-weight material had a sugar composition similar to that of O-antigen and was tentatively identified as capsular polysaccharide. Peaks 2 and 3 of the E. coli samples were analogous to those of the Salmonella isolates, containing lipopolysaccharide molecules with averages of 18 and 1 O-antigen repeat units, respectively. These lipopolysaccharide molecules did not completely dissociate during electrophoresis, and multimers were detected as distinct, anomalous, slow-migrating bands. Increasing the concentration of sodium dodecyl sulfate in the gels resulted in the dissociation of these multimers.     

Accelerated Adsorption of Bacteriophage T5 to Escherichia coli F, Resulting from Reversible Tail Fiber-Lipopolysaccharide Binding

A dual specificity for phage T5 adsorption to Escherichia coli cells is shown. The tail fiber-containing phages T5(+) and mutant hd-3 adsorbed rapidly to E. coli F (1.2 x 10(-9) ml min(-1)), whereas the adsorption rate of the tail fiber-less mutants hd-1, hd-2, and hd-4 was low (7 x 10(-11) ml min(-1)). The differences in adsorption rates were due to the particular lipopolysaccharide structure of E. coli F. Phage T4-resistant mutants of E. coli F with an altered lipopolysaccharide structure exhibited similar low adsorption for all phage strains with and without tail fibers. The same held true for E. coli K-12 and B which also differ from E. coli F in their lipopolysaccharide structures. Only the tail fiber-containing phages reversibly bound to isolated lipopolysaccharides of E. coli F. Infection by all phage strains strictly depended on the tonA-coded protein in the outer membrane of E. coli. We assume that the reversible preadsorption by the tail fibers to lipopolysaccharide accelerates infection which occurs via the highly specific irreversible binding of the phage tail to the tonA-coded protein receptor. The difference between rapid and slow adsorption was also revealed by the competition between ferrichrome and T5 for binding to their common tonA-coded receptor in tonB strains of E. coli. Whereas binding of T5(+) to E. coli K-12 and of the tail-fiber-less mutant hd-2 to E. coli F and K-12 was inhibited 50% by about 0.01 muM ferrichrome, adsorption of T5 to E. coli F was inhibited only 40% by even 1,000-fold higher ferrichrome concentrations.     

Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobcter freundii and Citrobacter sedlakii

A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freunndii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [ 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.     

A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains.

The modal distribution of O-antigen chain length is determined by the Wzz (Cld/Rol) protein in those cases in which it has been studied. The system of O-antigen synthesis in Escherichia coli serotypes O8 and O9 is different from that reported for most other bacteria, and chain length distribution is thought not to be determined by a Wzz protein. We report the existence in E. coli O8 and O9 strains of wzz genes which are very similar to and have sequences within the range of variation of those which determine the chain length of typical O antigens. We also find that wzz genes previously identified by their effect on O-antigen chain length, when cloned and transferred to O8 and O9 strains, affect the chain length of a capsule-related form of LPS, K(LPS). We conclude that in at least some O8 and O9 strains there is a wzz gene which controls the chain length of K(LPS) but has no effect on the O8 or O9 antigen.     

大肠埃希菌脂多糖诱导的血小板降解及急性休克:O抗原在LPS诱导的免疫反应中的作用

目的探讨LPS中的0抗原部分与其它部分在血小板反应中的作用。方法给BALB／c小鼠注人大肠埃希菌野生株E．coli O8、O9、K-12（不含有O抗原）及2株重组变异的K-12株（携带编码O8、O9的O抗原rfb基因）。结果K-12的LPS引起血小板反应及急性休克能力较弱,O8及O9引起一定的反应,而这2种重组的LPS,即在K-12的LPS上带有O8或O9的O抗原．显示出极强的活性。静脉注入补体C5的阻止剂后,重组株LPS的作用消失了。而且在缺乏补体C5小鼠DBA／2中,重组的LPS能引起血小板的聚集但不能降解,也不能引起休克症状。结论诱导血小板反应及急性休克的能力依赖于LPS结构;O抗原及R核心抗原是表现活性的必要结构;LPS诱导的血小板反应及急性休克依赖补体系统。     

Structural and genetic relationships of two pairs of closely related O-antigens of Escherichia coli and Salmonella enterica: E. coli O11/S. enterica O16 and E. coli O21/S. enterica O38

The O-antigen is a part of the lipopolysaccharide molecule present in the outer membrane of Gram-negative bacteria, and is essential for the full function of the microorganisms. Salmonella enterica and Escherichia coli are taxonomically closely related species. In this study, the O-antigen structures of S. enterica O16 and O38 and E. coli O11 were determined. Salmonella enterica O38 and E. coli O21 were found to have identical O-antigen structures, whereas S. enterica O16 and E. coli O11 had closely related structures, differing only in the presence of a lateral glucose residue and O-acetylation of a mannose residue in the former. The O-antigen gene clusters of S. enterica O16 and O38 and E. coli O11 were sequenced and analyzed together with that of E. coli O21 retrieved from the GenBank. Each S. enterica/E. coli pair was found to contain the same set of genes organized in the same manner and to share 56-78% overall DNA identity. These data suggest that the O-antigen gene clusters of each pair studied originated from a common ancestor. Thus, it has become evident that in the past, the degree of relatedness between the O-antigens of S. enterica and E. coli was underestimated.     

Immunoprotective murine monoclonal antibodies specific for the outer-core polysaccharide and for the O-antigen of Escherichia coli 0111:B4 lipopolysaccharide (LPS)

The antigen specificity of two immunoprotective monoclonal antibodies derived from mice immunized with Escherichia coli 0111:B4 bacteria and boosted with purified lipopolysaccharide (LPS) were investigated. One of the antibodies, B7, was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunostaining to bind to the O-antigen containing LPS species, whereas the other antibody, 5B10, reacted with both O-antigen containing homologs and the O-antigen-deficient LPS. 5B10 did not bind to LPS from E. coli J5, an Rc mutant of E. coli 0111:B4 that lacks both the O-antigen and outer core sugars. 5B10 did not cross-react with LPS from several other E. coli strains. Thus 5B10 appeared to recognize a type-specific epitope in the outer core of LPS exclusive of Rc determinants. The monoclonal antibody specific for the polymeric O-antigen is of the IgG3 subclass, and the monoclonal antibody 5B10 specific for the outer core of LPS is an IgG2a. Although B7 and 5B10 were equally able to protect mice from a lethal challenge of E. coli 0111:B4 organisms, the outer core-specific IgG2a antibody was much more efficient at mediating the binding of human complement C3 than the O-antigen-specific IgG3 monoclonal antibody.     

Molecular analysis of Shigella boydii O1 O-antigen gene cluster and its PCR typing

Shigella is an important human pathogen and is closely related to Escherichia coli. O-antigen is the most variable part of the lipopolysaccharide on the cell surface of Gram-negative bacteria and plays an important role in pathogenicity. The O-antigen gene cluster of S. boydii O1 was sequenced. The putative genes encoding enzymes for rhamnose synthesis, transferases, O-unit flippase, and O-unit polymerase were identified on the basis of homology. The O-antigen gene clusters of S. boydii O1 and E. coli O149, which share the same O-antigen form, were found to have the same genes and organization by adjacent gene PCR assay. Two genes specific for S. boydii O1 and E. coli O149 were identified by PCR screening against E. coli- and Shigella-type strains of the 186 known O-antigen forms and 39 E. coli clinical isolates. A PCR sensitivity of 103 to 104 CFU/mL overnight culture of S. boydii O1 and E. coli O149 was obtained. S. boydii O1 and E. coli O149 were differentiated by PCR using lacZ- and cadA-based primers.     

Comparative characteristics of antigenic conversion induced by Shigella flexneri and Escherichia coli 0129 phages

The interaction of S. flexneri converting phages PE5, P90 and fV with E. coli antigenic variant O129, E. coli O129 converting phage VB with the above antigenic variant and with S. flexneri y-variant was studied. Phage PE5 and phage VB were found to induce the conversion of O-antigen in E. coli antigenic variant 0129 and in S. flexneri y-variant with the detection of antigens V and 7,8. Phages P90 and fV induced no conversion of O-antigen. Changes in the antigenic properties of convertants were confirmed by the results obtained in the agglutination test and in the agglutination adsorption test.     

Structural elucidation of the O-antigenic polysaccharides from Escherichia coli O21 and the enteroaggregative Escherichia coli strain 105.

The structure of the O-antigen polysaccharide of the lipopolysaccharide from an enteroaggregative Escherichia coli (strain 105) has been elucidated, using primarily one-dimensional and two-dimensional NMR experiments. The sequence of residues was deduced with heteronuclear multiple-bond correlation and NOESY experiments. The structure of the repeating unit of the polysaccharide from the enteroaggregative E. coli is as follows:[sequence: see text] The structure of the O-antigen from enteroaggregative E. coli strain 105 was shown to be identical with that of E. coli O21 by sugar and methylation analyses as well as by 1H-NMR and 13C-NMR spectroscopy.     

Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O164 cross-reacting with Shigella dysenteriae type 3.

The structure of the O-antigen polysaccharide from Escherichia coli O164 has been determined. Nuclear magnetic resonance spectroscopy together with component and methylation analyses of lipid free polysaccharide were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity NMR experiments. It is concluded that the polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [structure: see text]. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on intact lipopolysaccharide and from the resulting molecular mass, the O-antigen part was estimated to contain approximately 24 repeating units. The nature of the previously reported cross-reactivity of this O-antigen to those of Escherichia coli O124 and Shigella dysenteriae type 3 is discussed.     

Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/ F41) and the genetic relationship to other O101 rfb loci

The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex. Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

Defective O-antigen polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress

We have previously shown that the TolA protein is required for the correct surface expression of the Escherichia coli O7 antigen lipopolysaccharide (LPS). In this work, delta tolA and delta pal mutants of E. coli K-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding the Klebsiella pneumoniae O1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis of Salmonella enterica O:54). Both DeltatolA and delta pal mutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression of K. pneumoniae O1 LPS and S. enterica O:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in a wzy (polymerase)-dependent manner (like those of E. coli O7 and O16) but not for O antigens assembled by wzy-independent pathways (like K. pneumoniae O1 and S. enterica O:54). Furthermore, we show that the reduced surface expression of O16 LPS in delta tolA and delta pal mutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intact tolA and pal genes, respectively. Using derivatives of W3110 delta tolA and W3110 delta pal containing lacZ reporter fusions to fkpA and degP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses in tol+ and pal+ genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.     

Genetic analysis of the Cronobacter sakazakii O4 to O7 O-antigen gene clusters and development of a PCR assay for identification of all C. sakazakii O serotypes

The Gram-negative bacterium Cronobacter sakazakii is an emerging food-borne pathogen that causes severe invasive infections in neonates. Variation in the O-antigen lipopolysaccharide in the outer membrane provides the basis for Gram-negative bacteria serotyping. The O-antigen serotyping scheme for C. sakazakii, which includes seven serotypes (O1 to O7), has been recently established, and the O-antigen gene clusters and specific primers for three C. sakazakii serotypes (O1, O2, and O3) have been characterized. In this study, the C. sakazakii O4, O5, O6, and O7 O-antigen gene clusters were sequenced, and gene functions were predicted on the basis of homology. C. sakazakii O4 shared a similar O-antigen gene cluster with Escherichia coli O103. The general features and anomalies of all seven C. sakazakii O-antigen gene clusters were evaluated and the relationship between O-antigen structures and their gene clusters were investigated. Serotype-specific genes for O4 to O7 were identified, and a molecular serotyping method for all C. sakazakii O serotypes, a multiplex PCR assay, was developed by screening against 136 strains of C. sakazakii and closely related species. The sensitivity of PCR-based serotyping method was determined to be 0.01 ng of genomic DNA and 10(3) CFU of each strain/ml. This study completes the elucidation of C. sakazakii O-antigen genetics and provides a molecular method suitable for the identification of C. sakazakii O1 to O7 strains.     

Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β- d -Gal p NAc-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p NAc-(1→4)-β- d -Gal p -(1→3)-α- d -Gal p NAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution of d -GlcNAc for d -Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 ( d -GlcNAc) and O117 ( d -Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.     

Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1).

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.     

In vitro reconstruction of the chain termination reaction in biosynthesis of the Escherichia coli O9a O-polysaccharide: the chain-length regulator, WbdD, catalyzes the addition of methyl phosphate to the non-reducing terminus of the growing glycan

The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located at the cytoplasmic membrane. An ABC-transporter subsequently exports the polymer to the periplasm where it is assembled onto lipopolysaccharide prior to translocation to the cell surface. The chain length of the O9a O-PS is regulated by the dual kinase/methyltransferase activity of the WbdD enzyme and modification of the polymer is crucial for binding and export by the ABC-transporter. Previous biochemical data provided evidence for phosphorylation/methylation at the non-reducing end of the O9a O-PS but the structure of the terminus has not been determined. Here, we describe the exploitation of a synthetic O9a O-PS repeating unit carrying a fluorescent tag as an acceptor for in vitro phosphorylation and methylation by a purified soluble form of WbdD. Phosphorylation of the acceptor was evident by both a mobility shift in thin layer chromatography and radiolabeling of the acceptor using [γ-(33)P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-(3)H]adenosyl-methionine as a substrate, in the presence of ATP. NMR spectroscopic and mass spectrometric methods were used to determine the precise structure of the terminal modification, leading to the conclusion that WbdD catalyzes the addition of a novel methyl phosphate group to the 3-position of the non-reducing terminal mannose of the O9a O-PS repeating unit.     

Structural studies utilizing 13C-enrichment of the O-antigen polysaccharide from the enterotoxigenic Escherichia coli O159 cross-reacting with Shigella dysenteriae type 4.

The structure of the O-antigen polysaccharide from Escherichia coli O159 has been determined using primarily NMR spectroscopy of the 13C-enriched polysaccharide. The sequence of the sugar residues could be determined by heteronuclear multiple bond connectivity NMR experiments. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [sequence: see text] Matrix assisted laser desorption ionization mass spectrometry was performed on intact lipopolysaccharide and from the resulting molecular mass the O-antigen part was estimated to contain approximately 23 repeating units. Cross-reactivity of this O-antigen to that of Shigella dysenteriae type 4 was confirmed using enzyme-linked immunoabsorbant assay.