Biosynthesis of enterobacterial common antigen.

Cultures of Salmonella typhimurium pulse-labeled with N-acetyl-D-[3H]glucosamine ([3H]GlcNAc) incorporated isotope into a GlcNAc-linked lipid that was tentatively identified as GlcNAc-pyrophosphorylundecaprenol. The incorporation of [3H]GlcNAc into this compound was abolished when cells were pulse-labeled in the presence of the antibiotic tunicamycin. Tunicamycin also abolished the in vivo synthesis of the haptenic form of enterobacterial common antigen (ECA) in S. typhimurium as determined by the passive hemagglutination test. These data indicated that the synthesis of the GlcNAc-linked lipid is related to ECA synthesis. Support for this conclusion was provided by the following observations. Cultures of Escherichia coli and S. typhimurium incorporated [3H]GlcNAc into cell envelope components that migrated as a homologous series of polymers when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The [3H]GlcNAc-labeled polymers were not detected in mutants of E. coli and S. typhimurium defective in ECA synthesis due to lesions in either the rfe or rff gene clusters. These polymers were identified as ECA based on Western blot analyses employing anti-ECA monoclonal antibody. The incorporation of [3H]GlcNAc into ECA polymers was abolished by tunicamycin when the drug was added to cultures to give a minimum concentration of 3 micrograms/ml. In addition, pulse-chase experiments provided evidence for a precursor-product relationship between the GlcNAc-linked lipid and ECA. These results strongly suggest that the GlcNAc-linked lipid is involved in the biosynthesis of ECA in a manner analogous to the role of carrier lipid in the biosynthesis of O-antigen and peptidoglycan.     

Localization of enterobacterial common antigen in Yersinia enterocolitica by the immunoferritin technique.

Rabbits were immunized with the enterobacterial common antigen (ECA)-immunogenic strain Escherichia coli F470. ECA-specific antiserum was obtained by absorbing the resulting antisera with the genetically closely related ECA-negative strain E. coli F1283. These two strains also served as positive and negative controls in the localization study of ECA in Yersinia enterocolitica strain 75, smooth and rough forms (Ye75S and Ye75R), by the indirect immunoferritin technique. Cells of Ye75S grown at 22 degrees C showed no labeling with ferritin after treatment with the ECA-specific antiserum and subsequent ferritin-conjugated goat anti-rabbit antibodies. If the cells were grown at 40 degrees C, however, most of the cells showed weak ferritin labeling. At this higher growth temperature, the lipopolysaccharide of this strain contains less O-specific chains (6-deoxy-L-altrose), as was shown in a previous study. The rough mutant Ye75R, which lacks O-specific chains completely, showed denser labeling with ferritin. These results indicate that ECA on the cell surface of Ye75S is covered by O-specific chains of the lipopolysaccharide if grown at 22 degrees C and is therefore not accessible to ECA antibodies. It becomes accessible, however, when O-chains are lacking (R mutants) or when they are reduced in size or amount (growth at 40 degrees C).     

ECA, the enterobacterial common antigen

Enterobacterial common antigen (ECA) is a family-specific surface antigen shared by all members of the Enterobacteriaceae and is restricted to this family. It is found in freshly isolated wild-type strains as well as in laboratory strains like Escherichia coli K-12. The family specificity of ECA can be used for taxonomic and diagnostic purposes. ECA is located in the outer leaflet of the outer membrane. It is a glycophospholipid built up by an aminosugar heteropolymer linked to an L-glycerophosphatidyl residue. In a few rough mutants, in addition, the sugar chain can be bound to the complete lipopolysaccharide (LPS) core. Recently, for Shigella sonnei a lipid-free cyclic form of ECA was reported. The genetical determination of ECA is closely related to that of lipopolysaccharide. For biosynthesis of ECA and LPS partly the same sugar precursors and the same carrier lipid is used.     

Adhesion of Proteus mirabilis and Proteus vulgaris to uroepithelial cells following exposure to various antimicrobial agents.

The effect of subinhibitory concentrations of netilmicin, ceftriaxone, cefotaxime, aztreonam and piperacillin on the adherence of Proteus species to uroepithelial cells was examined. Bacterial adhesion to human uroepithelial cells, measured microscopically, was affected by all five antibiotics but to different extents. The most effective was netilmicin. There was a correlation between the decreased rate of bacterial attachment and morphological changes in the drug-exposed bacteria.     

Lipoprotein from Proteus mirabilis.

The biosynthesis of a Proteus mirabilis outer membrane protein of molecular weight of approximately 7,000 was found to be relatively resistant to puromycin and rifampin, as is the case for the Escherichia coli liporotein. Furthermore, the existence of the lipoprotein in P. mirabilis was indicated by a comparison of the amino acid compositions of the purified free and bound forms of this protein with those of the E. coli free and bound lipoproteins.     

肠杆菌共同抗原的研究进展

肠杆菌共同抗原(Enterobacterial common antigen,ECA)是由多糖重复单元组成的多聚糖,几乎表达于所有肠杆菌细菌外膜,具有生物学功能。ECA由多基因协同作用而合成,这些基因在肠杆菌细菌基因组上成簇存在,形成ECA抗原基因簇。ECA是重要的毒力因子,在肠杆菌细菌入侵宿主、体内存活等过程中有一定作用。同时,ECA在维持细菌外膜渗透屏障、鞭毛表达、群集运动及抗胆酸胆盐等方面也有重要作用。此外,锚定在细菌脂多糖核心区的ECALPS还是细菌重要的表面抗原,能激发宿主产生高水平抗体,可以作为疫苗研究的靶点。结合笔者的研究,文中对ECA纯化、基因结构和合成、免疫特性、生物学功能及应用等方面进行了综述。     

Novel bacterial membrane surface display system using cell wall-less L-forms of Proteus mirabilis and Escherichia coli

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.     

Localization of enterobacterial common antigen immunoreactivity in the ribosomal cytoplasm of Escherichia coli cells cryosubstituted and embedded at low temperature.

The application of two on-section immunogold labeling techniques, the Lowicryl K4M (progressive lowering of temperature) procedure and the cryosection technique of Tokuyasu, in a previous work to study the topology of enterobacterial common antigen (ECA) biosynthesis revealed the presence of label on the outer membrane and in areas associated with the inner side of the cytoplasmic membrane. However, labeling was also observed in the ribosomal cytoplasm. The question of whether the cytoplasmic label was a result of ECA displacement during the more slowly acting aldehyde fixation or whether cytoplasmic ECA precursors are true constituents of the ribosomal cytoplasm could not be resolved from these results. In the study described here, cells of the same Escherichia coli F470 strain were reinvestigated by comparison of the progressive lowering of temperature and improved cryosubstitution-low-temperature embedment techniques. The latter procedure, applied directly to nonpretreated and noncentrifuged cells, led to superior ultrastructural preservation of the cytoplasmic organization, with little opportunity for cytoplasmic antigen displacement after the primary cryofixation step; the label distribution obtained supports the conclusion that N-acetylmannosaminuronic acid (ManNAcA)-containing ECA precursors are real constituents of the ribosomal cytoplasm. Results from tunicamycin inhibition studies of ECA biogenesis in the E. coli mutant 2465 suggested that even the ECA precursor UDP-ManNAcA alone or a chemically unidentified product(s) generated from accumulated ManNAcA residues may react with the monoclonal antibody used, leading to weak but clearly positive cytoplasmic labeling. The relatively intense labeling obtained with cells grown in the absence of the drug can be explained by the reactivity of further ManNAcA-containing ECA precursors with the monoclonal antibody used.     

Transposon mutagenesis in Proteus mirabilis.

A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.     

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins. Received: 17 February 1998 / Accepted: 30 June 1998     

Structural studies on the immunogenic form of the enterobacterial common antigen.

It has been shown that enterobacterial common antigen is chemically linked to the hexose region of the R1-type lipopolysaccharide fo the Escherichia coli strain F470 which is immunogenic for this antigen. The number of R core stubs substituted is very small but it is a-parently sufficient to induce antibody formation to the enterobacterial common antigen in the rabbit.