Localization of enterobacterial common antigen: Proteus mirabilis and its various L-forms.

An investigation of Proteus mirabilis wild-type strains and their various derived L-forms shows that the enterobacterial common antigen (ECA) is localized in the outer membrane of the cell envelope of these strains. In strains where the outer membrane is lacking (stable protoplast L-forms) or where its amount is reduced (spheroplast UL19) no ECA or only reduced amounts of it are detected by serological tests or by ferritin-labeling techniques.     

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.     

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。     

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.     

The effect of antibodies to the enterobacterial common antigen (ECA) on experimental mouse salmonellosis

Abstract The protective capacity of antibodies to the enterobacterial common antigen (ECA) was tested in experimental mouse salmonellosis after intraperitoneal challenge by moderately virulent smooth Salmonella typhimurium . No evidence could be found for a role of anti-ECA in protection or opsonization in assays in which homologous anti- Salmonella antiserum was strongly positive.     

Characterization of an enterobacterial common antigen (ECA) epitope recognized by anti-ECA-tetanus toxoid conjugate serum

The antigenic reactivity of both native and chemically modified enterobacterial common antigen (ECA) with anti-ECA-tetanus toxoid (TT) conjugate serum was investigated. The results obtained suggest that reduction of the carboxyl group of the mannosaminuronic acid component of ECA diminishes, but does not destroy its antigenic reactivity. Each of the sugar components were found to contribute to its reactivity with anti-ECA-TT conjugate serum, indicating that the trisaccharide repeating unit represents the ECA epitope. A nonasaccharide (trimer of the ECA repeating unit) inhibited antibody binding better than the hexasaccharide dimer, a finding which suggests that oligosaccharide conformation also makes a contribution to its inhibitory activity.     

Identification and biosynthesis of cyclic enterobacterial common antigen in Escherichia coli

Phosphoglyceride-linked enterobacterial common antigen (ECA(PG)) is a cell surface glycolipid that is synthesized by all gram-negative enteric bacteria. The carbohydrate portion of ECA(PG) consists of linear heteropolysaccharide chains comprised of the trisaccharide repeat unit Fuc4NAc-ManNAcA-GlcNAc, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The potential reducing terminal GlcNAc residue of each polysaccharide chain is linked via phosphodiester linkage to a phosphoglyceride aglycone. We demonstrate here the occurrence of a water-soluble cyclic form of enterobacterial common antigen, ECA(CYC), purified from Escherichia coli strains B and K-12 with solution nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and additional biochemical methods. The ECA(CYC) molecules lacked an aglycone and contained four trisaccharide repeat units that were nonstoichiometrically substituted with up to four O-acetyl groups. ECA(CYC) was not detected in mutant strains that possessed null mutations in the wecA, wecF, and wecG genes of the wec gene cluster. These observations corroborate the structural data obtained by NMR and ESI-MS analyses and show for the first time that the trisaccharide repeat units of ECA(CYC) and ECA(PG) are assembled by a common biosynthetic pathway.     

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.     

Assembly of cyclic enterobacterial common antigen in Escherichia coli K-12

We describe here the purification and quantification of a water-soluble cyclic form of enterobacterial common antigen (ECA(CYC)) from Escherichia coli K-12 as well as information regarding its subcellular location and the genetic loci involved in its assembly. Structural characterization of purified ECA(CYC) molecules obtained from E. coli K-12 revealed that they uniformly contained four trisaccharide repeat units, and they were substituted with from zero to four O-acetyl groups. Cells from overnight cultures contained approximately 2 microg ECA(CYC) per milligram (dry weight), and cell fractionation studies revealed that these molecules were localized exclusively in the periplasm. The synthesis and assembly of ECA(CYC) were found to require the wzxE and wzyE genes of the wec gene cluster. These genes encode proteins involved in the transmembrane translocation of undecaprenylpyrophosphate-linked ECA trisaccharide repeat units and the polymerization of trisaccharide repeat units, respectively. Surprisingly, synthesis of ECA(CYC) was dependent on the wzzE gene, which is required for the modulation of the polysaccharide chain lengths of phosphoglyceride-linked ECA (ECA(PG)). The presence of ECA(CYC) in extracts of several other gram-negative enteric organisms was also demonstrated; however, it was not detected in cell extracts of Pseudomonas aeruginosa. These data suggest that in addition to ECA(PG), ECA(CYC) may be synthesized in many, if not all, members of the Enterobacteriaceae.     

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).     

Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen.

It is known that only certain strains of the family of Enterobacteriaceae, notably rough (R) mutants with the type R1 or R4 core, evoked antibodies in high titers against the common enterobacterial antigen (CA) after immunization of rabbits with heated cell suspensions. The present investigation deals with genetic and immunochemical aspects of certain R1 and R4 mutants isolated from Escherichia coli 08 and various Shigella serotypes which, unexpectedly, do not induce CA antibody formation. Immunochemical and genetical (transduction and conjugation) experiments revealed that the rough phenotype of these special mutants was evoked by a mutation of pyrE-linked rfa gene, called rfaL, which is involved in translocation of O-specific polysaccharides onto the lipopolysaccharide core. The transduction of the defective rfaL, allele into appropriate rough recipients results in transductants which have simultaneously lost the ability to evoke CA antibodies. This finding suggests that a close connection exists between the function of the rfaL gene and the expression of CA immunogenicity in R1 and R4 mutants. One of the strains synthesized neither O-hapten nor CA, suggesting a mutation in a region equivalent to the rfe genes of Salmonella.     

Role for Salmonella enterica enterobacterial common antigen in bile resistance and virulence

Passage through the digestive tract exposes Salmonella enterica to high concentrations of bile salts, powerful detergents that disrupt biological membranes. Mutations in the wecD or wecA gene, both of which are involved in the synthesis of enterobacterial common antigen (ECA), render S. enterica serovar Typhimurium sensitive to the bile salt deoxycholate. Competitive infectivity analysis of wecD and wecA mutants in the mouse model indicates that ECA is an important virulence factor for oral infection. In contrast, lack of ECA causes only a slight decrease in Salmonella virulence during intraperitoneal infection. A tentative interpretation is that ECA may contribute to Salmonella virulence by protecting the pathogen from bile salts.