Localization of Enterobacterial Common Antigen: Immunogenic and Nonimmunogenic Enterobacterial Common Antigen-Containing Escherichia coli

In rabbits immunized with intact bacteria, the immune response to the enterobacterial common antigen (ECA) predominantly consists of the production of immunoglobulin M antibodies. This is not dependent on whether the animals are immunized for a short (2 weeks) or a long (3 months) period of time. The highest ECA-specific immunoglobulin G titers were observed after a short immunization with living bacteria. ECA-specific antisera were obtained by absorption with appropriate ECA-negative mutants. The absorbed antisera were then separated on Sephadex G-200. The resulting immunoglobulin G fractions were conjugated to ferritin by glutardialdehyde and used to visualize the distribution of ECA in E. coli. Bacterial strains either possessing the immunogenic form of ECA (F470, 2387) or solely the nonimmunogenic form (F614) or being devoid of both (ECA-negative mutants F1283 and F1327) were labeled with the conjugates. Freezeetchings of ferritin-labeled strains showed a dense labeling of the outer membrane in case of ECA-immunogenic strains, an essentially weaker labeling of the non-immunogenic ECA mutant and, as expected, no labeling of ECA-negative mutants. Comparable results were obtained with the indirect immunofluorescence technique: the whole cell envelope of strain F470 showed a brilliant fluorescence, whereas a much lesser, spotty distribution of fluorescence was noted with strain F614 and none at all was noted with the ECA-negative strains. These data show that ECA is localized in the outer membrane of ECA-containing strains and further demonstrate that there is more in the immunogenic strains than in the nonimmunogenic ones.     

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.     

伤寒杆菌和甲型副伤寒杆菌的稳定L型外膜蛋白和染色体DNA酶切研究

非高渗培养基传代培养的伤寒杆菌和甲型副伤寒杆菌的稳定L型丧失了主要外膜蛋白、特异性表面抗原和染色体DNA部分片段,保留了沙门氏菌共同的内部抗原和形成L型独特的表面抗原。提示伤寒杆菌和甲型副伤寒杆菌的稳定L型多种特性的丧失同其基因或主要外膜蛋白的缺失有关。     

Lipid composition of Staphylococcus aureus and its derived L-forms.

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.     

Strain-specific variation in the protein and lipopolysaccharide composition of the group B meningococcal outer membrane.

Variation in the protein and lipopolysaccharide composition of the meningococcal outer membrane may be due to either serotype differences or to changes in cultural conditions. There are 12 antigenically distinct serotypes of group B meningococci, and these are associated with distinct major outer membrane protein patterns on sodium dodecyl sulfate-polyacrylamide gels. In most strains the predominant outer membrane protein carries the serotype-specific determinant. Certain strains, when grown under similar conditions in different media showed an altered membrane composition. The type 2 strain, M986, grown in modified Frantz medium-A, had a reduced amount of the major 41,000-dalton protein while a 28,000-dalton protein predominated. The altered protein composition may be related to changes in cell metabolism as reflected by the pH of the medium after growth. Growth of the organism in Frantz medium-B caused a negligible drop in pH and the 41,000-dalton protein remained predominant. There was also variation associated with changes in the growth rate. Increasing the aeration caused a concomitant increase in growth rate and cell yield. We observed two quantitative changes in outer membrane proteins in four of seven strains examined: (i) where only a single major protein changed (three strains), and (ii) where an increase in one protein component was associated with a decrease in another protein (one strain). When the strains were grown in tryptic soy broth (Difco Laboratories, Detroit, Mich.) with either high or low aeration, the total protein in the outer membrane remained constant. In contrast, with high aeration there was a significant increase in lipopolysaccharide. These studies suggest that the cell surface proteins may be altered by the organism to meet a variety of environmental conditions.     

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。     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

Mutation in a gene required for lipopolysaccharide and enterobacterial common antigen biosynthesis affects virulence in the plant pathogen Erwinia carotovora subsp. atroseptica.

Spontaneous bacteriophage-resistant mutants of the phytopathogen Erwinia carotovora subsp. atroseptica (Eca) SCRI1043 were isolated and, out of 40, two were found to exhibit reduced virulence in planta. One of these mutants, A5/22, showed multiple cell surface defects including alterations in synthesis of outer membrane proteins, lipopolysaccharide (LPS), enterobacterial common antigen (ECA), and flagella. Mutant A5/22 also showed reduced synthesis of the exoenzymes pectate lyase (Pel) and cellulase (Cel), major virulence factors for this pathogen. Genetic analysis revealed the pronounced pleiotropic mutant phenotype to be due to a defect in a single gene (rffG) that, in Escherichia coli, is involved in the production of ECA. We also show that while other enteric bacteria possess duplicate homologues of this gene dedicated separately to synthesis of LPS and ECA, Eca has a single gene.     

Immunocytochemical localization of enterobacterial common antigen in Escherichia coli and Yersinia enterocolitica cells.

Enterobacterial common antigen (ECA) was localized on Lowicryl K4M sections and on ultrathin cryosections by using either a mouse monoclonal antibody or an absorbed rabbit polyclonal immune serum with the corresponding gold-labeled secondary antibodies. Comparable results were obtained with both monoclonal antibody and polyclonal immune serum. Controls with two ECA-negative mutants revealed the ECA specificity of both labeling systems. On Lowicryl K4M sections, good labeling of the outer membrane and of membrane-associated areas in the cytoplasm was obtained. Unexpectedly, however, the ribosome-containing areas of the cytoplasm also showed significant labeling. On ultrathin cryosections, labeling of the cytoplasmic areas was much weaker, although the density of label in the outer membrane was comparable to that obtained with the Lowicryl K4M sections. With the techniques used, it cannot be completely excluded that the appearance of ECA in the cytoplasm is due to displacement of ECA-reactive sites during the preparation procedure.     

Detection of enterobacterial common antigen on bacterial cell surfaces by colony-immunoblotting: effect of its linkage to lipopolysaccharide

Abstract A colony-immunoblotting procedure is described which allows a quick screening of high numbers of bacteria for their Enterobacterial Common Antigen phenotype. In this assay the intensity of reaction is dependent on the carrier to which ECA is linked and which anchors ECA in the outer membrane. Bacteria containing LPS-linked ECA react stronger in this and in other immunoassays than bacteria containing only the phospholipid-linked ECA.     

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.     

Antigenic structure of Salmonella L forms studied by an immunoferritin method

The antigenic structure of the L-forms of salmonellae (S. typhimurium and S. typhi) in comparison with that of the initial bacterial cultures and revertant cultures was studied with the use of the immunoferritin method. The L-forms of salmonellae were found to retain an insignificant amount of O-antigen, as well as to have K-antigen on the surface of the cytoplasmic membrane, but in a lesser amount than the initial strains. In the cultures reverting from the L-forms of S. typhimurium and S. typhi the quantitative and qualitative characteristics of O- and K-antigens were completely restored.     

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.     

Protein alterations in isolated outer membranes of polymyxin-resistantPseudomonas aeruginosa isolates

Isolated outer membranes fromPseudomonas aeruginosa strains resistant to polymyxin were compared with isolated outer membranes from polymyxin-sensitive strains as to their protein composition as determined by slab polyacrylamide gel electrophoresis. Both the porin protein and the H1 protein were decreased greatly in concentration in the outer membranes from the polymyxin-resistant strains. This confirms previous findings reduced concentrations of these proteins in cell envelopes of these strains. No evidence was found for these decreases being the result of an artifactual loss of these proteins from the outer membrane due to conditions used for growth or for preparation of cell envelopes. Nevertheless, the role of these outer membrane proteins in mediating polymyxin resistance is uncertain.     

Enterobacterial common antigen in rfb deletion mutants of Salmonella typhimurium.

The his-rfb deletion series of Salmonella typhimurium mutants characterized previously by Nikaido et al. was examined for the presence of the enterobacterial common antigen (ECA). All deletions not extending further to the left than the genes for cytidine phosphoabequose synthesis were ECA positive, whereas longer deletions (extending to the genes for thymidine diphosphorhamnose synthesis or further) were ECA negative. When these long-his-rfb deletion strains were studied further, it became clear that they (four out of four studied) had accumulated a second mutation, called rff, close to ilv, which prevented the synthesis of ECA. When rff- was replaced by rff+, the recombinants, now having the his-rfb deletion only, produced traces of ECA, showed reduced viability, increased sensitivity to sodium dodecyl sulfate (SDS) and to a lesser extent, to other anionic detergents, and accumulated secondary "suppressor" mutations upon storage. Such suppressor-containing mutants could be isolated by selecting for resistance to 1% SDS. Thirty of 46 SDS-resistant mutants studied had a second mutation, which alone prevented the synthesis of ECA, close to ilv. This ilv-linked mutation was similar to the rff mutation of the strains studied originally. The new rff mutation was similar to previously described rfe mutations in its close linkage to ilv and association with an ECA-negative phenotype. It differed from rfe, however, by not affecting the synthesis of the O antigens (O-6,7) of group C1. In Salmonella group C1, all ECA genes identified thus far are linked to ilv (rfe and/or rff) and none is linked to rfb.     

Assembly of outer membrane lipoprotein in an Escherichia coli mutant with a single amino acid replacement within the signal sequence of prolipoprotein.

We have compared the rate of assembly of outer membrane proteins including the lipoprotein in a pair of isogenic mlpA+ (lpp+) and mlpA (lpp) strains by pulse-chase experiments. The rate of assembly of the mutant prolipoprotein into the outer membrane was slightly slower than that of the wild-type lipoprotein. The rate of assembly of protein I and protein H-2 was similar in the wild type and the mutant, whereas the rate of assembly of protein II into the outer membrane was slightly reduced in the mutant strain. The organization of outer membrane was slightly reduced in the mutant strain. The organization of outer membrane proteins in the mutant cells appeared not to be grossly altered, based on the apparent resistance (or susceptibility) of these proteins toward trypsin treatment and their resistance to solubilization by Sarkosyl. Like the wild-type lipoprotein, the mutant prolipoprotein in the outer membrane was resistant to trypsin. On the other hand, the prolipoprotein in the cytoplasmic membrane fraction of the mutant cell envelope was susceptible to trypsin digestion. We conclude from these data that proteolytic cleavage of prolipoprotein is not essential for the translocation and proper assembly of lipoprotein into outer membrane.     

Cross-reacting antigens of human thymus myoid cells and stable L-forms of group A streptococci

Indirect immunofluorescence has shown a similarity between the antigen components of group A streptococcus L-forms and human thymus myoid cells. An analogous antigen (or antigens) is present in the cytoplasmic membrane of human myocardial cell fibers. The depletion of antiserum to the streptococcal L-forms both by the culture of L-forms grown in meat or casein media and by the homogenate of the cardiac muscle leads to the inhibition of immunofluorescence. The depletion of serum by the homogenate of other tissues (liver) or by L-form culture does not virtually affect the immunofluorescence intensity. According to the authors' opinion, the similarity of antigens of group A streptococcus L-forms to the antigenic components of organ tissues is likely to be responsible for long-term persistence of the microorganisms under consideration and to favour, in some cases, the occurrence of autoantibodies. The latter circumstance might lead to pathological changes in organs containing cross-reacting antigens.     

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol-pal mutants

The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects the cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.     

Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

Role of outer membrane components in the nonspecific binding ofEscherichia coli to cellulose

The involvement of lipopolysaccharide and outer membrane proteins in the binding ofEscherichia coli to cellulose was investigated. Cellulose binding was assayed in defined strains with or without O-antigenic polysaccharide and in mutants with defects in lipopolysaccharide core synthesis. Binding was also tested in strains lacking major outer membrane proteins. Optimal cellulose binding was exhibited by rough strains and was reduced to various extents in the presence of different O-antigens. Core defects also reduced but did not abolish binding to cellulose. Reduced binding was also found in mutants lacking OmpC protein, but OmpC/OmpA double mutants orompB mutants lacking OmpC and OmpF were not affected. Mutants with reduced cellulose binding were also isolated directly through selection of nonbinding populations after chromatography on cellulose columns. Each of the independent isolates derived fromE. coli K12 with reduced cellulose binding had multiple mutations, with additional phenotypic changes such as phage resistance, increased sensitivity to bile salts, or altered patterns of outer membrane proteins. These results suggest that no single receptor that could be altered by mutation was responsible for the binding ofE. coli to cellulose. Rather, the nonspecific binding of cellulose was more likely to be due to interaction with, or the combined activity of, several integral outer membrane components that could be masked by O-antigen.