Detection of antigens from gram-negative bacilli in urine of children with urinary tract infections. I. Common antigen of Enterobacteriaceae.

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→, occurs as a cyclic form (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA(LPS) to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA(LPS). ECA(LPS) consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.

IntroductionBacteria of the genus Proteus are opportunistic pathogens and cause mainly urinary tract infections. They also play a role in the pathogenesis of reactive arthritis (RA). Patients suffering from Yersinia-triggered RA often carry high titers of antibodies specific to enterobacterial common antigen (ECA). The immunogenicity of ECA has not received much attention thus far and studies have focused mainly on the ECA of Escherichia coli and Yersinia enterocolitica. In this paper the ECA-immunogenicity of Proteus mirabilis is elucidated using two wild-type strains (S1959 and O28) as well as their rough (R) derivative strains R110/1959, which expresses lipopolysaccharide (LPS) with a full core, and R4/O28, which expresses LPS with only an inner core.Materials and MethodsRabbit polyclonal antisera were produced by immunization with boiled suspensions of the four P. mirabilis strains. The antisera were tested for the presence of antibodies specific to ECA by Western blotting using glycerophospholipid- linked ECA (ECAPG) of Salmonella montevideo as antigen. Lipopolysaccharide (LPS) was isolated from the four strains by the hot phenol/water procedure in which ECAPG is co-extracted with LPS and by the phenol/chloroform/petroleum ether extraction that results in the isolation of LPS and/or LPS-linked ECA (ECALPS) free of ECAPG. The LPS preparations were tested for the presence of ECA by Western blotting using ECA-specific antibodies.ResultsThe results demonstrated that all four P. mirabilis strains were ECA immunogenic. The rabbit antisera immunized by the four strains all contained ECA-specific antibodies. Analysis of the LPS preparations demonstrated that the P. mirabilis wild-type strains O28 and S1959 and the Ra mutant strain R110/1959 expressed ECALPS, suggesting that it induced the anti-ECA antibody responses. Only the presence of ECAPG could be demonstrated in the Rc mutant strain R4/O28.ConclusionsThese results therefore suggest that, similar to E. coli, LPS with a full core is also required as the acceptor of ECA for P. mirabilis strains to produce ECALPS. Since ECAPG is not immunogenic unless combined with some proteins, it is likely that ECAPG-protein complexes formed during the intravenous immunization with the Rc mutant strain R4/O28.

An enzyme immunoassay for detection of bacterial antigens in urine specimens from urinary tract infections (UTI) was developed. The antigens detected in infections by Enterobacteriaceae: Escherichia coli, Enterobacter aerogenes, and Proteus sp. were different from the enterobacterial common antigen (ECA). They were digested by trypsin and were characterized by remarkable thermostability; their molecular weights were approximately 34,000 and 31,000. They were detected by means of antisera to ECA or to suspension of E. coli. The frequency of positive results with urine specimens from enterobacterial UTI was 87-99%. In contrast, the urine specimens of UTI by Pseudomonas aeruginosa were positive in fewer than 20% with anti-E. coli sera, but were positive in more than 85% in tests with antiserum to P. aeruginosa. The possible diagnostic application of the described tests was discussed.

Pseudomonas aeruginosa produces a factor (PF) which affects the enterobacterial common antigen (ECA); resulting in failure of the antigen to modify erythrocytes for hemagglutination by ECA antibodies. In the present study the nature of PF was determined. Pronase treatment abolished its activity, indicating the protein nature of PF. PF-treated ECA no longer coated erythrocytes but still reacted with ECA antibodies in immunoelectrophoresis tests with monospecific antiserum to ECA, although differences were noted between the precipitation patterns of PF-treated and untreated ECA. Therefore, PF does not significantly affect the antigenic determinant of ECA but rather affects its lipid carrier, an L-glycerophosphatide. Accordingly, differences in the sugar chain could not be detected by high-voltage paper electrophoretic examinations of partial hydrolysates of PF-treated and untreated ECA. PF liberates all fatty acids from ECA, similarly to commercial lipases, as evidenced by the liberation of unsubstituted glycerol upon HF degradation at 0 degrees C of PF-treated ECA. The lipase activity of PF is indicated also by the observation that a strain of P. aeruginosa with reduced lipase production and an exolipase-negative strain affect ECA either less or not at all. We conclude that PF is a lipase acting on the lipid moiety of ECA, which is responsible for the coating of erythrocytes, but not significantly on the serological determinant, the amino sugar chain.

The passive hemagglutination test for antibodies against the enterobacterial common antigen (ECA) of Kunin was standardized for diagnostic purposes. Human erythrocytes were coated with a soluble ECA+ preparation from Salmonella typhimurium or, as specificity controls, with a similar ECA- preparation from congenic ECA-negative bacteria with saline, and the hemagglutination assay was performed on microtiter plates. The specificity of the test was ascertained further by inhibition assays with purified ECA and with crude ECA+ and ECA- preparations. The reproducibility of the tests was 96.4%; on this basis, a fourfold or larger difference in titers was regarded as significant. The anti-ECA titers in 649 serum samples from healthy persons ranged from less than 4 to 8,192. The titers increased with age, so that th geometric mean titers were 57 at 1.5 years of age and 201 at 45 years of age. After this, the titers decreased again, to a low of 52 in persons more than 70 years old. Women had higher titers than men up to the age of 40 years.

A solid-phase radioimmunoassay (RIA) procedure was compared with the indirect fluorescent antibody (IFA) test in a serological study of 76 female adults with urinary tract infections. Relative serum antibody activity was determined against patients' homologous infecting enterobacteria by RIA and IFA and against heterologous enterobacterial common antigen (Escherichia coli O14) by RIA. There was marked correlation between results of the IFA and RIA methods using the homologous system; 22 of 51 patients (43%) with pyelonephritis had significantly elevated serum antibody activity by both IFA (titers greater than or equal to 512) and RIA (binding ratio greater than or equal to 2.0) when compared with normal serum controls; three had significant antibody activity detectable by RIA only. Eighteen (72%) of 25 patients with pyelonephritis had RIA binding ratios of greater than or equal to 2.0 against their homologous bacterial isolates and the enterobacterial common antigen; an additional 6 patients had binding ratios of greater than or equal to 2.0 against the antigen only. All 25 patients with cystitis had low serum antibody levels by IFA and RIA when tested against their own isolate as well as enterobacterial common antigen. The RIA procedure was objective, quantitative, and less tedious to perform than IFA.

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway. Consisting of the membrane proteins Wzx, Wzy and Wzz, the Wzy-dependent pathway is the most common polysaccharide biosynthetic pathway in Gram-negative bacteria where it is most notably involved in LPS O antigen (Oag) biosynthesis. As such, the majority of research directed towards these proteins has been orientated towards Oag biosynthetic homologues with little directed towards ECA homologues. Belonging to the Shape, Elongation, Division and Sporulation (SEDS) protein family, Wzy proteins are polymerases, and are characterized as possessing little or no peptide homology among homologues as well as being polytopic membrane proteins with functionally relevant residues within periplasmic loops, as defined by C-terminal reporter fusion topology mapping. Here, we present the first the first major study into the ECA polymerase WzyE. Multiple sequence alignments and topology mapping showed that WzyE is unlike WzyB proteins involved with Oag biosynthesis WzyE displays high peptide conservation across Enterobacteriales. In silico structures and reporter mapping allowed us to identify possible functionally conserved residues with WzyESF's periplasmic loops, which we showed were crucial for its function. This work provides novel insight into Wzy proteins and suggests that WzyE is an optimal model to investigate Wzy proteins and the Wzy-dependent pathway.

The enterobacterial common antigen (ECA) is a common determinant shared by almost all members of the family Enterobacteriaceae. The antigen modifies erythrocytes for agglutination by ECA antibodies. Previously it was reported that Pseudomonas aeruginosa produces a factor (PF) which destroys the erythrocyte-modifying capacity of ECA. The present investigation was undertaken to determine whether other species of this genus also produce PF. The passive bacterial hemagglutination and hemagglutination inhibition tests were used. It was observed that 47 strains belonging to 8 species of the genus Pseudomonas produce this factor and 34 strains representing 12 other species do not. Multiple strains of a given species gave concordant results. Mucoid variants of P. aeruginosa produced more of this factor than did nonmucoid isolates recovered from the identical sputum specimens from patients with cystic fibrosis. ECA treated with PF no longer modifies erythrocytes for agglutination by ECA antibodies and exerts less antibody-neutralizing capacity than untreated antigen.

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.

The enterobacterial common antigen (ECA) is a highly conserved exopolysaccharide in Gram-negative bacteria whose role remains largely uncharacterized. In a previous work, we have demonstrated that disrupting the integrity of the ECA biosynthetic pathway imposed severe deficiencies to the Serratia marcescens motile (swimming and swarming) capacity. In this work, we show that alterations in the ECA structure activate the Rcs phosphorelay, which results in the repression of the flagellar biogenesis regulatory cascade. In addition, a detailed analysis of wec cluster mutant strains, which provoke the disruption of the ECA biosynthesis at different levels of the pathway, suggests that the absence of the periplasmic ECA cyclic structure could constitute a potential signal detected by the RcsF-RcsCDB phosphorelay. We also identify SMA1167 as a member of the S. marcescens Rcs regulon and show that high osmolarity induces Rcs activity in this bacterium. These results provide a new perspective from which to understand the phylogenetic conservation of ECA among enterobacteria and the basis for the virulence attenuation detected in wec mutant strains in other pathogenic bacteria.

Lipopolysaccharide (LPS) O-antigen and enterobacterial common antigen (ECA) play crucial roles in maintaining the outer membrane in Gram-negative bacteria. Mutations in the biosynthetic pathways of LPS and ECA may lead to accumulation of intermediates, resulting in morphological changes and activation of stress responses. However, the functional consequences of abrogation of both O-antigen and ECA synthesis in Salmonella enterica serovar Typhimurium (S. Typhimurium) are not well investigated. In this study, we generated single and double-deletion mutants of rfbB and rffG, encoding dTDP-glucose 4,6-dehydratase paralogs that are important in the synthesis of both O-antigen and ECA. Importantly, mutations in the dTDP-D-glucose 4,6-dehydratase encoding gene in humans are known to cause Catel-Manzke syndrome, a rare genetic disease. All four strains, i.e., wild type (WT), ΔrfbB, ΔrffG and ΔrfbBΔrffG, grew well in rich Luria Bertani (LB) liquid media at 37°C; however, the functional loss of both rfbB and rffG, but not in single-deletion strains, resulted in round cell morphology and smaller colony size in LB agar plates. There was no significant differences in the growth of the four strains in minimal media at 37°C (nutritional deficiency), in LB at 42°C (high temperature), acidic pH or LB with 3–4% NaCl (high osmolarity; however the ΔrfbBΔrffG strain was hypersensitive to bile and cell wall-targeting antibiotics). These results demonstrated that the ΔrfbBΔrffG strain was sensitive to some stress conditions. Interestingly, the ΔrfbBΔrffG strain displayed an altered LPS profile, autoaggregated rapidly compared to the WT and the single mutant strains and showed high N-phenylnaphthylamine (NPN) fluorescence indicating greater surface hydrophobicity. Furthermore, transcriptomic analysis identified flagellar and SPI-1 pathways to be highly downregulated in ΔrfbBΔrffG which led to impaired swimming as well as swarming motility, lower adhesion and invasion of HeLa cells. Importantly, the ΔrfbBΔrffG strain was less proficient in colonizing Peyer’s patches, spleen and liver, was unable to induce pro-inflammatory cytokines and was attenuated in both the oral and intraperitoneal models of S. Typhimurium infection in mice. Overall, this study highlights the importance of rfbB and rffG in maintaining cell wall and cell membrane integrity, colony and cellular morphology, motility and virulence in S. Typhimurium.

The rff genes of Salmonella typhimurium include structural genes for enzymes involved in the conversion of UDP N-acetyl-D-glucosamine (UDP-GlcNAc) to UDP N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), the donor of ManNAcA residues in enterobacterial common antigen (ECA) synthesis. An rff mutation (rff-726) of Escherichia coli has been described (U. Meier and H. Mayer, J. Bacteriol. 163:756-762, 1985) that abolished ECA synthesis but which did not affect the synthesis of UDP-ManNAcA or any other components of ECA. The nature of the enzymatic defect resulting from the rff-726 lesion was investigated in the present study. The in vitro synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), an early intermediate in ECA synthesis, was demonstrated by using membranes prepared from a mutant of E. coli possessing the rff-726 lesion. However, in vitro synthesis of the next lipid-linked intermediate in the biosynthetic sequence, ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II), was severely impaired. Transduction of wild-type rff genes into the mutant restored the ability to synthesize both lipid II and ECA as determined by in vitro assay and Western blot (immunoblot) analyses done with anti-ECA monoclonal antibody, respectively. Our results are consistent with the conclusion that the rff-726 mutation is located in the structural gene for the transferase that catalyzes the transfer of ManNAcA from UDP-ManNAcA to lipid I.

Background The emergence of resistance among the most important bacterial pathogens is recognized as a major public threat worldwide. The impermeable outer membrane (OM) of gram-negative bacteria plays a significant role in withstanding antibiotic stresses as it is impervious to both large and hydrophobic molecules, very much limiting the access of different antibiotics to cells. Most of the antibiotics that are used to treat gram-positive bacterial infections are ineffective against gram-negative bacteria. To make these antibiotics effective against gram-negative bacteria, mechanism of the OM permeability barrier needs to be revisited. Many factors of outer surface are involved in maintaining of OM-integrity. One of those factor is ECA (Enterobacterial Common Antigen). It is a conserved molecule which presents only in the Enterobacterales’s order. Mitchel et al. (2018) have reported a novel function of the cyclic ECA molecule in maintaining the permeability barrier of the OM in a YhdP (inner-membrane protein) dependent manner in E. coli. Nevertheless, we do not know the mechanism through which YhdP and ECA affect OM-permeability. We are trying to find this mechanism by exploring a ∆yhdP∆fadR double mutant. Methods Knockout of ΔyhdPΔfadR and other E. coli strains were generated by P1 phase transduction method. Growth curve was done in 96-well plate at different temperatures (30°C, 37°C, and 42°C). Antibiotic sensitivity was done by disc diffusion and EOPs methods. Suppressor mutants were identified by transposon mutagenesis approach. Results We have found that the ∆yhdP ∆fadR double mutant is very sick at 30°C as compared to wild type, a phenotype which is specific to lower temperatures. In addition, we have screened various antibiotics against them and found that the double mutant is sensitive to benzalkonium chloride (cationic surfactant) and bacitracin (polypeptide). Deletion of fadR alone has slight effect on the growth of E. coli and it is sensitive to benzalkonium chloride. In silico database suggests that YhdP interacts with YdgH (a periplasmic protein of unknown function). To find the defect of this double mutant (∆yhdP ∆fadR), we have isolated suppressor mutants. These suppressor mutants were generated by plating the double mutant on LB plate, and allowing growth at 30°C overnight. The isolated suppressor mutants can grow at 30°C. The identified mutants are uspB::Tn10, pitA::Tn10, leuO::Tn10, and hycC::Tn10.We are currently exploring these suppressor mutants. Conclusion YhdP-FadR interaction is essential for survival at 30°C. Generation of suppressor mutants make the ∆yhdP∆fadR mutant to survive at low temperature. One of the identified suppressor mutants, leuO plays an important role in low temperature (Klauck et al., 1997; Repoila & Gottesman, 2003).This indicates that LeuO might be involved in of bacterial growth at 30°C. These studies will lead to better understanding of resistance mechanisms in gram-negative bacteria and help in target-based designing of antibiotics.

Anatomical Locus of the Common Enterobacterial Antigen

Urinary tract infection (UTI) is a major global infectious disease affecting millions of people annually. Human urinary copper (Cu) content is elevated during UTI caused by uropathogenic Escherichia coli (UPEC). UPEC upregulates the expression of Cu efflux genes during clinical UTI in patients as an adaptive response to host-derived Cu. Whether Cu is mobilized to urine as a host response to UTI and its role in protection against UTI remain unresolved. To address these questions, we tested the hypothesis that Cu is a host effector mobilized to urine during UTI to limit bacterial growth. Our results reveal that Cu is mobilized to urine during UTI caused by the major uropathogens Proteus mirabilis and Klebsiella pneumoniae, in addition to UPEC, in humans. Ceruloplasmin, a Cu-containing ferroxidase, is found at higher levels in UTI urine than in healthy control urine and serves as the molecular source of urinary Cu during UTI. Our results demonstrate that ceruloplasmin decreases the bioavailability of iron in urine by a transferrin-dependent mechanism. Experimental UTI with UPEC in nonhuman primates recapitulates the increased urinary Cu content observed during clinical UTI. Furthermore, Cu-deficient mice are highly colonized by UPEC, indicating that Cu is involved in the limiting of bacterial growth within the urinary tract. Collectively, our results indicate that Cu is a host effector that is involved in protection against pathogen colonization of the urinary tract. Because urinary Cu levels are amenable to modulation, augmentation of the Cu-based host defense against UTI represents a novel approach to limiting bacterial colonization during UTI.