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Separation, characterization and biological significance of a common antigen in Enterobacteriacae

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Abstract

An antigen common to Enterobacteriaceae and closely associated with endotoxin fractions has been separated by chromatography on DEAE cellulose employing elution with a NaCl gradient. The purified common antigen fails to coat erythrocytes, is poorly, if at all antigenic, it is non-dialyzable and excluded from sephadex G-100 gel. It is composed of polysaccharide and polypeptide. The most important property of this antigen thus far determined appears to be its interference with the specificity of the hemagglutination test commonly employed to measure antibody to O antigen of Enterobacteriaceae. It may also have taxonomic significance in classification of this family of bacteria.
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... The inhibiting capacity is not affected by the ability or inability of ECA to sensitize RBC, to elicit an immune response, or to be precipitated. This inhibitory capacity has been successfully used in following ECA during purification steps (85,100,192). Until chemical identification of ECA has progressed further, HAI remains the principal method of quantitating ECA. ...
... Other Serological Methods Immunoprecipitation would be especially useful for monitoring an antigen during chemical purification and as a final test of purity and identity. It was therefore disappointing that in the initial studies of ECA the results were negative: no precipitation was seen in test tube or agar-gel diffusion (100)(101)(102). More recently it has become evident that the failure to detect precipitation is dependent on the antiserum usedsome sera contain precipitating antibodies, others do not. ...
... Agglutination of coated latex particles does not take place either. Latex particles adsorb both ECA and LPS from crude bacterial extracts (100,219), and the presence ofboth can be demonstrated by the ability of the coated particles to remove the corresponding antibodies from immune sera (37,146) and to be opsonized for phagocytosis (38). However, the latex particles become agglutinable by anti-LPS sera only (217). ...
... coli O14 rabbit serum with various rough and smooth E. coli strains. It was further demonstrated that only a few E. coli strains (serotype O14, O54, O124, and O144) elicited highly cross-reactive anti-ECA antibodies upon immunization of rabbits [6,12]. E. coli O14 was proven later on to be rough and synthesize LOS characterized by the R4 core chemotype [13,14]. ...
... The story of ECA began with the discovery of ECA LPS since the presence of ECA LPS was originally inferred from serological cross-reactions in hemagglutination assays between patients' sera and various E. coli O-serotypes during studies of urinary tract E. coli infections [5]. A few E. coli strains (serotypes O14, O54, O124, and O144) elicited highly cross-reactive antibodies in rabbits that could be removed from anti-O14 serum by absorption with extracts of any E. coli strain while retaining homological reactivity of the serum [6,12]. Described cross-reactivity was not related to O or K antigens. ...
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Enterobacterial common antigen (ECA) is a conserved surface antigen characteristic for Enterobacteriaceae. It is consisting of trisaccharide repeating unit, →3)-α-d-Fucp4NAc-(1→4)-β-d-ManpNAcA-(1→4)-α-d-GlcpNAc-(1→, where prevailing forms include ECA linked to phosphatidylglycerol (ECAPG) and cyclic ECA (ECACYC). Lipopolysaccharide (LPS)-associated form (ECALPS) has been proved to date only for rough Shigella sonnei phase II. Depending on the structure organization, ECA constitutes surface antigen (ECAPG and ECALPS) or maintains the outer membrane permeability barrier (ECACYC). The existence of LPS was hypothesized in the 1960–80s on the basis of serological observations. Only a few Escherichia coli strains (i.e., R1, R2, R3, R4, and K-12) have led to the generation of anti-ECA antibodies upon immunization, excluding ECAPG as an immunogen and conjecturing ECALPS as the only immunogenic form. Here, we presented a structural survey of ECALPS in E. coli R1, R2, R3, and R4 to correlate previous serological observations with the presence of ECALPS. The low yields of ECALPS were identified in the R1, R2, and R4 strains, where ECA occupied outer core residues of LPS that used to be substituted by O-specific polysaccharide in the case of smooth LPS. Previously published observations and hypotheses regarding the immunogenicity and biosynthesis of ECALPS were discussed and correlated with presented herein structural data.
... The ECA is a conserved polysaccharide found across all enterobacteria [137], although its biological function still remains rather enigmatic [138]. ECA can be found in the outer membrane via linkage to phosphoglyceride [139], as a cyclic form in the periplasm [140], and, in bacterial strains incapable of producing O-antigens, it can be bound to lipid A [141]. ...
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The acylation of sugars, most commonly via acetylation, is a widely used mechanism in bacteria that uses a simple chemical modification to confer useful traits. For structures like lipopolysaccharide, capsule and peptidoglycan, that function outside of the cytoplasm, their acylation during export or post-synthesis requires transport of an activated acyl group across the membrane. In bacteria this function is most commonly linked to a family of integral membrane proteins – acyltransferase-3 (AT3). Numerous studies examining production of diverse extracytoplasmic sugar-containing structures have identified roles for these proteins in O -acylation. Many of the phenotypes conferred by the action of AT3 proteins influence host colonisation and environmental survival, as well as controlling the properties of biotechnologically important polysaccharides and the modification of antibiotics and antitumour drugs by Actinobacteria. Herein we present the first systematic review, to our knowledge, of the functions of bacterial AT3 proteins, revealing an important protein family involved in a plethora of systems of importance to bacterial function that is still relatively poorly understood at the mechanistic level. By defining and comparing this set of functions we draw out common themes in the structure and mechanism of this fascinating family of membrane-bound enzymes, which, due to their role in host colonisation in many pathogens, could offer novel targets for the development of antimicrobials.
... ECA PG represents a major form of ECA and together with lipopolysaccharide (LPS) and ECA LPS , is located on the cell surface, contributing to antigenicity and outer membrane integrity. Serological observations suggested ECA LPS as the only immunogenic form of ECA capable to generate anti-ECA antibodies upon immunization [1,3,[11][12][13][14]. ECA CYC is located in the periplasm and has been recently pointed out as an important factor maintaining the outer membrane permeability barrier [15]. ...
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Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide − endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.
... Other antigens, both group-specific and nonspecific, have been described for E. coli (18,31,106,107,164,203,225,245), but these are not used for the classification of the group, and, in most cases, their significance is not known. ...
... Prevalence of ECA antibodies. Many early studies have reported a low titer of ECA antibodies present in human serum (33,59,60), with the caveat that these studies were conducted before the availability of an ECA knockout strain and so may report the combined titer of both ECA antibodies and antibodies to protein antigens shared among Enterobacterales (e.g., OmpA). These antibodies have been found in both healthy donors and, at higher levels, in patients with chronic urinary tract infections (61). ...
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The outer membrane (OM) of Gram-negative bacteria poses a barrier to antibiotic entry due to its high impermeability. Thus, there is an urgent need to study the function and biogenesis of the OM. In Enterobacterales , an order of bacteria with many pathogenic members, one of the components of the OM is enterobacterial common antigen (ECA). We have known of the presence of ECA on the cell surface of Enterobacterales for many years, but its properties have only more recently begun to be unraveled. ECA is a carbohydrate antigen built of repeating units of three amino sugars, the structure of which is conserved throughout Enterobacterales. There are three forms of ECA, two of which (ECA PG and ECA LPS ) are located on the cell surface, while one (ECA CYC ) is located in the periplasm. Awareness of the importance of ECA has increased due to studies of its function that show it plays a vital role in bacterial physiology and interaction with the environment. Here, we review the discovery of ECA, the pathways for the biosynthesis of ECA, and the interactions of its various forms. In addition, we consider the role of ECA in the host immune response, as well as its potential roles in host-pathogen interaction. Furthermore, we explore recent work that offers insights into the cellular function of ECA. This review provides a glimpse of the biological significance of this enigmatic molecule.
... In P. mirabilis, ECA can exist in many forms: linked to other lipids, in a circularized and soluble form, or surface-exposed and linked to the LPS core in the outer membrane (35,(38)(39)(40)(41)(42). Repeated efforts to confirm the presence of ECA via Western blotting with E. coli O14 serum (SSI Diagnostica, Hillerød, Denmark), which is reactive against E. coli-derived ECA (43,44), were unsuccessful. We instead characterized the overall LPS composition and cell envelope sensitivity to antibiotics. ...
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Proteus mirabilis swarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multifunctional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, the rffG -dependent moieties provide a novel attractive target for potential antimicrobials.
... The Enterobacterial Common Antigen (ECA) was first described in 1963 by Kunin [31] and is defined as a crossreactive antigen that is detectable in all genera of Enterobacteriaceae by several methods including using antisera to E. coli [32]. ECA was later found to be strictly family specific with diagnostic potential because of its universal presence in the family (see reviews [32]). ...
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Objectives: The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shiga toxin (Stx) and the E. coliouter membrane protein (Eae) or intimin, encoded by the stx and eaegenes, respectively. Although several polymerase chain reaction (PCR) protocols target these virulence genes, few aim at detecting all variants or have an internal amplification control (IAC) included in a multiplex assay. The objective of this work was to develop a simple multiplex PCR assay in order to detect all stxand eae variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. Results: The wecA gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of stx and eae were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the stx genes was achieved by subjecting the PCR products to restriction digestion and semi-nested duplex PCR, providing a simple screening assay for human diarrhoea diagnostic.
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Although discovered over 50 years ago, the physiological role of enterobacterial common antigen, a surface antigen produced by all members of the Enterobacteriaceae , has been poorly understood. In the work of Mitchell et al. (mBio 9:e01321-18, 2018, https://doi.org/10.1128/mBio.01321-18 ), the cyclized version of enterobacterial common antigen has been shown to play a role in maintaining the outer membrane permeability barrier, possibly through the inner membrane protein YhdP. This work also provides the tests needed to separate true effects from the numerous possible artifacts possible with mutations in enterobacterial common antigen synthesis.
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Gram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. In Enterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughout Enterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes in Escherichia coli K-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these, yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants of yhdP with loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability from yhdP deletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore, yhdP deletion changed cellular ECA levels and yhdP was found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability with yhdP deletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found with yhdP deletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.
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