Use of factor-H sera for the differentiation of the flagellar antigens of Escherichia coli serological variants

The use of factor sera permitting the differentiation of the variants, described in earlier. works, among the flagellar antigens of E. coli, formally denoted as H2, H4, H7, H10 and H34 in accordance with their official nomenclature, has made it possible to reveal that each of these variants is widely spread among E. coli and occurs in bacteria of different O-groups. Besides, this study has shown the possibility of subdividing a number of formal H: O types into 2 or more serovars on the basis of differences in the factor composition of their antigens. The results obtained in this study suggest that in the process of the evolution of E. coli H-antigen variants differing in their factor composition have been formed as independent varieties; therefore, these variants do not reflect the features characteristic of individual strains, but constitute one of the diagnostic signs of serological classification, i. e. the differentiation of the species into various serovars.     

Flagellar antigens of E. coli serologically interrlelated to H40, 41 and H41, 42, 43 flagellar antigens of Citrobacter. New H-antigeny E. coli i Citrobacter.]

The authors confirmed the reference of the test strains H13 (P6c) and H22 (A231a) of the international collection of E. coli to Citrobacter; their antigenic formula was established. As shown, strains P6c possessed a variety of the H-antigen which was not described in Citrobacter earlier, designated as H41a, 97. Three types of flagellar antigens characterized by the presence of an interrelationship with the partial factor H41 of the flagellar Citrobacter antigens were revealed in E. coli; the partial composition of H-antigenic components common for E. coli and Citrobacter was studied. Two of three new varieties of the E. coli H-antigen revealed was characterized by a cross correlation and a relation to the standard H19 E. coli antigen. The strain with the third variety of the H-antigen was capable of forming the H-antigenic mutants which acquired the antigenic component identical to the standard H16 E. coli antigen. E. coli strain is recommended for the replacement of the strain P6c in the International collection of E. coli.     

Comparison of the flagellins from different flagellar morphotypes of Escherichia coli.

The molecular weights of the flagellins of 13 strains of Escherichia coli, each with a different H antigen, were estimated using polyacrylamide gel electrophoresis. In each case only one major polypeptide was demonstrated, although some strains possessed apparently sheathed flagella. Considerable differences in the molecular weight of flagellin accompanied the previously described structural differences between flagella from strains with different H antigens. The relationship between flagellar diameter and the molecular weight of the corresponding flagellins was similar for both unsheathed and apparently sheathed flagella. Crosss-polymerization occurred between seed consisting of fragment of unsheathed flagella and flagellin solution from apparently sheathed flagella and vice versa. Co-polymerization of flagellin from unsheathed flagella and flagellin from apparently sheathed flagella was also demonstrated. These polymerization experiments indicate that the assembly pattern of flagellin molecules is probably the same in all E. coli flagella. The above and other evidence suggests that there is no true sheath, but that the differences in flagellar surface structure between different E. coli flagella are the result of differences in the superficial parts of the flagellin molecules.     

Morphological distinction between different H serotypes of Escherichia coli.

The structure of the flagellar filaments of 50 Escherichia coli strains, each with a different H antigen, was examined. Although the flagella within each strain were structurally identical, there was variability in flagellar surface pattern between strains with differrent H antigens. Investigation of additional strains confirmed that flagella structure was the same in all strains having the same H antigen. In three pairs of strains with cross-reacting H antigens, the antigenic relatedness was associated with identical flagella structure.     

A genomic islet mediates flagellar phase variation in Escherichia coli strains carrying the flagellin-specifying locus flk

The occurrence of unilateral flagellar phase variation was previously demonstrated in Escherichia coli strains carrying the non-fliC flagellin-specifying locus flk. In this study, we investigated the mechanism involved in this process. By using sequencing and sequence analysis, the flk region between the chromosomal genes yhaC and rnpB was characterized in all described flk-positive E. coli strains, including the H35 strain identified in this study (the other strains used are H3, H36, H47, and H53 strains), and this region was found to contain a putative integrase gene and flanking direct repeats in addition to the flk flagellin-specifying gene flkA and a fliC repressor gene, flkB, indicating that there is a typical genomic islet (GI), which was designated the flk GI. The horizontal transfer potential of the flk GI was indicated by detection of the excised extrachromosomal circular form of the flk GI. By generating fliC-expressing variants of H3 and H47 strains, unilateral flagellar phase variation in flk-positive strains was shown to be mediated by excision of the flk GI. The function of the proposed integrase gene was confirmed by deletion and a complementation test. The potential integration sites of the flk GI were identified. A general model for flagellar phase variation in flk-positive E. coli strains can be expressed as fliC(off) + flkA(on) --> fliC(on) + flkA(none). This is the first time that a molecular mechanism for flagellar phase variation has been reported for E. coli.     

Serodiagnosis of infection with verocytotoxin-producing Escherichia coli

Human sera (167) were screened for antibodies to lipopolysaccharide (LPS) prepared from strains of Verocytotoxin-producing Escherichia coli (VTEC) belonging to a range of serogroups, secreted proteins expressed by attaching and effacing VTEC, enterohaemolysin and H = 7 flagellar proteins. Twelve sera (about 7%) contained antibodies to the LPS of E. coli 05 (one), 026 (two), 0115 (two), 0145 (one), 0163 (one) and 0165 (five). Sera containing antibodies to the LPS of E. coli O26 and O145 also contained antibodies to secreted proteins of 100 and 40 kDa. An additional 34 sera, known to contain antibodies to the lipopolysaccharide of E. coli O157, were examined for antibodies to enterohaemolysin, H = 7 flagellar antigens and bacterial cell surface-associated proteins of 5, 6 and 22 kDa. Three sera contained antibodies to enterohaemolysin and one serum contained antibodies to flagellar proteins. Antibodies to membrane-associated proteins were not detected. It was concluded that enterohaemolysin, H = 7 flagellar proteins and the cell surface-associated proteins were unsuitable for use in immunoassays for providing evidence of infection with VTEC.     

Different alleles of the flagellin gene hagB in Escherichia coli standard H test strains

Abstract The genes determining flagellar antigen specificities H36, H47 and H53 in the respective E. coli standard H test strains were found to be alleles of the flagellin gene hagB . Until now, only the allele encoding the flagellar antigen H3 has been identified. The chromosomal regions of flagellin genes hagB in E. coli and H2 in Salmonella were non-homologous as these genes integrated at different sites in the E. coli K-12 chromosome and were unable to replace each other. The hagA allele encoding E. coli flagellar antigen H48 was insensitive to the repressor produced by Salmonella gene rhl or by its putative analog in E. coli .     

Expressed and cryptic flagellin genes in the H44 and H55 type strains of Escherichia coli

Bacterial H antigens are specified by flagellin molecules, which constitute the flagellar filament. Escherichia coli 781-55 and E2987-73 are the type strains for H44 and H55 antigens, respectively. Unlike E. coli K-12, they possess two flagellin genes, fliC and fllA, on their chromosomes. However, they are monophasic, expressing exclusively the fllA genes, which specify the type antigens. In this study, the flagellin genes were cloned from these strains and their structure and expression were analyzed. It was found that the fliC genes encode apparently intact flagellin subunits but possess inefficient sigma28-dependent promoters, which may result in these genes being silent. The chromosomal locations of the fllA genes are approximately, but not exactly, identical with that of the phase-2 flagellin gene, fljB, of diphasic Salmonella strains. However, unlike the Salmonella fljB gene, the invertible H segment and the fljA gene responsible for the control of flagellar phase variation are both absent from the fllA loci. The fllA genes are highly homologous to the E. coli fliC gene but distantly related to the Salmonella fljB gene. These results suggest a hypothesis that the fllA genes may have emerged by an intra-species lateral transfer of the fliC gene. This hypothesis is further supported by the observation that the fllA genes are flanked by several IS elements and located within cryptic prophage elements.     

Serotype-specific monoclonal antibodies against the H12 flagellar antigen of Escherichia coli

The flagellar filaments of morphotype E isolates of Escherichia coli characteristically possess an apparent helically arranged sheath structure, surrounding the central core of the filament. Re-examination of the type strains of H-serotypes belonging to morphotype E showed that all but serotype H34 possessed the expected morphology. Heterogeneity was observed in both the diameter of filaments from individual morphotype E strains and in the Mr of individual flagellins. There was no apparent correlation between these two features. Monoclonal antibodies (MAbs) of the IgM class were raised against serotype H12 flagella. In Western immunoblotting and agglutination tests, the MAbs recognized the H12 antigen of six isolates with different O:K antigen combinations. The MAbs were H-serotype-specific, with no significant reaction with the H-antigens of other morphotype E strains. The location of the serotype-specific H12 epitope(s) was studied by immunolabelling with colloidal gold markers. The epitope was surface-exposed and appeared to be helically arranged on the flagellar filament. The pattern of colloidal gold labelling was consistent with the possibility that the H12 serotype-specific epitope resides in the apparent sheath structure.     

Molecular serotyping of Escherichia coli O26:H11

Serotyping is the foundation of pathogenic Escherichia coli diagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens of E. coli O26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.     

Biochemical and Serological Characterization of Hydrogen Sulfide-Positive Variants of Escherichia coli

Over 200 H(2)S-positive, gram-negative rods have been characterized by standard biochemical and serological techniques. The results indicate that the isolates are H(2)S-positive variants of Escherichia coli. Comparison of the variants with biochemically typical E. coli suggests that they represent a rather limited subgroup within the species. The H(2)S-positive strains were more resistant to antibiotics than the typical strains; 54% of the H(2)S-positive isolates were resistant to three or more antibiotics compared with only 25% of the typical strains. Similar differences were also seen in the distribution of O and H antigens and in the results of certain biochemical tests.     

Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

Background  

Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.     

Molecular serotyping of Escherichia coli O111:H8

Accurate Escherichia coli serotyping is critical for pathogen diagnosis and surveillance of non-O157 Shiga-toxigenic strains, however few laboratories have this capacity. The molecular serotyping protocol described in this paper targets the somatic and flagellar antigens of E. coli O111:H8 used in traditional serotyping, and can be performed routinely in the laboratory.     

Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern.

Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins.The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.     

Monoclonal antibodies against serotype specific and conserved epitopes in morphotype E Escherichia coli flagellins

Monoclonal antibodies (mAbs) were used to examine the interrelationships between morphologically identical flagellar filaments from Escherichia coli H serotype strains belonging to morphotype E. Serotype specific mAbs recognised epitopes exposed on the surface of flagellar filaments from H1, H7, H23, H49 and H51, but were inaccessible to immunolabelling in H45. Several mAbs which recognised conserved epitopes were also examined. mAb 7-56.1 recognised an epitope present in all morphotype E flagellins but not expressed on the filament surface. Similarly, mAb 1-5.1 recognised an internal epitope shared only by serotypes H1 and H12. Serotype H23 expressed a surface epitope which was present but not surface exposed in H7, H1 and H45 filaments.     

Acquisition of the rfb-gnd cluster in evolution of Escherichia coli O55 and O157

The rfb region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The rfb locus is adjacent to gnd, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if rfb and gnd cotransfer, we sequenced gnd in five O55 and 13 O157 strains of Escherichia coli. E. coli O157:H7 has a gnd allele (allele A) that is only 82% identical to the gnd allele (allele D) of closely related E. coli O55:H7. In contrast, gnd alleles of E. coli O55 in distant lineages are >99.9% identical to gnd allele D. Though gnd alleles B and C in E. coli O157 that are distantly related to E. coli O157:H7 are more similar to allele A than to allele D, there are nucleotide differences at 4 to 6% of their sites. Alleles B and C can be found in E. coli O157 in different lineages, but we have found allele A only in E. coli O157 belonging to the DEC5 lineage. DNA 3' to the O55 gnd allele in diverse E. coli lineages has sequences homologous to tnpA of the Salmonella enterica serovar Typhimurium IS200 element, E. coli Rhs elements (including an H-rpt gene), and portions of the O111 and O157 rfb regions. We conclude that rfb and gnd cotransferred into E. coli O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic E. coli O55 and O157.     

Sequence Diversity of Flagellin (fliC) Alleles in Pathogenic Escherichia coli

To study the molecular evolution of flagellin, the protein subunit specifying flagellar (H) antigens, the fliC genes from 15 pathogenic strains of Escherichia coli were amplified by PCR and sequenced. Comparison of fliC sequences of H6 and H7 strains revealed that alleles have a mosaic structure indicating the occurrence of past horizontal transfer of DNA segments between strains. The close similarity of H7 sequences also indicates the exchange of an entire fliC H7 allele between distant clonal lineages. In addition, the ratio of silent substitutions to amino acid replacements suggests that a short segment in the central region of fliC has been under positive selection in the divergence of H6 and H7 alleles. Phylogenetic analysis demonstrates that the fliC sequences of O157:H7 and O55:H7 serotypes are nearly identical and highly divergent from those of E. coli strains expressing H6 and H2 flagellar antigens. A nonmotile clone of sorbitol-fermenting O157 has rapidly accumulated multiple mutations in fliC, presumably as a result of the silencing of flagellin expression.     

Structure and genetics of Shigella O antigens

This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.     

The expression of an R3 lipopolysaccharide-core by pathotypes of Escherichia coli

AIMS: To investigate the incidence of an R3 lipopolysaccharide (LPS)-core amplicon in a range of pathotypes of Escherichia coli, including Verocytotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC) and enteropathogenic E. coli (EPEC). METHODS AND RESULTS: A total of 100 strains of E. coli belonging to a range of pathotypes, including 41 strains of VTEC, were screened for the genes encoding the R3 LPS-core using PCR. Fifty-four per cent produced an amplicon with the R3 primer set. Of the 41 VTEC, 66% had an R3 LPS-core with a PCR product being observed with all strains belonging to serotypes O26:H11, O111ac:H- and O145:H25. However, 46% of enteroaggregative E. coli and 50% of enteropathogenic E. coli were also shown to have an R3 LPS-core structure. CONCLUSIONS: Strains with an R3 LPS-core are widely distributed within the species E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E. coli with an R3 LPS-core structure appear not to be associated with a specific pathotype.