Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesinencoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap⁺ and/or sfa⁺ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing a-haemolysin (Hly⁺) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesinencoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.La PCR a permis de détecter les opérons pap, sfa et afa chez 243 souches de Escherichia coli isolées d'infections de l'arbre urinaire. On observe que respectivement 54, 53 et 2% des souches sont du génotype pap, sfa et afa. Les souches pap⁺ et/ou sfa⁺ sont plus fréquentes dans les pyelonephritis aiguës (94%) que dans les cystites (67%) (P < 0,001) et dans les bactériuries asymptomatiques (57%) (P < 0,001). Les opérons pap et/ou sfa sont décelés chez 90% des souches MRHA⁺ (hémagglutination mannoserésistante) et 37% des souches MRHA⁻ (P < 0,001). La présence des opérons pap et sfa est spécialement significative chez les souches appartenant au type III de MRHA (100%) sans adhésines P, et au type IVa (97 %) exprimant la liaison Gai-Gal typique des adhésines P. Les opérons pap et sfa sont tous deux étroitement associés aux souches toxigéniques produisant l'α-hémolysine (Hly⁺) et le facteur cytotoxique nécrotique de type 1. Une corrélation apparaît entre les opérons pap et sfa et le sérogroupe: 93 % des souches appartenant aux groupes O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 et O83 possèdent ces opérons, et seulement 37 % des souches appartenant à d'autres sérogroupes (P < 0,001) les possèdent. Ces résultats confirment l'utilité de notre typage MRHA pour l'identification (présomptive) des souches pathogènes de E. coli porteuses de divers facteurs de virulence. Ainsi, 85 % des souches possédant à la fois les opérons pap et sfa codant les adhésines sont du type III ou IVa (de MRHA) en relation avec la virulence des souches responsables d'infections urinaires et de bactériémies.     

Genetic studies on the promoter of malT, the gene that encodes the activator of the Escherichia coli maltose regulon

We report the construction of a chromosomal malT-lacZ gene fusion that is expressed under the control of the malT promoter in Escherichia coli K12. The resulting hybrid protein is soluble and stable in crude cellular extracts, which allowed us to measure very low levels of malTp activity. In this note, we confirm and extend previous observations on the regulation of malTp. We show that the promoter is 40-times less active in the absence of cAMP receptor protein (CRP) than in its presence, that CRP works by binding to the site centred at position -70.5, and that all of the elements necessary and sufficient for the regulation by CRP are located downstream from position -122.     

Semi-automated rep-PCR for rapid differentiation of major clonal groups of Escherichia coli meningitis strains

DiversiLab, a semi-automated repetitive-sequence-based PCR (rep-PCR) device, is a highly integrated platform designed for rapid bacterial genotyping. Here, we evaluated the capacity of the DiversiLab system to determine the genetic relatedness of Escherichia coli neonatal meningitis (ECNM) strains and to identify clonal groups. We analyzed 80 isolates representative of the diversity of ECNM strains in Europe and North America and 52 E. coli reference (ECOR) strains belonging to phylogenetic groups A, D, and B2. All the strains had previously been characterized by means of multilocus sequence typing (MLST). The DiversiLab dendrogram clustered all but 8 of the strains according to their phylogenetic groups. After defining a rep-PCR type complex (RPTc) based on an average similarity threshold of 95% between rep-PCR types, we observed excellent agreement between RPTc and sequence type complexes (STc) in groups D and B2. In group A, rep-PCR typing was more discriminative than MLST, dividing the 25 ECOR group A strains into 19 RPTc, compared to only 10 STc. In the highly virulent clonal group B2₁, mainly composed of O1, O2, O18, and O45:K1 strains, the DiversiLab system individualized a particular subgroup of O2:K1 strains. In addition, among O18:K1 strains the system identified a particular genetic background associated with pathogenicity island II_J96-like domains. Thus, the DiversiLab system is a rapid and powerful tool for identifying and discriminating clonal groups among ECNM strains.     

Loss of λ prophage recombinogenicity in UV-irradiated Escherichia coli: the role of host genes ruvA, ruvB, ruvC, and recG

Earlier studies have revealed a radiation-induced process leading to the loss of λ prophage recombinogenicity. The process takes place in UV-irradiated Escherichia coli cells, and renders the prophage incapable of site-specific recombination with the host chromosome, and of general recombination with an infecting homologous phage. It was found that the inhibition of prophage recombinogenicity depends on functional RecBCD enzyme of E. coli. In this work, the role of ruvABC and recG genes in the inhibitory process was assessed. The products of these genes are known to act at the last step of homologous recombination and recombinational DNA repair by catalyzing the resolution of recombination intermediates (the Holliday junctions). Irradiated prophage retained its ability to recombine in ruvA, ruvB, ruvC, and recG mutants. These results suggest that in addition to RecBCD enzyme, RuvABC and RecG proteins are also involved in the inhibition of prophage recombinogenicity. We infer that RuvABC and RecG act in this process before RecBCD, probably by processing the Holliday junctions formed upon replication arrest, and thereby providing double-stranded DNA breaks as substrate for RecBCD-mediated recombinational repair of UV-damaged bacterial chromosome.     

Production of type 1 fimbriae by Escherichia coli HB101

Escherichia coli HB101 is frequently used as a host in the cloning of bacterial virulence genes because of its reported lack of virulence determinants such as fimbriae, adhesins and haemagglutinins. However, passage of HB101 in standing broth culture rapidly induced the production of fimbriae which mediated adhesion to HEp-2 cells and mannose-sensitive haemagglutination of human and guinea-pig erythrocytes. Fimbrial serology, morphology and pilin molecular mass of 18 kDa were consistent with those of type 1 fimbriae.     

The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity

Superoxide dismutases (SODs) are vital components that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous SODs, a manganese- and an iron-containing enzyme (Mn-SOD and Fe-SOD, respectively). In contrast, a single Mn-SOD is present in Bacillus subtilis. In E. coli, the absence of SODs was found to be associated with an increased sensitivity to cadmium, nickel and cobalt ions. Mutants lacking either sodA or sodB exhibited metal resistance to levels comparable to that of the wild-type strain. Although sod-deficient mutant cells were more resistant to zinc than their wild-type counterpart, no differences between the strains were observed in the presence of copper. In B. subtilis, the sodA mutation had no effect on cadmium and copper resistance. These results suggest that intracellular generation of superoxide by cadmium, nickel and cobalt is toxic in E. coli. They support the participation of sod genes in its protection against metal stress.     

Escherichia coli CS31A fimbriae: molecular cloning, expression and homology with the K88 determinant

CS31A is a plasmid-encoded K88-related fimbrial antigen. A Sau3AI library was constructed from p31A, a 180 kb CS31A encoding plasmid, in the pSUP202 vector. Bacterial recombinant clones expressing CS31A were isolated. A 8.5 kb EcoRI-HinIII DNA fragment from one of them was subcloned in pBR322 and pHSG575 vectors, leading to pAG315 and pEH524 recombinant plasmids respectively. Escherichia coli harboring pAG315 or pEH524 expressed CS31A fimbrial antigens on their cell surface. Analysis of these plasmids in minicells showed that at least seven mature polypeptides were encoded by the EcoRI-HindIII DNA fragment, with apparent molecular masses of 76,000, 54,000, 30,000, 29,000, 28,000, 15,500 and 13,500 daltons respectively. The genetic organization of the CS31A gene cluster was determined and showed to be similar to that of the K88 operon. The nucleotide sequence homology between CS31A and K88 determinants was investigated by Southern blot hybridization at high stringency. This indicated that extensive nucleotide sequence homology exists throughout both gene clusters except for the subunit structural genes.     

Production of the matrix protein of Nipah virus in Escherichia coli: Virus-like particles and possible application for diagnosis

The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (M_r) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 °C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.     

Serological response to type 1-like somatic fimbriae in diarrheal infection due to classical enteropathogenic Escherichia coli

Fimbriae from enteropathogenic Escherichia coli strain E2349/69 (0127:H6) and its plasmid-minus derivative, MAR20, were purified and characterized as type 1-like by their physicochemical and hemagglutination patterns. Sera from adult volunteers challenged with the diarrheagenic parent strain and the attenuated plasmid-minus derivative were examined to detect an immune response, using the purified fimbriae as antigens in an enzyme linked immunosorbent assay (ELISA) and immunoblot assay. An anti-fimbrial response was evident in sera of 7 of 10 volunteers fed the diarrheagenic parent strain E2348 but also in 8 of 9 individuals fed the attenuated, plasmid-cured, derivative MAR20. The antibody response appeared specific in that the sera failed to react in an ELISA and by immunoblot assay with type 1 fimbriae from other E. coli. These findings suggest that the type 1 fimbriae of this representative EPEC strain are antigenically distinct. The results of this investigation provide the first evidence of seroconversion to type 1-like fimbriae in infections caused by diarrheagenic E. coli.     

Cloning, expression and characterization of the 36 KDal Salmonella typhi porin gene in Escherichia coli

A recombinant plasmid containing the gene for the 36 KDal porin of Salmonella typhi has been identified in a cosmid library of S. typhi propagated in Escherichia coli. The recombinant clone was identified by its ability to endow E. coli with susceptibility to porin specific phages, and by the appearance in the outer membrane of E. coli containing the clone of a new protein of 36 KDal. While the porin confers upon a porinless mutant of E. coli an increased susceptibility to beta-lactam antibiotics, it does not react with serum from patients with typhoid fever in immunoblotting assays.     

Aspects of genome plasticity in pathogenic Escherichia coli

The species Escherichia coli comprises not only non-pathogenic or commensal variants that belong to the normal intestinal flora of most mammals, but also various pathogenic strains causing diverse intestinal and extraintestinal infections in man and animals. Virulence factors and mechanisms involved in pathogenesis have been successfully analyzed for many years resulting in a wealth of knowledge about many E. coli pathotypes. However, our knowledge on the genome content, diversity and variability between pathogenic and also non-pathogenic subtypes is only slowly accumulating. Pathotypes have been largely defined by the presence or absence of particular DNA segments that in most cases appear to have been acquired via horizontal gene transfer events. As these regions are frequently subjected to excisions, rearrangements, and transfers they contribute to the previously unexpected and underestimated rapid evolution of E. coli variants resulting in the development of novel strains and even pathotypes. In these studies various novel aspects of genome diversity and plasticity in extraintestinal and intestinal pathogenic E. coli pathotypes have been addressed and the results have been directly applied for the improvement of diagnostic methods.     

Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis

Pelargonium zonate spot virus (PZSV) is identified recently in tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is a necessity. The coat protein (CP) gene of a California isolate of PZSV was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC). The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3 mg refolded protein per 200 mL of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum was able to detect the recombinant protein, the PZSV coat protein and PZSV infected plant samples. The antiserum was successfully used in indirect-ELISA at dilutions of up to 1:16,000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.     

Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called ‘attaching and effacing’ lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eaeγ-tirα-espAα-espBα (65%), eaeβ-tirβ-espAβ-espBβ (29%), eaeα-tirα-espAα-espBα (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.     

Cloning of an outer membrane protein of Neisseria meningitidis in Escherichia coli

A gene bank of chromosomal DNA of Neisseria meningitidis group B was constructed in phage lambda EMBL3, and screened by rabbit polyclonal antibodies to major outer membrane (OM) proteins of the meningococcus. Several clones expressing a 28 kDa protein were found. The gene coding for the 28 kDa protein was subcloned into plasmid pUC18 in Escherichia coli. The protein was expressed in E. coli and located in the OM. Rabbit antibodies were raised to the 28 kDa protein purified from E. coli and used to localize the protein in the meningococcus. The antiserum recognized a minor protein of similar electrophoretic mobility in the outer membrane complex (OMC) of the meningococcus. The 28 kDa protein was found to be common to different Neisseria species; it was expressed by both pathogenic and nonpathogenic Neisseria.     

Impact of O-glycosylation on the molecular and cellular adhesion properties of the Escherichia coli autotransporter protein Ag43

Antigen 43 (Ag43) represents an entire family of closely related autotransporter proteins in Escherichia coli and has been described to confer aggregation and fluffing of cells, to promote biofilm formation, uptake and survival in macrophages as well as long-term persistence of uropathogenic E. coli in the murine urinary tract. Furthermore, it has been reported that glycosylation of the Ag43 passenger domain (α⁴³) stabilizes its conformation and increases adhesion to Hep-2 cells.We characterized the role of Ag43 as an adhesin and the impact of O-glycosylation on the function of Ag43. To analyze whether structural variations in the α⁴³ domain correlate with different functional properties, we cloned 5 different agn43 alleles from different E. coli subtypes and tested them for autoaggregation, biofilm formation, adhesion to different eukaryotic cell lines as well as to purified components of the extracellular matrix. These experiments were performed with nonglycosylated and O-glycosylated Ag43 variants. We show for the first time that Ag43 mediates bacterial adhesion in a cell line-specific manner and that structural variations of the α⁴³ domain correlate with increased adhesive properties to proteins of the extracellular matrix such as collagen and laminin. Whereas O-glycosylation of many α⁴³ domains led to impaired autoaggregation and a significantly reduced adhesion to eukaryotic cell lines, their interaction with collagen was significantly increased. These data demonstrate that O-glycosylation is not a prerequisite for Ag43 function and that the different traits mediated by Ag43, i.e., biofilm formation, autoaggregation, adhesion to eukaryotic cells and extracellular matrix proteins, rely on distinct mechanisms.     

Characterization of bovine septicemic, bovine diarrheal, and human enteroinvasive Escherichia coli that hybridize with K88 and F41 accessory gene probes but do not express these adhesins

Certain DNA probes derived from accessory genes of cloned K88 and F41 determinants hybridize with Escherichia coli strains that express K88 or F41 and with certain other E. coli strains that do not express these antigens. We found that these probes hybridized with human enteroinvasive E. coli, and with bovine E. coli isolates which produced a fatal septicemia in experimentally infected piglets. These strains did not hybridize with probes derived from the structural subunit genes encoding the K88 and F41 antigens. E. coli strains isolated from turkeys with septicemia, Shigella and Salmonella strains did not hybridize to the K88 and F41 accessory gene probes. The K88 and F41 accessory gene probes hybridized with a 200 kb plasmid which is required for invasion by human enteroinvasive E. coli. The K88 and F41 accessory gene homology in the bovine isolates was located on a 150 kb transmissible plasmid but was unrelated to plasmids encoding aerobactin, Vir, or colicin V, which are suspected virulence factors in septicemic E. coli. A common plasmid-encoded antigen was associated with bovine isolates that hybridized with the K88 and F41 accessory gene probes. This included strains which express CS31A, a surface antigen associated with bovine septicemic E. coli, which also hybridized with the K88 and F4 accessory gene probes. The results suggest that the K88 and F41 accessory gene probes hybridized with sequences that may be associated with a common mechanism of pilus expression in distinct groups of E. coli pathogens.     

Influence of cloned Escherichia coli hemolysin genes, S-fimbriae and serum resistance on pathogenicity in different animal models

The virulence of the uropathogenic E. coli strain 536 (O6:K15:H31) which produces the S-fimbrial adhesin (Sfa+), is serum-resistant (Sre+) and hemolytic (Hly+) and its derivatives were assessed in five different animal models. Cloned hemolysin (hly) determinants from the chromosomes of O6, O18 and O75 E. coli strains and from the plasmid pHly152 were introduced into the spontaneous Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains (Infect. Immun. 42: 57-63) the O18-hly determinant but not the plasmid-encoded hly determinant of pHly152 transformed into 536-31 contribute to lethality in a mouse peritonitis model. Similar results were obtained with both Hly- host strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 15 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. In contrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. In a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a level comparable to that of the parental 536 strain.     

Recombinant interleukin-1 stimulates clearance of Escherichia coli K1 bacteraemia

Enhancement of non-specific resistance to neonatal Escherichia coli K1 infection by inter-leukin-1 (IL-1) was analysed. Recombinant human IL-1 administered prophylactically to newborn LPS-non-responsive C3H/HeJ mice induced rapid clearance of E. coli 018:K1 bacteraemia. The effect was dose-dependent and was observed with mice treated immediately to 1 day before bacterial challenge, whereas treatment 2 days before challenge was ineffective. Clearance of intravenously injected radiolabelled 018:K1 E. coli suggested that IL-1 triggered defence mechanisms that contribute to bacterial sequestration and killing in the spleen and liver. Comparable increase in bacterial clearance occurred in naturally resistant LPS-responsive mice that had been subjected to transient E. coli K1 bacteraemia and showed increased resistance to reinfection. In the course of E. coli K1 bacteraemia a strong synthesis of acute phase reactants was observed in both susceptible and resistant mouse strains, which indicated that these proteins alone cannot confer natural resistance to E. coli K1. IL-1 induced a very rapid synthesis of acute phase proteins. The clearance of K1 E. coli when still viable in IL-1-treated animals suggested that acute phase proteins are not likely to be major mediators of the IL-1-enhanced non-specific resistance.