Characterization of serotypes and outer membrane protein profiles in enteroaggregative Escherichia coli strains.

Twenty-four Escherichia coli strains mainly isolated from children with diarrhea in São Paulo, and showing characteristics of enteroaggregative E. coli (EAEC), were characterized by serotyping and outer membrane protein (OMP) profiles. The relationship between these characteristics was evaluated, as well as the usefulness of OMP profiles in the clonal analysis of EAEC strains. All strains presented aggregative adherence to HeLa cells and were classified in two groups based on their interaction with the EAEC DNA probe. A diversity of serotypes and OMP profiles was observed in both groups studied. Although no significant correlation between serotypes and OMP profiles was observed, unique OMP profiles were identified in 80% of the probe-positive strains which were distributed in only 4 OMP profiles. This result may indicate the presence of a few clones in the probe-positive group. On the other hand, probe-negative strains seem to constitute a more diverse group. In general, the observed heterogeneity in serotypes and OMP profiles described in the present study suggest a great genetic diversity in EAEC isolates of either the same or different serotypes and in strains presenting the same EAEC markers identified in our community.     

Characterization of typical and atypical enteroaggregative Escherichia coli in Kagoshima,Japan: biofilm formation and acid resistance

EAEC is increasingly recognized as an emerging enteric pathogen. Typical EAEC expressing the AggR regulon have been proven to be an important cause of childhood diarrhea in industrialized countries as well as in the developing world, while atypical EAEC without this regulon have not been thoroughly investigated. To investigate the bacteriological characteristics of EAEC, including both typical and atypical strains in Kagoshima, Japan, 2417 E. coli strains from Japanese children with diarrhea were screened by a quantitative biofilm assay to detect possible EAEC strains, resulting in the identification of 102 (4.2%) of these strains by the HEp‐2 cell adherence test. Virulence gene patterns, PFGE analysis and O‐serogrouping demonstrated the heterogeneity of the EAEC. The EAEC strains were classified into two groups: typical EAEC with aggR (74.5%, 76/102) and atypical EAEC without aggR (25.5%, 26/102). There was no significant difference between the typical EAEC strains (median OD₅₇₀= 0.73) and the atypical strains (median OD₅₇₀= 0.61) in biofilm formation (P= 0.17). Incidences of resistance against ampicillin, cefotaxime and tetracycline were significantly higher in the typical EAEC strains than the atypical EAEC strains (84.2% vs. 53.8%, 36.8% vs. 7.7% and 93.4% vs. 73.1%, respectively, P < 0.05). The typical EAEC strains showed significantly higher resistance ratios against HCl and lactate than the atypical strains (94.7% vs. 61.5% and 92.1% vs. 57.7%, respectively, P < 0.001). To investigate the pathogenicity of not only typical but also atypical EAEC, further bacteriological and epidemiologic studies including atypical EAEC are needed.     

Pathogenesis of enteroaggregative Escherichia coli infection

Enteroaggregative Escherichia coli (EAEC) is emerging as a significant diarrheal pathogen in multiple population groups. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in adults including HIV-positive patients and travelers and has been a cause of food-borne outbreaks in the industrialized world. Current data suggest that one set of virulence elements is not associated with all EAEC strains, but that combinations of multiple factors prevail. Pathogenesis is believed to be initiated with adherence to the terminal ileum and colon in an aggregative, stacked-brick-type pattern by means of one of several different hydrophobic aggregative adherence fimbriae. Some strains of EAEC may then elaborate cytotoxins including the plasmid-encoded toxin and the enterotoxins, EAST1 and ShET1. An AraC homolog termed AggR regulates several genes contributing to fimbrial biogenesis in 'typical EAEC strains'. AggR has now also been shown to regulate genes on a chromosomal island. Sequencing of the EAEC type strain 042 completed at the Sanger Center has revealed two other chromosomal islands that are being explored for their pathogenetic potential. This article reviews these virulence elements and presents on-going areas of research in EAEC pathogenesis.     

Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic E. coli

To analyze whether Escherichia coli strains that cause urinary tract infections (UPEC) share virulence characteristics with the diarrheagenic E. coli (DEC) pathotypes and to recognize their genetic diversity, 225 UPEC strains were examined for the presence of various properties of DEC and UPEC (type of interaction with HeLa cells, serogroups and presence of 30 virulence genes). No correlation between adherence patterns and serogroups was observed. Forty-five serogroups were found, but 64% of the strains belonged to one of the 12 serogroups (O1, O2, O4, O6, O7, O14, O15, O18, O21, O25, O75, and O175) and carried UPEC virulence genes (pap, hly, aer, sfa, cnf). The DEC genes found were: aap, aatA, aggC, agg3C, aggR, astA, eae, ehly, iha, irp2, lpfA(O113), pet, pic, pilS, and shf. Sixteen strains presented aggregative adherence and/or the aatA sequence, which are characteristics of enteroaggregative E. coli (EAEC), one of the DEC pathotypes. In summary, certain UPEC strains may carry DEC virulence properties, mostly associated to the EAEC pathotype. This finding raises the possibility that at least some faecal EAEC strains might represent potential uropathogens. Alternatively, certain UPEC strains may have acquired EAEC properties, becoming a potential cause of diarrhoea.     

A new understanding of enteroaggregative Escherichia coli as an inflammatory pathogen

Enteroaggregative Escherichia coli (EAEC) is an important cause of endemic and epidemic diarrheal disease worldwide. Although not classically considered an inflammatory pathogen in the style of Shigella and Salmonella species, clinical data from patients suggests that inflammatory responses may play an important role during EAEC disease. However, the specific role of inflammation during EAEC pathogenesis has not been investigated in detail. To better understand how EAEC may induce inflammation, we have focused our attention on the intimate interactions between EAEC and the host epithelium and the subsequent induction of host cell signaling events leading to innate immune responses. Here, we discuss our recent findings on the signaling pathway by which EAEC promotes transepithelial migration of polymorphonuclear leukocytes (PMNs), the role of aggregative adherence fimbriae in triggering this event and the implementation of human intestinal xenografts in immunodeficient mice for studying EAEC pathogenesis in vivo. Our findings suggest that EAEC shares conserved mechanisms of inducing PMN recruitment with other intestinal pathogens, providing new insight into the potential pathological consequences of EAEC-induced inflammation.     

Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.     

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs

Abstract We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA ( E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA⁺ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA⁻ or eaeA⁺ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli . In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and efficacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli .     

Escherichia coli K12 arabinose-binding protein mutants with altered transport properties.

The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild-type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and K(en) (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal Ven values, 3 mutants had Ven and K(en) values twice wild type, and 11 mutants had Ven and K(en) values 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.     

Characterization of enteroaggregative Escherichia coli isolates

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.     

Solution structure of the novel dispersin protein of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface.     

Site-directed mutagenesis of penicillin-binding protein 3 of Escherichia coli: Role of Val-545

Abstract Val545 of the Escherichia coli penicillin-binding protein 3 is essential to the acyl transfer mechanism through which the active-site serine 307 is acylated by benzylpenicillin and cephalexin and to the mechanism through which the protein allows rapidly growing cells to divide.     

Role of type I fimbriae in the aggregative adhesion pattern of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is distinguished by its characteristic aggregative adherence (AA) pattern to cultured epithelial cells. In this study we investigated the role of type I fimbriae (TIF) in the AA pattern to HEp-2 cells and in biofilm formation. Accentuation of this pattern was observed when the adherence assay was performed in the absence of mannose. This effect was observed in the prototype EAEC strain 042 (O44:H18), O128:H35 strains and for other EAEC serotypes. Antiserum against TIF decreased AA by 70% and 90% for strains 042 and 18 (O128:H35 prototype strain), respectively. A non-polar knockout of fimD, the TIF usher, in strains 042 and 18 resulted in inhibition of the accentuated AA pattern of approximately 80% and 70% respectively, and biofilm formation diminution of 49% for 042::fimD and 76% for 18::fimD. Our data evidence a role for TIF in the AA pattern and in EAEC biofilm formation, demonstrating that these phenotypes are multifactorial.     

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.     

The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin

Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the blood stream and eventually invasion of the meninges. The latter two aspects have been well characterized at a molecular level in the last decade. Less is known about the early stages of pathogenesis, i.e. adhesion to and invasion of gastrointestinal cells. Here, the characterization of the Hek protein is reported, which is expressed by neonatal meningitic E. coli (NMEC) and is localized to the outer membrane. It is demonstrated that this protein can cause agglutination of red blood cells and can mediate autoaggregation. Escherichia coli expressing this protein can adhere to and invade epithelial cells. So far, this is the first outer membrane protein in NMEC to be directly implicated in epithelial cell invasion.     

Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

Abstract Outer membranes of Escherichia coli K-12 were used to isolate hybridoma cell lines that produce monoclonal antibodies against the FhuA (TonA) protein. Two monoclonal antibodies were obtained from independent immunization and fusion experiments. The antibodies belonged to the subclass IgG₁ and κ, and IgG_2b and κ, respectively. The latter antibody was purified by affinity chromatography on protein A-Sepharose. The culture supernatants of the hybridoma cell lines and the isolated antibody inhibited adsorption of the phages T5 and T1 to E. coli cells while binding of phage ø80, which also uses the FhuA protein as a receptor, remained unaffected. The specificity of the antibodies to the FhuA protein was supported by the prevention of killing of cells by colicin M and by the lack of inhibition of colicin B and of phage BG23. Transport of iron(III) as ferrichrome complex was not inhibited by the isolated antibody. However, partial competition with the adsorption of the phages T2, TuIb and T6 was observed which may indicate an organization of certain functional phage receptors into clusters.     

Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram‐negative bacteria and have a relevant role in bacteria–host interactions. Using 1D SDS–PAGE and highly sensitive LC–MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain‐linked genes and 57 were common to pathogen‐derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic‐derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 ( http://proteomecentral.proteomexchange.org/dataset/PXD000367 ).     

Role of multiple efflux pumps in Escherichia coli in indole expulsion

Escherichia coli chromosome encodes several multidrug transporters. Despite their protective function against antibacterial agents, the specific physiological actions of these transporters are not fully understood. E. coli produces indole, a metabolite of tryptophan, under physiological conditions. Defined inactivation of the acrEF gene, the product of which is known as an energy-dependent multiple drug efflux pump, decreased indole excretion while reintroduction of the acrEF gene restored it. A DeltaacrEF mutant accumulated more intracellular indole than the parent. This mutant was more susceptible to the growth-inhibitory effect of indole than the parent. These results indicate that the AcrEF system plays a significant role in indole efflux.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.