EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation

Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ ehaA , z0469/ ehaB , z3487/ ehaC and z3948/ ehaD ) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655 flu background. Preliminary characterization revealed that ehaA , ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes ( ehaA ) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H^– did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.     

Presence and characterization of a mosaic genomic island which distinguishes sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H(-) from EHEC O157:H7.     

The type 4 pili of enterohemorrhagic Escherichia coli O157:H7 are multipurpose structures with pathogenic attributes

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 produces long bundles of polar type 4 pili (T4P) called HCP (for hemorrhagic coli pili) that form physical bridges between bacteria associating with human and animal epithelial cells. Here, we sought to further investigate whether HCP possessed other pathogenicity attributes associated with T4P production. Comparative studies performed with wild-type EHEC EDL933 and an isogenic hcpA mutant revealed that HCP play different roles in the biology of this organism. We found that in addition to promoting bacterial attachment to host cells, HCP mediate (i) invasion of epithelial cells, (ii) hemagglutination of rabbit erythrocytes, (iii) interbacterial connections conducive to biofilm formation, (iv) specific binding to host extracellular matrix proteins laminin and fibronectin but not collagen, and (v) twitching motility. Nonadherent laboratory E. coli strain HB101 complemented with hcpABC genes on plasmid pJX22, which specifies for HCP overproduction in EDL933, became hyperadherent and invasive and produced a thick biofilm, suggesting that the presence of HCP confers HB101(pJX22) new attributes otherwise not exhibited by HB101. Analogous to other bacteria in which T4P are involved in the pathogenesis of several infectious diseases, our data strongly suggest that HCP display multiple functions that may contribute to EHEC colonization of different hosts and to virulence, survival, and transmission of this food-borne pathogen.     

Comparative proteomic analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli strains and their ihf and ler mutants

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli

It had been suggested that the flagella of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC fliC deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.     

Design and evaluation of PCR primers which differentiate Escherichia coli O157:H7 and related serotypes

Aims:  To develop methods to differentiate Escherichia coli O157:H7 and related serotypes by the use of amplicon length polymorphism (ALP) analysis based on identifying DNA sequence deletions within highly homologous regions of three sequenced E. coli strains.
Methods and Results:  Potential primer locations along the ancestral genomic backbone were identified and evaluated against three sequenced genomes and then applied to a reference set of pathogenic E. coli strains. All 16 primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes.
Conclusions:  This study defines a collection of primers distributed along the length of the E. coli genome that were applied to ALP analysis methods to successfully differentiate between serotypes of E. coli O157:H7 and other E. coli serotypes.
Significance and Impact of the Study:  ALP-PCR analysis method was validated as an independent method of classification when compared with that of rep-PCR. The principles underlying ALP analysis can be readily applied for the detection and differentiation of other closely related microbial species because of the abundance of complete DNA sequence data for a large number of microbial genomes.     

Comparative Proteomic Analysis of Extracellular Proteins of Enterohemorrhagic and Enteropathogenic Escherichia coli Strains and Their ihf and ler Mutants

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.     

EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V

In this study, we identified and characterized a novel secreted protein, the extracellular serine protease EspP, which is encoded by the large plasmid of enterohaemorrhagic Escherichia coli (EHEC) O157:H7. The corresponding espP gene consists of a 3900 bp open reading frame that is able to encode a 1300-amino-acid protein. EspP is synthesized as a large precursor which is then processed at the N- and C-termini during secretion. It can be grouped into the autotransporter protein family. The deduced amino acid sequence of EspP showed homology to several secreted or surface-exposed proteins of pathogenic bacteria, in particular EspC of enteropathogenic E. coli and IgA1 proteases from Neisseria spp . and Haemophilus influenzae . Hybridization experiments and immunoblot analysis of clinical EHEC isolates showed that EspP is widespread among EHEC of the serogroup O157 and that it also exists in serogroup O26. A specific immune response against EspP was detected in sera from patients suffering from EHEC infections. Functional analysis showed that EspP is a protease capable of cleaving pepsin A and human coagulation factor V. Degradation of factor V could contribute to the mucosal haemorrhage observed in patients with haemorrhagic colitis.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Avian- and mammalian-derived antibodies against adherence-associated proteins inhibit host cell colonization by Escherichia coli O157:H7

AIM: To evaluate the potential for polyclonal antibodies targeting enterohaemorrhagic Escherichia coli (EHEC) virulence determinants to prevent colonization of host cells by E. coli O157:H7. METHODS AND RESULTS: Rats and laying hens were immunized with recombinant proteins from E. coli O157:H7, EspA, C-terminal intimin or EscF. Rat antisera (IgG) or chicken egg powders (IgY) were assessed for their ability to inhibit growth and colonization-associated processes of E. coli O157:H7. Mammalian antisera with antibodies to intimin, EspA or EscF effectively reduced adherence of the pathogen to HeLa cells (P<0.05) and prevented type III secretion of Tir. Similarly, HeLa cells treated with chicken egg powder containing antibodies against intimin or EspA were protected from EHEC adherence (P<0.05). Neither egg nor rat antibody preparations had any antibacterial effect on the growth of EHEC (P>0.05). CONCLUSIONS: Antibody preparations targeting EHEC adherence-associated factors were effective at preventing adhesion and intimate colonization-associated events. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates that immunotherapy with anti-adherence antibodies can reduce E. coli O157:H7 colonization of host cells. Passive immunization with specific antibodies may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle or humans.     

Characterization of a novel two-partner secretion system in Escherichia coli O157:H7

Gram-negative bacteria contain multiple secretion pathways that facilitate the translocation of proteins across the outer membrane. The two-partner secretion (TPS) system is composed of two essential components, a secreted exoprotein and a pore-forming beta barrel protein that is thought to transport the exoprotein across the outer membrane. A putative TPS system was previously described in the annotation of the genome of Escherichia coli O157:H7 strain EDL933. We found that the two components of this system, which we designate OtpA and OtpB, are not predicted to belong to either of the two major subtypes of TPS systems (hemolysins and adhesins) based on their sequences. Nevertheless, we obtained direct evidence that OtpA and OtpB constitute a bona fide TPS system. We found that secretion of OtpA into the extracellular environment in E. coli O157:H7 requires OtpB and that when OtpA was produced in an E. coli K-12 strain, its secretion was strictly dependent on the production of OtpB. Furthermore, using OtpA/OtpB as a model system, we show that protein secretion via the TPS pathway is extremely rapid.     

The Escherichia coli ycbQRST operon encodes fimbriae with laminin-binding and epithelial cell adherence properties in Shiga-toxigenic E. coli O157:H7

The human pathogen Shiga-toxigenic Escherichia coli (STEC) O157:H7 contains a ycbQRST fimbrial-like operon, which shares significant homology to the family of F17 fimbrial biogenesis genes f17ADCG found in enterotoxigenic E. coli . We report that growth of STEC O157:H7 strain EDL933 in minimal Minca medium at 37°C and during adherence to epithelial cells led to the production of fine peritrichous fimbriae, which were found to be composed of a major subunit of 18 kDa whose N-terminal amino acid sequence matched the predicted protein product of the ycbQ gene; and showed significant homology to the F17a-A fimbrin. Similar to the F17 fimbriae, the purified STEC fimbriae and the recombinant YcbQ protein fused to a His peptide tag bound laminin, but not fibronectin or collagen. Thus, we propose the name E . coli YcbQ l aminin-binding f imbriae (ELF) to designate the fimbriae encoded by the ycbQRST operon. The role of ELF as an adherence factor of STEC to cultured epithelial cells was investigated. We provide compelling evidence demonstrating that ELF contributes to adherence of STEC to human intestinal epithelial cells and to cow and pig gut tissue in vitro . Deletion in the fimbrin subunit gene elfA (or ycbQ ) in STEC strain EDL933 led to an isogenic strain, which showed significant reduction (60%) in adherence to HEp-2 cells in comparison with the parental strain. In addition, antibodies against the purified ELF also partially blocked adherence of two STEC O157:H7 strains. These observations suggest that ELF functions as an accessory adherence factor that, along with other known redundant adhesins, contributes to the overall adhesive properties of STEC O157:H7 providing these organisms with ecological advantages to survive in different hosts and in the environment.     

Characterization of the NleF effector protein from attaching and effacing bacterial pathogens

Enterohemorrhagic Escherichia coli (EHEC) is a water- and food-borne pathogen that causes hemorrhagic colitis. EHEC uses a type III secretion system (T3SS) to translocate effector proteins that subvert host cell function. T3SS-substrates encoded outside of the locus of enterocyte effacement are important to E. coli pathogenesis. We discovered an EHEC secreted protein, NleF, encoded by z6020 in O-island 71 of E. coli EDL933 that we hypothesized to be a T3SS substrate. Experiments are presented that probe the function of NleF and its role in virulence. Immunoblotting of secreted and translocated proteins suggest that NleF is secreted by the T3SS and is translocated into host cells in vitro where it localizes to the host cytoplasm. Infection of HeLa cells with E. coli possessing or lacking nleF and transient expression of NleF-GFP via transfection did not reveal a significant role for NleF in several assays of bacterial adherence, host cytoskeletal remodeling, or host protein secretion. However, competitive coinfection of mice with Citrobacter rodentium strains possessing or lacking nleF suggested a contribution of NleF to bacterial colonization. Challenge of gnotobiotic piglets also revealed a role for NleF in colonization of the piglet colon and rectoanal junction.