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.     

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.     

Designed Coiled-Coil Peptides Inhibit the Type Three Secretion System of Enteropathogenic Escherichia coli

Background

Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry.

Methods/Principal Findings

We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled–coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments.

Conclusions

Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis.     

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker,Antibody and Method

Background

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.

Methodology

First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.

Principal findings

EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.

Conclusion

RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.     

Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69

Enteropathogenic Escherichia coli (EPEC) was the first pathovar of E. coli to be implicated in human disease; however, no EPEC strain has been fully sequenced until now. Strain E2348/69 (serotype O127:H6 belonging to E. coli phylogroup B2) has been used worldwide as a prototype strain to study EPEC biology, genetics, and virulence. Studies of E2348/69 led to the discovery of the locus of enterocyte effacement-encoded type III secretion system (T3SS) and its cognate effectors, which play a vital role in attaching and effacing lesion formation on gut epithelial cells. In this study, we determined the complete genomic sequence of E2348/69 and performed genomic comparisons with other important E. coli strains. We identified 424 E2348/69-specific genes, most of which are carried on mobile genetic elements, and a number of genetic traits specifically conserved in phylogroup B2 strains irrespective of their pathotypes, including the absence of the ETT2-related T3SS, which is present in E. coli strains belonging to all other phylogroups. The genome analysis revealed the entire gene repertoire related to E2348/69 virulence. Interestingly, E2348/69 contains only 21 intact T3SS effector genes, all of which are carried on prophages and integrative elements, compared to over 50 effector genes in enterohemorrhagic E. coli O157. As E2348/69 is the most-studied pathogenic E. coli strain, this study provides a genomic context for the vast amount of existing experimental data. The unexpected simplicity of the E2348/69 T3SS provides the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context.     

In Vitro Evolution of an Archetypal Enteropathogenic Escherichia coli Strain

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal^r) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nal^r strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str^r) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK. Another, in gyrA, is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD, the lysogenization regulator, to purB. The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal^r clone has a lower growth rate than the Str^r isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB. Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal^r clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro.     

Effects of Psidium guajava leaf extract on secretion systems of gram‐negative enteropathogenic bacteria

In this study, 672 plant‐tissue extracts were screened for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, an extract from the leaves of Psidium guajava (guava) was found to inhibit secretion of EPEC‐secreted protein B (EspB) from EPEC and EHEC without affecting bacterial growth. Guava extract (GE) also inhibited EPEC and EHEC from adhering to, and injecting EspB into, HEp‐2 cells. GE seemed to block translocation of EspB from the bacterial cells to the culture medium. In addition, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped secreting Yersinia outer proteins and was unable to induce apoptosis of mouse bone marrow‐derived macrophages. S. typhimurium exposed to GE stopped secreting Sip proteins and was unable to invade HEp‐2 cells. GE inhibited secretion of EspC, the type V secretion protein of EPEC, but not secretion of Shiga toxin 2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used to treat and prevent gram‐negative enteropathogenic bacterial infections.

     

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.     

Cloning and characterization of the eae gene from a dog attaching and effacing Escherichia coli strain 4221

We have cloned and determined the nucleotide sequence of the eae gene from a dog attaching and effacing (A/E) Escherichia coli (DEPEC) strain 4221. When comparing the predicted amino acid sequence of the eae_DEPEC to that of the Eae proteins from enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli O157:H7 (EHEC), Citrobacter freundii biotype 4280, and a swine A/E E. coli strain O45 (PEPEC), the overall sequence identity was 84, 81, 83 and 83%, respectively, with the greatest divergence at the C-terminal end, the putative receptor-binding portion. Interestingly, the DEPEC Eae shares the greatest identity at the C-terminal region with the Citrobacter freundii Eae protein. We have constructed and purified a maltose-binding fusion protein (MBP) containing the product of the entire eae gene of the DEPEC strain 4221. Binding of MBP-Eae_DEPEC fusion protein to HEp-2 cells was demonstrated by immunofluorescence microscopy. In addition, the Eae protein of DEPEC (4221) demonstrated a strong serological relationship with that of EPEC (E2348/69) as observed using a polyclonal antiserum against MBP-Eae_DEPEC fusion protein.     

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E. coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E. coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E. coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E. coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.     

Transduction of Porcine Enteropathogenic Escherichia coli with a Derivative of a Shiga Toxin 2-Encoding Bacteriophage in a Porcine Ligated Ileal Loop System

In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Φ3538 (Δstx₂::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with Φ3538 (Δstx₂::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions.     

EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium. Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export. Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion. CesD shows sequence homology to chaperone proteins from other type III secretion pathways. Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC. A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA. The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis. CesD was shown to interact with EspD but not EspB or EspA. CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane. Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.     

Mice vaccinated with enteropathogenic Escherichia coli ghosts show significant protection against lethal challenges

Aim: To prepare enteropathogenic Escherichia coli (EPEC) E2348/69 ghosts and investigate whether immunization with EPEC bacterial ghosts can elicit protective immune responses. Methods and Results: A recombinant plasmid with double λPL/PR‐cI857 temperature‐sensitive regulatory cassettes was constructed. The lysis gene E and/or the staphylococcal nuclease A (SNA) gene were separately inserted downstream of the two regulatory cassettes to construct the lysis plasmids pBV220::E and pBV220::E::CI‐P‐SNA. An EPEC reference strain E2348/69 (serotype O127:H6) was transformed with the lysis plasmids to produce EPEC ghosts. Mice injected with bacterial ghosts EGE (EPEC ghosts produced using lysis protein E) or EGES (EPEC ghosts produced using a combination of lysis protein E and SNA) gained weight normally and showed no clinical signs of disease. Vaccination trials showed that mice immunized with EGE or EGES were significantly protected against subsequent challenge with the wild‐type virulent parent strain, EPEC E2348/69 (42/50 and 45/50 survival, respectively); in contrast, none of the 30 control mice survived. Conclusions: Immunization with EPEC ghosts can elicit protective immune responses in BALB/c mice. Significance and Impact of the Study: EPEC ghosts may represent a promising new approach for vaccination against EPEC infection.     

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.