Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies.
Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice.
Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC.
Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.
Diarrheal diseases are responsible for more than 1.5 million deaths annually in developing countries. Enterotoxigenic E. coli (ETEC) are among the most common bacterial causes of diarrhea, accounting for an estimated 300,000–500,000 deaths each year, mostly in young children. There unfortunately is not yet a vaccine that can offer sustained, broad-based protection against ETEC. While most vaccine development effort has focused on plasmid-encoded finger-like ETEC adhesin structures known as colonization factors, additional effort is needed to identify conserved target antigens. Epidemiologic studies suggest that immune responses to uncharacterized, chromosomally encoded antigens could contribute to protection resulting from repeated infections. Earlier studies of immune responses to ETEC infection had identified a class of surface-expressed molecules known as autotransporters (AT). Therefore, available ETEC genome sequences were examined to identify conserved ETEC autotransporters not shared by the commensal E. coli HS strain, followed by studies of the immune response to these antigens, and tests of their utility as vaccine components. Two chromosomally encoded ATs, identified in ETEC, but not in HS, were found to be immunogenic and protective in an animal model, suggesting that conserved AT molecules contribute to protective immune responses that follow natural ETEC infection and offering new potential targets for vaccines.
Citation: Harris JA, Roy K, Woo-Rasberry V, Hamilton DJ, Kansal R, et al. (2011) Directed Evaluation of Enterotoxigenic Escherichia coli Autotransporter Proteins as Putative Vaccine Candidates. PLoS Negl Trop Dis 5(12): e1428. doi:10.1371/journal.pntd.0001428
Editor: Elizabeth Angelica Leme Martins, Instituto Butantan, Brazil
Received: April 25, 2011; Accepted: October 25, 2011; Published: December 6, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: These studies were funded by Merit Review funding from the Department of Veterans Affairs (JMF), by NIH grant R01 AI089894-01 (JMF) and by the NIH Medical Student Research Fellowship Program (NIDDK, 5T35DK007405-25). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in developing countries where these organisms cause hundreds of millions of infections and an estimated 300,000–500,000 deaths in young children each year . ETEC are perennially by far the most common cause of traveler's diarrhea . Disease caused by ETEC is highly endemic in regions plagued by inadequate sanitation and a lack of clean drinking water, and prevention of ETEC is a high priority , . ETEC are genetically heterogeneous pathogens that share the ability to colonize the small intestine where they deliver the cholera toxin-like heat-labile toxin (LT) and/or small peptide heat-stable (ST) toxins that ultimately result in diarrhea .
In the classic paradigm for ETEC pathogenesis, small intestinal colonization requires plasmid-encoded colonization factors (CFs) . A variety of more than 25 antigenically distinct fimbrial, or fibrillar CFs have been described to date , . These antigens, along with LT, remain central to ETEC vaccine development . However, CF antigens are not appreciably cross-protective, and many ETEC strains do not appear to produce CFs , . Moreover, LT alone (or the homologous cholera toxin) do not appear to afford complete sustained protection , while ST, typically only 19 amino acids in its mature form, is not suitably immunogenic.
These constraints, as well as a growing appreciation of the complexity of ETEC pathogenesis , , have prompted searches for additional surface-expressed antigens. Use of classical genetic approaches including TnphoA mutagenesis to find novel molecules exposed on the surface of ETEC, recently led to the identification of several putative virulence loci, including the etpBAC two-partner secretion locus , responsible for secretion of the EtpA adhesin molecule , and the autotransporter (AT) protein EatA .
EatA and other AT proteins contain three essential domains: an amino terminal signal peptide, the secreted “passenger” domain, and a third carboxy-terminal beta barrel domain inserted into the outer membrane . The variable passenger portion of the protein may be cleaved by surface proteases and freely secreted as in the case of EatA, or remain attached to the transport domain. The surface expression of AT passenger domains proteins make them attractive targets for vaccine development, while only limited portions of the beta regions are predicted to be exposed .
While a broadly protective ETEC vaccine remains outstanding, one approach currently being explored is a protein subunit vaccine based on multiple ETEC antigens. Present acellular pertussis vaccines , , , subunit formulations containing a two-partner secretion (TPS) exoprotein adhesin (filamentous hemagglutinin, FHA) , , the pertactin autotransporter , , and pertussis toxoid offer a potential strategy that might be adopted to guide ETEC vaccine development. Indeed, recent investigations of EtpA , , , , an ETEC TPS exoprotein adhesin, were prompted by its similarity to FHA. Recent immunoproteomic studies of ETEC H10407 independently identified EtpA as well as several AT proteins including EatA, TibA and antigen 43 suggesting that these proteins are expressed during both experimental infection in mice and in humans .
Interestingly, it appears clear that children repeatedly exposed to ETEC infections are ultimately protected against subsequent symptomatic infections . However, the precise composition of the protective antigens remains uncertain , , . The present studies were performed to examine the possible contribution of conserved, chromosomally-encoded AT proteins to protective ETEC immune responses, and to evaluate passenger domains of these ATs as possible candidates for ETEC vaccine development.
Bacterial strains and plasmids
A complete list of strains and plasmids employed in these studies is included in table 1. ETEC strains H10407 and E24377A were originally provided by Marcia Wolf and Stephen Savarino, respectively, from cGMP lots maintained at Walter Reed Army Institute of Research.
In silico analyses
A number of parallel bioinformatics approaches were used to identify candidate AT genes in recently sequenced ETEC strains. Strains B7A and E24377A were searched for highly conserved autotransporter domains using the Pfam database including the autotransporter beta domain (http://pfam.sanger.ac.uk//family/PF03797) and the pertactin domain (http://pfam.sanger.ac.uk//family/PF03212). The resulting sequences containing these domains were used to identify additional autotransporters in the genome of ETEC strain H10407 available in un-annotated form via the Sanger Institute (http://www.sanger.ac.uk/Projects/E_coli_H10407/), which was facilitated by interrogating the available sequence using the National Microbial Pathogen Database Resource (NMPDR)  on the Rapid Annotation Subsystem Technology (RAST) server (http://RAST.nmpdr.org/) . SignalP (http://www.cbs.dtu.dk/services/SignalP/) was used to identify potential signal peptide encoding regions of the predicted AT coding sequences. BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Pfam domain searches of each putative AT peptide were used to define the conserved beta barrel transport domain (TIGR01414, and pfam03797, in the NCBI Conserved Domain Database http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml and Pfam databases, respectively). Peptide sequence alignments of AT proteins common to the three ETEC strains used ClustalW  and MUSCLE  alignment algorithms performed locally (Mac OS 10.5.8) with an additional alignment plug-in (v1.06) for CLC Main Workbench software (v 5.5).
Molecular cloning of autotransporter passenger domains
Regions corresponding to the majority of each passenger domain (region between the end of the putative signal peptide encoding sequence and the beginning of the beta barrel domain) were amplified using primers bearing attB flanking sequences by high fidelity PCR (Platinum PCR SuperMix, Invitrogen). A complete list of primers used in these studies is included in table 2. Resulting amplicons were agarose gel-purified (Ultra Clean 15, MOBIO). Amplicons containing attB flanking sequences were cloned by recombination with the lambda attP sites on the entry vector pDONR221 (BP Clonase II, Invitrogen), and transformed into DH10B One Shot ccdB Survival T1 Chemically Competent E. coli (Invitrogen). Colonies were selected and patched onto kanamycin and chloramphenicol plates. Plasmid DNA extracted from kanamycin-resistant, chloramphenicol-sensitive colonies was analyzed by restriction digest to confirm presence of the appropriate insert. pDONR221 entry clones were then recombined with pDEST17 (LR Clonase II) placing the passenger domains in-frame with an amino-terminal polyhistidine tag in the resulting expression plasmid. Cloning reactions were used to transform DH5α to ampicillin resistance. Plasmid DNA from ampicillin-resistant, chloramphenicol-sensitive colonies was analyzed first by restriction endonuclease digestion, then sequenced using T7 promoter primer 5′-TAATACGACTCACTATAGG-3′ to confirm that the 6His- and passenger encoding sequences were in-frame. Resulting plasmids were then used to transform E. coli expression strain BL21-A1.
Production of recombinant polyhistidine-tagged passenger proteins
To produce recombinant polyhistine-tagged passenger proteins cultures of BL21-A1 containing pDEST17-encoded 6His-passenger clones were grown overnight at 37°C in Luria broth containing ampicillin (100 µg/ml). After diluting 1:100 in 100 ml of fresh media, cultures were grown at 37°C, 225 rpm for approximately 3 hours to an OD600 of approximately 0.6, then induced by the addition of L-arabinose to a final concentration of 0.02%. After approximately 2 hours, cultures were centrifuged for 5 minutes (4°C, 10,000×g), and the resulting pellet saved and frozen at −80°C for subsequent processing. Pellets were thawed on ice, and resuspended in binding buffer containing 20 mM Tris, 8 M Urea, 500 mM NaCl, 5 mM imidazole, protease inhibitor cocktail (1×, Sigma P8465) and PMSF (1 mM), at pH 8.0. After lysis on a rotator for 30 minutes, the suspension was centrifuged for 10 minutes at 10,000×g at 4° C. Recombinant polyhistidine-tagged autotransporter proteins (rATp) were then purified from clarified lysates using immobilized metal affinity chromatography (IMAC) in small-scale (His SpinTrap columns (GE Healthcare) or in larger scale by using Ni-Sepharose columns (HisTrap HP, GE Healthcare). Proteins were eluted over an imidazole gradient produced on a low-pressure chromatography system (BioLogic LP, BioRad). Purity of recombinant proteins was assessed by SDS-PAGE. Where necessary, fractions containing the protein of interest were further purified by ion exchange (HiTrap Q) or by SDS-PAGE and subsequent electroelution (Mini Whole Gel Eluter, BioRad).
Antibody production and affinity purification
Polyclonal rabbit antisera were produced as previously described (Rockland Immunochemicals, Gilbertsville, PA). Mouse polyclonal antibodies were obtained upon sacrifice of mice following completion of intranasal immunization with either adjuvant control or adjuvant and recombinant protein of interest as described below. Antibodies were affinity-purified as previously described , . Briefly, antibodies were absorbed onto immobilized antigen of interest on nitrocellulose, then eluted in 100 mM glycine, pH 2.5 followed by neutralization with 1 M Tris, pH 8.0.
To detect polyhistidine-tagged rATp proteins transferred onto nitrocellulose, blocking was performed for one hour in 5% skim milk in tris-buffered saline (pH 7.2) containing 0.05% Tween-20 (TBS-T), and subsequent immunoblotting used purified rabbit polyclonal anti-His serum (1:500) and goat anti-rabbit immunoglobulin G (Fc)-Horseradish peroxidase (HRP) (1:10,000). All wash and incubation steps were performed in Tris-Buffered Saline (pH 7.2)- 0.05% Tween 20. Detection used luminol-based chemiluminescent substrate.
Assessment of immune responses to individual rATp proteins was carried out in a similar fashion using pooled primary convalescent sera (1:500) from mice following infection with ETEC strain H10407. Pooled, pre-immune sera (1:500) from the same mice was used as a primary antibody control. Sera obtained from ETEC-infected children (ICDDR,B) Dhaka, Bangladesh, and uninfected (age-matched) controls (LeBonheur Children's Medical Center, Memphis, TN), were tested at a 1:2048 dilution. Primary antibody binding was detected using an HRP-labeled secondary (anti-mouse or anti-human) antibody that detects IgA, IgM and IgG (total Ab) and developed using sensitive chemiluminescent substrate (SuperSignal West Femto, Thermo Scientific).
Kinetic ELISA assays were performed as previously described . (For an in-depth discussion comparing kinetic and standard end-point ELISA techniques, the reader is referred to an earlier review available at http://www.biotek.com/resources/docs/KineticAppNoteFinal.pdf) . ELISA wells were coated overnight at 4°C with (100 µl/well) of individual rATp proteins diluted to a final concentration of 4 µg/ml in 0.1 M NaHCO3 buffer (pH 8.6). Plates were washed three times with TBS-T, blocked with TBS-T containing 1% BSA (Blocker, Thermo Scientific) for 1 hour at 37°C. After washing briefly with TBS-T, plates were incubated with dilutions of primary antibody in TBS-T/1% BSA for 1 hour at 37°C, and again washed as above. Next, plates were incubated (1 h, 37°C) with goat anti-human or goat anti-mouse secondary antibody, which recognizes IgA, IgM, and IgG, at a final concentration of 1:10000 in TBS-T-BSA. After washing, plates were developed with TMB (3,3′,5,5′-Tetramethylbenzidine) peroxidase substrate (Kirkgaard and Perry Laboratories). Kinetic absorbance measurements  were determined at 650 nm, acquired at 60 second intervals (Molecular Devices Spectramax 340PC microplate reader). SoftMax Pro v5.0.1 was used for data recording and processing and the rate of substrate development was expressed as the Vmax in milli-units/min.
Immunization of mice
Mice were vaccinated intranasally with candidate antigens as previously described . Briefly, groups of 10 ICR  mice received either IVX908 (Protollin®)  (7.5 µg) alone (controls), or IVX908 (7.5 µg) + rATp domains (20 µg) on days 0, 14, 28. Sera were collected on day 0 prior to immunization (pre-immune) and again 7–14 days after the final vaccination (post-immune). Prior to bacterial challenge (day 42), 5–6 fresh fecal pellets were collected from each mouse and immediately suspended in 1.5 ml of extraction buffer containing Tris (10 mM), NaCl (100 mM), Tween-20 (0.05%), and sodium azide (5 mM) pH 7.4.
Intestinal colonization studies
Approximately 1 week after completion of the immunization schedule, mice were challenged with jf876, a derivative of ETEC H10407 containing a KmR marker in the lacZ gene as previously described , . Two independent challenge studies were completed; each involved a total of 30 mice including 10 controls (IVX908 only), and groups of 10 mice vaccinated with either of the two rATp proteins. Briefly, mice were treated with streptomycin (5 g/liter) in their drinking water to eliminate colonization resistance from competing normal enteric flora. Food was then withheld 12 hours prior to ETEC challenge and sterile water was used to replace the streptomycin solution. All mice received cimetidine (50 mg/kg via intraperitoneal injection) 2 hours prior to administration of bacteria. Mice were then challenged by gavage administration of bacterial strain jf876 with total dose between 104 and 105 cfu/mouse. Previous studies have demonstrated that at 24 hours after inoculation with ETEC in the murine model, the number of colonizing colony forming units in the small intestine parallel values obtained at later time points (72 hours) . Therefore, in these studies, approximately 24 hours after challenge mice were sacrificed, samples of ileum were harvested, then solubilized in saponin solution to release bacteria, and finally plated onto Luria agar plates containing kanamycin (25 µg/ml). Colonies were counted after incubation overnight at 37°C. Intestinal lysates with no bacteria recovered were assigned a value of 1 cfu (the lower limit of detection) as previously described . Care was taken to minimize the use of animals in these experiments where possible. When necessary, appropriate steps were taken to ameliorate suffering of mice during vaccination and testing. The Institutional Animal Care and Use Committees of the University of Tennessee Health Sciences Center, and the VA Medical Center approved studies presented here. All procedures involving mice complied with Public Health Service guidelines, and The Guide for the Care and Use of Laboratory Animals.
To evaluate surface expression of autotransporter passenger domains, suspensions of H10407 in phosphate buffered saline (PBS, pH 7.2) were first fixed with 2% paraformaldehyde for 15 minutes. After washing twice with PBS, cells were blocked with 1% BSA in PBS for 30 minutes. The resulting cell suspension was incubated with either pre- or post-immune mouse sera (diluted 1:50 in blocking buffer) for 1 hour at room temperature (RT). After washing three times with PBS, cells were incubated with Alexa-Fluor (488)-labeled anti-mouse IgG [1:250] for 1 hour at RT, washed three times, and resuspended in PBS. Analysis of cell-bound fluorescence by flow cytometry used a BD FACSCalibur 4-color, dual-laser flow cytometer equipped with a FACStation data management system.
Identification of additional ETEC autotransporter genes
In silico analysis of available ETEC genomes using searches for one or more of the known highly conserved AT domains identified multiple autotransporters in the genome of each ETEC strain. From these, we selected AT genes that were present in ETEC, but absent in the recently sequenced E. coli HS commensal isolate , for additional study (table 3). All three genomes shared at least one copy of both antigen43, and the pAT autotransporter genes (figure 1). Similar to other pathovars , we identified two copies of the agn43 gene in both H10407, and E24377A, but a single copy in B7A. The amino acid sequence of the passenger domains of these Ag43 molecules was highly conserved (supporting figure S1a), while the pAT passenger domains exhibited approximately 70% identity and 80% similarity (supporting figure S1b).
a. location of antigen 43 (agn43) genes in the chromosomes of ETEC strains (H10407, E24377A, and B7A) and nonpathogenic E. coli strains (MG1655, and HS). Genes are shaded by similarity. Individual autotransporter genes are depicted in blue. Putative full-length autotransporter genes include their assigned ncbi protein reference numbers. Mobility elements are depicted in green. Open arrows represent hypothetical genes. b. location of pAT genes (in blue). Figures are based on RAST annotations (http://rast.nmpdr.org/).
Although genes encoding proteins similar to pAT were found in the Enterobacteriaceae by BLAST searches of the prototype pAT molecule from H10407 (Uniprot designation E3PFJ1) in the UniprotKB database, and were widely distributed among various pathovars of E. coli as well as Salmonella, and Shigella species (taxonomic distribution of these proteins is included in supporting figure S2), only those in E. coli exhibited more than 80% identity. As demonstrated in table 4, pAT homologues were identified in other diarrheagenic E. coli pathotypes including enterohemorrhagic strains (EHEC), enteropathogenic isolates (EPEC), and uropathogenic E. coli (UPEC), and extraintestinal pathogenic E. coli (ExPEC) isolated from meningitis. However, we also identified potential pAT homologues in non-HS commensal isolates from humans  as well as animals , suggesting that while pAT may not be essential for commensalism, it is not unique to pathogenic strains.
Surface expression of autotransporter passenger domains
Passenger domains of autotransporter proteins are exported to the bacterial surface where they may be cleaved and appear in the supernatant as in the case of EatA , or remain largely associated with bacterial cell surface as has been described for antigen 43 . Antibodies raised against the recombinant passenger domains of Ag43, pAT, and EatA were tested in kinetic ELISA assays against the corresponding antigen as well as each of the other rATp domains. These studies revealed little or no cross-reactivity between antigens, suggesting that these proteins are immunologically distinct (figure 2a). Flow cytometry detected significant amounts of passenger domains of Ag43 and pAT associated with the bacterial cell surface of ETEC H10407, but not with the HS commensal strain (figure 2b). Likewise, pAT (figure 2c) and Ag43 (figure 2d) were identified on the surface of both the E24377A, and B7A ETEC strains.
a. specificity of antibodies directed against ETEC autotransporter passenger domains. Data shown are kinetic ELISA data obtained at a 1:1024 dilution of all primary antisera. b. flow cytometry study comparing the presence of pAT and Ag43 AT passenger domains on the surface of ETEC strain H10407 compared to the HS E. coli commensal strain. Pre-immune (NI for non-immune) sera, as well as unstained bacteria (no primary antibody) are shown as controls c. Examination of pAT surface expression by ETEC strains B7A and E24377A relative to the H10407 (+ control) and the commensal HS (negative control); unstained control used no primary antibody. d. Examination of Ag43 surface expression by ETEC strains B7A and E24377A relative to the H10407 and HS controls. Flow cytometry histograms depict intensity of fluorescence signal on the abscissa (x-axis) while the relative frequency of bacteria counted is depicted on the ordinate (y-axis).
Passenger domains of conserved chromosomally-encoded autotransporter proteins are recognized during infection
Because passenger domains of autotransporter proteins are exported to the bacterial surface, they often elicit an immunologic response in the host during the course of infection , , . Furthermore, recent immuno-proteomic studies have indicated that autotransporters are recognized during the course of experimental infection in mice . Therefore, we examined the immune responses to conserved, chromosomally encoded autotransporter proteins identified in ETEC, using sera obtained from mice following experimental infection and from humans following natural ETEC infections.
Immunoblotting studies of both of the H10407 autotransporters tested H10407_Ag43, and H10407_pAT, demonstrated that the passenger domain of both of these antigens is recognized during the course of experimental intestinal infection in mice (figure 3 a). This was also true of TibA and EatA passenger domains (not shown) as predicted by earlier proteomic studies, however we chose to focus here on the conserved, chromosomally encoded antigens, Ag43 and pAT. Subsequent studies demonstrated that both Ag43 and pAT are recognized during the course of human infections with ETEC (figure 3 b, c).
a. passenger domains of autotransporters pAT and Ag43 are recognized by during the course of experimental murine infection with ETEC H10407. Shown are metal affinity chromatography-purified antigens (MAC) used in analysis, followed by corresponding immunoblots using pooled sera obtained from mice before and after intestinal challenge with ETEC H10407. b. human ETEC convalescent sera (from patients infected with ETEC obtained at ICDDR,B), but not age-matched sera from uninfected controls (from LeBonheur Children's Hospital, Memphis) recognize the ETEC H10407 autotransporter passenger domain of pAT (RAST designation 459). c. human ETEC convalescent sera, but not age-matched control sera recognize passenger domain of the ETEC H10407 autotransporter Ag43 (RAST designation 2318).
Immunization with passenger proteins protects against ETEC colonization
To explore the utility of ETEC autotransporter proteins as vaccine candidates, we examined both the immunogenicity and protective efficacy of individual passenger domains for Ag43 and pAT in a murine model of ETEC infection. For these studies, we used the mucosal adjuvant Protollin (ivx908), a mixture of Shigella flexneri 2a LPS, and meningococcal outer membrane proteins. When delivered intranasally, Protollin (IVX908) elicits high levels of S. flexneri LPS-specific fecal IgA , and when combined as an adjuvant with other proteins, it promotes similarly robust immune responses to target antigens , . Immunization with either of the passenger domains for antigen 43 or pAT resulted in significant increases in total serum and fecal antibody levels in immunized mice relative to adjuvant-only (ivx908) controls (figure 4a–c). Importantly, immunization with either rPATp or rAg43p resulted in significant increases in fecal IgA directed at the respective antigens (figure 4d).
a. kinetic ELISA data showing serologic responses of animals vaccinated with passenger domain of autotransporter designated pATp (closed circles) relative to adjuvant-only controls (ivx908, open circles). b. kinetic ELISA data for serologic responses of animals immunized with Ag43 passenger (Ag43p, closed triangles) relative to ivx908-only controls. c. kinetic ELISA of fecal antibodies (total IgG, IgM, IgA) obtained from pATp and Ag43p immunized mice (closed symbols) relative to adjuvant-only controls (open symbols); antigen (in shaded region) on the x-axis refers to the antigen used to coat ELISA wells. d. kinetic ELISA of fecal IgA antibody following vaccination with either the pAT or Ag43 passenger domains (closed symbols) relative to adjuvant-only controls (ivx908, open symbols) e. KmR-bacteria recovered from intestinal lysates following challenge with (1.2×104 cfu/mouse) of jf876 (ΔlacZYA::KmR mutant of ETEC strain H10407). Dashed horizontal lines reflect geometric means. All statistical comparisons were performed using two-tailed Mann Whitney analysis.
Immunization with either the antigen43 or pAT recombinant passenger domains provided significant protection against subsequent colonization with ETEC relative to adjuvant-immunized controls (figure 4e).
Despite the global importance of enterotoxigenic E. coli infections, there is presently no vaccine against these pathogens that would offer sustained, broad-based protection . Vaccine development for ETEC is a challenge for a number of reasons. First, the inherent plasticity of E. coli genomes makes discovery of conserved, pathotype-specific antigens difficult , . In addition, much of the ETEC vaccine development effort to date has focused on the plasmid-encoded colonization factors (CFs). Unfortunately, antigenic heterogeneity and lack of appreciable cross protection between CFs have been impediments to this approach. To date, over twenty-five different CFs have been identified in ETEC , and many strains do not appear to make any of the known antigens , . Finally, carefully conducted epidemiologic studies of natural ETEC infections have suggested that LT and perhaps other as yet unidentified chromosomally-encoded antigens , in addition to the plasmid-encoded CFs, could be involved in protective immune responses.
Because we have recently demonstrated that several autotransporter proteins are recognized following experimental and natural ETEC infections , we chose to investigate the possible contribution of conserved chromosomally-encoded autotransporter proteins. The studies here suggest that the passenger domains of these autotransporters are recognized during the course of both experimental infections in animals and naturally-occurring infection in humans, and they validate recent immunoproteomic data obtained with the prototype H10407 ETEC strain using sera from infected mice or human convalescent sera .
Two additional autotransporters have previously been examined in ETEC pathogenesis. These include the chromosomally-encoded TibA adhesin ,  protein and, EatA , a plasmid-encoded member of the SPATE family (serine protease autotransporters of the Enterobacteriaceae). Both proteins are recognized during the course of experimental and naturally occurring infections . Interestingly, the EatA protein appears modulate adhesion and colonization by digesting another recently described virulence molecule, the EtpA two-partner secretion exoprotein, an adhesin , . In turn, this modulation of adherence appears to be required for optimal delivery of heat-labile toxin (LT), a critical ETEC virulence molecule . Although recent data suggest that both EtpA and EatA are reasonably conserved within the ETEC pathovar , , , the inherent plasticity of E. coli genomes, and the relative paucity of pathovar-specific virulence genes  identified to date suggests that additional effort is warranted to explore the potential utility of other highly conserved surface structures as vaccine candidates.
Although it is likely that autotransporters contribute the overall fitness of ETEC as a pathogen, neither of the proteins under study here has been shown to contribute to the pathogenesis of ETEC. Antigen 43 does however appear to be of importance in the pathogenesis of other E. coli pathovars, including uropathogenic E. coli (UPEC). In the urinary tract, Ag43 is expressed in intracellular biofilm-like pods , and particular variants appear to contribute to biofilm formation, and colonization of the urinary tract . Similarly, immunoproteomic studies demonstrate that this antigen is also expressed  and recognized  during the course of E coli urinary tract infections in humans.
Interestingly, in a study of an E. coli laboratory isolate, Ag43 contributed to biofilm formation in vitro, but did not appear to play a role in intestinal colonization in a murine model . Nevertheless, some studies have suggested that specific Ag43 alleles segregate with diarrheagenic E. coli pathogens compared to other isolates from other pathovars , and that in general Ag43 was more commonly found in pathogens than in commensal strains.
Assessing the precise contribution of given antigens to the protective immune responses that develop following infection, or even following vaccination can be challenging , . While serologic responses to some CFs such as CFA/I have previously been correlated with a protective immune response to ETEC , it is likely that protection seen following natural infections reflects a composite response to a number of antigens.
Additional studies will be needed to determine the utility of these antigens as well as other autotransporters in ETEC vaccines. The surface expression of the autotransporter passenger domains, their immunogenicity, and preliminary data presented here support the concept that this class of molecules could serve as protective antigens. Although the inherent plasticity of E. coli genomes  in general poses an impediment to vaccine development for ETEC, important data emerging from the DNA sequencing of multiple ETEC genomes does suggest that these pathogens maintain a core subset of relatively pathovar-specific genes, such as the eatA autotransporter gene, that might serve as suitable targets , . The suggestion that relatively few genes separate the ETEC pathovar from commensal E. coli  is an important consideration in moving forward with putative ETEC vaccines. The data presented here suggest that other autotransporters not unique to the ETEC pathovar contribute to intestinal colonization, a critical step in ETEC pathogenesis as well as the host immune response to these important pathogens. Whether the more widely distributed ATs such as Ag43 and pAT are truly dispensable for the commensal E. coli similar to the HS prototype strain will be an important consideration in designing both subunit and live-attenuated vaccine strategies.
Conservation of antigen 43 and pAT passenger domains. Shown are MUSCLE alignments for predicted AT passenger regions selected from three sequenced ETEC strains, (H10407, E24377A, and B7A): a. Ag43 autotransporters H10407_Ag43.1, H10407_Ag43.2, E24377A_Ag43.1, and E24377A_Ag43.2 and B7A_Ag43. (arrowhead shows predicted signal peptide cleavage site) b. pAT from H10407, E24377A, and B7A.
Distribution of potential pAT-like proteins in the Enterobacteriaceae. The distribution of potential pAT-like proteins was defined by BLASTP searches of Enterobacteriaceae with pAT from H10407 [http://www.uniprot.org/uniprot/E3PFJ1] using the UniProtKB database (threshold E value of 0.0001; filtered for low regions of complexity). The degree of identity varied from 89% (for enterohemorrhagic E. coli strain 12009) to less than 50% for the other Enterobacteriaceae. Only E. coli proteins were more than 80% identical to pAT. The closest homologues in E. coli are shown in table 4.
IVX908 was acquired by Dr. James Dale from ID Biomedical, and was kindly provided to our laboratory for use in these studies. We appreciate the assistance of Ramy Aziz of the National Microbial Pathogen Data Resource (NMPDR) in implementing RAST analysis of ETEC genomes.
Conceived and designed the experiments: JMF. Performed the experiments: JAH KR VW-R DJH RK. Analyzed the data: FQ JMF. Contributed reagents/materials/analysis tools: FQ. Wrote the paper: JAH JMF.
- 1. WHO (2006) Future directions for research on enterotoxigenic Escherichia coli vaccines for developing countries. Wkly Epidemiol Rec 81: 97–104.
- 2. Al-Abri SS, Beeching NJ, Nye FJ (2005) Traveller's diarrhoea. Lancet Infect Dis 5: 349–360.
- 3. (2008) Enterotoxigenic Escherichia coli: advances in technical and laboratory aspects of research and development of vaccines. Wkly Epidemiol Rec 83: 92–95.
- 4. Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, et al. (2010) Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect 12: 89–98.
- 5. Qadri F, Svennerholm AM, Faruque AS, Sack RB (2005) Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev 18: 465–483.
- 6. Sjoling A, Wiklund G, Savarino SJ, Cohen DI, Svennerholm AM (2007) Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. Journal of clinical microbiology 45: 3295–3301.
- 7. Walker RI, Steele D, Aguado T (2007) Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 25: 2545–2566.
- 8. Oyofo BA, Subekti DS, Svennerholm AM, Machpud NN, Tjaniadi P, et al. (2001) Toxins and colonization factor antigens of enterotoxigenic Escherichia coli among residents of Jakarta, Indonesia. Am J Trop Med Hyg 65: 120–124.
- 9. Peruski LF Jr, Kay BA, El-Yazeed RA, El-Etr SH, Cravioto A, et al. (1999) Phenotypic Diversity of Enterotoxigenic Escherichia coli Strains from a Community-Based Study of Pediatric Diarrhea in Periurban Egypt. J Clin Microbiol 37: 2974–2978.
- 10. Clemens JD, Sack DA, Harris JR, Chakraborty J, Neogy PK, et al. (1988) Cross-protection by B subunit-whole cell cholera vaccine against diarrhea associated with heat-labile toxin-producing enterotoxigenic Escherichia coli: results of a large-scale field trial. J Infect Dis 158: 372–377.
- 11. Croxen MA, Finlay BBMolecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol 8: 26–38.
- 12. Fleckenstein JM, Roy K, Fischer JF, Burkitt M (2006) Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli. Infect Immun 74: 2245–2258.
- 13. Roy K, Hilliard GM, Hamilton DJ, Luo J, Ostmann MM, et al. (2009) Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells. Nature 457: 594–598.
- 14. Patel SK, Dotson J, Allen KP, Fleckenstein JM (2004) Identification and molecular characterization of EatA, an autotransporter protein of enterotoxigenic Escherichia coli. Infect Immun 72: 1786–1794.
- 15. Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala'Aldeen D (2004) Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68: 692–744.
- 16. Wells TJ, Tree JJ, Ulett GC, Schembri MA (2007) Autotransporter proteins: novel targets at the bacterial cell surface. FEMS Microbiol Lett 274: 163–172.
- 17. Gustafsson L, Hallander HO, Olin P, Reizenstein E, Storsaeter J (1996) A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N Engl J Med 334: 349–355.
- 18. Greco D, Salmaso S, Mastrantonio P, Giuliano M, Tozzi AE, et al. (1996) A controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. Progetto Pertosse Working Group. N Engl J Med 334: 341–348.
- 19. Ward JI, Cherry JD, Chang SJ, Partridge S, Lee H, et al. (2005) Efficacy of an acellular pertussis vaccine among adolescents and adults. N Engl J Med 353: 1555–1563.
- 20. Relman DA, Domenighini M, Tuomanen E, Rappuoli R, Falkow S (1989) Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci U S A 86: 2637–2641.
- 21. Alonso S, Reveneau N, Pethe K, Locht C (2002) Eighty-kilodalton N-terminal moiety of Bordetella pertussis filamentous hemagglutinin: adherence, immunogenicity, and protective role. Infect Immun 70: 4142–4147.
- 22. Charles IG, Dougan G, Pickard D, Chatfield S, Smith M, et al. (1989) Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis. Proc Natl Acad Sci U S A 86: 3554–3558.
- 23. Novotny P, Chubb AP, Cownley K, Charles IG (1991) Biologic and protective properties of the 69-kDa outer membrane protein of Bordetella pertussis: a novel formulation for an acellular pertussis vaccine. J Infect Dis 164: 114–122.
- 24. Roy K, Hamilton D, Ostmann MM, Fleckenstein JM (2009) Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model. Vaccine 27: 4601–4608.
- 25. Roy K, Hamilton D, Allen KP, Randolph MP, Fleckenstein JM (2008) The EtpA exoprotein of enterotoxigenic Escherichia coli promotes intestinal colonization and is a protective antigen in an experimental model of murine infection. Infect Immun 76: 2106–2112.
- 26. Roy K, Bartels S, Qadri F, Fleckenstein JM (2010) Enterotoxigenic Escherichia coli elicits immune responses to multiple surface proteins. Infect Immun 78: 3027–3035.
- 27. Cravioto A, Reyes RE, Trujillo F, Uribe F, Navarro A, et al. (1990) Risk of diarrhea during the first year of life associated with initial and subsequent colonization by specific enteropathogens. Am J Epidemiol 131: 886–904.
- 28. Rao MR, Wierzba TF, Savarino SJ, Abu-Elyazeed R, El-Ghoreb N, et al. (2005) Serologic correlates of protection against enterotoxigenic Escherichia coli diarrhea. J Infect Dis 191: 562–570.
- 29. Steinsland H, Valentiner-Branth P, Gjessing HK, Aaby P, Molbak K, et al. (2003) Protection from natural infections with enterotoxigenic Escherichia coli: longitudinal study. Lancet 362: 286–291.
- 30. McNeil LK, Reich C, Aziz RK, Bartels D, Cohoon M, et al. (2007) The National Microbial Pathogen Database Resource (NMPDR): a genomics platform based on subsystem annotation. Nucleic Acids Res 35: D347–353.
- 31. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, et al. (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9: 75.
- 32. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
- 33. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.
- 34. Harlow E, Lane D, Harlow E (1999) Using antibodies : a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
- 35. Goodrich W (2006) The Kinetic ELISA Advantage in Quantitative Assays. Winooski, Vermont.
- 36. Tsang VC, Wilson BC, Maddison SE (1980) Kinetic studies of a quantitative single-tube enzyme-linked immunosorbent assay. Clin Chem 26: 1255–1260.
- 37. Roy K, Hamilton D, Ostmann MM, Fleckenstein JM (2009) Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model. Vaccine.
- 38. Allen KP, Randolph MM, Fleckenstein JM (2006) Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains. Infect Immun 74: 869–875.
- 39. Chabot S, Brewer A, Lowell G, Plante M, Cyr S, et al. (2005) A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice. Vaccine 23: 1374–1383.
- 40. Dorsey FC, Fischer JF, Fleckenstein JM (2006) Directed delivery of heat-labile enterotoxin by enterotoxigenic Escherichia coli. Cellular Microbiology 8: 1516–1527.
- 41. Rasko DA, Rosovitz MJ, Myers GS, Mongodin EF, Fricke WF, et al. (2008) The pan-genome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates. J Bacteriol.
- 42. van der Woude MW, Henderson IR (2008) Regulation and function of Ag43 (flu). Annu Rev Microbiol 62: 153–169.
- 43. Toh H, Oshima K, Toyoda A, Ogura Y, Ooka T, et al. (2010) Complete genome sequence of the wild-type commensal Escherichia coli strain SE15, belonging to phylogenetic group B2. Journal of bacteriology 192: 1165–1166.
- 44. Yi H, Cho YJ, Hur HG, Chun J (2011) Genome sequence of Escherichia coli AA86, isolated from cow feces. Journal of bacteriology 193: 3681.
- 45. Al-Hasani K, Navarro-Garcia F, Huerta J, Sakellaris H, Adler B (2009) The immunogenic SigA enterotoxin of Shigella flexneri 2a binds to HEp-2 cells and induces fodrin redistribution in intoxicated epithelial cells. PLoS One 4: e8223.
- 46. Turner DP, Marietou AG, Johnston L, Ho KK, Rogers AJ, et al. (2006) Characterization of MspA, an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in Neisseria meningitidis. Infect Immun 74: 2957–2964.
- 47. Litwin CM, Rawlins ML, Swenson EM (2007) Characterization of an immunogenic outer membrane autotransporter protein, Arp, of Bartonella henselae. Infect Immun 75: 5255–5263.
- 48. Fries LF, Montemarano AD, Mallett CP, Taylor DN, Hale TL, et al. (2001) Safety and immunogenicity of a proteosome-Shigella flexneri 2a lipopolysaccharide vaccine administered intranasally to healthy adults. Infect Immun 69: 4545–4553.
- 49. Jones T, Cyr S, Allard F, Bellerose N, Lowell GH, et al. (2004) Protollin: a novel adjuvant for intranasal vaccines. Vaccine 22: 3691–3697.
- 50. Boedeker EC (2005) Vaccines for enterotoxigenic Escherichia coli: current status. Curr Opin Gastroenterol 21: 15–19.
- 51. Rasko DA, Rosovitz MJ, Myers GS, Mongodin EF, Fricke WF, et al. (2008) The pangenome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates. Journal of bacteriology 190: 6881–6893.
- 52. Sahl JW, Steinsland H, Redman JC, Angiuoli SV, Nataro JP, et al. (2011) A comparative genomic analysis of diverse clonal types of enterotoxigenic Escherichia coli reveals pathovar-specific conservation. Infection and immunity 79: 950–960.
- 53. Qadri F, Das SK, Faruque ASG, Fuchs GJ, Albert MJ, et al. (2000) Prevalence of Toxin Types and Colonization Factors in Enterotoxigenic Escherichia coli Isolated during a 2-Year Period from Diarrheal Patients in Bangladesh. J Clin Microbiol 38: 27–31.
- 54. Chowdhury F, Rahman MA, Begum YA, Khan AI, Faruque AS, et al. (2011) Impact of Rapid Urbanization on the Rates of Infection by Vibrio cholerae O1 and Enterotoxigenic Escherichia coli in Dhaka, Bangladesh. PLoS neglected tropical diseases 5: e999.
- 55. Elsinghorst EA, Weitz JA (1994) Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein. Infect Immun 62: 3463–3471.
- 56. Lindenthal C, Elsinghorst EA (2001) Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells. Infect Immun 69: 52–57.
- 57. Roy K, Kansal R, Bartels SR, Hamilton DJ, Shaaban S, et al. (2011) Adhesin Degradation Accelerates Delivery of Heat-labile Toxin by Enterotoxigenic Escherichia coli. The Journal of biological chemistry 286: 29771–29779.
- 58. Crossman LC, Chaudhuri RR, Beatson SA, Wells TJ, Desvaux M, et al. (2010) A commensal gone bad: complete genome sequence of the prototypical enterotoxigenic Escherichia coli strain H10407. Journal of bacteriology 192: 5822–5831.
- 59. Del Canto F, Valenzuela P, Cantero L, Bronstein J, Blanco JE, et al. (2011) Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands. Journal of clinical microbiology 49: 3198–3203.
- 60. Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, et al. (2003) Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301: 105–107.
- 61. Ulett GC, Valle J, Beloin C, Sherlock O, Ghigo JM, et al. (2007) Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in long-term persistence in the urinary tract. Infect Immun 75: 3233–3244.
- 62. Alteri CJ, Mobley HL (2007) Quantitative profile of the uropathogenic Escherichia coli outer membrane proteome during growth in human urine. Infect Immun 75: 2679–2688.
- 63. Hagan EC, Mobley HL (2007) Uropathogenic Escherichia coli outer membrane antigens expressed during urinary tract infection. Infect Immun 75: 3941–3949.
- 64. de Luna MG, Scott-Tucker A, Desvaux M, Ferguson P, Morin NP, et al. (2008) The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization. FEMS Microbiol Lett 284: 237–246.
- 65. Restieri C, Garriss G, Locas MC, Dozois CM (2007) Autotransporter-encoding sequences are phylogenetically distributed among Escherichia coli clinical isolates and reference strains. Appl Environ Microbiol 73: 1553–1562.
- 66. Plotkin SA (2008) Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis 47: 401–409.
- 67. Plotkin SA (2010) Correlates of protection induced by vaccination. Clin Vaccine Immunol 17: 1055–1065.
- 68. Ouyang-Latimer J, Ajami NJ, Jiang ZD, Okhuysen PC, Paredes M, et al. (2010) Biochemical and genetic diversity of enterotoxigenic Escherichia coli associated with diarrhea in United States students in Cuernavaca and Guadalajara, Mexico, 2004–2007. The Journal of infectious diseases 201: 1831–1838.
- 69. Levine MM, Nalin DR, Hoover DL, Bergquist EJ, Hornick RB, et al. (1979) Immunity to enterotoxigenic Escherichia coli. Infect Immun 23: 729–736.
- 70. Dupont H, Formal S, Hornick R, Snyder M, Libonati J, et al. (1971) Pathogenesis of Escherichia coli diarrhea. N Engl J Med 285: 1–9.
- 71. Evans DG, Silver RP, Evans DJ Jr, Chase DG, Gorbach SL (1975) Plasmid-controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect Immun 12: 656–667.
- 72. Evans DJ Jr, Evans DG (1973) Three characteristics associated with enterotoxigenic Escherichia coli isolated from man. Infect Immun 8: 322–328.
- 73. Levine MM, Ristaino P, Marley G, Smyth C, Knutton S, et al. (1984) Coli surface antigens 1 and 3 of colonization factor antigen II-positive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans. Infect Immun 44: 409–420.
- 74. Ogura Y, Ooka T, Iguchi A, Toh H, Asadulghani M, et al. (2009) Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 106: 17939–17944.
- 75. Levine MM, Nataro JP, Karch H, Baldini MM, Kaper JB, et al. (1985) The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. The Journal of infectious diseases 152: 550–559.
- 76. Paulozzi LJ, Johnson KE, Kamahele LM, Clausen CR, Riley LW, et al. (1986) Diarrhea associated with adherent enteropathogenic Escherichia coli in an infant and toddler center, Seattle, Washington. Pediatrics 77: 296–300.
- 77. De Rycke J, Comtet E, Chalareng C, Boury M, Tasca C, et al. (1997) Enteropathogenic Escherichia coli O103 from rabbit elicits actin stress fibers and focal adhesions in HeLa epithelial cells, cytopathic effects that are linked to an analog of the locus of enterocyte effacement. Infection and immunity 65: 2555–2563.
- 78. Roos V, Ulett GC, Schembri MA, Klemm P (2006) The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine. Infection and immunity 74: 615–624.
- 79. Stapleton A, Moseley S, Stamm WE (1991) Urovirulence determinants in Escherichia coli isolates causing first-episode and recurrent cystitis in women. The Journal of infectious diseases 163: 773–779.
- 80. Chen SL, Hung CS, Xu J, Reigstad CS, Magrini V, et al. (2006) Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach. Proceedings of the National Academy of Sciences of the United States of America 103: 5977–5982.
- 81. Korhonen TK, Valtonen MV, Parkkinen J, Vaisanen-Rhen V, Finne J, et al. (1985) Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis. Infection and immunity 48: 486–491.