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Infection and Immunity, April 2007, p. 1916-1925, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01269-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli{triangledown} ,{dagger}

Lionel Durant,1 Arnaud Metais,1 Coralie Soulama-Mouze,1 Jean-Marie Genevard,1 Xavier Nassif,2 and Sonia Escaich1*

Mutabilis SA, 93230 Romainville,1 Unité INSERM 570, Faculté de médecine Necker-Enfants Malades, 75015 Paris, France2

Received 8 August 2006/ Returned for modification 22 September 2006/ Accepted 20 November 2006


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ABSTRACT
 
Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause a large spectrum of infections. The majority of ExPEC strains are closely related to the B2 or the D phylogenetic group. The aim of our study was to develop a protein-based vaccine against these ExPEC strains. To this end, we identified ExPEC-specific genomic regions, using a comparative genome analysis, between the nonpathogenic E. coli strain K-12 MG1655 and ExPEC strains C5 (meningitis isolate) and CFT073 (urinary tract infection isolate). The analysis of these genomic regions allowed the selection of 40 open reading frames, which are conserved among B2/D clinical isolates and encode proteins with putative outer membrane localization. These genes were cloned, and recombinant proteins were purified and assessed as vaccine candidates. After immunization of BALB/c mice, five proteins induced a significant protective immunity against a lethal challenge with a clinical E. coli strain of the B2 group. In passive immunization assays, antigen-specific antibodies afforded protection to naive mice against a lethal challenge. Three of these antigens were related to iron acquisition metabolism, an important virulence factor of the ExPEC, and two corresponded to new, uncharacterized proteins. Due to the large number of genetic differences that exists between commensal and pathogenic strains of E. coli, our results demonstrate that it is possible to identify targets that elicit protective immune responses specific to those strains. The five protective antigens could constitute the basis for a preventive subunit vaccine against diseases caused by ExPEC strains.


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INTRODUCTION
 
Escherichia coli is a well-known bacterial species and a common isolate in clinical microbiology laboratories. In terms of biological significance to humans, E. coli strains are grouped into three categories: (i) commensal strains that represent a large part of the normal flora, (ii) intestinal pathogenic strains that cause diseases when ingested in sufficient quantities, and (iii) pathogenic strains causing extraintestinal infections (extraintestinal pathogenic E. coli [ExPEC]) (60). ExPEC strains can be part of the normal intestinal flora and are isolated in 11% of healthy individuals (22). ExPEC strains do not cause gastroenteritis in humans, but their main feature is their capacity to colonize extraintestinal sites and to induce infections in diverse organs or anatomical sites. They are involved in urinary tract infections (UTI), septicemia, diverse abdominal infections, and meningitis (33, 34, 82). Bacteremia can arise (26) with a risk of severe sepsis (11, 25), which ranks as the 10th overall cause of death in the United States (40). ExPEC strains are also the most frequently isolated bacteria in nosocomial septicemic infections (16% of cases) (11). Phylogenetic relationship analysis has led to the classification of E. coli strains into four phylogenetic groups designated A, B1, B2, and D (30). Experimental (15, 54) and epidemiological (12) data have shown that extraintestinal infections due to E. coli strains were mostly due to the B2 and D groups. Since ExPEC strains are the major cause of most extraintestinal infections due to gram-negative bacteria, prevention is desirable from both a medical and an economical point of view (63).

In recent years, the resistance of the ExPEC strains to various classes of antibiotics has become a major concern both in hospitals and in the community. For example, the resistance of E. coli to trimethoprim-sulfamethoxazole, the drug of choice for the treatment of uncomplicated cystitis, has been reported worldwide with some variations but is still increasing yearly (28, 29, 65). A high degree of resistance to common antibiotics, such as ampicillin and gentamicin, has also been described among E. coli strains causing sepsis in neonates (7, 17). The antimicrobial resistance will make the future management of extraintestinal E. coli infections more difficult, and an alternative strategy would be very useful to counteract these infections.

Vaccines represent a rational alternative approach for the prevention of these infections. In this case, the challenge is to selectively prevent a subtype of E. coli strains that is not normally part of the commensal flora. Therefore, it is of great importance to find some specific genetic traits of these ExPEC strains. In the past, some studies have attempted to elaborate subunit vaccines from known virulence factors. Hemolysin, capsule, or fimbrial proteins were shown to protect against ExPEC strains causing UTI (35, 37, 46) or systemic infections (66) in experimental models. The fimbrial adhesin known as FimH, a critical determinant for cystitis, was the most promising against UTI and has begun clinical trials. However, by using DNA array to detect E. coli pathotypes, FimH was found not to be unique to pathogenic strains but was also detected in nonpathogenic strains (10).

In recent studies, the genetic comparison of E. coli strains causing extraintestinal infections indicated a high degree of variability in virulence gene contents (21, 42). Since an ideal vaccine antigen needs to be highly specific for pathogenic strains and, in the meantime, needs to be conserved among the extraintestinal E. coli strains, the analysis of additional specific genetic traits of ExPEC strains is necessary to find suitable conserved protective antigens specific to E. coli strains responsible for extraintestinal infections.

In this study, we report the identification of proteins as putative antigens from ExPEC-specific genomic sequences. In an animal model of lethal sepsis, the protective effect of immunization with these antigens was demonstrated, allowing the identification of five antigens as vaccine candidates against an extraintestinal E. coli infection. Since these antigens were shown, by using comparative genomic hybridization techniques, to be associated with a majority of B2 and/or D clinical isolates of E. coli, we suggest that they could be used to prevent most of the severe infections due to extraintestinal E. coli.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions. All E. coli clinical isolates used in this study were generously provided by S. Bonacorsi (Hôpital Robert Debre, France). These E. coli strains were recovered from the cerebrospinal fluid (CSF) of neonates with meningitis (Table 1) and belong to various phylogenetic groups. The O18:K1:H7 E. coli neonatal meningitis (ECNM) strain S26 was used in the animal model. Strains RS218 (78) and C5 (1) are of O18:K1:H7 serotype and were also isolated from the CSF of neonates with meningitis. E. coli strain CFT073 was originally isolated from the blood and urine of a woman with acute pyelonephritis and was classified as an uropathogenic E. coli strain (41). These four ExPEC strains belong to the B2 phylogenetic group. All strains were grown under aerobic conditions on Luria agar or in Luria-Bertani broth (LB; Difco Laboratories) supplemented with or without ampicillin (100 µg/ml). Iron chelator, 2-2' dipyridyl (Sigma, St. Louis, MO), was used to induce the expression of siderophore receptor and was added to bacterial culture at a final concentration of 0.2 mM.


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  		TABLE 1. E. coli strains used in this study

Isolation of ExPEC-specific sequences. The nucleotide sequence of 259 C5-specific DNA fragments isolated from a subtractive hybridization experiment between E. coli strain C5 and nonpathogenic E. coli strains was obtained from a previous work of Bonacorsi and colleagues (16). To define the open reading frames (ORFs) including these DNA fragments in E. coli CFT073, the nucleotide sequences were compared to the complete chromosomal sequence of E. coli strain CFT073 (80) using the BlastN algorithm (4). The genomic sequences located upstream and downstream from the CFT073 BLAST hit sequences were then isolated and submitted for direct comparison with the complete genome sequence of E. coli strain K-12 (13). The genomic comparison ended when the alignment of the two genomic sequences produced an exact match over a sequence of at least 150 bp in size. This strategy led to the isolation of sequences specifically present on the chromosome of E. coli strain CFT073 and partially common to ECMN strain C5 and probably to other ExPEC strains.

Bioinformatics sequence analysis. To identify potential vaccine candidates, CodonUse 3.5.5 software (an unpublished program by Conrad Halling) was used to identify all the putative ORFs. The corresponding amino acid sequences were analyzed for the presence of a leader peptide at the N terminus of the protein using the SignalP program (44). Additional amino acid motifs and characteristics of proteins localized in the outer membrane of the bacteria or of secreted proteins were sought on protein sequences, using public data processing programs such as ProDom (http://protein.toulouse.inra.fr/prodom/current/html/form.php), Pfam (http://www.sanger.ac.uk/Software/Pfam/), TMpred (http://www.ch.embnet.org/software/TMPRED_form.html/), and Blocks (http://blocks.fhcrc.org). In parallel, ORFs were analyzed for homology to known surface or secreted proteins previously described in the genomic databases (blastN and TblastN algorithms).

The hydrophilic regions of the protein sequences were predicted using the VectorNTI software (version 7.0; Informax) based on the hydrophobicity plot (Kyle and Doolittle algorithm) and the polarity profile (Zimmerman algorithm).

Production and use of DNA arrays. The primers for PCR experiments were designed, according to the CFT073-specific sequences, to selectively amplify each predicted ORF. The maximum size of amplicon was 800 bp. If predicted genes were longer than 800 bp, they were amplified in sections of about 800 bp until the entire gene was covered, which allows homogeneity at the level of the hybridization signal. In addition, primers corresponding to genes which have been implicated in the virulence of pathogenic E. coli were generated on the basis of publicly available sequences. These genes included cnf1 (NCBI accession no. X70670), ompR (NCBI accession no. X12374), hra (NCBI accession no. AAC13754), iha (NCBI accession no. AF126104), fyuA (NCBI accession no. CAA21395), and sfaS (NCBI accession no. AAB25046). Two insertion sequences, IS100 (NCBI accession no. X78302) and IS630 (NCBI accession no. X05955), were added. The PCR products obtained from the CFT073 chromosomal DNA were spotted robotically (MicroGrid; Biorobotics Ltd., Cambridge, United Kingdom), in duplicate, onto nylon membranes and fixed by treatment with alkali.

The chromosomal DNA of 29 E. coli strains belonging to various phylogenetic groups was prepared using a standard molecular biology protocol (Promega). The membranes were hybridized with [{alpha}-33P]dCTP-radiolabeled DNA (Amersham Pharmacia Biotech, United Kingdom) overnight under stringent conditions. After hybridization, the membranes were washed as previously described (18) before being exposed in a hybridization cassette (Molecular Dynamics). Images were revealed with a STORM PhosphorImager (Molecular Dynamics) and analyzed using the XDotsReader software (COSE, Dugny, France) to quantify the intensity of the signal associated with each spot. A positive result is given when the intensity is above the threshold value defined by the intensity obtained with the DNA from control strains, as previously described (52) (see Table S1 in the supplemental material for the complete list of primers and PCR and hybridization conditions).

Cloning, expression, and purification. The chromosomal DNA of strain S26 was used as the source of DNA for expression of predicted surface antigens. PCR was performed using the proofreading Pfu DNA polymerase (Promega, Madison, WI) and primers designed to specifically amplify DNA fragments in the genes. After purification (PCR purification kit; QIAGEN), the PCR products were introduced into plasmid expression vectors to generate proteins fused with His6 either at the N terminus or at the C terminus of the protein sequence. The resulting plasmids were introduced into E. coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA) by following the manufacturer's instructions (see Table S2 in the supplemental material for the complete list of primers and plasmid expression vectors).

For protein expression, overnight cultures were used to inoculate a fresh LB medium supplemented with ampicillin (100 µg/ml). Bacteria were grown at 37°C under aerobic conditions, and isopropyl-ß-D-thiogalactopyranoside (IPTG; Qbiogene, Vista, CA) was added (1 mM final) to the bacterial culture in the exponential phase (optical density at 600 nm = 0.5). After a further incubation for 3 h, cells were harvested by centrifugation.

Purification of recombinant proteins was performed by affinity chromatography on nickel-nitrilotriacetic acid (Ni-NTA) agarose by following the manufacturer's instructions (QIAGEN). Briefly, when the proteins were expressed as insoluble inclusion bodies, the cell pellet was solubilized for 2 h in buffer A (100 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0) containing 6 M guanidium hydrochloride. The cell suspension was centrifuged at 10,000 x g for 30 min at 6°C, and the supernatant was added to Ni-NTA agarose. After extensive washing, His-tagged proteins were eluted under acid conditions using buffer A containing 8 M of urea. Elution fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining. Fractions containing the recombinant protein were pooled, and the urea was removed by dialysis as previously described (55).

When proteins were soluble, pellets were resuspended in buffer L (50 mM NaH2PO4, 300 mM NaCl) containing lysozyme (1 mg/ml, final concentration) and incubated for 60 min in ice. The bacterial suspension was sonicated (6 cycles of 30 s separated by 1 min of cooling on ice) (Branson Sonifier). The supernatant was collected by centrifugation and added to Ni-NTA agarose. The washing and elution steps were performed using buffer L containing increasing concentrations of imidazole. Finally, imidazole was removed from the eluted material by dialysis against phosphate-buffered saline (PBS).

Finally, recombinant proteins were concentrated using a Centriprep filter 10 kDa (Millipore, Billerica, MA), and protein concentrations were determined using the Bradford protein assay (Pierce, Rockford, IL).

Active immunization and challenge. Purified recombinant proteins were used to immunize groups of 6-week-old BALB/c@Rj mice (Janvier Laboratories, France). Each mouse was injected subcutaneously with 20 µg of recombinant protein emulsified in complete Freund's adjuvant (Sigma) on day 1. Three weeks later (day 21), the mice were given a boosting injection with 10 µg of recombinant protein emulsified in incomplete Freund's adjuvant. A control group was included in each experiment that consisted of mice injected on days 1 and 21 with PBS and adjuvant alone. Blood samples were drawn from control and immunized mice on day 41, and sera were examined for antigen-specific antibody response. Control and immunized groups of mice were challenged on day 42 by intraperitoneal injection of E. coli S26 at a dose that caused death in 50% of the mouse population (LD50) (5 x 105 CFU/mouse). The survival of mice was monitored for 2 days after challenge. The survival rate in the vaccinated group was compared to the one obtained in the control group.

Immunoglobulin preparation and passive immunization. Preimmune and hyperimmune sera containing antibodies directed against protective antigens were obtained from Covalab after an immunization schedule of New Zealand rabbits injected with purified antigens in complete Freund's adjuvant (first injection) or incomplete Freund's adjuvant (boosting injection). The immunoglobulins were purified and concentrated by affinity chromatography using protein A-agarose (Pierce). Briefly, after loading the serum, the column was washed with 6 column volumes (CV) of 20 mM Tris, pH 8.0, followed by 8 CV of 20 mM glycine, pH 5.0. The antibodies were then eluted with 3 to 5 CV of 0.1 M glycine, pH 2.5, and collected in 1-ml fractions into microtubes containing a 1/10 volume of 1 M Tris, pH 8.0. The protein concentrations were determined using the Bradford protein assay (Pierce).

For passive immunization assays, 3-week-old BALB/c@Rj mice (Janvier Laboratories, France) were immunized by intravenous administration with 20 to 25 µg of protein A-purified rabbit immunoglobulin, preimmune purified rabbit serum, or PBS. Twenty-four hours later, immunized mice were challenged by intraperitoneal injection with 3 x 104 CFU of E. coli S26 (LD50). The survival of mice was monitored for 2 days after challenge. The survival rate of the mice treated with the antibodies was compared to the one obtained in the control group injected with PBS only.

Isolation of LPS from E. coli strains. Lipopolysaccharide (LPS) was prepared from S26 or BL21 E. coli strains as previously described (31). Briefly, overnight colonies were washed in PBS and resuspended in a protein lysis buffer. The suspension was boiled for 5 min at 95°C, and proteinase K (Sigma) was added to 0.3 mg/ml. Following 1 h of incubation at 55°C, treated lysates were centrifuged (30 min, 10,000 x g) and the supernatants were stored at –20°C until use.

Human sera. Human serum samples were obtained from the Hôpital Necker-Enfants Malades (France). Blood samples were drawn from patients 3 to 4 days after diagnosis of an E. coli bacteremia and during treatment with an antibiotic adapted to the susceptibility of the isolated E. coli strain. The serum preparation was achieved by blood agglutination and centrifugation. Sera from various patients were pooled and then diluted to 1:100 to perform Western blot experiments.

Measurement of antibody titers in mouse antisera. Specific antibody titers were determined by the end point dilution method using an enzyme-linked immunosorbent assay (ELISA). Microtiter plates (MaxiSorp; Nunc AS, Roskilde, Denmark) were coated with recombinant proteins (2 µg/ml in 0.1 M carbonate buffer, pH 9.6) for 60 min at 37°C followed by an overnight incubation at 4°C. After saturation of coated plates for 60 min with PBS containing 3% bovine serum albumin (BSA; Sigma), immune sera were serially diluted in PBS containing 1% BSA and incubated for 2 h with recombinant protein-coated plates. After washing with PBS containing 0.1% Tween 20 (PBST), horseradish peroxidase-coupled goat anti-mouse antibody (Sigma) (1:6,000 in PBS-0.1% Tween 20-1% BSA) was added to the microplates and incubated for 60 min at room temperature. After extensive washing with PBST, orthophenylenediamine (Sigma) was used to develop the reaction by following the manufacturer's instructions. The reaction was stopped by the addition of 2 M HCl. The absorbance was measured at 490 nm in an ELISA spectrophotometer (Sanofi Pasteur diagnostic PR2100).

The specific antibody titer of immune serum was expressed as the reciprocal of the serum dilution that gave an A490 value above the cutoff defined as twice the absorbance value of the control wells.

SDS-PAGE and Western blot. Protein samples were denatured using the Laemmli protocol (48). Samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Schleicher and Schuell) using a semiliquid transfer protocol (Blotter Biometra). Membranes were saturated with 5% nonfat dry milk in PBST for 60 min and then incubated with the desired immune serum for 60 min at room temperature. After washing with PBST, the membranes were incubated with the conjugate solution (dilution, 1:4,000) (Sigma) for 60 min at room temperature, and after extensive washing with PBST, bound conjugate antibodies were detected by the addition of diaminobenzidine (Sigma) and H2O2 as substrate. The reaction was stopped with distilled water.

Whole-cell membrane preparation. Bacteria grown to exponential phase were harvested by centrifugation and resuspended in 50 mM Tris, pH 7.0, containing 2% Triton X-100, 1 mM MgCl2. After incubation for 30 min at 4°C, the cells were sonicated (three cycles of 30 s each separated by 1 min on ice) (Branson Sonifier). The cell suspension was centrifuged (4°C, 6,000 x g, 15 min) to eliminate cellular debris or unbroken cells. The supernatant was then centrifuged for 3 h at 20,000 x g, 4°C, and the pellet containing whole-cell membranes was resuspended in a Tris buffer (50 mM, pH 7.0) and stored at –20°C until use.

Purification of membrane fractions from E. coli S26. Inner and outer membranes were separated by sucrose density gradient centrifugation as previously described (74). Briefly, 1 liter of E. coli culture, grown with or without the iron chelator 2-2' dipyridyl (Sigma) was washed three times in PBS and resuspended in 5 ml of 50 mM Tris, pH 8.0. The bacteria were then passed twice through a French pressure cell at 15,000 lb/in2. After the elimination of unbroken cells by centrifugation, the supernatant was loaded on a discontinuous sucrose gradient and submitted to centrifugation for 2 h at 180,000 x g at 15°C. The crude extract membrane fraction was collected and adjusted to 30% sucrose with 3 mM EDTA, pH 8.0, followed by a separation on a second discontinuous sucrose gradient. After centrifugation for 18 h at 150,000 x g at 15°C, fractions of 1 ml were collected by piercing the bottom of the tube. To detect the inner membrane fractions, lactate dehydrogenase activity was measured as previously described (23, 47). LPS was used as marker of the outer membrane fractions and was detected by silver staining on Tris-Tricine SDS-PAGE.

Statistical analysis. Fisher's exact test was used to analyze the statistical significance of the lethal challenge data.


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RESULTS
 
Isolation of genomic sequences specific to CFT073. The 259 DNA fragments previously obtained from a subtractive hybridization experiment between the pathogenic E. coli strain C5 and two nonpathogenic E. coli strains from the ECOR collection (16) were mapped on the genome of the uropathogenic E. coli strain CFT073 (80). The genomic sequences of strain CFT073 located upstream and downstream of each target sequence were analyzed for the presence of any homologous sequences on the E. coli K-12 MG1655 chromosome (13). We found that 154 sequences among the 259 sequences initially identified as specific to E. coli strain C5 were also present on the E. coli CFT073 genome. Starting from these sequences, 716 kb of genomic sequences specific for E. coli CFT073 were isolated. These sequences are scattered throughout 83 discrete regions of DNA all over the genome of CFT073. The sizes of those regions are very heterogeneous and range from 0.6 to 48 kb, with a majority of genomic regions having a size between 1 kb and 10 kb (59 regions). In addition, 50 regions were greater than 4 kb in size (see Table S3 in the supplemental material).

Identification and selection of putative antigens. It is commonly admitted that valuable antigens aimed at preventing infections due to extracellular pathogens should be accessible to functional antibodies. Therefore, it is important for an antigen to be localized at the cell surface or secreted. The isolated specific genomic regions were analyzed to predict cell surface or secreted proteins. First, the nucleotide sequences were submitted to homology searching using the BLASTN algorithm on public databases. BLAST results revealed that 12 of 83 regions contain virulence-associated genes corresponding to known pathogenicity islands or sequences homologous to some prophage sequences. The remaining 71 regions did not possess any known pathogenic loci (e.g., pathogenicity island or phage sequences) and were called the "B2 core genome." The DNA sequences of these regions were scanned to find all putative ORFs encoding proteins of more than 100 amino acids in size (see Materials and Methods), leading to the identification of 650 ORFs. Next, by using computer-assisted programs, ORFs were selected on the basis of specific motives present on protein sequences allowing export or secretion through the general secretory pathway (i.e., signal sequence, TAT motif) (73). Additional features typical to outer membrane proteins were also sought (e.g., transmembrane domains, outer membrane-anchoring motives, and host cell binding domains). In this manner, 54 ORFs were selected from the sequences of the "B2 core genome." In addition, two genes from the pool of pathogenic related genomic regions were retained and corresponded to iroE and iroN, which are part of the enterobactin-like operon. This group of ORFs was completed with three virulence determinants associated with extraintestinal E. coli strains of the B2 group: the S fimbrial adhesin, sfaS (68), the heat-resistant agglutinin, hra (72), and the yersiniabactin receptor, fyuA (69).

We then analyzed the prevalence of each selected gene among a set of E. coli clinical isolates. To this end, DNA arrays spotted with the PCR products corresponding to ORFs identified in specific CFT073-derived genomic regions were produced (detailed in Materials and Methods). The chromosomal DNA of 29 E. coli strains belonging to most of the phylogenetic groups (with the exception of the B1 group) was extracted and used to hybridize the DNA arrays. In detail, six strains were from group A, five were from group D, and the majority (18 strains) belonged to the B2 phylogenetic group. Among these strains, 21 represent clinical isolates recovered from neonates with meningitis (Table 1).

As expected, all the DNA spots corresponding to the selected genes hybridized with chromosomal DNA fragments from CFT073. In the meantime, none of them produced a positive result when DNA from E. coli K-12 was used as the probe. The results of the hybridization experiments showed that two-thirds (40 ORFs) of the ORFs encoding putative antigens were more frequently associated with E. coli strains belonging to the B2 and/or D groups than to the A group (Table 2). Among them, 17 (42%) ORFs were present both in the strains of the B2 and D groups. Twenty-three ORFs (58%) were found to be strictly specific to the strains of the B2 group and were detected in 50 to 100% of the B2 population. None of the selected genes was found only in strains of the D group (Table 2). Altogether, 40 genes encoding putative antigens and present in several extraintestinal pathogenic strains of the B2 and D groups were selected and analyzed for their capacity to induce a protective immunity in mice.


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  		TABLE 2. Distribution of putative antigens in E. coli strains according to phylogenetic group

Expression and purification of selected antigens. Due to their peculiar physicochemical properties, the transmembrane proteins are difficult to obtain in soluble native forms. Each of the forty protein sequences to be expressed was scanned using computer algorithms to delineate regions of the protein that were more likely to be hydrophilic and, thus, soluble. With these data, primers were designed and selected DNA fragments were cloned in expression vectors and expressed in E. coli BL21. Thirty-three proteins (80%) were recovered from E. coli after IPTG induction, and His-tagged recombinant proteins were purified by affinity chromatography. Despite efforts made to predict ordered regions in the protein sequence, only three recombinant proteins were purified as cytoplasmic and soluble forms (IroN, C4214, and C4424). The 30 other proteins were obtained as inclusion bodies, purified in nonnative forms, and partially refolded as previously described (55).

Vaccine efficacy in a mouse model of lethal sepsis. To assess the protective effect of the purified proteins against lethal challenge, a murine model of lethal sepsis was set up. Twelve-week-old BALB/c mice were injected with various clinical strains isolated from meningitis, including the prototypical ECNM strains C5 and RS218. E. coli S26 was found to be the most virulent strain of all tested. After intraperitoneal injection, the dose that killed 50% of the mouse population corresponded to 5 x 105 CFU/mouse at 48 h postinfection. Bacteremia and dissemination in various organs were measured in infected mice. As shown in Fig. 1, E. coli S26 became rapidly bacteremic (4 h) in a dose-dependent manner, with a bacterial count that reached up to 107 CFU/ml in the blood at 24 h postinfection. At this time point, strain S26 was recovered from the spleen, liver, and brain, indicating that the bacteria induce a rapid systemic infection in the mouse (Fig. 1). Therefore, the S26 E. coli isolate was used in subsequent challenge experiments.


Figure 1
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  		FIG. 1. Time course of E. coli infection in BALB/c mouse. Mice were inoculated intraperitoneally with 3.5 x 104 CFU (dark square) or with 4.7 x 105 CFU (open circle) of the E. coli strain S26. (A) Animals were sacrificed at 0, 4, 10, or 24 h postinjection. The number of CFU in the blood was determined using 10-fold serial dilutions and the plating method. Each point represents the number of E. coli CFU in blood. (B) Mice were sacrificed at 24 h postinfection, and the liver, spleen, and brain were harvested and homogenized. E. coli CFU were enumerated in the various organs by serial dilution and plating of homogenates on LB agar. Limit of detection, 6 CFU/mg organ.

Each of the 33 recombinant polypeptides was used to immunize groups of 6-week-old BALB/c mice. Blood samples were drawn after immunization, and sera were examined for protein-specific antibody response by ELISA and Western blotting. As shown in Fig. 2, recombinant proteins, when injected into mice, induced a massive humoral response characterized by high antibody titers and a good specificity for the corresponding recombinant protein.


Figure 2
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  		FIG. 2. Analysis of the antibody response to recombinant proteins after immunization of BALB/c mice. Blood samples were drawn from mice after immunization, and sera were examined for the total antibody response to recombinant proteins by ELISA using serial dilution starting from 1:4,000. In the meantime, the reactivity of protein-specific antibodies was evaluated in Western blot experiments. FyuA (A), IroN (B), C0393 (C), C4424 (D), and C3389 (E) were detected using serum samples tested at dilutions of 1:2,000. Values of the titration curves are the means (± standard deviations) of the four serum samples of immunized mice. PM, protein marker (kDa); OD, optical density.

Since antibodies directed against LPS could elicit an effective protection against E. coli challenge (56) and since contamination by LPS in the protein preparation could have been undetected, the mouse antisera raised against the protective antigens were tested for the presence of antibodies to LPS. To this end, LPS was prepared from strains S26 and BL21 and was used in immunoblot experiments. The results indicate that sera from immunized mice were devoid of any anti-LPS antibodies (data not shown). We could, therefore, rule out the idea that the protection observed in the mice could be due to anti-LPS antibodies.

At day 42, the immunized and control groups of mice were challenged by intraperitoneal injection with 5 x 105 CFU of E. coli S26 (LD50). Using this systematic screening, five antigens were shown to significantly increase the survival rate among the immunized mice compared to the control group (P < 0.05). The number of mice surviving the lethal challenge was increased by 32% in the case of C0393 to 82% in the case of IroN (Table 3). Some virulence factors, such as SfaS or Hra, were inefficient in protecting mice from this lethal challenge (data not shown). Surprisingly, among the proteins having a homology to iron-regulated receptors, FyuA and IroN but not ChuA, a hemin receptor associated with ExPEC strains (43), were able to confer a protective immunity to mice (Table 3).


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  		TABLE 3. Active immunization of BALB/c mice with recombinant E. coli proteins in Freund's adjuvant protects mice from lethal challenge with ExPEC strain S26

Passive immunization induced by antigen-specific antibodies. To further confirm that protection was mediated by specific antibodies, 3-week-old BALB/c mice were passively immunized with purified rabbit antibodies raised against each one of the five protective antigens. Groups of mice injected with purified preimmune serum or PBS were also included in the experiments. Challenges of 3 x 104 CFU of E. coli S26 were administrated intraperitoneally, and the survival rate of the mice was assessed 48 h later. The results indicate that injection of antigen-specific antibodies afforded protection to mice against a lethal challenge with E. coli S26 compared to PBS-injected mice. A significant high level of protection was obtained in all cases, with protection values ranging from 66% in the case of C4424 to 100% complete protection in the case of C3389 (Table 4) (P < 0.05). By contrast, the rabbit preimmune serum did not protect from the infection with strain S26, indicating that the protection of naive mice against a lethal challenge with E. coli S26 is strictly dependent on the injection of antigen-specific immunoglobulins.


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  		TABLE 4. Protection of mice from lethal challenge with ExPEC strain following passive immunization with purified rabbit polyclonal sera raised against recombinant E. coli proteins

Analysis of vaccine candidates. The sequence analysis of protective antigens revealed that they could be grouped into two functional classes. The first group comprised the FyuA, IroN, and C0393 proteins, which are part of the iron-regulated outer membrane protein family. Iron-restricted mediums result in up-regulation of fyuA expression in ExPEC (71). The fyuA gene is part of the high-pathogenicity island initially described for Yersinia (24). Mutation in the fyuA gene has been shown to impair virulence of ExPEC strains in mice (70).

The expression of iroN is also dependent on the free iron available in the medium (62). During the infection of the urinary tract, iroN is up-regulated (71), and some studies have shown that this gene contributed to the urovirulence of E. coli CP9 (62) and to the virulence of E. coli C5 in an infant rat model of meningitis (43).

C0393 showed 78% sequence identity to the hemoglobin protease (Hbp) of E. coli (accession no. O88093). The Hbp protein contributed to the degradation of hemoglobin and to the binding of heme (49). In vivo, hbp is highly expressed during peritonitis induced by the E. coli strain EB1 (O8:K43 serotype) and plays a key role in the intra-abdominal infection of bacteria in synergy with Bacteroides fragilis (50).

The second class is composed of antigens with unknown functions. The sequence analysis of the c3389 gene product indicates that it contains an ompA-like domain located at the C terminus of the protein that could be responsible for the binding to peptidoglycan in the periplasm of the gram-negative bacteria (27), but in actual fact, no function could be assigned to the c3389 gene product.

The putative function of the c4424 gene product was only inferred by homology. BLAST analysis revealed that this antigen was similar to SapB (60% sequence identity, accession no. CAC14217), a putative autotransporter of Salmonella enterica (51). A YadA-like motif is present at the C terminus of the protein also found in YadA of Yersinia spp. (32) and now characterizing a family of surface proteins. The presence of additional motives related to the adhesins suggested that the c4424 gene product might be implicated in the adhesion processes.

The nucleotide sequences of the DNA fragment encoding the protective antigens were determined in a set of five clinical isolates of E. coli (C5, S14, S53, S132, and S26) used in the DNA array experiments. The amino acid sequences of the antigens deduced from DNA sequencing were identical in the five E. coli strains analyzed (data not shown). The conservation of these proteins suggests that these antigens might induce a protective immunity against several other ExPEC strains.

Antigen expression in E. coli S26. The fact that the immune response elicited by the vaccine candidates was protective constitutes evidence that these antigens are recognized by antibodies and supports the idea that they are localized at the surface of the bacteria. To test this hypothesis, E. coli S26 was grown in LB with or without the iron chelator 2-2'-dipyridyl. Western blot experiments were performed with mouse antisera on total cell lysates and demonstrated that IroN and FyuA were specifically detected on the total cell extracts of E. coli S26 grown in iron-depleted medium (Table 5), which is in agreement with the function assigned to the two proteins (53, 64) and the presence of a "FUR" box (8) in the promoter of the two genes. The subcellular fractionation of E. coli S26 grown under the same conditions indicates that the two proteins are localized in the membrane fraction and particularly in the outer membrane of the bacteria (Table 5).


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  		TABLE 5. Detection of protective antigens by mouse or human immune serum

However, in the same in vitro conditions, the other antigens were not detected in the membrane preparations even though the challenge strain carried the protein coding sequence (Table 5). These results suggest that C0393, C4424, or C3389 antigens are not expressed by the bacteria during in vitro growth or that they are expressed at a very low level undetectable by this assay. To assess their expression during the infectious process, immunoblot experiments were performed using human sera drawn from patients with a diagnosis of bacteremia due to ExPEC. As shown in Table 5, pooled human sera reacted with recombinant FyuA and IroN proteins. Interestingly, human sera also detected C0393, C4424, and C3389 antigens. These results indicate that native proteins are expressed during the infectious process and that the antigens can induce an antibody response in the host.


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DISCUSSION
 
In this study, we used genomic data from E. coli to identify vaccine candidates that protect against an ExPEC strain in a model of lethal sepsis. The strategy for antigen selection was based upon two criteria: (i) proteins with vaccine potential are mainly located at the cell surface or are secreted and (ii) extraintestinal E. coli strains shared common features with commensal strains and, thus, efficient vaccine candidates would only correspond to proteins that are specific to extraintestinal pathogenic strains.

The availability of the genomic sequences of many pathogens has led to the development of a new strategy to identify vaccine candidates, termed "reverse vaccinology" (2). The concept relies upon the mining of genomic sequences to find putative antigen coding genes. This technology has been successfully used for the identification of vaccine candidates against several bacterial pathogens (6, 55, 59, 81). With the recent sequencing of the E. coli CFT073 genome, a urovirulent E. coli strain, the application of reverse vaccinology to E. coli became possible. One major difficulty of such studies resides in the identification of pathogen-specific sequences common to several ExPEC strains. In the present study, starting from short specific sequences of the meningitis-causing C5 E. coli strain, we were able to identify more than 700 kb of DNA specific to E. coli CFT073 using in silico genomic comparisons, which were defined as the B2 core genome. This total amount of ExPEC-specific DNA is different from the 1.3 Mb of DNA that was reported in a study comparing the genome of E. coli CFT073 and E. coli K-12 (80). However, the distribution of these CFT073-specific sequences revealed 50 genomic regions of more than 4 kb (Table 2) which represent 83% of 60 unique segments of more than 4 kb previously identified in CFT073 (80). These results indicate that additional specific sequences of CFT073 could be isolated, but it is not known if they are shared by other ExPEC strains.

The comparison of pathogenic strains of E. coli has shown a great diversity in their gene contents. In particular, the virulence-associated genes, described in the literature as specific to ExPEC, are not equally distributed among the virulent strains (21, 42). The sequences of the B2 core genome were used for comparative genomic hybridizations to determine the prevalence of the genes encoding putative antigens among clinical isolates of E. coli responsible for extraintestinal infections. However, the comparative genomic hybridization results indicated the presence of homologous genes that could have some genetic variability (21) or could be nonfunctional. We sequenced the genes encoding protective antigens in five different NMEC strains and observed a high degree of conservation. Since we did not observe any genetic polymorphism, this result indicates that the antigenic variability may not occur for the antigens we identified. So far, in E. coli, antigenic variation has only been described for fimbriae (77, 79). Moreover, we indirectly demonstrated that homologous genes were functional in the E. coli S26 clinical isolate because specific antibodies against their product inhibit this infection. It remains to be determined whether these vaccine candidates also present in the CFT073 genome are protective in a UTI model.

More than half of the protective antigens were related to iron metabolism. This observation could be explained by the model of infection that was used to screen for vaccine candidates. Because the infectious model is a rapid dissemination of the bacteria from the peritoneal site in 24 h, resulting in the killing of the host in less than 48 h, the antibodies which recognize the essential factors for bacterial survival and multiplication in the peritoneum and the blood will be the most effective.

This hypothesis would explain the fact that SfaS, the adhesin of the S fimbriae (68) implicated in the adhesion of bacteria to endothelial vascular cells (75) and reported to be expressed in CSF and blood of infected rats (67), is unable to protect mice from lethal sepsis. However some adhesins implicated in the early stage of bacterial colonization in UTI have been shown to induce protection against this type of infection. Indeed, vaccination with FimH, the adhesin of the type 1 fimbriae, or PapG, the adhesin of P fimbriae, could protect against cystitis and pyelonephritis, respectively (37, 38, 58, 76). The identification of vaccine candidates depends greatly on the experimental model of infection used to evaluate the protective efficacy.

Iron is an important growth factor for pathogenic bacteria. In the host, a very low concentration of free iron is available. Bacteria have developed several strategies to uptake and store iron present within the host by producing siderophore receptors (9, 19, 61, 70) or iron uptake systems involving proteins which release iron from host-iron complexes (5). We found that the IroN, FyuA, and C0393 recombinant proteins have the ability to protect mice from a lethal sepsis. Recently, a protective effect has been described for IroN in a UTI model (64) as well as a contribution of the protein to the virulence of ExPEC strains of different pathotypes (43, 62). The implication of FyuA in the virulence of ExPEC strains has already been demonstrated (70). However, the role of FyuA as a protective antigen has never been explored, and we report for the first time a protective effect associated with this protein. The high identity between Hbp and C0393 (78%) suggests that the C0393 protein may act as a hemoglobin protease with heme-binding properties (49). In addition to the role of the Hbp in the pathogenesis of extraintestinal E. coli strains, the protein has been shown to protect mice against the formation of abscesses following a challenge with E. coli and B. fragilis (50). Therefore, our results are the first to provide experimental evidence for the vaccine potential of IroN and an Hbp-like protein in a systemic model of extraintestinal infection.

We also examined the role of the ChuA protein, a known hemin receptor associated with ExPEC strains (43), as a vaccine candidate. We found that, unlike the other iron-regulated proteins, ChuA was unable to protect mice in our model. The data presented in this study are in agreement with the idea that a vaccine target related to iron metabolism should be essential to the bacterial pathogenesis, since, in contrast to other proteins, ChuA is dispensable for the virulence in E. coli (43). However, one cannot exclude expression of ChuA in later stages of the infectious process or a mechanism of compensation by the induction of another hemin receptor following ChuA inhibition by functional antibodies. Many years ago, Bolin and Jensen revealed that antibodies directed against uncharacterized iron-regulated outer membrane proteins were able to protect from E. coli septicemia (14), and our findings provide some insight into the nature of these proteins.

In the present study, a direct correlation has been made between the protection elicited by FyuA or IroN and the detection of the native protein in the outer membrane fractions (Table 4). In the meantime, we were not able to define in vitro culture conditions that would permit the detection of the c0393, c4424, or c3389 gene products in such fractions. However, results of immunoblot experiments using pooled human sera from infected patients combined with results of passive immunization assays gave some indirect evidence that these three genes are expressed in vivo during the infection and that they are located in a cellular compartment that renders them accessible to functional antibodies. Experiments using extraintestinal E. coli have shown that, in the host, the bacteria express a number of genes implicated in various cellular processes, with many of these genes being specifically induced in vivo (36, 57). The differential fluorescence induction technique has identified genes of CFT073 that are induced after contact with the peritoneal cavity (57). None of the antigens we identified were found in that study. Our data indicate that c0393, c4424, and c3389 might be expressed specifically in other organs or at later stages of the infectious process including bloodstream dissemination.

The sequencing of the bacterial genomes revealed that 30 to 40% of all genes belong to hypothetical or unknown protein families. Because our approach identifies putative antigens independent of their functions, some antigens may have no assigned function. In the present study, two protective antigens fall into that case. This observation has already been reported in other "reverse vaccinology" studies (39, 55). The investigation of such unknown proteins has helped in the identification of new bacterial functions (3, 20). Determination of the functions associated with C4424 or C3389 antigens will allow for a better understanding of the ExPEC physiology, including the identification of new virulence determinants.

In conclusion, using genome-based information from E. coli C5 and CFT073, we identified 59 proteins predicted to be localized at the surface of the bacteria; 40 of them were demonstrated to be conserved in the clinical isolates belonging to the B2 and/or D phylogenetic groups. In a mouse model of lethal sepsis, five antigens provided a significant protection against lethal sepsis due to E. coli strain S26. Among these antigens, three are associated with iron metabolism and two represent new proteins not previously described. Since reverse vaccinology relies on genomic information to predict putative vaccine candidates, our results further confirm that this technique constitutes a powerful tool for the identification of vaccine candidates which would be hidden to conventional vaccinology approaches. The findings of specific ExPEC antigens show that it is possible to design a vaccine for these strains despite the relative heterogeneity of the ExPEC population. These antigens constitute the basis for a preventive subunit vaccine against severe infections due to extraintestinal E. coli strains, and further investigations are in progress to improve the vaccinal efficacy of such vaccines.


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ACKNOWLEDGMENTS
 
We thank Etienne Carbonnelle for supplying the human serum and Jean-Luc Beretti for his technical assistance for the purification of recombinant proteins.

This work was supported by the Association Nationale de la Recherche Technique (grant no. 20030631).


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FOOTNOTES
 
* Corresponding author. Mailing address: Mutabilis SA, 102 route de Noisy, 93230 Romainville, France. Phone: 33 1 57 14 05 21. Fax: 33 1 57 14 05 24. E-mail: sonia.escaich{at}mutabilis.fr. Back

{triangledown} Published ahead of print on 4 December 2006. Back

Editor: A. D. O'Brien

{dagger} Supplemental material for this article may be found at http://iai.asm.org/. Back


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Infection and Immunity, April 2007, p. 1916-1925, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01269-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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