Abstract
Escherichia coli O157:H7 is an important zoonotic pathogen, causing hemolytic uremic syndrome (HUS). The colonization of cattle and human hosts is mediated through the action of effectors secreted via a type III secretion system (T3SS). The structural genes for the T3SS and many of the secreted effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE). We cloned and expressed the genes coding for 66 effectors and purified each to measure the cross-reactivity of type III secreted proteins from Shiga toxin-producing Escherichia coli (STEC) serotypes. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with type III secreted proteins (T3SPs) from four STEC serotypes, experimentally infected cattle, and human sera from six HUS patients. Twenty proteins were recognized by at least one of the STEC T3SP-vaccinated rabbits by Western blotting. Several structural proteins (EspA, EspB, and EspD) and a number of effectors (Tir, NleA, and TccP) were recognized by O26-, O103-, O111-, and O157-specific sera. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA, and NleA were recognized by the majority of the samples tested. A number of other proteins also were recognized by individual serum samples. Overall, proteins such as Tir, EspB, EspD, NleA, and EspA were highly immunogenic in vaccinated and naturally infected subjects and could be candidates for a cross-protective STEC vaccine.
INTRODUCTION
Shiga toxin-producing Escherichia coli (STEC) strains are an important group of zoonotic pathogens that are responsible for hemorrhagic colitis and hemolytic uremic syndrome (HUS) (12, 13, 23). Hemolytic uremic syndrome is attributed to the action of Shiga toxins (Stx1 and Stx2), identified first in Shigella dysenteriae, produced by STEC either alone or in combination (15, 25). Hemolytic uremic syndrome is also the leading cause of acute renal failure in children worldwide.
The most common STEC serotype in North America is O157, where an estimated 73,000 illnesses occur each year in the United States, resulting in 2,000 hospitalizations and 60 deaths (21). The most frequent non-O157 serotypes responsible for disease are O26, O103, O111, and O145, with numerous outbreaks reported worldwide. In Denmark it is estimated that 68% of STEC infections resulted from non-O157 serotypes (24). It also is estimated that 58% of all cases in Argentina, which has the highest reported frequency of HUS worldwide, result from infection by non-O157 serotypes (19, 28).
Cattle are the main reservoir for STEC, and the organism does not cause clinical disease in them. The colonization of both cattle and human hosts is mediated through the action of effector molecules secreted through a type III secretion system (T3SS) (7, 8). These effectors contribute to the formation of attaching and effacing (A/E) lesions, which are the hallmark of STEC infection (9). The genes which express the structural proteins of the T3SS and many of its effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE) (4). Many of these proteins, such as Tir, EspA, EspB, and EspD, are critical for the virulence of STEC (7, 22). However, the discovery of non-LEE effectors, such as NleA, TccP, and NleB, whose genes are located in small pathogenicity islands, and prophages, also has been shown to play an important role in the colonization and virulence of STEC (6, 14, 22, 27, 31).
A number of experimental vaccines based on LEE proteins have been tested. The vaccination of pregnant dams using intimin from STEC O157 protected suckling piglets against challenge (2), yet a cross-protective vaccine based on this protein would be challenging, as more than 17 serologically distinct variants have been identified (5). Potter and colleagues demonstrated that vaccination with secreted proteins from STEC O157:H7 was able to significantly reduce the number of bacteria shed, as well as the number of shedding animals in an experimental setting (26). However, vaccination using T3SPs appears to be serotype specific (1, 20). At present, the repertoire of T3SPs in the supernatant which are harvested for vaccination, as well as their immunogenic properties, are unknown. In a natural infection, it is unclear which T3SPs are secreted by STEC and recognized by cattle and human hosts.
In this study, we have cloned and expressed the genes coding for 66 structural and effector proteins, which include 37 LEE-encoded proteins and 29 non-LEE effectors to assess their immunological cross-reactivity using sera from vaccinated and naturally infected animals as well as human sera from HUS patients.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
Bacterial strains used in this study included E. coli EDL933 (O157:H7) (29), CL101 (O111:NM), CL9 (O26:H11), and N01-2454 (O103:H2) (11). Strains were stored at −70°C in 30% glycerol and were grown in Luria-Bertani (LB) agar and in LB broth at 37°C. All non-O157 STEC strains were kindly provided by the Laboratory for Food-Borne Zoonoses, Guelph, Ontario, Canada.
Cloning of LEE and non-LEE genes.
The STEC O157:H7 strain EDL933 was used as the source of DNA. The desired region of chromosomal DNA was amplified by PCR, allowing for the introduction of unique restriction sites cloned into the pQE-30 plasmid (Qiagen) for 6×His-tagged proteins (Qiagen) and the pGEX-5X-1 plasmid for glutathione S-transferase (GST)-fused proteins. Ligations were completed using the Rapid DNA ligation kit as described by the manufacturer (Fermentas). Plasmids were chemically transformed into E. coli JM105 cells (pQE-30) and E. coli BL21 cells (pGEX-5X-1). Primers and restriction sites for genes cloned can be found in Table S1 in the supplemental material.
Expression and purification of His-tagged LEE and non-LEE proteins.
An overnight LB culture was inoculated at 1:100 into fresh LB supplemented with ampicillin (100 μg/ml). The culture was grown at 37°C with shaking to an absorbance of 0.6 at 600 nm and induced for 3 h with 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside). Bacteria were pelleted, and His-tagged proteins were purified with nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) under denaturing conditions using the protocol from QIAexpressionist (Qiagen). The purity of proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie brilliant blue staining (16).
Expression and purification of GST fusion proteins.
GST-fused proteins were expressed and purified as described previously (10). Briefly, a culture containing 500 ml LB, ampicillin (100 μg/ml), and chloramphenicol (50 μg/ml) was inoculated with 3 ml of an overnight culture containing the desired plasmids in BL21 cells. Bacteria were grown at 37°C with shaking to an absorbance of 0.2 at 600 nm, at which point IPTG was added at a concentration of 0.25 mM and cultures were incubated for an additional 3 h at 30°C. Bacteria were pelleted and resuspended in binding buffer (540 mM NaCl, 2.7 mM KCl, 10.15 mM Na2HPO4, 1.75 mM KH2PO4, 10 mM MgCl2, 1% [vol/vol] Triton X-100, 50 μg of DNase I, 30 μg/ml phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 10 μg/ml leupeptin [pH 7.4]), followed by sonication (three times for 30 s with a 1-s pulse and a 12-mm probe at maximum power; Vibra-Cell, Sonics & Materials Inc., Dansbury, CT). GST-fused proteins were purified by adding 1 ml of a 1:1 slurry of glutathione-Sepahrose 4B beads (Amersham) in phosphate-buffered saline (PBS) to 10 ml of cleared lysate. The beads then were washed four times with 15 ml of binding buffer. The purity of proteins was visualized following SDS-PAGE using Coomassie brilliant blue staining (16).
SDS-PAGE and Western blot analysis.
Proteins were separated by SDS-PAGE and visualized by staining with Coomassie brilliant blue (16). Proteins were transferred to nitrocellulose membranes by electroblotting as described by the manufacturer (Bio-Rad Laboratories), and Western blot analysis was carried out using rabbit anti-T3SPs, STEC (O157, O26, O111, and O103) antibodies, anti-6×His monoclonal antibody (Becton, Dickinson), and bovine anti-STEC O157 polyclonal antibodies as described previously (16, 18).
Production of T3SPs and rabbit anti-T3SP polyclonal antibodies.
Type III secreted proteins from all STEC serotypes were prepared as described previously (18). Briefly, overnight cultures of STEC serotypes were diluted 100-fold in M9 minimal medium containing 0.4% glucose, 0.1% Casamino Acids, 44 mM NaHCO2, 8 mM MgSO4, and 45 mM KHCO3. Cultures then were incubated without shaking at 37°C in a 5% CO2 environment to an optical density at 600 nm (OD600) of 0.6 to 0.8. Bacteria were pelleted by centrifugation, and the supernatant proteins were concentrated by precipitation with 10% trichloroacetic acid. One-ml quantities containing 50 μg of STEC T3SPs and 30% Emulsigen-D (MVP Laboratories) were used to immunize female New Zealand White rabbits by the subcutaneous route. The animals received two boosts 3 weeks apart before being euthanized. Rabbits were bled according to the guidelines provided by the University Council on Animal Care (UCAC), under protocol number 1994-213. The blood collected was centrifuged, and the serum was stored at −20°C until further use.
Experimentally infected cattle.
Two yearling mixed-breed cattle were obtained from farms in Saskatchewan and housed at the University of Saskatchewan. Both were fed a barley-based finishing ration with free-choice roughage. Both animals were screened prior to challenge for shedding and existing titers against T3SPs from STEC O157:H7. Cattle received two oral challenges, 21 days apart, of 500 ml containing 109 CFU/ml of STEC O157:H7. Serological conversion was measured by ELISA and Western blotting.
Sera from human HUS patients.
Sera were obtained from six human HUS patients, with informed consent, who developed HUS as a result of STEC O157:H7 infections. All serum samples were collected roughly 1 week after the onset of clinical signs of disease. Naïve sera were collected from individuals with no history of recent diarrhea.
ELISA.
Enzyme-linked immunosorbent assays (ELISA) using rabbit anti-T3SP serotype STEC (O157, O26, O111, and O103) and polyclonal antibodies against 6×His-tagged LEE and non-LEE proteins were completed as described previously (18), with the following modifications. Plates were blocked for 1 h with 1% nonfat dried milk (CO-OP, Canada) in Tris-buffered saline, pH 7.6, with the addition of 0.05% Tween 20 (Sigma) (TBST). Serial dilutions were completed for primary antibodies and incubated for 2 h at room temperature. After washes, labeled goat anti-rabbit IgG (Kirkegaard & Perry Laboratories) at a dilution of 1:2,000 in TBST was incubated for 1 h at room temperature, and the alkaline phosphate activity was detected using p-nitrophenyl phosphate (PNPP) (Sigma). The OD was measured at 405 nm using a Bio-Rad model 1350 microplate reader. Data are reported as median values and their ranges (Table 1; also see Table S2 in the supplemental material).
Table 1.
ELISAs using anti-T3SP E. coli O157:H7 and non-O157 sera against recombinant STEC O157 LEE proteinsa
| Protein | ELISA titer after exposure to: |
||||
|---|---|---|---|---|---|
| Preimmune | O157 | O26 | O103 | O111 | |
| EscC | 1,525 (A) | 6,684 (B) | 22,390 (C) | 7,304 (B) | 17,222 (D) |
| SepD | 265 (A) | 6,453 (B) | 760 (C) | 197,773 (D) | 1,381 (C) |
| Tir | 419 (A) | 567,692 (B) | 522,874 (C) | 130,202 (D) | 489,475 (C) |
| EspA | 234 (A) | 637,500 (B) | 297,646 (C) | 299,648 (C) | 395,028 (B, C) |
| EspD | 224 (A) | 412,851 (B) | 5,694 (A) | 384,520 (B) | 288,509 (B) |
| EspB | 179 (A) | 511,393 (B) | 99,719 (C) | 474,865 (B) | 497,104 (B) |
| EspF | 332 (A) | 937 (B) | 6,995 (C) | 25,672 (D) | 86,078 (E) |
| EspG | 398 (A) | 386,863 (B) | 5,643 (C) | 1,123 (D) | 422,629 (B) |
| NleA | 128 (A) | 460,507 (B) | 63,889 (C) | 55,801 (D) | 20,062 (D) |
| NleE | 1,177 (A) | 6,345 (B) | 1,224 (A) | 2,443 (C) | 5,714 (D) |
| NleF | 313 (A) | 1,362 (B) | 392 (A) | 512,229 (C) | 4,155 (D) |
| EspRl | 3,225 (A) | 156,521 (B) | 6,543 (C) | 4,407 (A) | 6,891 (C) |
| NleH | 4,283 (A) | 24,373 (B) | 5,997 (C) | 19,138 (D) | 2,919 (E) |
| Niel | 413 (A) | 5,738 (B) | 1,757 (C) | 19,969 (D) | 5,310 (E) |
| NleG2-1 | 388 (A) | 4,566 (B, C) | 2,587 (C) | 121,235 (D) | 6,563 (B) |
| NleG2-2 | 953 (A) | 6,719 (B) | 2,574 (A, B) | 84,860 (C) | 7,027 (B) |
| TccP | 368 (A) | 1,447 (B) | 132,429 (C) | 27,261 (D) | 6,875 (E) |
| EspY1 | 1,958 (A) | 1,874 (A) | 7,273 (B) | 8,455 (B) | 6,318 (B) |
| NleG6-1 | 395 (A, C) | 4,386 (B) | 1,360 (A, C) | 1,502 (B)(C) | 1,399 (C) |
| Map | 381 (A) | 4,542 (B) | 469 (C) | 1,271 (D) | 1,530 (E) |
All proteins which had a positive reaction by Western blotting using rabbit anti-T3SP E. coli O157, anti-T3SP E. coli O26, anti-T3SP E. coli O103, and anti-T3SP E. coli O111 sera also were tested with the same sera in ELISAs for quantitative measure. A total of 20 proteins were tested, including 18 proteins that were positive by Western blotting (Table 2) and 2 negative proteins (NleG6-1 and Map). Preimmune, preimmune sera; O157, rabbit anti-O157 T3SP polyclonal antibodies; O26, rabbit anti-O26 T3SP polyclonal antibodies; O103, rabbit anti-O103 T3SP polyclonal antibodies; O111, rabbit anti-O111 T3SP polyclonal antibodies. Values represent ELISA titers. Responses were grouped statistically (indicated by A, B, C, D, and E; differences in antisera reactivity were examined using one-way ANOVA in which the means of raw or transformed data were compared using the least-significant-difference method; differences were considered significant when P < 0.05).
For human sera, single-well dilutions were completed for each protein (in duplicate). Duplicate wells using naive human sera were calculated to measure the background for each protein. Data were modified by subtracting the median value (from uninfected individuals) from the values of the HUS-positive human sera. Duplicate values were averaged, and three standard deviations were calculated before subtraction. This subtraction method was selected instead of the traditional titer presentation due to limited volumes of HUS-positive human sera. The subtraction method also was used with the experimentally infected cattle sera to compare results to those of HUS-positive human sera.
Statistical analysis.
Differences in antisera reactivity with each protein were examined using one-way analysis of variance (ANOVA). When data were normally distributed, the ANOVA was performed on the raw data. Nonnormally distributed data were rank or log transformed, and then the ANOVA was performed on the data. Means of raw or transformed data were compared using the least-significant-difference method. Differences were considered significant when P < 0.05.
RESULTS
Immune responses against STEC O157:H7 secreted proteins determined by Western blotting.
Bovine and rabbit sera raised against STEC O157:H7 T3SPs were tested against 37 LEE-purified proteins to investigate if the pattern of reactive proteins was similar. Both rabbit and bovine sera reacted with the same proteins (Tir, EspA, EspB, EspG, and EspD), and the only difference observed was in band intensity (Fig. 1B and G). Rabbit anti-O26-, anti-O103-, anti-O111-, and anti-O157-specific sera raised against T3SPs also were tested against the 37 LEE proteins to determine which cross-reactive serotype proteins were present in the bacterial culture (Fig. 1C to E and Tables 2 and 3). The pattern of LEE proteins by Western blotting was comparable, where Tir, EspA, EspB, and EspD reacted with sera from all serotypes. The EspF and EspG proteins were detected by the majority of the sera, and SepD and EscC were detected by at least one individual serum tested.
Fig. 1.
Western blots using anti-T3SP E. coli O157:H7 and non-O157 sera against recombinant STEC O57 LEE proteins. In total, 40 LEE genes were selected for overexpression and purification. All genes were cloned and sequenced using the Qiagen pQE-30 6×His-tagged vector cloning system (except EscS and Rorf1, which were cloned into pGEX-5X-1 for GST fusion purification). Thirty-seven proteins were purified and tested against sera. (A) Proteins visualized by SDS-PAGE using Coomassie brilliant blue. (B) Western blots using rabbit anti-T3SP E. coli O157. (C) Western blots using rabbit anti-T3SP E. coli O26. (D) Western blots using rabbit anti-T3SP E. coli O103. (E) Western blots using rabbit anti-T3SP E. coli O111. (F) Western blots using rabbit preimmune sera. (G) Western blots using bovine anti-T3SP E. coli O157. (H) Western blots using anti-6×His monoclonal antibody. #, GST-fused proteins; +, membrane proteins not purified; *, Tir protein supplied by Bioniche Life Sciences. The following shading was used in the arrows: small dots, orf of unknown function; large dark circles, chaperone; diagonal lines, intimin; vertical lines, secreted protein; mesh, regulator; white dots on a black background, translocator protein; horizontal lines, structural protein.
Table 2.
Summary of reactive recombinant STEC O157 T3SPs against rabbit O26-, O103-, O111-, and O157-specific seraa
| Protein | Reactivity after exposure to: |
|||
|---|---|---|---|---|
| O157 | O26 | O103 | 111 | |
| LEE | ||||
| EscC | + | |||
| SepD | ++ | |||
| Tir | ++ | ++ | +++ | ++ |
| EspA | +++ | +++ | +++ | ++ |
| EspD | +++ | ++ | +++ | +++ |
| EspB | +++ | ++ | +++ | ++ |
| EspF | ++ | ++ | ++ | |
| EspG | ++ | + | +++ | |
| Non-LEE | ||||
| NleA | +++ | ++ | +++ | +++ |
| NleE | + | + | ||
| NleF | ++ | |||
| EspRl | ++ | |||
| NleH | + | ++ | + | |
| Niel | ++ | |||
| NleG2-l | ++ | |||
| NleG2-2 | + | |||
| TccP | + | ++ | +++ | + |
| EspYl | + | ++ | ||
Shown are LEE and non-LEE proteins which reacted against O26-, O103-, O111-, and O157-specific sera in Western blots. O157, rabbit anti-O157 T3SP polyclonal antibodies; O26, rabbit anti-O26 T3SP polyclonal antibodies; O103, rabbit anti-O103s T3SP polyclonal antibodies; O111, rabbit anti-O111 T3SP polyclonal antibodies.
A plus sign indicates reactivity.
Table 3.
Summary of reactive recombinant STEC O157 T3SPs against sera from O157-experimentally infected and O157-vaccinated cattlea
| Protein | Reactivity against sera: |
|
|---|---|---|
| Vaccinated with STEC O157:H7 TTSPs | Experimentally infected with STEC O157:H7 | |
| LEE | ||
| Tir | ++ | + |
| EspA | ++ | + |
| EspD | + | ++ |
| EspB | ++ | +++ |
| EspG | + | |
| Non-LEE | ||
| EspM2 | + | |
| NleA | + | + |
| TccP | + | |
Shown are LEE and non-LEE proteins which reacted against sera from O157-experimentally infected and O157-vaccinated cattle.
A plus sign represents reactivity.
A total of 29 non-LEE purified STEC O157:H7 secreted proteins also were tested for reactivity with anti-rabbit O26, O103, O111, and O157 secreted proteins (Tables 2 and 3; also see Fig. S1 and S2 in the supplemental material). This pattern of recognized proteins varied, where only NleA and TccP reacted with all sera, NleE and NleH with the majority, and EspY1, NleG2-1, NleG2-2, NleI, EspR1, and NleF with at least one individual serum sample tested. Overall, O157-specific serum reacted to the least number of purified proteins (8 of 66), while O103-specific serum reacted to the most proteins (15 of 66). Rabbit preimmune serum was used as a negative control against all proteins (Fig. 1F; also see Fig. S1F and S2F in the supplemental material), as well as an anti-6×His monoclonal antibody (Fig. 1H; also see Fig. S1H and S2H in the supplemental material).
Immune responses against STEC O157:H7 secreted proteins determined by ELISA.
To obtain a quantitative measure of the response against STEC T3SPs, ELISAs were used to analyze the reactivity of purified STEC O157:H7 secreted proteins with rabbit sera raised to STEC O157, O26, O103, and O111 T3SPs. A total of 20 proteins were tested, including 18 proteins which reacted positively in Western blots (Table 1) and 2 proteins (NleG6-1 and Map) that did not react. The majority of proteins gave similar findings compared to the Western blotting results described above (Tables 2 and 3). Antisera which were reactive in Western blotting gave ELISA titers that were significantly higher than those of both the preimmune and nonreactive sera. The two proteins used that did react to any of the sera tested in Western blotting showed low readings by ELISA compared to those of the reactive proteins, even though some of the antisera were significantly different from the preimmune serum. However, a number of proteins which were positive by Western blotting with O26-, O103-, O111-, and O157-specific sera demonstrated low titers by ELISA (NleE, NleH, and EspY1). Statistical differences were considered significant when P < 0.05.
Immune responses against STEC O157:H7 secreted proteins determined by Western blotting and ELISA.
Sera collected from cattle which had been experimentally infected with STEC O157:H7 were tested against the 66 purified STEC O157:H7 secreted proteins. Using Western blot analysis, 6 of these 66 proteins reacted against the sera from infected cattle (Tables 2 and 3). Four of these six proteins also reacted with sera from cattle vaccinated with STEC O157:H7 T3SPs (Tir, EspA, EspB, and EspD) (Fig. 1G). In addition, sera from infected cattle also recognized TccP and EspM2 (Tables 2 and 3), while vaccinated cattle recognized NleA and EspG (Fig. 1G and Tables 2 and 3; also see Fig. S2G in the supplemental material). ELISA was completed using sera from experimentally infected cattle. A total of seven proteins gave an ELISA OD of greater than 0.050 after the subtraction of the values from the preimmune control sera (Fig. 2; also see Fig. S3 in the supplemental material). Five of the seven proteins resulted in significant reactivity when the sera from experimentally infected or vaccinated animals against T3SPs were used (Tir, EspA, EspD, EspB, and NleA).
Fig. 2.
Antibody response of sera from STEC O157:H7 experimentally infected cattle and human sera from HUS patients against STEC O157 secreted proteins. Sixty-six purified proteins were tested, and only reactive proteins (ELISA OD higher than 0.050) were graphed. Negative proteins not shown on the graph consist of Ler, Orf2, CesA/B, Orf4, Orf5, EscT, Rorf13, GrlR, CesD, EscC, SepD, EscJ, Orf8, SepZ, Orf12, EscN, Orf16, EspH, CesF, Map, CesT, EscD, SepL, CesD2, EscF, Orf29, EspF, NleB, NleB2-1, NleC, NleE, NleG, NleH1-2, NleI, NleG2-2, NleG3, NleG5-1, NleG6-1, NleG8-2, NleG9, EspK, EspL2, EspR1, TccP, EspV, EspW, EspX2, EspX7, EspY1, EspY2, and EspY3. Single-well dilutions of sera were used for each protein. Preimmune cattle sera were used to calculate background values against each protein. The graphed ELISA OD represents the means plus standard deviations from samples (six samples of human HUS patients and two samples from experimentally infected cattle) which were calculated by subtracting the preimmune value from the infected cattle value. Duplicate values were averaged, and three standard deviations were calculated before subtraction. Solid grey bars, sera from experimentally infected cattle; dotted bars, sera from human from HUS patients.
Reactivity of human sera from HUS patients against recombinant purified STEC O157:H7 secreted proteins.
To compare the profile of T3SPs recognized by sera from HUS patients previously infected with STEC relative to those of cattle described above, we tested the reactivity of sera from six individuals against the 66 purified STEC O157:H7 secreted proteins. Twelve proteins gave a positive ELISA reading relative to those of naive controls. Four proteins (Tir, EspD, EspA, and NleA) reacted with the majority of sera tested (Fig. 2; also see Fig. S4 in the supplemental material), while EspG, EspB, Rorf1, and EscS demonstrated significant levels of reaction to at least one individual serum sample. The highest level of reactivity was seen with the Tir protein, where all six samples demonstrated elevated readings.
DISCUSSION
The production and secretion of T3SPs by STEC strains is essential for colonization, as these proteins are involved in the formation of A/E lesions, which are critical for bacterial persistence in both bovine and human hosts. Type III secreted proteins have been shown to have protective properties, since vaccination with a culture supernatant containing T3SPs significantly reduced the number of animals shedding STEC, as well as the number of STEC O157:H7 organisms shed in individual fecal samples, after an experimental challenge and under field conditions (26). At this time, only a small number of LEE and non-LEE secreted proteins have been identified as being present in the supernatant used for immunization (27, 31). In this study, a total of 66 LEE and non-LEE proteins were studied to determine their immunogenicity and serological cross-reactivity and to investigate their presence in the bacterial supernatant previously used to vaccinate against STEC O157:H7.
Initially we attempted to express and purify all proteins coded for by genes found within the LEE pathogenicity island, excluding intimin. However, we were unable to express and purify three structural proteins (EscR, EscU, and EscV), and therefore these were omitted from the analyses. These proteins are believed to be part of the inner membrane structure of the type III apparatus and are not thought to be crucial in protection against infection.
The majority of LEE proteins that reacted with serum raised against STEC O157:H7 secreted proteins consisted of secreted effectors and structural proteins involved in the assembly of the external portion of the secretion apparatus. These results were expected, since the majority of regulators and inner membrane and periplasmic structural proteins are not exposed on the cell surface but instead remain within the bacterial cell. The reaction of structural and regulatory proteins (SepD and EscC) with sera raised against T3SPs could be a result of bacterial lysis leading to the contamination of the supernatant with these proteins, although there was no evidence of other intracellular proteins present in the vaccine formulation.
Several LEE proteins, including Tir and EspA, have been shown to be cross-reactive with sera raised against T3SPs from non-O157 serotypes (1). In this study, we have identified several other LEE and non-LEE proteins which also are cross-reactive with O26-, O103-, O111-, and O157-specific sera. The initial cross-reactivity observed with Tir and EspA was due to their sequence homology with other STEC serotypes. We believe that the observed cross-reactivity in this study also could be related to sequence homology among the serotypes.
The ELISA results presented in Table 1 were consistent with the Western blotting results for the majority of proteins which reacted with the STEC T3SP antisera (Tables 2 and 3). However, proteins such as NleE, which were positive for STEC O111 antisera by Western blotting, did not strongly react using an ELISA procedure. Two other proteins, NleH and EspY1, both were negative by Western blotting and positive by ELISA. These results could be linked to the greater sensitivity of the ELISA procedure or the level of protein denaturation that occurs during SDS-PAGE.
The ELISA data in Table 1 also demonstrate how STEC strains can be grouped based on reactivity and secretion profiles. For example, proteins such as Tir, EspA, EspB, and NleA all are secreted and appear to be cross-reactive with sera specific for all serotypes tested. Other proteins, such as EspF, TccP, NleG2-1, and NleG2-2, are cross-reactive with a number of non-O157 serotypes, such as STEC O103, but not with STEC O157 serum. The lack of reactivity observed with STEC O157 sera could be a result of low secretion levels of the specific proteins. However, further testing is required to confirm this possibility. For the majority of proteins which showed low reactivity with non-O157 sera, we are unable to conclude if these results are due to reduced homology, secretion levels, or the presence or absence of specific epitopes, since all proteins were expressed from STEC O157 genes. We previously showed with the Tir protein that the location of reactive epitopes differed significantly by serotype (unpublished results). To properly address this question, all proteins tested also would have to be purified from the non-O157 serotypes used.
The main reservoir for STEC is ruminants, and cattle are considered the most important source of human infection. These animals are colonized by highly virulent STEC strains without ever causing overt disease. Interestingly, STEC still are able to cause A/E lesions in cattle intestinal epithelium (3). In humans, STEC infection involving A/E lesions leads to hemorrhagic colitis, which results in complications such as HUS and TTP (7). In this study, we compared sera from experimentally infected cattle and human HUS patients against LEE and non-LEE T3SPs to determine if there were major differences in the host response. In general, the majority of immunogenic proteins were recognized by both bovine and human sera. While the bovine response against the purified proteins was fairly consistent, the magnitude of the response by the human HUS sera appeared to differ. The Tir protein gave the highest response of all tested proteins, which was not unexpected based on previous studies (26). The bulk of the immunogenic proteins were structural components involved in the secretion of T3SPs. These results, along with those for A/E lesions previously reported in humans and cattle, confirm that protein secretion is functional during the natural infection of both hosts. In addition, the immunogenic proteins reported here are found in genetic mobile elements which have been highlighted by Karmali and colleagues to be useful in the characterization of serotypes into seropathotypes A through E based on their occurrence in human disease, including outbreaks and HUS cases (11). We must note that although all preimmune antisera (human and cattle) were prescreened against STEC proteins, the preimmune antisera used in this assay may not be a truly negative control, considering that many cattle and humans have preexisting antibody to STEC proteins as a result of exposure. Based on the similarity of proteins recognized by both human and cattle sera, the potential to use measurements of serological responses to STEC proteins such as Tir, EspA, EspD, and NleA to detect or diagnose past or present infections in cattle and humans also should be considered.
The majority of purified immunogenic proteins were LEE encoded (Tir, EspB, EspD, and EspA) and have been shown to participate in colonization by STEC serotypes. Purified EspB, EspA, and Tir have been tested previously with sera from HUS patients and shown to be recognized by the host during natural infection (18). The only purified non-LEE protein that appears to be highly immunogenic is NleA. This protein has been shown previously to be involved in the modulation of virulence by the A/E pathogen Citrobacter rodentium (17). Recently, this protein also has been shown to play a role in the disruption of host intestinal tight junctions (30). However, the role of NleA in the colonization of humans and cattle remains unknown.
Interestingly, many of the secreted proteins were recognized by antibodies from experimentally infected cattle and human HUS patients even though these proteins are injected into the host cell and remain intracellular. It is possible that epitopes from a number of secreted proteins could be presented on the surface of the infected cell much like virus antigen, resulting in a cell-mediated response. However, in this study we focused on the humoral response to secreted proteins. In future studies it may be critical to also investigate the role of cell-mediated responses against the colonization and infection of STEC serotypes.
Supplementary Material
ACKNOWLEDGMENTS
This research was supported by the National Science and Engineering Research Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR), and Bioniche Life Sciences.
We thank D. Wilson and the Clinical Research Group at VIDO for assistance with animal experiments and M. Karmali for kindly providing STEC strains. A.A.P. is the holder of an NSERC Senior Industrial Research Chair.
Footnotes
Supplemental material for this article may be found at http://cvi.asm.org/.
Published ahead of print on 18 May 2011.
REFERENCES
- 1. Asper D. J., Sekirov I., Finlay B. B., Rogan D., Potter A. A. 2007. Cross reactivity of enterohemorrhagic Escherichia coli O157:H7-specific sera with non-O157 serotypes. Vaccine 25:8262–8269 [DOI] [PubMed] [Google Scholar]
- 2. Dean-Nystrom E. A., Gansheroff L. J., Mills M., Moon H. W., O'Brien A. D. 2002. Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection. Infect. Immun. 70:2414–2418 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Dean-Nystrom E. A., Stoffregen W. C., Bosworth B. T., Moon H. W., Pohlenz J. F. 2008. Early attachment sites for Shiga-toxigenic Escherichia coli O157:H7 in experimentally inoculated weaned calves. Appl. Environ. Microbiol. 74:6378–6384 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Elliott S. J., et al. 2000. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 68:6115–6126 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Garrido P., et al. 2006. STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains. Clin. Chem. 52:192–201 [DOI] [PubMed] [Google Scholar]
- 6. Gruenheid S., et al. 2004. Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7. Mol. Microbiol. 51:1233–1249 [DOI] [PubMed] [Google Scholar]
- 7. Gyles C. L. 2007. Shiga toxin-producing Escherichia coli: an overview. J. Anim. Sci. 85:E45–E62 [DOI] [PubMed] [Google Scholar]
- 8. Jarvis K. G., Kaper J. B. 1996. Secretion of extracellular proteins by enterohemorrhagic Escherichia coli via a putative type III secretion system. Infect. Immun. 64:4826–4829 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Jerse A. E., Gicquelais K. G., Kaper J. B. 1991. Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli. Infect. Immun. 59:3869–3875 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Johnson J. S., et al. 2004. Adenovirus protein VII condenses DNA, represses transcription, and associates with transcriptional activator E1A. J. Virol. 78:6459–6468 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Karmali M. A., et al. 2003. Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. J. Clin. Microbiol. 41:4930–4940 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Karmali M. A., et al. 2004. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. 1985. J. Infect. Dis. 189:556–563 [DOI] [PubMed] [Google Scholar]
- 13. Karmali M. A., Petric M., Lim C., Fleming P. C., Steele B. T. 1983. Escherichia coli cytotoxin, haemolytic-uraemic syndrome, and haemorrhagic colitis. Lancet ii:1299–1300 [DOI] [PubMed] [Google Scholar]
- 14. Kelly M., et al. 2006. Essential role of the type III secretion system effector NleB in colonization of mice by Citrobacter rodentium. Infect. Immun. 74:2328–2337 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Kulkarni H., Goldwater P. N., Martin A., Bettelheim K. A. 2002. Escherichia coli′O' group serological responses and clinical correlations in epidemic HUS patients. Comp. Immunol. Microbiol. Infect. Dis. 25:249–268 [DOI] [PubMed] [Google Scholar]
- 16. Laemmli U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [DOI] [PubMed] [Google Scholar]
- 17. Lee S. F., et al. 2008. A C-terminal class I PDZ binding motif of EspI/NleA modulates the virulence of attaching and effacing Escherichia coli and Citrobacter rodentium. Cell Microbiol. 10:499–513 [DOI] [PubMed] [Google Scholar]
- 18. Li Y., Frey E., Mackenzie A. M., Finlay B. B. 2000. Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors. Infect. Immun. 68:5090–5095 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Lopez E. L., et al. 1989. Hemolytic uremic syndrome and diarrhea in Argentine children: the role of Shiga-like toxins. J. Infect. Dis. 160:469–475 [DOI] [PubMed] [Google Scholar]
- 20. McNeilly T. N., et al. 2010. Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge. Vaccine 28:1422–1428 [DOI] [PubMed] [Google Scholar]
- 21. Mead P. S., Slutsker L., Griffin P. M., Tauxe R. V. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:841–842 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Misyurina O., et al. 2010. The role of Tir, EspA, and NleB in the colonization of cattle by Shiga toxin producing Escherichia coli O26:H11. Can. J. Microbiol. 56:739–747 [DOI] [PubMed] [Google Scholar]
- 23. Morrison D. M., Tyrrell D. L., Jewell L. D. 1986. Colonic biopsy in verotoxin-induced hemorrhagic colitis and thrombotic thrombocytopenic purpura (TTP). Am. J. Clin. Pathol. 86:108–112 [DOI] [PubMed] [Google Scholar]
- 24. Nielsen E. M., Scheutz F., Torpdahl M. 2006. Continuous surveillance of Shiga toxin-producing Escherichia coli infections by pulsed-field gel electrophoresis shows that most infections are sporadic. Foodborne Pathog. Dis. 3:81–87 [DOI] [PubMed] [Google Scholar]
- 25. Paton J. C., Paton A. W. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11:450–479 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Potter A. A., et al. 2004. Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins. Vaccine 22:362–369 [DOI] [PubMed] [Google Scholar]
- 27. Roe A. J., et al. 2007. Analysis of the expression, regulation and export of NleA-E in Escherichia coli O157: H7. Microbiology 153:1350–1360 [DOI] [PubMed] [Google Scholar]
- 28. Su C., Brandt L. J. 1995. Escherichia coli O157:H7 infection in humans. Ann. Intern. Med. 123:698–714 [DOI] [PubMed] [Google Scholar]
- 29. Tarr P. I., et al. 1989. Genotypic variation in pathogenic Escherichia coli O157:H7 isolated from patients in Washington, 1984–1987. J. Infect. Dis. 159:344–347 [DOI] [PubMed] [Google Scholar]
- 30. Thanabalasuriar A., et al. 2010. The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli. Cell Microbiol. 12:31–41 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Tobe T., et al. 2006. An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination. Proc. Natl. Acad. Sci. U. S. A. 103:14941–14946 [DOI] [PMC free article] [PubMed] [Google Scholar]
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