HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by CHART, H. Articles by WILLSHAW, G. A. Search for Related Content PubMed PubMed Citation Articles by CHART, H. Articles by WILLSHAW, G. A. Agricola Articles by CHART, H. Articles by WILLSHAW, G. A.

Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E. coli

Laboratory of Enteric Pathogens, Division of Gastrointestinal Infections, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT

Corresponding author: Dr H. Chart (e-mail: hchart{at}phls.org.uk).

Received 5 Feb. 2002; accepted 26 June 2002.

	Abstract

TOP Abstract Introduction Materials and methods Results Discussion References

 
Antibody–antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E. coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157. Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined. These common LPS-core epitopes were also present on strains of E. coli O26 which did not produce verocytotoxin. These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E. coli O157.

	Introduction

TOP Abstract Introduction Materials and methods Results Discussion References

 
Verocytotoxin-producing Escherichia coli (VTEC), and particularly strains of serotype O157:H7, are recognised as a major cause of diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome (HUS) in North America [1] and the UK [2]. Certain other VTEC, and in particular those belonging to serogroups O26, O103, O111 and O145 [3], also cause a disease characterised by bloody diarrhoea, which may develop into HUS [4]. These strains, together with O157 strains, have been termed enterohaemorrhagic E. coli (EHEC) [1, 5].

Patients infected with VTEC O157 produce serum antibodies to the O157 lipopolysaccharide (LPS) antigens [6–9], and these antibodies form the basis for routine serological tests providing evidence of infection in the absence of faecal isolates of E. coli O157 [10, 11]. The interpretation of serological results relies on a knowledge of antibody cross-reactions with the LPS antigens of other members of the Enterobacteriaceae and these have been examined extensively [12–16].

Strains of E. coli express LPS with a core region comprising one of five different structures, termed R1, R2, R3, R4 and K-12 [17], and it has been suggested that the majority of EHEC express the R3 LPS-core structure [17]. Based on this, it was reported that human serum antibodies binding to R3 core oligosaccharides of E. coli O157 LPS might cause antibody cross-reactions between the LPS of E. coli O157 and other EHEC [18] and, because of this, antibodies to E. coli O157 LPS detected in patients’ sera should be interpreted with caution. In the present study, LPS was prepared from a range of EHEC, including strains belonging to serogroups O26, O103, O111, O145 and O157, to examine the binding of human serum antibodies to epitopes thought to be located on the R3 LPS-core.

	Materials and methods

TOP Abstract Introduction Materials and methods Results Discussion References

 
Bacterial strains and culture conditions

Strains of VTEC used in this study (Table 1) were isolated from cases of HUS and some of these have been described previously [19]. Strains of E. coli belonging to serogroup O157 express LPS producing one of three different LPS profiles – A, B and C – by SDS-PAGE [20]; only strains expressing profile C produce VT [20]. E. coli strains O157:H19 (E10964) and E. coli O157:H45 (E59055) were VT-negative strains expressing O157 LPS profiles A and B respectively. These two strains express LPS profiles which bind rabbit antibodies used for detecting strains of E. coli belonging to serogroup O157. Ten further strains of E. coli O26:H11 were also used (Table 2) [21], five of these strains were VTEC and five did not express VT [21]. Strains of Yersinia enterocolitica O9 (E4610) [14], Vibrio cholerae O1 Inaba (SC1074) [15], Citrobacter freundii (E69366) [16] and Salmonella enterica serotype Urbana (S127490) [16] were also used to prepare LPS. These strains had been shown to express LPS which shared epitopes with E. coli O157 [14, 16]. The strain of S. Urbana was a representative of the group N Salmonella [16]. All bacteria were from the culture collection held by the Laboratory of Enteric Pathogens (LEP). Bacteria were grown on nutrient agar (Oxoid) at 37°C for 16 h.

Sera

Sera with antibodies to the LPS of E. coli O157 were from 10 patients with clinical HUS and from whom faecal O157 VTEC had been isolated and form part of the LEP collection. Six were from female (mean age: 3 years 6 months) and three were from male (mean age: 3 years 3 months) patients. Sera from two girls with HUS (1 year 8 months, E134825; 9 years 6 months, E160315) were also used in this study and O26 VTEC was isolated from the faeces of both patients. Sera from 10 blood donors were provided by the North London Blood Transfusion Centre and used as controls.

Antisera were prepared in New Zealand White rabbits to LEP type-strains of E. coli belonging to serogroups O26, O103, O111, O145 and O157 [22]. Rabbits received 0.5 ml (i.v.) of a heat-killed bacterial suspension (10⁹ bacteria/ml) on day 1; 1 ml (i.v.) on days 5 and 10, and 2 ml (i.v.) on days 15 and 20. Rabbits were bled on day 30. All sera were stored at -30°C.

LPS

LPS was prepared by digesting whole bacterial cells with proteinase-K [6]. Bacteria were placed in pre-weighed 1.5-ml plastic tubes and the cells were suspended in SDS-PAGE sample buffer [23] to give a concentration of 1 mg/30 µl before incubation at 100°C for 10 min. After cooling, samples were mixed with an equal volume of SDS-PAGE buffer containing proteinase K (Sigma) 100 µg/30 µl and incubated at 60°C for 1 h.

SDS-PAGE and immunoblotting

For SDS-PAGE, preparations containing 500 µg of digested cell mass were used in each lane. Samples were loaded on to gels (4.5% stacking gel and 12.5% separation gel) and electrophoresis was performed at 50 mAmp for 2.25 h [6]. Gels were either stained with silver to show LPS profiles [24] or profiles were transferred on to nitrocellulose paper (NCP) electrophoretically (0.50 Amp, 1 h) [6]. NCP sheets were blocked with skimmed milk 3% in phosphate-buffered saline (PBS) for 30 min and exposed to sera (30 µl/lane) for 60 min. After washing with PBS three times (10 min each), profiles were treated with either goat anti-human polyvalent Ig conjugated with alkaline phosphatase (Sigma) 5 µl/lane or goat anti-rabbit IgM (Southern Biotechnology Associates, USA) 5 µl/lane in skimmed milk-PBS for 60 min. After washing, as above, profiles were placed in substrate buffer (0.1 M Tris, 0.09 M NaCl, 0.15 M Mg₂Cl.6H₂O) for 10 min. For colour development, NCP strips were placed in 10 ml of substrate buffer containing 45 µl of nitroblue tetrazolium (Sigma; 75 mg/ml in 70% aqueous dimethyl formamide) and 35 µl of 5-bromo-4-chloro-3-indolylphosphate. Na₂ (Sigma; 50 mg/ml in de-ionised water).

	Results

TOP Abstract Introduction Materials and methods Results Discussion References

 
SDS-PAGE and immunoblotting

Initially, five strains of E. coli belonging to serotypes O26:H11 (E36039), O103:H2 (E05276), O111:H^- (E52849), O145:H25 (E38938) and O157:H^- (E32511) were examined; all expressed long-chain LPS as detected by SDS-PAGE and silver staining (Fig. 1, lanes 1–5). When these profiles were exposed to sera from patients infected with O157 VTEC, antibodies bound to the long-chain LPS components of O157 LPS (Fig. 1, lane 10) in sera from all 10 patients. However, six of the patients were also shown to have antibodies that bound to the core region only of the remaining LPS preparations (Fig. 1, lanes 6–9).

View larger version (81K): [in this window] [in a new window]   Fig. 1. The five strains of E. coli belonging to serogroups O26, O103, O111, O145 and O157 expressed long-chain LPS as detected by SDS-PAGE and silver staining (lanes 1–5). Patients’ serum antibodies bound to the core LPS of all five strains (arrow) (lanes 6–10), but bound only to long-chain LPS expressed by E. coli O157 (lane 10).

Profiles of the five preparations of LPS were also treated with rabbit antibodies prepared to E. coli O26, O103, O111, O145 and O157. The sera reacted with the homologous LPS preparations only and antibodies did not bind to the core region of the heterologous LPS preparations (not shown). Human serum antibodies binding to the core region of EHEC LPS did not bind to the core LPS of VT-negative strains of O157:H19 (E10964) expressing O157 LPS profile A, or E. coli O157:H45 (E59055) expressing O157 LPS profile B (not shown). Similarly, the core-binding antibodies did not bind to the LPS profiles from strains of Y. enterocolitica O9 (E4610), V. cholerae O1 Inaba (SC1074), S. Urbana (S127490) or C. freundii (E69366). The 10 control sera did not contain antibodies to long-chain LPS or core regions of any of the LPS preparations used.

The LPS preparations from the remaining 23 strains of EHEC listed in Table 1 were also exposed to six sera from patients with antibodies to E. coli O157 LPS containing antibodies to the core region of five LPS types described above. The core-LPS of 13 of the 23 strains bound antibodies present in the patients’ sera (Table 1). These 13 strains comprised five strains belonging to serogroup O111, and strains belong to serogroups O26, O104, O105ac, O145(2), O153 and O165. The antibodies also bound to the core-LPS of strain E31705 that did not express long-chain LPS.

Immunoblots, resulting from the routine serodiagnosis of 600 patients shown not to have produced serum antibodies to O157 LPS, were examined for antibodies binding to the LPS core-region, thought to be R3. Only two were detected (E134825 and E160315). Both were from young girls with HUS and VTEC O26 had been isolated from both patients. When LPS profiles prepared from E. coli strains O26, O103, O111, O145 and O157 were exposed to these sera, antibodies were found to bind to the core LPS of all five strains, but only to the long-chain LPS of E. coli O26. The presence of the common LPS-core region, thought to be R3, in strains of E. coli O26:H11 was examined further with five strains of EHEC and five non-VTEC strains of O26:H11 (Table 2). All 10 strains were found to bind human antibodies to the LPS R3 core region.

	Discussion

TOP Abstract Introduction Materials and methods Results Discussion References

 
The observation that patients’ antibodies also bound to the core of the LPS prepared from the non-O157 EHEC strains examined supported the theory that certain EHEC have shared core components, thought to be the R3 core region [17, 18, 25]. These antibodies did not bind to the core LPS from E. coli O157:H19 (E10964) and E. coli O157:H45 (E59055) showing that the core epitopes were associated with EHEC O157 and were not merely a common property of strains of E. coli belonging to this serogroup.

The five EHEC LPS profiles prepared from strains of O26, O103, O111, O145 and O157 were shown to bind homologous antibodies raised in rabbits [26], but these rabbit sera did not contain antibodies to the shared LPS-core epitopes. The type-strains of E. coli used to prepare the rabbit sera to O26, O103, O111 and O145 were isolated in the early 1950s and 1960s, and the precise source of these strains is unknown; the type-strain of E. coli O157 was isolated from a piglet [27]. These type-strains did not carry the genes encoding VT.

In addition to the five E. coli strains belonging to serotypes O26:H11, O103:H2, O111:H^-, O145:H25 and O157:H^-, 13 other strains also bound core-LPS antibodies present in six of the 10 human sera and these included five belonging to serogroup O111, and strains of serogroups O26, O104, O105ac, O145, O153, O165 and O-rough. Ten of the 28 strains did not bind the antibodies directed at core-LPS, indicating that not all VTEC or EHEC have the common (R3) core epitopes. The strain of O55:H10 used in this study did not bind antibodies directed at LPS-core, although Amor et al. [17] detected R3 core-LPS in a strain of VTEC belonging to serotype O55:H7. A study examining antibody cross-reactions between E. coli O157 and 19 strains of E. coli O55 with a range of flagellar types [28] did not detect binding of human serum antibodies specific for O157 LPS to the LPS of any of the O55 strains tested. This suggested that the R3 core might be expressed only rarely by strains of E. coli O55. The patients’ antibodies did not bind to the LPS of strains of Y. enterocolitica O9, V. cholerae O1 Inaba, S. Urbana or C. freundii, indicating that the epitopes on the R3 core were not present on these bacteria either.

Two sera were shown to contain antibodies binding to the LPS core, thought to be R3, but not to long-chain O157 LPS. The patients from whom these two sera were obtained both developed HUS and were subsequently found to have been infected with strains of E. coli O26:H11 with genes encoding VT2, and VT1 and VT2, respectively. Strains of VTEC and non-VTEC O26:H11 were shown to have the cross-reacting LPS core region, indicating that this was a common feature of strains belonging to this serotype, irrespective of whether they were VTEC or not.

The R1, R2, R3, R4 and K-12 LPS core structures have been partially elucidated [17, 29] and strains of E. coli with R3 core share an inner core sugar – 3-deoxy-D-manno-oct-2-ulsonic acid (Kdo) – with strains of Salmonella spp. [29]. However, as patients’ serum antibodies binding to EHEC LPS did not bind to the core of S. Urbana, this moiety of the core was not thought to be involved in antibody binding. The outer-core region of R3 E. coli LPS contains arrangements of glucose and N-acetyl-glucosamine [28] which are likely to be involved in antibody binding; however, the precise role of these components in forming the common R3 epitope(s) remains to be determined.

Sera from patients infected with O157 VTEC did not contain antibodies binding to the long-chain LPS of O26, O103, O111 and O145; therefore, it was concluded that the presence of shared R3 epitopes would not influence the results of routine serology. Furthermore, because only two of 600 sera from patients shown not to have antibodies to O157 LPS expressed antibodies binding to the LPS core-region, it is concluded that human serum antibodies to the R3 core, other than that of O157 VTEC, are rare. It was demonstrated in this study that routine serodiagnosis of infections with E. coli O157, with an established immunoblotting procedure [8], continues to be a reliable method of providing evidence of infection with E. coli O157 in the absence of faecal isolates of O157 VTEC.

	References

TOP Abstract Introduction Materials and methods Results Discussion References