Sequence analysis.

Sequencing was performed with the ABI PRISM Dye Terminator cycle sequencing kit (Perkin-Elmer, Foster City, Calif.). Gels were run by the Biomedical Instrumentation Center at the Uniformed Services University of the Health Sciences. Sequence comparisons were done with the Wisconsin Sequence Analysis package from the Genetics Computer Group, Inc.

His-tagged intimin protein purification.

His-tagged intimin protein RIHisEae and His-tagged intimin protein fragments were induced in late-log-phase cultures with 1 to 2 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The His-tagged proteins were then purified over nickel affinity columns essentially as described previously (47), except that after elution from the columns, the proteins suspended in elution buffer were dialyzed sequentially against 6, 4, and 2 M urea buffers and finally against phosphate-buffered saline (PBS). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of His-tagged intimin protein fragments is shown in Fig. ​Fig.11.

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FIG. 1

(A) His-tagged intimin constructs used in this study. Full-length intimin contains 934 amino acids. The RIHisEae, His-IntN_1/3, and His-IntN_2/3 proteins lack the N-terminal 35 amino acids of the native protein (47). Each of the intimin fragments has a six-histidine tag at the amino terminus. The construction of the plasmids encoding these proteins is described in Materials and Methods; the plasmid encoding RIHisEae was described previously (47). (B) Coomassie-stained SDS-PAGE gel of His-tagged intimin fragments designed for this study after purification by nickel affinity chromatography. Lane 1, His-IntN_1/3; lane 2, His-IntMid_1/3; lane 3, His-IntN_2/3; lane 4, His-IntC_1/3; lane 5, His-IntC_1/2. The stained gel was scanned with a Hewlett-Packard ScanJet 4C scanner, and the figure was labeled with Adobe Photoshop version 5.0.2 for Macintosh.

Production of antisera.

For preparation of antisera against RIHisEae, protein was sent to Duncroft, Inc. (Lovettsville, Va.), where a goat was immunized with the material emulsified in Freund's adjuvant. For preparation of antisera against the C-terminal half of intimin, protein was sent to Cocalico Biologicals, Inc. (Reamstown, Pa.), where a rabbit was immunized with the material emulsified in Freund's adjuvant. For preparation of antisera against the C-terminal third of intimin, we immunized four mice with the protein and TiterMax (CytRx Corp., Norcross, Ga.) as an adjuvant. For all animals, prebleeds were obtained before immunization.

Affinity purification of antibodies against different fragments of intimin.

Antibodies specific to different regions of the intimin protein were purified from the goat anti-RIHisEae serum. This serum contained antibodies that recognized N-terminal, central, and C-terminal portions of intimin, as observed by reactivity on an immunoblot with the various intimin protein fragments (data not shown). Affinity columns were prepared. Each column contained a different His-tagged intimin protein fragment coupled to Affi-Gel immunoaffinity support resin (Bio-Rad, Hercules, Calif.) according to instructions from the manufacturer. The antigen columns were prewashed with the buffer to be used for antibody elution, 100 mM glycine (pH 2.5), to remove any contaminants before passage of the serum over the columns, and the columns were then neutralized with Tris-HCl (pH 7.5). Passage of the sera over the immunoaffinity columns and elution of the affinity-purified antibodies were done according to protocols described by Harlow and Lane (26) and the Bio-Rad instructions.

The anti-His-IntN_2/3 antibody preparation was concentrated by ultrafiltration with a Microsep 30K concentrator (Pall Filtron Corp., Northborough, Mass.) to bring it to a concentration similar to that of the anti-C-terminal antibody fraction. Both antibody preparations were dialyzed against tissue culture grade PBS (BioWhittaker, Walkersville, Md.) for use in the HEp-2 cell adherence assay. The concentration of the anti-His-IntN_2/3 antibody preparation, as determined by the method of Bradford (Bio-Rad protein assay reagent) with bovine serum albumin as the standard, was 0.24 mg/ml; the concentration of the anti-His-IntC_1/3 preparation was 0.27 mg/ml. The antibody preparations were also analyzed by SDS-PAGE to confirm that they had similar concentrations (data not shown). To assess blocking capacity in adherence assays (described below), equal microgram quantities of each antibody fraction were compared; PBS was added to the anti-His-IntC_1/3 antibody samples so that equal volumes of the anti-N- and anti-C-terminal antibodies were mixed with bacteria.

Adherence assays.

Adherence of EHEC to HEp-2 (human laryngeal epithelial) cells was assessed essentially as described previously (10, 47), with slight modifications. For the assays, 6 × 10⁴ HEp-2 cells were seeded per well into eight-well plastic chamber slides (Nalge Nunc International, Naperville, Ill.) and grown for 20 to 24 h to form subconfluent (approximately 80% confluency) monolayers. Freshly grown single bacterial colonies were inoculated into 0.5-ml Luria broth cultures and grown overnight statically. Preimmune sera and sera from immunized animals were heated to 56°C for 30 min to inactivate complement prior to the assay. Equal volumes of preimmune and postimmunization sera were compared for each strain. An equal volume of PBS buffer was added to an additional well for comparison. Bacteria and serum (or PBS) were preincubated in 300 μl of adherence medium (Eagle minimum essential medium [BioWhittaker], 1% mannose, 0.4% sodium bicarbonate) at 37°C for 35 min, and tubes were mixed gently three times during this period. HEp-2 cell monolayers were washed twice with Hanks balanced salt solution (with Mg²⁺ and Ca²⁺), the preincubated solutions of bacteria were added to the cells, and HEp-2 cells and bacteria were incubated for 3 h at 37°C. The wells were washed once with Dulbecco's PBS (with Mg²⁺ and Ca²⁺), and fresh medium containing antisera (or PBS buffer) was added for a second 3-h incubation. The monolayers were then washed six times with PBS. Cells were fixed and stained with the Hema-3 staining solution kit according to instructions of the manufacturer (Biochemical Sciences, Inc., Swedesboro, N.J.).

To assess the effect of the purified antibodies on the adherence assay, several minor modifications were made to optimize the above procedure. First, fetal bovine serum, preheated to inactivate complement factors, was added to the adherence medium to a 0.5% final concentration. Second, the bacteria were preincubated in adherence medium for 18 to 20 min before addition of antibodies. An additional well that contained PBS without antibodies was included as a control.

To evaluate the degree of bacterial adherence in each assay, slides were first scored by blinded microscopic observation, i.e., without a key as to which wells contained which antiserum or PBS. Following this, to quantitate the adherence of the bacteria, eight (or more) randomly selected fields from each chamber slide well that contained either preimmune serum or postimmunization serum were photographed and the bacteria and HEp-2 cells were counted. Quantitation was done by this method because, as previously reported, visual observation of the bacteria in situ on the HEp-2 cells was a more sensitive method for detection of differences between strains than viable counts of bacteria eluted from the wells (47). Since this may be due to excess bacteria that were observed to be nonspecifically adherent to the plastic along the very edges of the wells, we did not include fields at the very edges of wells for quantitation. For each independent assay, the total number of HEp-2 cells counted in each set of eight photographs for a serum-containing well was at least 100. For each assay done with affinity-purified antibodies, photographs of 11 or 12 random fields for each well were taken; at least 160 HEp-2 cells per antibody or buffer-alone sample were counted, and at least 3,500 bacteria were counted for the anti-His-IntN_2/3 samples. Statistical analysis of the data was performed as described in Tables ​Tables2 and2 and ​and33.

TABLE 3

Adherence of strains of different serotypes in the presence of polyclonal serum that contains antibodies againstintimin_O157

Straina	Serotype	Assay	Avg no. of adherent bacteria ± 2SEM/10 HEp-2 cells in the presence ofb:		Avg fold reduction in adherence by anti-intiminO157 serum (range)c
			Preimmune serum	Anti-intimin serum
86-24	O157:H7	I	106±25	10±5	8.1(4.7–10.9)
		II	50±13	6±2
		III	158±26	34±10
EDL933	O157:H7	I	263±66	30±10	5.7(3.7–8.9)
		II	195±25	43±12
		III	220±39	60±21
97-3256	O55:H7	I	109±22	11±6	10.3(4.9–16.4)
		II	187±49	11±4
		III	136±34	28±10
85-3007	O111:NM	I	57±19	16±12	3.0(1.7–3.7)
		II	29±9	17±13
		III	61±19	17±5
H19	O26:H11	I	58±13	25±7	1.9(1.6–2.3)
		II	67±17	37±13
		III	65±15	41±16

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^aThe numbers of bacteria added to the assay mixtures with respect to strain 86-24 (n = 1.2 × 10⁷) were as follows: EDL933, n/2; 97-3256, n/15; 85-3007, n; H19, n/5. Serum dilutions were 1:140 for strains 86-24 and EDL933 and 1:90 for strains 97-3256, 85-3007, and H19. 

^bThree independent assays were performed. For each assay, bacteria and HEp-2 cells were counted in 8 to 14 microscope fields per sample. The total number of HEp-2 cells counted in the set of fields observed for each sample was at least 100. The difference between the levels of adherence of the O157:H7 and O55:H7 strains in the presence of preimmune serum and polyclonal anti-intimin serum was significant, as calculated by Student's unpaired t test; P < 0.001 for each assay. For O111:NM strain 85-3007, the difference between the sera in assay II was not significant (P > 0.05); the P values for assays I and III were <0.01 and <0.001, respectively. For O26:H11 strain H19, the P values were <0.001, <0.02, and <0.05, respectively, for assays I to III. 

^cThe fold reduction in adherence in the presence of anti-intimin antiserum was calculated with respect to the preimmune serum control and was computed before rounding the numbers for each sample. The average fold reductions and ranges for the three assays are presented. For strains 95-3208, 97-3250, and E2348/69 there was no anti-intimin_O157-specific reduction in adherence. 

Polyclonal sera raised against C-terminal fragments of intimin reduced adherence of strain 86-24.

As one way to investigate the importance of the C-terminal region of intimin in the adherence-reducing capacity of anti-intimin antisera, we immunized a rabbit with the His-tagged C-terminal half fragment (His-IntC_1/2) and four mice with the His-tagged C-terminal one-third fragment (His-IntC_1/3). Intimin fragments are shown in Fig. ​Fig.1.1. As assessed by microscopic observation, serum samples from the rabbit immunized with His-IntC_1/2 reduced adherence of 86-24 to a greater extent than did the preimmune serum (Table ​(Table2).2). The antisera from three of the four immunized mice also reduced adherence of 86-24 to a greater extent than did the preimmune sera from the respective mice, although to different degrees (Table ​(Table2).2). No difference between pre- and postimmunization sera from one of the mice was observed; this result is addressed in Discussion.

An affinity-purified antibody fraction containing antibodies to the C-terminal third of intimin reduced adherence of strain 86-24 to HEp-2 cells.

Since serum contains other factors in addition to antibodies, we sought to test our hypothesis that the reduction in adherence in the presence of anti-intimin_O157 antisera was due to the presence of anti-intimin antibodies. Additionally, we sought to investigate by another method the importance of antibodies to the C-terminal region of intimin, suggested by our results above, for the effect on adherence. We thus purified two antibody fractions from the goat anti-intimin_O157 antiserum for use in the adherence assays: an antibody fraction that contained antibodies that recognized the C-terminal third of intimin, His-IntC_1/3, and another fraction that contained antibodies that recognized the N-terminal two-thirds of intimin, His-IntN_2/3. Since the antibodies were stored in a saline-containing buffer, as recommended by Harlow and Lane (26), we chose to limit the volume of antibody preparations added to the adherence assay mixture because we subsequently observed that adherence could also be affected by excess salt in the assay mixture, at amounts approximately two to three times what we added in these experiments (data not shown). It is potentially relevant to the latter finding that we repeated the experiment on which the published observation (18) that purified EHEC intimin protein can bind to HEp-2 cells was based (data not shown), and observed that this intimin-cell interaction could be disrupted by the addition of similar amounts of excess salt (59). The amounts of antibody tested in the bacterial adherence assays for this study were limited to a range of 0.8 to 4.0 μg.

The anti-His-IntC_1/3 antibody fraction reduced the adherence of strain 86-24 to HEp-2 cells compared to that of control samples that received an equal volume of PBS buffer alone, but the antibody fraction that contained antibodies to the N-terminal two-thirds portion of intimin did not block adherence at all at any amount tried. While 0.8 μg of the anti-His-IntC_1/3 antibody fraction did not show a consistent effect on adherence, the 2.4- and 4-μg amounts both showed reproducible effects, with the effect of 4 μg about twice that of 2.4 μg. Data for 4 μg of the anti-His-IntC_1/3 antibody fraction are presented in Table ​Table2;2; in three independent experiments, performed on different days, the anti-His-IntC_1/3 antibody fraction reduced adherence of strain 86-24 to HEp-2 cells by an average of 4.4-fold compared to adherence levels seen in the presence of the anti-His-IntN_2/3 antibodies. The results observed with the fraction of antibodies containing reactivity to the C-terminal third of intimin indicate that anti-intimin antibodies can reduce adherence to HEp-2 cells and that epitopes in the C-terminal third of intimin are critical for this effect.

Immunoblot analysis with whole-cell lysates of E. coli strains demonstrated the presence of reactivity in these antibody fractions to the 97-kDa intimin protein in the pathogenic E. coli examined. The immunoblots revealed additional reactivity to one other band, of approximately 15 kDa, that was present in both clinical and nonpathogenic laboratory strains. However, for reasons discussed below, we think that the antibodies to the C-terminal domain of intimin caused the reduction in adherence of strain 86-24.

Polyclonal antisera raised against intimin from O157:H7 strain 86-24 reduced the HEp-2 cell adherence of another O157:H7 strain and that of some, but not all, strains of other serotypes.

Next, we sought to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of EHEC strains of other serotypes. For these experiments, we chose to analyze (i) an additional strain of the O157:H7 serotype to complement our studies on 86-24; (ii) an O55:H7 strain, since this serotype is closely related to O157:H7 strains (54, 67, 68); and (iii) strains from the O26 and O111 serogroups, since these are the most frequent non-O157:H7 isolates from hemorrhagic colitis and HUS patients (58). We analyzed two independently isolated O26:H11 strains and two independently isolated O111:NM strains. Additionally, we tested one EPEC strain, O127:H6 strain E2348/69. All of the strains were isolated either from human patients or from contaminated food associated with an outbreak and are described in Table ​Table1.1. Since some of these strains, especially the recent isolates, had not been characterized with respect to A/E lesion formation, we first tested all of the strains in HEp-2 cell assays for the capacity to rearrange cellular actin, a hallmark of the A/E lesion and thus of intimin function. We used the standard fluorescence actin staining (FAS) test (33); as expected, all of these eae⁺ strains were FAS positive (data not shown).

Trial assays were done to assess the adherence capacity of each eae⁺ strain. Most of the strains displayed a greater level of adherence to HEp-2 cells than did strain 86-24 (data not shown). For more convenient comparison with strain 86-24, cultures of these other strains were diluted prior to the assays, as noted in Table ​Table3.3. Additionally, we further titrated down the number of bacteria in an attempt to optimize the adherence-blocking effects for strains that did not show a strong effect initially. To observe a reduction of adherence by the goat anti-intimin_O157 serum, some strains also required the addition of more serum than was necessary to see an effect on strain 86-24. Data are presented in Table ​Table33 for three independent assays performed on different days.

Addition of anti-intimin_O157 antisera to the HEp-2 cell assay mixture reduced adherence of some of the strains compared with their adherence in the presence of preimmune serum; despite some day-to-day variation in the assay, the greatest fold reductions were seen with the two O157:H7 strains and the EHEC O55:H7 strain (Table ​(Table3). Somewhat3). Somewhat lesser effects, on average, were seen for strain 85-3007 (O111:NM) and for strain H19 (O26:H11). The reduction in adherence seen with these strains was readily apparent by microscopic observation even before bacterial counts were assessed. However, there was no specific anti-intimin_O157 serum effect on adherence of strains 95-3208 (EHEC O111:NM), 97-3250 (EHEC O26:H11), and E2348/69 (EPEC O127:H6), as assessed by microscopic observation, in at least two independent assays in which different amounts of sera and bacteria were tested. Thus, adherent bacteria were not counted for these strains.

We also tested our rabbit anti-His-IntC_1/2 polyclonal antisera for the capacity to reduce adherence of the strains for which an effect was seen with the anti-intimin_O157 serum, as described above. These included O157:H7 strains 86-24 and EDL933, O55:H7 strain 97-3256, O111:NM strain 85-3007, and O26:H11 strain H19. In support of the above results, the rabbit polyclonal serum reduced adherence of these strains compared to the preimmune serum control, as assessed by microscopic observation; the effects on adherence were most readily observed with the two O157:H7 strains, 86-24 and EDL933 (data not shown).

This work was supported by grant AI20148-16 from the National Institutes of Health and by grant 97-35201-4578 from the U.S. Department of Agriculture.

We are grateful to N. Strockbine for providing us with strains 97-3250, 97-3256, 85-3007, and 95-3208 and for helpful discussion. We thank W. D. Roof and R. Young for the generous gift of strain RY2844. We are grateful to T. S. Whittam and S. Plock for the determination of the intimin subtypes of strains 97-3250, 97-3256, 85-3007, and 95-3208 and for communication of these unpublished results. We thank Angela Melton-Celsa for helpful discussion and Clare Schmitt and James Sinclair for critical reading of the manuscript.