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Infection and Immunity, January 2009, p. 170-179, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00943-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cytotoxic Necrotizing Factor Type 1-Neutralizing Monoclonal Antibody NG8 Recognizes Three Amino Acids in a C-Terminal Region of the Toxin and Reduces Toxin Binding to HEp-2 Cells

Kerian K. Grande,¹ Karen C. Meysick,² Susan B. Rasmussen,¹ and Alison D. O'Brien^1*

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814,¹ FDA/CBER, Bethesda, Maryland 20892²

Received 29 July 2008/ Returned for modification 17 September 2008/ Accepted 22 October 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are toxins of pathogenic Escherichia coli that share 85% identity over 1,014 amino acids. Although both of these toxins modify GTPases, CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and, despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than CNF2. Here we exploited the differences between CNF1 and CNF2 to define the epitope on CNF1 to which the CNF1-specific neutralizing monoclonal antibody (MAb) (MAb NG8) binds and to determine the mechanism by which MAb NG8 neutralizes CNF1 activity on HEp-2 cells. For these purposes, we generated a panel of 21 site-directed mutants in which amino acids in CNF1 were exchanged for the amino acids in CNF2 between amino acids 546 and 869 and vice versa. This region of CNF1 not only is recognized by MAb NG8 but also is involved in binding of this toxin to HEp-2 cells. All the mutants retained the capacity to induce multinucleation of HEp-2 cells. However, the CNF1 double mutant with D591E and F593L mutations (CNF1_{D591E F593L}) and the CNF1_H661Q single mutant displayed drastically reduced reactivity with MAb NG8. A reverse chimeric triple mutant, CNF1_{E591D L593F Q661H}, imparted MAb NG8 reactivity to CNF2. MAb NG8 neutralized CNF2_{E591D L593F Q661H} activity in a dose-dependent manner and reduced the binding of this chimeric toxin to HEp-2 cells. Taken together, these results pinpoint three amino acids in CNF1 that are key amino acids for recognition by neutralizing MAb NG8 and further help define a region in CNF1 that is critical for full toxin binding to HEp-2 cells.

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Cytotoxic necrotizing factor type 1 (CNF1), a toxin made by many uropathogenic and other extraintestinal isolates of Escherichia coli, is a 115-kDa member of the Rho family of GTPase-activating toxins (for reviews, see references 2, 3, 6, 19, and 26). CNF1 deamidates glutamine 63 (Q63) of RhoA and glutamine 61 (Q61) of Rac1 and Cdc42; these modifications lead to constitutive activation of the target GTPases (1), which in turn results in downstream effects on the mammalian cell phenotype and cell cycle. The typical phenotype associated with CNF1 intoxication in cell cultures is multinucleation, as has been reported previously for HEp-2 cells (14, 15), HeLa cells (13), and Swiss 3T3 cells (23). In addition, this toxin can be cytotoxic to cell lines such as 5637 human bladder cells (25). CNF1-intoxicated cells may also display formation of stress fibers, focal adhesions, lamellipodia, and filopodia and rearrangement of the actin cytoskeleton (16, 20, 30). In vivo, CNF1 evokes necrosis when it is injected intradermally into rabbits (9). The contribution of CNF1 to uropathogenic E. coli virulence has also been demonstrated using a rat model of acute prostatitis (28), as well as a mouse model of ascending urinary tract infection (11, 29, 33). In the latter model, intraurethral infection with a CNF1-positive strain leads to an increased inflammatory response compared to the response with a CNF1-negative strain (28, 33). CNF1 is also responsible for submucosal edema in uropathogenic E. coli-infected mouse bladders and for modification of the phagocytic and killing activities of murine polymorphonuclear cells (11, 28).

CNF1 shares 61% amino acid identity with CNFY from Yersinia pseudotuberculosis and 70% identity with CNF3 from necrotoxigenic E. coli (21, 27). CNF1 more closely resembles CNF2, another GTPase-activating cytotoxin (85% amino acid identity and 90% similarity over the entire toxin molecule [1]). Although CNF1 and CNF2 have a high degree of sequence similarity and have the same catalytic residues (C866 and H881), these toxins are produced by different E. coli isolates and have disparate effects in animal models and in cell culture. Notably, CNF1-intoxicated HeLa cells are enlarged, rounded, and multinucleate and may display filamentous tendrils, whereas CNF2-treated HeLa cells, although enlarged, are elongated and only moderately multinucleate (4, 12). The different effects of CNF1 and CNF2 on HeLa cells may be attributed to differences in the preference of these two toxins for small GTPase substrates (34). Furthermore, McNichol et al. (22) recently showed that the capacity of CNF2 to bind to HEp-2 cells was significantly reduced but that CNF2 bound to the laminin receptor precursor protein (LRP) at levels similar to those of CNF1. Together, these results provide evidence that small regions of CNF1 and CNF2 may significantly influence the phenotype of each toxin (24).

Several attempts have been made to analyze the relationship between the structure and function of CNF1. Meysick et al. (24) generated a panel of monoclonal antibodies (MAbs) that were used in conjunction with a series of CNF1 deletion constructs to identify the functional regions of this toxin and epitopes unique or common to CNF1 and CNF2. One of the MAbs in this panel, CNF1-specific MAb NG8, was found to bind to a region of the toxin from amino acids 546 to 869, and loss of MAb NG8 reactivity was apparent after deletion of amino acids 373 to 783. In a more recent study performed by Hoffman et al. (18), chimeric constructs were designed to exchange functional regions between CNF1 and a homologous toxin, CNFY, to determine the regions of the toxins responsible for the difference in the cellular phenotypes observed. At that time, the N terminus of CNF1 was believed to be important for cell binding, the middle portion of CNF1 was believed to be important for translocation, and the C terminus was believed to be important for catalysis. In fact, two specific residues in the C terminus of CNF1, C866 and H881, had previously been shown to be essential for deamidation activity (7). In a third study, McNichol et al. (22) proposed that there are two receptor-binding domains for CNF1, the first located in the N terminus (amino acids 135 to 164) and the second located in the C-terminal portion of CNF1 (amino acids 683 to 730). These domains were hypothesized to interact and form a three-dimensional receptor-binding domain.

In this study, we exchanged disparate amino acids in a region of CNF1 and CNF2 (amino acids 546 to 869) in order to identify the specific amino acids that comprise the neutralizing epitope of MAb NG8 and to determine the mechanism of toxin neutralization. Since this region of CNF1 included one of the proposed receptor-binding domains of CNF1 (22), we also investigated whether the amino acids in the MAb NG8 epitope and the amino acids responsible for differences in CNF1 and CNF2 HEp-2 cell binding were the same. We found that when three noncontiguous amino acids from CNF1 were exchanged for the amino acids present at the same positions in CNF2, MAb NG8 recognized the chimeric CNF2 molecule with the E591D, L593F, and Q661H mutations (CNF2_{E591D L593F Q661H}) and had the capacity to neutralize CNF2 intoxication of HEp-2 cells. In addition, we showed that the extent of binding of the CNF2_{E591D L593F Q661H} mutant toxin to HEp-2 cells was reduced in the presence of MAb NG8.

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Bacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. E. coli strain XL1-Blue was used for maintenance of plasmids. The E. coli strains were grown at 37°C with shaking in Luria-Bertani (LB) broth supplemented with 100 µg ml^–1 ampicillin and/or 25 µg ml^–1 kanamycin. E. coli strain M15(pREP4) was used for expression of histidine (His)-tagged proteins.

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TABLE 1. Bacterial strains and plasmids

Construction of CNF1 and CNF2 site-directed mutants. Constructs designed to exchange one to three amino acids that were different in CNF1 and CNF2 were generated by using a QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Briefly, two complementary primers that were 36 to 51 bases long and had the desired mutations were synthesized by the Biomedical Instrumentation Center at the Uniformed Services University of the Health Sciences (USUHS) (Table 2). Both of these primers were used in a PCR with plasmid pCNF24 or p2CNF as the template. High-fidelity PFU Turbo and PFU Ultra polymerases (Stratagene) along with reduced amounts of template (10 ng) and a reduced number of cycles (16 cycles) were used according to the manufacturer's instructions to amplify the plasmid products with the desired mutations and to minimize extraneous mutations. Restriction enzyme DpnI was then added to the amplification reaction mixture to eliminate the parental plasmid template. Plasmids were transformed into E. coli XL1-Blue according to the manufacturer's instructions and were purified with a QiaprepSpin miniprep kit (Qiagen). To verify all mutations, plasmids were sequenced at the Biomedical Instrumentation Center at the USUHS.

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TABLE 2. Primers used for site-directed mutagenesis

Protein purification. E. coli strain M15(pREP4) transformed with recombinant CNF plasmids was grown at 37°C with shaking in LB broth that contained 100 µg ml^–1 ampicillin and 25 µg ml^–1 kanamycin. Cultures were grown at 37°C to an optical density at 600 nm of 0.6 to 0.7, induced with 0.1 mM (final concentration) isopropyl-beta-D-thiogalactopyranoside (IPTG), and then incubated for an additional 4 h at 37°C before the bacteria were harvested by centrifugation. Bacteria were resuspended in binding buffer (pH 7.8) (50 mM sodium phosphate [pH 7.8], 300 mM NaCl, 5 mM imidazole) that contained protease inhibitors (Complete Mini EDTA-free protease inhibitor cocktail; Roche Diagnostics, Mannheim, Germany) and lysed by sonication. CNF1, CNF2, and mutant toxins were purified from bacterial lysates by fast protein liquid chromatography (FPLC) using a HisTrap Ni²⁺ affinity column with an AKTA system (GE Healthcare) according to the manufacturer's instructions. Buffer exchange from 250 mM imidazole to 20 mM Tris-HCl (pH 7.8) was done by performing FPLC with HiTrap HP desalting columns (GE Healthcare). The purity of the toxins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an 8% Tris-glycine gel, and the gel was stained with Coomassie blue G-250 (Bio-Rad) or analyzed by Western blotting (see below).

Recombinant RhoA was also purified by FPLC. Briefly, recombinant plasmid pGEX-2T-wtRhoA, which encodes a glutathione S-transferase-tagged RhoA GTPase (a gift from Alan Hall, University College London, London, United Kingdom [32]), was transformed into E. coli DH5. Strain DH5/pGEX-2T-wtRhoA was grown in LB broth that contained 100 µg ml^–1 ampicillin at 37°C for 1 h, induced with 0.1 mM IPTG, and grown for an additional 4 h. Bacteria were subsequently concentrated by centrifugation and lysed by sonication. RhoA was purified from clarified bacterial lysates over a GSTrap HP affinity column (GE Healthcare) used according to the manufacturer's instructions.

The concentrations of all purified proteins were determined with a bicinchoninic acid protein assay kit (Pierce). Comparisons of toxins in various assays were done by using equal protein concentrations.

Western blotting. Western blot analysis was carried out as previously described (24), with the following modifications. Equal concentrations of purified toxins were subjected to SDS-PAGE (8% Tris-glycine gels; Invitrogen, Carlsbad, CA). After electrophoretic separation, the proteins were transferred to 0.45-µm nitrocellulose membranes with a Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad), and the membranes were then blocked overnight in phosphate-buffered saline (PBS) that contained 5% skim milk and 0.1% Tween 20 (PBS-T). Membranes were incubated with CNF1 MAb NG8 or an RGS His-tagged MAb (Qiagen), washed in PBS-T, and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Roche). Reactive proteins were detected by enhanced chemiluminescence (GE HealthCare, Buckinghamshire, United Kingdom).

Assessment of MAb NG8 reactivity using ELISAs. The capacity of site-directed mutant toxins to react with MAb NG8 was assessed by using enzyme-linked immunosorbent assays (ELISAs). Briefly, 5 µg of purified CNF1, CNF2, or mutant toxin in 20 mM Tris-HCl (pH 7.8) was used to coat ELISA plates for 1 h at room temperature. A hybridoma culture supernatant of MAb NG8 was used as the primary antibody at a dilution of 1:10, and goat anti-mouse immunoglobulin G(H+L) [IgG(H+L)]-peroxidase F(ab')₂ (Roche) was used as a secondary antibody at a dilution of 1:3,000 in PBS-T with 3% bovine serum albumin. Antibodies were incubated for 1 h at room temperature. Color was developed with 3,3',5,5'-tetramethylbenzidine peroxidase (EIA substrate kit; Bio-Rad) used according to the manufacturer's instructions, and the reaction was stopped by addition of 1 N H₂SO₄. The final optical densities at 405 nm of samples were determined after subtraction of the value for a 20 mM Tris-HCl sample blank. All ELISAs were done in triplicate.

Cell lines and media. HEp-2 cells (human laryngeal cell line; ATCC CCL-23) were grown in an atmosphere containing 5% CO₂ at 37°C in Eagle's minimal essential medium with Earle's balanced salt solution (Lonza) supplemented with 10% fetal bovine serum (Biosource), 2 mM L-glutamine (Invitrogen), 10 µg ml^–1 gentamicin, 10 U ml^–1 penicillin, and 10 µg ml^–1 streptomycin.

HEp-2 cell multinucleation assay. HEp-2 cell multinucleation assays were performed as previously described (25), with the following modifications. Cells were seeded at a concentration of 5 x 10³ cells/well into 96-well plates (Costar) and incubated for 4 h at 37°C with 5% CO₂. Dilutions of purified CNF1, CNF2, and mutant toxins were applied to cells at a starting concentration of 330 ng per well. Cells were incubated for 72 h at 37°C in the presence of 5% CO₂, fixed, and stained with Hema-3 (Fisher Scientific), and the degree of multinucleation was determined by light microscopy.

In vitro neutralization assay. Purified toxins (0.33 µg/well) were incubated for 2 h at 37°C with 5 µg of purified MAb NG8 (IgG2a; Hycult, The Netherlands), with 1:6 dilutions of MAb NG8, or with one of the following negative control antibodies: 5 µg of MAb 11E10, an anti-Shiga toxin type 2 A-subunit MAb (IgG1; Hycult); or 1 µg of nonneutralizing anti-CNF1/CNF2 MAb GC2 (IgG2a hybridoma culture supernatant prepared in our laboratory). After incubation, toxin-MAb samples (100 µl) were added to a microtiter plate that was seeded with 5 x 10³ HEp-2 cells/well, and the multinucleation assay was performed as described above.

Modification of Rho GTPases. Deamidation assays were performed by using the method of Schmidt et al. (30), with the following modifications. Purified RhoA GTPase and toxin at a molar ratio of 16:1 were incubated in deamidation buffer (50 mM Tris-HCl [pH 7.4], 5 mM MgCl₂, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride) for 2.5 h at 37°C. Dihydrofolate reductase and buffer (20 mM Tris-HCl, pH 7.4) were used as negative controls. After toxin treatment, samples were precipitated by addition of 15% trichloroacetic acid and incubation for 2 h at 4°C. Precipitated proteins were pelleted, washed with acetone, air dried, and resuspended in 20 mM Tris-HCl (pH 7.4). Samples were subjected to SDS-PAGE with 12% acrylamide, and proteins were transferred to 0.45-µm nitrocellulose membranes with a Trans-Blot semidry electrophoretic transfer cell. The membranes were blocked overnight at 4°C in PBS-T, and then they were incubated with one of the following antibodies: mouse anti-RhoA MAb (1:1,500; Santa Cruz Biotechnology) or rabbit anti-RhoA polyclonal serum (1:2,000) that was raised against the deamidated RhoA peptide (22, 23, 34). Reactive proteins were subsequently detected with either HRP-conjugated goat anti-mouse IgG (1:7,500; Roche) or goat anti-rabbit IgG (1:3,000; Bio-Rad), followed by visualization with enhanced chemiluminescence (GE Healthcare). To determine the capacity of MAbs to neutralize CNF1 deamidation activity, the toxin was preincubated with 5 µg of either MAb NG8 or nonneutralizing CNF1 MAb GC2 hybridoma supernatants and the assay was performed as described above.

Binding of toxin to HEp-2 cells. Toxin-HEp-2 cell binding ELISAs were performed by using the protocol of McNichol et al. (22), with the following modifications. Approximately 2 x 10⁴ HEp-2 cells/well were seeded into 96-well plates and incubated overnight at 37°C with 5% CO₂. Cells were fixed with 3% formalin in PBS for 30 min at room temperature, washed with PBS, and then blocked for 1 h at 37°C with 1x PBS-3% bovine serum albumin. The wells were washed again, and purified toxins were added at a concentration of 50 µg/ml (5 µg/well). To determine the degree of nonspecific binding to cells, the following set of controls was included: no primary antibody, no secondary antibody, and no toxin. The plates were incubated for 1 h at 37°C to allow toxin to bind, the cells were washed to reduce nonspecific binding, and then the plates were incubated for 1 h at 37°C with goat anti-CNF1 polyclonal serum (1:5,000) (25) and for 1 h at room temperature with anti-goat HRP-conjugated IgG (1:7,500; Roche). Color was developed with 3,3',5,5'-tetramethylbenzidine peroxidase, and the reaction was stopped by addition of 1 N H₂SO₄. Samples (100 µl) were transferred to a second 96-well plate and read with an EL_x800 plate reader at 405 nm. One-way analysis of variance followed by Dunnett's two-sided post hoc test was performed to compare each mutant to CNF1.

Immunofluorescence studies were also performed to assess toxin binding to HEp-2 cells in the presence or absence of antibody, as follows. HEp-2 cells were seeded into Lab-Tek II eight-well glass chamber slides (Thomas Scientific) at a concentration of 2 x 10⁵ cells/well, and the slides were incubated for 24 h at 37°C in the presence of 5% CO₂. The slides were then chilled to 4°C and washed with cold Hanks' balanced salt solution (Invitrogen/Gibco) prior to addition of the toxin or toxin-antibody mixture (prepared as follows). Purified CNF1, CNF2, or toxin mutants diluted to obtain a final concentration of 10 µg/well in cold binding buffer without maltose (17, 25) were incubated with MAb NG8, MAb 11E10, or PBS for 2 h at 37°C. Control wells received binding buffer in the absence of toxin. Cells were incubated with toxin for 30 min at 4°C, washed with PBS, and fixed with 3% formalin for 20 min at room temperature. Wells were then blocked with 3% bovine serum albumin in PBS for 1 h at room temperature and incubated for 1 h at room temperature with goat anti-CNF1 polyclonal serum (1:500) (25). After washing with PBS, AlexaFluor 488-conjugated chicken anti-goat IgG (1:300; Invitrogen-Molecular Probes) was added and incubated for 1 h at room temperature. Finally, wells were washed again and fixed a second time for 10 min at room temperature, which was followed by incubation for 15 min with 0.1% Evans blue as a nonspecific protein stain. Prior to examination, slides were treated with SlowFade antifade kit solutions (Molecular Probes, Carlsbad, CA) and coverslips were applied. Cells were visualized at a magnification of x40, and images were taken by using the same exposure times with an Olympus BX60 microscope. Images were processed with LSM Image 5 Browser software. Punctate green signals indicative of bound toxin were enumerated visually, and the results were compared to the results for controls that included cells treated with only primary or secondary antibody or cells incubated with both primary and secondary antibodies but no toxin. MAb GC2, which reacts with but does not neutralize CNF1 or CNF2 (24), did not reduce the binding of CNF1 or the mutant toxins to HEp-2 cells in this assay (data not shown). A statistical analysis was performed by using randomized block analysis of variance with four preplanned post hoc comparisons, using Fisher's protected least significant difference. A significant overall treatment effect was indicated by a P value of 0.01, and the individual P values were calculated by using a pooled standard error of 1.36.

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Evaluation of CNF1 site-directed mutant toxins. We sought to define the specific amino acids that recognize the toxin-neutralizing MAb NG8 in CNF1 but not the highly related toxin CNF2. For this purpose, we generated a series of site-directed mutations in the genes that encode the CNF1 or CNF2 toxin (Fig. 1). As shown in Fig. 2A, all of the mutant toxins appeared to be relatively pure and could be detected at levels similar to those of the CNF1 and CNF2 parent toxins. To obtain an indication of conformational integrity, mutant toxins were initially examined to determine their capacity to induce multinucleation of HEp-2 cells, a phenotype that allows differentiation between intoxication with CNF1 and intoxication with CNF2. Each mutant toxin was found to possess multinucleation activity (data not shown) at a level similar to that of its parent toxin (CNF1 or CNF2); therefore, each toxin was considered to have maintained the native structure.

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FIG. 1. CNF1 and CNF2 site-directed mutants. Twenty-one CNF1 and CNF2 site-directed mutant toxins were generated, in which differential amino acids were exchanged in a functionally important region of CNF1 (gray boxes) (amino acids 546 and 869; the positions of the catalytic amino acids are indicated). All toxins contained an N-terminal histidine tag and maintained the capacity to multinucleate HEp-2 cells at levels similar to those observed with the parent toxin. The positions of the amino acids exchanged (open boxes, CNF1; black boxes, CNF2) and the reactivities of chimeric toxins with MAb NG8 (+ or –) are indicated. Most of the CNF1-derived mutant toxins are numbered (1 to 20); the exceptions are CNF1D704N T707I R709H, CNF1S720N, and CNF1K726 S727L F729L, which are indicated by an asterisk. Preliminary Western analysis (data not shown) revealed that these mutants did not contain the MAb NG8 epitope, and the toxins were not included in subsequent analyses.

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FIG. 2. Recognition of purified toxins by MAb NG8. (A) SDS-PAGE and Western blot analyses of toxins. Purified wild-type and CNF1 and CNF2 site-directed mutant toxins were analyzed by using Coomassie blue-stained SDS-PAGE gels and by Western blotting to determine reactivity with anti-RGS-His and MAb NG8, a CNF1-specific MAb. The gel lanes correspond to the toxin numbers shown in Fig. 1. Lanes containing CNF1- and CNF2-derived toxins are indicated by brackets. (B) ELISA to assess the reactivity of MAb NG8 with mutant toxins. The sample numbers correspond to the toxin and lane numbers in Fig. 1 and panel A, respectively. Toxins (5 µg) were used to coat ELISA plates and then incubated with MAb NG8 or 20 mM Tris HCl (blank) and the appropriate secondary antibody. ELISAs were done in triplicate. The error bars indicate ±1 standard deviation from the mean.

Identification of amino acids in CNF1 essential for MAb NG8 reactivity. Mutant toxins were also assessed to determine their capacity to bind to the CNF1-specific neutralizing antibody MAb NG8 by Western blot analysis (Fig. 2A). Most CNF1-based toxins were recognized by the control anti-RGS-His MAb, and similar levels were also detected with MAb NG8; the exceptions were mutant toxins CNF1_{D591E F593L} and CNF1_H661Q (Fig. 2A, lanes 7 and 9). Note that MAb NG8 also reacted to various degrees with 80-kDa truncated forms of the CNF1-based toxins (except CNF1_{D591E F593L} and CNF1_H661Q) (Fig. 2A, lanes 7 and 9), like the full-length toxins. The finding that truncated forms of CNF1 are present in purified preparations of the toxin has been reported previously by workers in our laboratory (22, 25).

As substitution of amino acids at positions 591, 593, and 661 led to loss of reactivity with MAb NG8, we reasoned that these amino acids might be part of the CNF1 MAb NG8 epitope. To determine whether recognition by CNF1-specific MAb NG8 could be imparted to CNF2, site-directed E591D, L593F, and Q661H mutants were constructed in a CNF2 toxin background. Like the findings for reciprocal mutants of CNF1, no reactivity with MAb NG8 was observed. From these results, we concluded that either all of the amino acids necessary for MAb NG8 reactivity were not identified or the MAb NG8 epitope was conformational and toxins had partially refolded in Western blots. To address the second possibility, toxins were examined for reactivity with MAb NG8 using ELISAs (Fig. 2B). Consistent with the results obtained in the Western blot analysis, CNF1 mutant toxins CNF1_{D591E F593L} and CNF1_H661Q showed little or no reactivity with MAb NG8. Several other mutant toxins (CNF1_{T548P P551S}, CNF1_{G585R S588N}, CNF1_D776N, and CNF1_N780D) also showed partial reductions in MAb NG8 reactivity; however, the diminished antibody recognition observed with these toxins was not as dramatic as that seen with the CNF1_{D591E F593L} and CNF1_H661Q mutants.

From both Western blotting and ELISA, it appeared that three amino acids, the amino acids at positions 591, 593 and 661, were strongly associated with recognition by MAb NG8. To confirm that together these amino acids are a critical component of the MAb NG8 epitope, a CNF2 triple mutant, CNF2_{E591D L593F Q661H}, was constructed and examined to determine its MAb NG8 reactivity. This mutant was recognized by MAb NG8 in Western blots (Fig. 2A, lane 20) and showed reactivity similar to that of wild-type CNF1 in an ELISA (Fig. 2B). Together, these findings indicate that the three amino acids in CNF1 (D591, F593, and H661) are essential for MAb NG8 recognition.

In vitro neutralization assays. Next, we tested the capacity of MAb NG8 to neutralize the effects of CNF1, CNF2, and CNF2 mutant toxin CNF2_{E591D L593F Q661H} on HEp-2 cells (Fig. 3). Purified toxins were incubated with MAb NG8 or irrelevant MAbs 11E10 (data not shown) and GC2. Toxin-antibody samples were then added to HEp-2 cell monolayers, and the cell phenotype was assessed by microscopy 3 days later. As shown in Fig. 3, HEp-2 cells in PBS alone were small and compact (Fig. 3A). CNF1 induced significant multinucleation of HEp-2 cells compared to the effect of CNF2 (Fig. 3B and C), whereas CNF2 (Fig. 3C) exhibited greater cytotoxicity against HEp-2 cells than CNF1 exhibited (Fig. 3B). Examination of the CNF2-derived CNF2_{E591D L593F Q661H} mutant (Fig. 3D) indicated that this toxin produced a cell phenotype similar to produced by CNF2 (Fig. 3C). Incubation of either wild-type toxin with MAb GC2 had no neutralization effect (Fig. 3E and data not shown); however, dramatic differences were observed when wild-type toxins were incubated with CNF1-specific MAb NG8. In the case of CNF1, MAb NG8 substantially reduced the amount of cell enlargement and multinucleation, while in the presence of CNF2 MAb NG8 showed no protection against cell cytotoxicity (compare Fig. 3B and F and Fig. 3C and G, respectively). Although CNF2 mutant toxin CNF2_{E591D L593F Q661H} (Fig. 3D) induced a cell phenotype comparable to the cell phenotype induced by CNF2 in the presence of MAb GC2 (Fig. 3H), incubation with MAb NG8 neutralized the toxin activity of this mutant (Fig. 3D and I), like the results observed with CNF1 but not CNF2 in the presence of MAb NG8. Furthermore, neutralization of both CNF1 and CNF2 mutant toxin CNF2_{E591D L593F Q661H} with MAb NG8 occurred in a dose-dependent manner (Fig. 3J to L).

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FIG. 3. Neutralization of CNF activity on HEp-2 cells by MAb NG8. Purified toxin was incubated with MAb NG8 or an irrelevant MAb (MAb GC2). Each toxin-MAb sample was added to a semiconfluent monolayer of HEp-2 cells. Samples were incubated for 72 h, after which cells were fixed and stained. For each panel the sample applied to the HEp-2 cells is indicated, and a scale bar for all images is shown in panel L. Magnification, x10.

Toxin-mediated RhoA deamidation. Since MAb NG8 could neutralize the phenotypic effects on HEp-2 cells induced by CNF1, we next assessed whether MAb NG8 could specifically neutralize the enzymatic activity of CNF1 (namely, the deamidation of RhoA). For these studies, CNF1 was preincubated with MAb NG8 or MAb GC2 (24; data not shown) prior to addition to RhoA deamidation reaction mixtures. RhoA was deamidated in the presence of CNF1 alone, as well as when the toxin was incubated in the presence of MAb NG8 (data not shown). This finding strongly suggests that the capacity of MAb NG8 to neutralize toxin activity on HEp-2 cells was not the result of a block in the enzymatic activity of CNF1.

HEp-2 cell binding. The capacity of CNF1, CNF2, and selected mutant toxins to bind to HEp-2 cells was quantified by a cell binding ELISA (Fig. 4A). Toxins were incubated with fixed HEp-2 cells, and bound toxin was detected with an anti-CNF1 polyclonal serum that recognizes CNF1 and CNF2 equally (22). As previously demonstrated by McNichol et al. (22), CNF1 bound to HEp-2 cells to a greater extent (P = 0.047, t test) than CNF2. Most mutant toxins derived from CNF1 were comparable to the parent toxin in terms of the extent of HEp-2 cell binding, with three notable exceptions. CNF1-derived mutant toxin CNF1_{G585R S588N} exhibited a slight reduction in binding to HEp-2 cells (P = 0.008; the overall P value was significant at the P = 0.001 level), but the CNF1_D776N and CNF1_N780D mutants showed a drop in cell binding that was statistically significant (P = 0.002 and P < 0.001, respectively). These observations suggest that the differences in HEp-2 cell binding between CNF1 and CNF2 observed may have been related to these specific amino acids. As expected, CNF2-derived mutant toxins CNF2_{E591D L593F} and CNF2_Q661H bound at levels similar to the levels observed for the parent toxin (P < 0.001 for both toxins).

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FIG. 4. Interaction of mutant toxins with HEp-2 cells. (A) Toxin-HEp-2 cell binding ELISAs. The sample numbers correspond to the toxin, lane, and sample numbers in Fig. 1 and 2. CNF1, CNF2, and mutant toxins (5 µg/well) were allowed to bind to a confluent layer of fixed HEp-2 cells, and samples were then assessed by performing ELISA with goat anti-CNF1 polyclonal serum as the primary antibody and an appropriate secondary antibody. Negative controls included wells with PBS and without primary or secondary antibodies. The optical densities of the negative controls were subtracted from the values for samples and therefore are not shown. Assays were done in triplicate. The error bars indicate the standard deviations. (B) Inhibition of toxin binding to HEp-2 cells. CNF1, CNF2, and CNF2-derived mutant toxin CNF2E591D L593F Q661H (10 µg/well) were incubated in the presence or absence of MAb NG8 or 11E10 (irrelevant anti-Shiga toxin type 2) (data not shown) and then allowed to bind to HEp-2 cells. Bound toxin was detected with an anti-CNF1 polyclonal serum and an AlexaFluor 488-conjugated secondary antibody (green), while cells were counterstained with Evans blue (red). Cells were examined with an Olympus BX60 fluorescence microscope, and the quantity of bound toxin was determined by enumeration of punctuate green signals in three fields from two independent experiments. The error bars indicate the 95% confidence intervals of the means, and P values are indicated for comparisons.

Inhibition of HEp-2 cell binding. Since MAb NG8 did not appear to affect the enzymatic activity of CNF1, we reasoned that this neutralizing MAb might block toxin binding to eukaryotic cells. To address this possibility, immunofluorescence microscopy was used to examine the binding of CNF1, CNF2, and the CNF2-derived mutant toxin CNF2_{E591D L593F Q661H} to HEp-2 cells. As expected based on a previously published report (22), on average, CNF1 showed greater binding to HEp-2 cells than CNF2 showed (P < 0.001) (Fig. 4B). In addition, the CNF2-derived toxin mutant CNF2_{E591D L593F Q661H} exhibited HEp-2 cell binding that was greater than that of the CNF2 parent toxin, but the increase in adherence was not statistically significant (P = 0.079). In the presence of MAb NG8, CNF1 binding to HEp-2 cells was significantly reduced compared to CNF1 binding in buffer alone (P = 0.002). The observation that MAb NG8 reduced CNF1 adherence to HEp-2 cells was consistent with the previous finding by McNichol et al. (22) that there was a trend in this direction, but, perhaps because of differences in the assay methods (ELISA in the previous study versus immunofluorescence in this study), the impact of MAb NG8 on toxin binding was statistically significant in this study. As expected, the binding capacity of CNF2 was not reduced in the presence of MAb NG8, nor was the binding of CNF1 reduced after preincubation with MAb 11E10 or PBS (data not shown). However, after incubation with MAb NG8, the CNF2 triple mutant showed a fourfold reduction in cell binding compared to the toxin in the absence of antibody (P = 0.001). Based on these findings, it appears that the CNF2_{E591D L593F Q661H} mutant contains a unique epitope recognized by MAb NG8 that can specifically block toxin binding to HEp-2 cells.

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In a previous study, Meysick et al. (24) identified MAb NG8, which reacts with and neutralizes CNF1 but not CNF2. Since neutralizing antibodies often delineate regions that are functionally important in a toxin, the primary goals of this study were to more precisely map the MAb NG8 epitope of CNF1, which has previously been reported to be between amino acids 546 and 869 (22, 24), and to determine the mechanism by which the interaction with MAb NG8 leads to neutralization of CNF1 activity. By using Western blotting, ELISAs, and HEp-2 cell neutralization assays, three amino acids unique to CNF1, at positions 591, 593, and 661, which were essential for recognition of this toxin by MAb NG8, were identified. Based on the results of in silico analysis (data not shown), we expect that homologous toxins CNFY and CNF3, which have disparate amino acids at those sites (ETQ and ETK, respectively), would also show no reactivity with MAb NG8, like CNF2 (ELQ). Exchange of the three CNF2-specific amino acids with the corresponding amino acids in CNF1 permitted MAb NG8 to recognize and neutralize the CNF2 chimeric toxin, which provided additional evidence that substantiated the importance of these amino acids for MAb NG8 reactivity. The finding that the three amino acids (D591, F593, and H661) which comprise the MAb NG8 epitope are in the same general region of the toxin but are not contiguous supports the idea that the MAb NG8 epitope is conformational rather than linear. We also deduced, using a series of toxin cell binding and RhoA deamidation assays, that the mechanism by which MAb NG8 neutralizes CNF1 activity involves interference with the adherence of CNF1 to eukaryotic cells rather than inhibition of the toxin enzymatic function. This conclusion is consistent with previous observations reported by McNichol et al. (22) showing that the MAb NG8 epitope is within a binding domain in the C-terminal half of CNF and that MAb NG8 interferes with toxin access to receptors on HEp-2 cells.

As indicated in previous studies, truncated forms of CNF1 cannot intoxicate cells and are highly sensitive to degradation during purification (22, 25; data not shown). For this reason we chose to construct mutants with specific point mutations in either CNF1 or CNF2 to reexamine the epitope of MAb NG8. Overall, we found that the mutants with point mutations were more stable than CNF1 deletion constructs; however, an 80-kDa product that was also reactive with MAb NG8 was visible on a Coomassie blue-stained gel. We reasoned that the truncated toxins were inactive, like all other truncated CNF1-derived toxins that we have generated to date. Therefore, truncations should not have affected the activity assays used in this study.

In general, a neutralizing antibody can confer protection against intoxication by blocking the intoxication process at multiple stages, including toxin binding, translocation, and enzymatic activity. To determine the mechanism by which MAb NG8 neutralizes CNF1, we considered the information that was known about the steps in the CNF1 intoxication pathway. The crystal structure of the C terminus of CNF1 has been solved (amino acids 720 to 1014); however, no structural analysis has been performed for CNF2 (7). The C terminus of CNF1 contains the catalytic site with residues C866 and H881 specifically required for deamidase activity (31). One possible mechanism of neutralization is that an antibody could bind at or near the catalytic pocket of the toxin and sterically hinder the capacity of the toxin to interact with and modify GTPases. However, this scenario does not appear to be the case for MAb NG8 since CNF1 retained the capacity to modify the RhoA GTPase in the presence of the antibody.

Little is known about the possible mechanism(s) by which MAbs could specifically block or perturb toxin translocation. A more common and well-documented mechanism by which MAbs can mediate neutralization is through inhibition of toxin interactions with the receptor. LRP (37 kDa) has been identified in human brain microvascular endothelial cells as the receptor for CNF1 produced by an E. coli meningitis isolate (10), and heparin sulfate proteoglycan has been implicated as a potential coreceptor for CNF1 (5). Recently, McNichol et al. (22) used recombinant LRP to define two regions of CNF1, one in the N terminus and one in the C terminus, that are necessary for the maximum binding of the toxin to LRP on the surface of HEp-2 cells. This group also reported that a truncated form of the toxin that contains only the C-terminal half of CNF1 exhibits diminished binding to HEp-2 cells in the presence of MAb NG8, a finding which suggests that the MAb NG8 epitope may contain the toxin binding site. Finally, McNichol et al. (22) showed that CNF2 binds equivalently to LRP but less efficiently to HEp-2 cells than CNF1, an observation that coincided with the results obtained in this study.

As the binding capacity of CNF2 with the E591D, L593F, and Q661H mutations was increased compared to that of the parent toxin, CNF2, we surmised that these amino acids not only comprised the MAb NG8 epitope but are also located in the C-terminal cell binding region of CNF1. The fact that the binding to HEp-2 cells by the CNF2 triple mutant was consistently greater than the binding to HEp-2 cells by wild-type CNF2 but the difference was not statistically significant (P = 0.079) suggests that in CNF2 the presence of the MAb NG8 epitope alone is not sufficient for complete toxin binding. In addition, single and double point mutations of the MAb NG8 epitope in a CNF1 background did not affect toxin binding.

The precise mechanism by which MAb NG8 inhibits binding of CNF1 to HEp-2 cells remains to be determined; however, it is possible that the interaction of MAb NG8 with CNF1 residues D591, F593, and H661 could interfere directly with toxin binding to HEp-2 cells by steric hindrance or could indirectly inhibit interaction of the C- and N-terminal binding domains during creation of a potential combined receptor binding site. Both these hypotheses are based on the crystal structure of the catalytic domain of CNF1 (amino acids 720 to 1014), which is composed of a domain of mixed beta-sheets surrounded by alpha-helices (Fig. 5B). The predicted flexible loop regions of CNF1 have been implicated in the restriction of substrate access to the catalytic site, in the recognition of RhoA, and in toxin binding to cells (8). Based on this information, we speculate that some of these exposed loop regions may also be involved in receptor recognition and binding. Moreover, the fact that CNF2 binds less efficiently to HEp-2 cells than CNF1 suggests that there may be differences in the cell binding regions of these toxins that include the amino acids identified in this study.

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FIG. 5. Alignment of the amino acids between residues 546 and 869 of CNF1 and CNF2 and crystal structure of the C terminus of CNF1. (A) Amino acid alignment was performed with the Clone Manager Professional Suite 8 program (Scientific and Educational Software, Durham, NC) by using the global-ref alignment procedure and the scoring matrix BLOSUM 62. The accession numbers for CNF1 and CNF2 are X70670 and AAA18229, respectively. The first amino acid in each line of the alignment is numbered, as are the functional amino acids identified in this study. Green letters indicate similar amino acids, while red letters indicate unique amino acids. Amino acids important for cell binding and MAb NG8 reactivity are indicated by an arrow. The green underlined sequence is the region of CNF1 that has been crystallized. (B) Crystal structure of the C-terminal enzymatic region of CNF1 (amino acids 720 to 1014). Catalytic residues are green, and the loop region is indicated by an asterisk. The X-ray crystallographic coordinates of CNF1 were modeled using the program DeepView/Swiss-PDB viewer. The Protein Data Bank code for the crystal structure is 1HQ0_A (7).

 
We thank Mike Flora of the USUHS Biomedical Instrumentation Center for oligonucleotide synthesis and sequencing services, Susan Kim for assistance with protein purification, and Cara Olsen of the USUHS Biostatics Consulting Center for statistical analysis. We also thank Amanda Sweeney of the FDA/CBER for technical assistance.

This work was supported by grant AI38281 from the National Institutes of Health.

The opinions or assertions in this paper are those of the authors and are not to be construed as views of the Department of Defense.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Phone: (301) 295-3400. Fax: (301) 295-3773. E-mail: aobrien{at}usuhs.mil

Published ahead of print on 27 October 2008.

Editor: V. J. DiRita

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Infection and Immunity, January 2009, p. 170-179, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00943-08
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