Epitope mapping of monoclonal antibodies capable of neutralizing cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli

Abstract

Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.

FIG. 1

Reactivity of MAbs with CNF1 and cross-reactivity with CNF2 as demonstrated by Western blot analyses. Toxin-containing bacterial lysates were separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with individual MAb hybridoma supernatants. Reactive proteins were visualized with appropriate HRP-conjugated secondary antibodies and either DAB (all IgG MAbs) or ECL substrates (MAb BF8). (A) CNF1 immunoblot. The position of CNF1 is indicated with an arrow. (B) CNF2 immunoblot.

FIG. 2

Schematic of the CNF1 toxin deletion mutants used to localize the epitopes recognized by the CNF1 MAbs (left) and Western blot immunoreactivity patterns obtained for each MAb (right). Amino acid residues deleted from each of the constructs were as follows: pΔC469, amino acids 547 to 1014; pΔN545, amino acids 1 to 546; pΔ442, amino acids 373 to 783; pΔC146, amino acids 869 to 1014; pΔN63, amino acids 1 to 62; and pΔC25, amino acids 990 to 1014. The pCNF24 construct expressed wild-type His-tagged toxin. Immunoreactive toxin proteins are indicated (+), as are those that were nonreactive (−).

FIG. 3

Epitope mapping of the CNF1-neutralizing MAbs JC4, BF8, and NG8 by Western blot analyses with N- and C-terminal CNF1 deletion proteins. (A) Schematic of the CNF1 deletion derivatives used as antigens. The designation of each construct is given on the left, and the deleted portion of the toxin is indicated on the right. (B to D) Immunoblots of E. coli lysates that contained wild-type toxin and either the N- or the C-terminal truncated toxin molecules probed with hybridoma supernatants of MAb JC4 (B), MAb BF8 (C), or MAb NG8 (D). Reactive proteins were detected with appropriate HRP-conjugated secondary antibodies and DAB (MAbs JC4 and NG8) or ECL substrates (MAb BF8).

FIG. 4

Proposed epitope map of the anti-CNF1 MAbs. The epitope of each MAb is denoted as a solid black bar below the linear sequence of CNF1. The putative CNF1 binding and enzymatic domains are indicated above the toxin molecule. The three neutralizing MAbs are shown in italics. Note that MAb BF8 also neutralized CNF2.