Characterization of the binding specificity of K88ac and K88ad fimbriae of enterotoxigenic Escherichia coli by constructing K88ac/K88ad chimeric FaeG major subunits

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) fimbriae are the major cause of diarrhea in young pigs. Three antigenic variants of K88 fimbriae (K88ab, K88ac, and K88ad) have been identified among porcine ETEC strains. Each K88 fimbrial variant shows a unique pattern in binding to different receptors on porcine enterocytes. Such variant specificity in fimbrial binding is believed to be controlled by the major subunit (FaeG) of the K88 fimbriae, because the genes coding for the only other fimbrial subunit are identical among the three variants. Uniqueness in binding to host receptors may be responsible for differences in the virulence levels of porcine diarrhea disease caused by K88 ETEC strains. To better understand the relationships between the structure of FaeG proteins and fimbrial binding function, and perhaps virulence in disease, we constructed and expressed various K88ac/K88ad faeG gene chimeras and characterized the binding activity of each K88 chimeric fimbria. After verifying biosynthesis of the chimeric fimbriae, we examined their binding specificities in bacterial adherence assays by using porcine brush border vesicles that are specific to either the K88ac or K88ad fimbria. Results showed that each fimbria switched binding specificity to that of the reciprocal type when a peptide comprising amino acids 125 to 163 was exchanged with that of its counterpart. Substitutions of a single amino acid within this region negatively affected the binding capacity of each fimbria. These data indicate that the peptide including amino acids 125 to 163 of the FaeG subunit is essential for K88 variant-specific binding.

FIG. 1.

Constructions of the K88ac/K88ad chimeric and single-amino-acid-mutated FaeG proteins and their binding activities to phenotype B and D brush borders and illustrations of constructions of K88ac and K88ad chimeras. Up to 20 individual brush borders for each construct were blindly examined for bound bacteria. * indicates that for chimera c/d-200, the first 200 amino acid residues at the N terminus are of K88ac FaeG and the rest of the amino acids (201 to 264) are of K88ad FaeG. @ indicates a mutant K88 fimbria with a single amino acid substitution at that particular position. For example, c→d@V133A means that the fimbria was K88ac, but its amino acid residue at position 133 (valine) was replaced by that (alanine) of the K88ad fimbria. The mean and standard deviations of the numbers of bacteria bound to each brush border vesicle are shown. P values from the Student t test are shown in parentheses.

FIG. 2.

Expression of chimeric FaeG proteins illustrated by a Western blot analysis. Total protein samples from strains 8189 (K88ac), 8190 (K88ad), 8191 (c/d-124), 8192 (d/c-200), 8193 (c/d/c-124/200), 8194 (d/c/d-124/200), 8199 (c/d/c-124/163), 8200 (d/c/d-124/163), and 8197 (c/d-200) were separated by 10% SDS-PAGE. A mixture of hybridoma supernatants (1:500) from MAbs 36/41 (K88ac), 30/17 (K88ac), 17/44 (K88ac and K88ad), 99/150 (K88ad), and 221/38 (K88ac and K88ad) was used as the primary antibody and horseradish peroxidase-conjugated goat-anti-mouse IgG (1:5,000) as the secondary antibody to recognize the chimeric FaeG proteins.

FIG. 3.

TEM images showing E. coli cells expressing K88ac/K88ad chimeric fimbriae recognized by anti-K88ac and -K88ad MAbs. Overnight-grown bacterial culture was coated on a 200-mesh copper grid, incubated with a mixture of anti-K88ac and -K88ad MAbs (36/41, 30/17, 17/44, 99/150, and 221/38) and goat-anti-mouse gold-conjugated (20 nm) IgG, and examined using a Joel-1210 transmission electron microscope (×30,000). TEM images from construct 8189 (K88ac), 8190 (K88ad), 8191 (c/d-124), 8192 (d/c-200), 8193 (c/d/c-124/200), 8194 (d/c/d-124/200), 8199 (c/d/c-124/163), and 8200 (d/c/d-124/163) illustrated biosynthesis of K88 chimeric fimbriae. K-12:C600 was used as the negative control.