Functional Consequences of Sequence Variation in Bundlin, the Enteropathogenic Escherichia coli Type IV Pilin Protein

Abstract

The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is an important virulence factor. We examined the role of divergent alleles of bfpA encoding bundlin, the BFP pilin protein, in pilus biogenesis, pilus interactions, and immune responses. We found that the BFP biogenesis machine from an EPEC strain that expresses one bundlin type is capable of assembling all other bundlin types. Furthermore, we found that EPEC strains expressing divergent bundlin types are capable of forming mixed autoaggregates, suggesting that different pilin types can intertwine. However, we found that there was a marked difference between alleles in immunogenicity in both rabbits and mice of a peptide derived from a region of bundlin undergoing apparent diversifying selection. In addition, despite a high degree of cross-reactivity between divergent bundlin proteins, in both mice and rabbits responses appeared to be stronger against the homologous pilin protein than against the heterologous protein. This result was verified using sera from a volunteer study, which demonstrated that the human antibody responses after an initial challenge with live EPEC were stronger against the homologous bundlin protein than against a divergent bundlin protein. However, a repeat challenge induced equivalent responses. These results are consistent with the hypothesis that human immune responses against bundlin exert selective pressure on bfpA sequence divergence.Type IV pili (Tfps) are surface appendages that are expressed by diverse gram-negative species. Tfps play numerous roles in pathogenesis, including roles in colonization, adherence, autoaggregation, biofilm formation, horizontal gene transfer, motility, and virulence (5,8,21,22,24,28,32,36,38,(41)(42)(43). The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is an excellent model for the study of Tfps as expression of the 14-gene bfp cluster in a laboratory strain of E. coli is sufficient for BFP biogenesis and function (30,40). EPEC is an important cause of serious diarrhea in infants in developing countries (1,12,16,19). Volunteer studies have confirmed the importance of BFP expression for full virulence of EPEC (5). BFP are composed of repeating subunits of the pilin protein bundlin, the product of the bfpA gene. Antibodies against bundlin are found in volunteers convalescing from experimental EPEC infection and in breast milk of mothers and serum of infants in developing countries (15,25,33). Whether these antibodies confer protection against subsequent infection is not known.The Tfps expressed by different strains in a species may vary in sequence. In Neisseria gonorrhoeae, transformation and recombination from multiple silent pilus loci encoding variant pilin proteins result in abundant antigenic variation in pilin expression (29). This extremely dynamic process can lead to the expression of several pilin variants during infection of a single host and may help the bacteria avoid the immune response and cause persistent infection (39). Pilin sequence v...

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin

“…In these experiments, we demonstrated that neither the b-4 nor the b-5 bundlin-expressing strains could bind to HEp-2 cells (Fig. 6A), a phenotype that was not surprising given the Fernandes (2007) lack of bundlin expression by these two strains (Blank et al, 2000). The b-1, -2 and -3 bundlin-expressing strains were capable of binding as microcolonies to HEp-2 cells after 45 min (Fig.…”

“…8, lanes C, D and E), BFP (Fig. 7D, E and F) and AA (Fernandes, 2007), none of these strains displayed LA on HEp-2 cells in the 45 min LA assay ( Fig. 9) but did express the LA phenotype after 3 h (Fig.…”

“…UMD901 was complemented with a-1 bfpA (pRPA100), b-1 bfpA (pXLW13), b-2 bfpA (pXLW16) and b-3 (pXLW17). These plasmids each contain a bfpA allele cloned behind the native promoter from strain E2348/69 into the lowcopy-number vector pWKS30 as described previously (Fernandes, 2007;Anantha et al, 2000). Soluble bundlin from strain E2348/69 lacking the first 24 hydrophobic residues that form an extended N-terminal helix responsible for BFP polymerization was purified as described previously (Ramboarina et al, 2004).…”

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

“…Western blotting. Western blotting for bundlin, BfpB, and BfpU was performed as previously described (23,27,69). SDS-PAGE gels were run according to the manufacturer's instructions (Bio-Rad) and transferred at 21 V for 80 min at 4°C to Immobilon polyvinylidene fluoride (Micropore, catalog no.…”

Interactions of Enteropathogenic Escherichia coli with Pediatric and Adult Intestinal Biopsy Specimens during Early Adherence

“…The ␣-bundlin proteins are N-acetyllactosamine (LacNAc) lectins, whereas the ␤-bundlins are not, since they lack the carbohydrate binding domain found in the ␣-bundlins (13,16). The divergence between bundlins may be the result of selective immunological pressure, since the ␤-bundlins are less immunogenic in rabbits and mice than are the ␣-bundlins (8). It appears that ␣ 1 -bundlin is the only adhesin mediating early (45-min) LA of EPEC strain E2348/69 to HEp-2 cells, since replacing the ␣ 1 -bundlin allele in this strain with ␤-bundlin alleles 1 to 3 abolishes early adherence despite the expression of BFP (16).…”