Characterization of Escherichia coli Type 1 Pilus Mutants with Altered Binding Specificities

Abstract

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.Type 1 pili are filamentous proteinaceous appendages produced by many members of the family Enterobacteriaceae. In Escherichia coli, the biosynthesis and binding properties of these pili have been well studied (reviewed in reference 30). Pili are made principally of a repeating monomer, FimA, the product of the fimA gene (13), that is arrayed helically to form a hollow-cored fiber (2). There are at least three minor pilus proteins that are organized into structures seen on the ends of pili (12) and may also be present in the pilus fiber (22). One of these minor components, FimH, the product of the fimH gene, is the molecule that actually binds to mannose-containing receptors on eucaryotic cells (14). The precise nature of the affinity of FimH for mannose is unclear. However, it has been known for some time that different arrangements of mannose monomers and substituent groups affect the affinity of FimH for these substrates (5). It is also known that other pilus components, ostensibly interacting with FimH, also affect the specificity of the interaction of FimH with mannose (16).In this study, we have identified fimH mutants with changes in binding specificity. One especially novel feature of both mutants is the ability to bind and agglutinate yeast cells at parental levels but failed to bind macrophages any better than a fimH insertion mutant.Bacterial strains, plasmids, and growth conditions. The bacterial strains, which were all E. coli K-12 derivatives, and plasmids used are listed in Table 1. Media consisted of L broth, L agar (18), and maltose-tetrazolium agar (27). Antibiotic concentrations were as described previously (20).Receptor specificity mutant isolation. Plasmid pORN163, containing the fimH gene flanked by the EcoRI and SalI restriction endonuclease sites, was used as a template for generating fimH PCR amplicons with a high proportion of mutations. Amplicons, obtained after 30 amplification cycles using a threefold-higher concentration of MgCl 2 than called for by standard PCR conditions (32), were ligated into EcoRI-and SalI-cleaved pBR322. This mutant amplicon pool was subsequently introduced (by transformation [15]) into a ⌬fim strain (ORN201) containing a plasmid, pORN307, carrying all the genes for fimbriation except fimH. In a typical experiment, approximately 8,000 transformant colonies were pooled and subjected to enrichment for receptor specificity mutants (defined as those mutants that bound guinea pig erythrocytes in the presence of either of two inhibitors: 250 mM fructose or 0.25 mM p...

The Distinct Binding Specificities Exhibited by Enterobacterial Type 1 Fimbriae Are Determined by Their Fimbrial Shafts

“…Most work to date has directly related differential binding among E. coli type 1 fimbriae to alterations in the primary sequence of FimH (52)(53)(54)(55). Sequence variations that diminish the mannose-binding ability of FimH were found to be located within or close to the sequences that make up the FimH binding pocket as defined by the FimH crystal struc- ture (23,40,54), although the location of the FimH binding pocket in intact fimbriae may vary somewhat from that determined by the crystal structure of FimH complexed with only its chaperone, FimC (23).…”

Unexpected Similarities betweenBordetella aviumand Other Pathogenic Bordetellae

“…Overnight cultures were tested for their ability to agglutinate guinea pig erythrocytes in plate agglutination assays (16). Logarithms (base 2) of the reciprocal value of the agglutination titers were compared after normalization to that for the parental strain (100% The predicted B. avium FHA protein had an extended (72-amino-acid) signal sequence and the attachment motifs (RGD, RRARR, and CRD), secretion motifs (NPNL and NPNG), and proline-rich region described for B. pertussis (22).…”

Design and synthesis of theranostic antibiotic nanodrugs that display enhanced antibacterial activity and luminescence

“…The compounds in the nanoaggregate form generally resulted in lower MICs than the soluble forms (Table 1) Table S3), a nalidixic acid (Nal)-resistant variant from E. coli K-12 (44). These data demonstrate that the PND strategy could indeed lead to enhanced antibacterial activity of the free drug.…”