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Journal of Bacteriology, July 2001, p. 4099-4102, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.4099-4102.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606

Received 19 January 2001/Accepted 13 April 2001

	ABSTRACT

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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.

	TEXT

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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).

View this table: [in this window] [in a new window]   TABLE 1.   Bacterial strains, phage, and plasmids used in the study

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 MgCl₂ 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-nitrophenyl -D-manno-pyranoside [N-phenyl mannose]). Enrichment was accomplished by mixing 0.5 ml of the transformant pool (ca. 5 × 10⁸ cells) with 100 µl of settled guinea pig erythrocytes in phosphate-buffered saline (PBS) containing an inhibitor. After a 10-min room temperature incubation, erythrocytes were isolated by a 1-s centrifugation in a microcentrifuge and the supernatant was aspirated. The pellet was gently resuspended in 1.0 ml of PBS containing an inhibitor, and the erythrocytes were reisolated by centrifugation. After five more washing steps, the pelleted erythrocytes were resuspended in 1.0 ml of distilled water and diluted with 1.5 ml of L broth containing chloramphenicol and ampicillin, and the erythrocyte-bound population was expanded by overnight growth (with shaking) at 37°C. A second and third enrichment were typically employed. At the end of the procedure, broth cultures were streaked for colony isolation on L agar plates containing chloramphenicol and ampicillin. Individual colonies were screened for the ability to agglutinate erythrocytes in the presence of an inhibitor (10). Plasmids bearing candidate mutant fimH alleles were isolated (4) and reintroduced into strain ORN201 harboring pORN307 by transformation to confirm that the fimH-containing plasmid conferred the altered binding phenotype. One mutant per experiment was kept to ensure independent origin.

Characterization of the plasmid-borne alleles and introduction of fimH mutant alleles into the chromosome. Twenty-five plasmid-borne fimH specificity mutants were initially isolated (15 using fructose and 10 using N-phenyl mannose). Nineteen of the fimH alleles conferring the strongest phenotypes were completely sequenced employing the methods of Russell and Orndorff (24). Seventeen of the alleles were unique, but 13 of the 17 had more than one mutation in the fimH coding sequence. Six of the plasmid-borne mutant alleles that had the fewest mutations were cloned into pKAS32 and introduced into the chromosome of ORN208 by allelic exchange (28) (diagrammed in Fig. 1).

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Diagram of allelic exchange between a mutant fimH allele (fimH*) carried on the nonreplicating vector pKAS32 and the fim region on the chromosome of strain ORN208. Following introduction of the plasmid by mating (28), recombinants were selected and scored for the phenotypic characteristics noted. Recombinants that had deletions () of the vector and the original fimH insertion mutation in ORN208 were identified by selection on streptomycin-containing agar and scored for the appropriate antibiotic and hemagglutination phenotype as indicated. Each fimH lesion introduced was more than 98% linked (by P1 transduction [18]) to the tetR insertion adjacent to fimH. Phenotypic designations denote sensitivity (s) or resistance (r) to antibiotics as follows: Nal, nalidixic acid; Kan, kanamycin; Tet, tetracycline; Amp, ampicillin; Str, streptomycin. Erythrocyte or yeast cell agglutination is collectively denoted phenotypically as Hag. Streptomycin sensitivity is conferred by the rpsL gene, kanamycin resistance is conferred by the neoR gene, ampicillin resistance is conferred by the bla gene, and tetracycline resistance is conferred by the tetR gene.

Interestingly, only two of the six fimH mutant alleles (both identified by enrichment in 250 mM fructose) conferred an altered binding specificity phenotype when chromosomally located even though all six of the chromosomal alleles were confirmed to have sequences identical to the starting plasmid-encoded alleles (as indicated by the sequencing of PCR-generated amplicons of the entire fimH gene; UNC Automated DNA Sequencing Facility, Chapel Hill, N.C.). The rest of the mutants produced either the parental phenotype (one of six) or a null binding phenotype (three of six) with regard to yeast cell and erythrocyte agglutination.

Altered phenotypes conferred by the two chromosomal fimH mutant alleles. Both of the fimH chromosomal mutations that conferred altered binding specificities had single missense mutations predicted to cause an amino acid substitution within the first half of the mature FimH protein (alleles fimH165 and fimH166 from strains ORN209 and ORN210, respectively) (Table 2). Electron-microscopic examination using the methods of Hamrick et al. (8) revealed that strains ORN209 and ORN210 were similar to the parental strain in terms of pilus number per cell and in pilus morphology (data not shown). Also, they were similar to the parent in terms of the ability to agglutinate yeast cells in simple titration tests (Table 2). In contrast to the parent, the mutants retained approximately 50% of this ability in the presence of either of the two inhibitors used to initially isolate the mutants (Table 2). Both mutants also showed a somewhat reduced level of FimH cross-reacting material in antiserum agglutination reactions and a reduced ability to agglutinate guinea pig erythrocytes, and they were unable to bind to macrophages any better than a fimH insertion mutant (Table 2).

View this table: [in this window] [in a new window]   TABLE 2.   Summary of chromosomal fimH lesions and phenotypes associated with strains derived from the enrichment procedurea

Conclusions. Our results describe the utilization of mannose analogs to enrich mutants with different FimH binding specificities. The isolated mutants retained yeast cell binding activity in the presence of normally inhibitory mannose analogs and were additionally altered in the range of cell types they normally bound. Most striking was the complete loss of macrophage binding and the contrasting retention of parental levels of yeast cell agglutination.

Neither of the two single-site fimH mutations described here (fimH165 and fimH166) have been previously reported. However, the lesion at amino acid position 142 (fimH166) has been noted by Schembri et al. (26). Unfortunately, the allele described by Schembri et al. had an additional lesion. Consequently, the two alleles cannot be directly compared. Also, Schembri et al. utilized recombinant plasmid-borne fimH alleles. In the mutants we isolated, only one-third displayed the same phenotype when the plasmid-borne lesions were introduced into the chromosome. It is possible that overproduction (or other effects) attendant with transcription from a recombinant plasmid, when combined with certain lesions, effected an altered binding phenotype that was not exhibited in the chromosome. We chose to examine only those fimH alleles whose mutant binding phenotypes were manifested in the chromosome.

Of the two chromosomal fimH mutants described here, the one with the fimH165 allele (a LeuPro substitution at position 58 of FimH) was of particular interest because a mutant with a lesion at the same nucleotide (conferring a LeuArg change) has been noted to confer a temperature-dependent binding phenotype (8). The present LeuPro substitution effected a complete absence of macrophage binding but the mutant retained full ability to agglutinate yeast and partial ability to agglutinate erythrocytes. In contrast, the LeuArg mutant retains macrophage binding ability at the restrictive temperature (albeit with an altered specificity) but erythrocyte binding ability is lost (8). The previous and present results indicate that the type of amino acid at position 58 influences two important traits: (i) the conditions under which FimH is active and (ii) the specificity of that interaction. The lesion we found at amino acid position 142 (fimH166) changes an amino acid predicted to line the FimH binding pocket (3). The location of this lesion would appear to make a good deal of structural sense (i.e., a change in receptor specificity attributable to an amino acid lining the binding pocket).

Quite a variety of eucaryotic cells bind type 1 piliated E. coli via FimH. However, the FimH receptor on each eucaryotic cell type differs (6, 7, 11). Consequently, FimH mutants of the type reported here (i.e., those that have retained the affinity for one cell type but not another) may be particularly useful in better understanding issues such as bacterial tissue trophism (23) and intra- and intercellular signaling initiated by the interaction of FimH with particular types of eucaryotic cells (11).

Nucleotide sequence accession numbers. The fimH165 and fimH166 alleles have been submitted to GenBank. They have been given the following accession numbers: fimH165, AF306535; and fimH166, AF306536.

	ACKNOWLEDGMENTS

We thank Craig Altier for a critical reading of the manuscript.

This work was supported by a grant from the National Institutes of Health (AI22223).

	FOOTNOTES

^* Corresponding author. Mailing address: College of Veterinary Medicine, 4700 Hillsborough St., Raleigh, NC 27606. Phone: (919) 513-6207. Fax: (919) 513-6455. E-mail: Paul_Orndorff{at}ncsu.edu.

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