Role of BfpF, a member of the PilT family of putative nucleotide-binding proteins, in type IV pilus biogenesis and in interactions between enteropathogenic Escherichia coli and host cells

Abstract

Adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells is dependent on a type IV fimbria, termed the bundle-forming pilus (BFP). A cluster of 14 genes is required for expression of BFP. The eighth gene in the cluster, bfpF, encodes a putative nucleotide-binding protein which resembles the PilT protein of Pseudomonas aeruginosa. It has been proposed that PilT is required for the retraction of the P. aeruginosa pilus, which results in twitching motility. To test the role of BfpF in BFP function and EPEC pathogenesis, two different mutations were constructed in the bfpF gene, one in the cloned gene cluster in a laboratory E. coli strain and one in wild-type EPEC. Neither mutation affected prepilin synthesis, leader sequence processing, or pilus biogenesis. However, both mutations resulted in increased localized adherence. In addition, the EPEC bfpF mutant displayed increased aggregation. The EPEC bfpF mutant was not deficient in attaching and effacing activity or invasion capacity. These results suggest that BfpF decreases aggregation and adherence by EPEC but that subsequent steps in EPEC pathogenesis do not require this protein.

FIG. 1

Map of the bfp gene cluster illustrating construction of bfpF mutants. (A) The entire bfp gene cluster is shown. Arrows represent bfp genes. Selected restriction enzyme sites shown in bfpF were used for cloning. (B) The bfpEFP genes are magnified to illustrate the construction of pRPAF1. A 72-bp KpnI site was deleted from a subcloned fragment containing bfpF as shown in the line below the map. The SacI-DraIII fragment from this construct was then used to replace the SacI-DraIII fragment of pKDS135 to generate pRPAF1. (C) The bfpEFP genes are magnified to illustrate the construction of strain UMD916. A nonpolar kanamycin resistance cassette containing the aphA3 gene was inserted via its flanking SmaI sites into the BbrPI site in a subcloned fragment containing bfpF. B/S and S/B represent the junctions of ligated fragments that had BbrPI and SmaI sites. A fragment containing this insert was cloned into positive-selection suicide vector pCVD442 for allelic exchange with the wild-type strain.

FIG. 2

Analysis of the bfpF mutant and wild-type EPEC strains by PCR. Fragments of bfpA (lanes 1 to 3), bfpF (lanes 4 to 6), and bfpI (lanes 7 to 9) genes were amplified by PCR from wild-type EPEC strain E2348/69 (lanes 1, 4, and 7), bfpF mutant strain UMD916 (lanes 2, 5, and 8), and EAF plasmid-cured strain JPN15 (lanes 3, 6, and 9). PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and photographed. The positions of molecular size markers (in kilobases) are indicated on the left.

FIG. 3

Competitive RT-PCR on the bfpP transcripts from wild-type EPEC and the bfpF mutant strain. Competitive RT-PCR on bfpP was performed on total RNA from wild-type EPEC and bfpF mutant strain UMD916. Plasmid pDN19PBΔ, containing bfpP with a 100-bp deletion, was used as the competitor DNA in the PCRs. Positive controls include PCR products of reactions using cDNA from wild-type EPEC (lane 1) and strain UMD916 (lane 2) and 0.2 μg of pDN19PBΔ DNA (lane 3) as a control for the competitor fragment. RNA from wild-type EPEC (lane 4) and strain UMD916 (lane 5) which was boiled immediately after addition of reverse transcriptase for cDNA synthesis were run as negative controls. The remaining lanes consisted of either wild-type EPEC cDNA (lanes 6 to 12) or strain UMD916 cDNA (lanes 13 to 19), with the following amount of pDN19PBΔ DNA added: 2 μg (lanes 6 to 13), 0.2 μg (lane 7 and 14), 0.1 μg (lanes 8 and 15), 0.05 μg (lanes 9 and 16), 0.025 μg (lanes 10 and 17), 0.0125 μg (lanes 11 and 18), or 4 × 10⁻⁶ μg (lanes 12 and 19). The positions of molecular size markers of 1,018 bp and 507 bp are indicated on the left.

FIG. 4

Expression and processing of bundlin in EPEC and in HB101 strains containing the cloned gene cluster. Whole-cell lysates of strains grown in LB were prepared, separated by SDS-PAGE on a 15% polyacrylamide gel, and analyzed by immunoblotting with an antibundlin antiserum. Lane 1, bfpA mutant strain UMD901; lane 2, wild-type strain E2348/69; lane 3, bfpF mutant UMD916; lane 4, BL21(DE3) with plasmid pMSD205, which contains bfpA alone; lane 5, HB101 with plasmids pKDS135 and pJPN14; lane 6, HB101 with plasmids pRPAF1 (bfpF) and pJPN14. The positions of molecular weight markers (in thousands) are indicated on the left.

FIG. 5

Expression of BFP by EPEC and by E. coli HB101 strains containing the cloned bfp gene cluster. Strains were grown directly on Formvar-copper-coated grids, stained with phosphotungstic acid, and analyzed by electron microscopy. (A) Wild-type EPEC strain E2348/69. (B) bfpF mutant strain UMD916. (C) E. coli HB101 containing the complete bfp gene cluster in plasmid pKDS135 and plasmid pJPN14, which is also required for BFP expression. (D) HB101 containing pRPAF1, which is identical to pKDS135 except for a 72-bp bfpF deletion, and pJPN14. Bundles of flexible fimbriae are apparent in each frame. Bars indicate scale, as marked.

FIG. 6

Autoaggregation of EPEC strains. Overnight cultures of EPEC strains were diluted 1:25 in DMEM and incubated for 4 h. Samples were spotted on microscope slides and examined by phase-contrast microscopy. (A) Wild-type EPEC strain E2348/69. (B) bfpF mutant strain UMD916. (C) Strain UMD916 containing complementing plasmid pRPA102. (D) Strain UMD916 containing control vector pWKS30.

FIG. 7

Localized adherence of EPEC and of E. coli HB101 strains containing the cloned bfp gene cluster to HEp-2 cells. HEp-2 cells were incubated for 3 h with bacteria, fixed, and examined by light microscopy. (A) Wild-type EPEC strain E2348/69. (B) bfpF mutant strain UMD916. (C) E. coli HB101 with pKDS135, which contains the complete bfp gene cluster, and plasmid pJPN14, which is also required for BFP expression. (D) HB101 containing pRPAF1, which is identical to pKDS135 except for a 72-bp bfpF deletion, and pJPN14.

FIG. 8

Transmission electron micrographs of isogenic EPEC strains adhering to HEp-2 cells. HEp-2 cells grown on Transwell filters were infected with eae mutant EPEC strain CVD206 (A) or eae mutant strain CVD207 (pUMD916) (B), which also carries the bfpF::aphA-3 mutation. Thin sections were cut and processed for electron microscopy. The distance between the cells and the bacteria in the layer closest to the cells was measured in these and similar photomicrographs. Bar, 500 nm.