Transcriptional regulation of subclass 5b fimbriae

Abstract

Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of infant and child mortality in developing countries. This enteric pathogen causes profuse watery diarrhea by elaborating one or more enterotoxins that intoxicate eukaryotic cells and ultimately leads to a loss of water to the intestinal lumen. Virulence is also dependent upon fimbrial adhesins that facilitate colonization of the small intestine.

Results: The expression of CS1 fimbriae is positively regulated by Rns, a member of the AraC/XylS superfamily of transcriptional regulators. Based on fimbrial protein homology, CS1 fimbriae have been categorized as subclass 5b along with CS17, CS19, and PCFO71 fimbriae. In this study we show that Rns positively regulates the expression of these other subclass 5b members. DNase I footprinting revealed a Rns binding site adjacent to the -35 hexamer of each fimbrial promoter. The CS17 and PCFO71 fimbrial promoters carry a second Rns binding site centered at -109.5, relative to the Rns-dependent transcription start site. This second binding site is centered at -108.5 for the CS19 promoter. Mutagenesis of either site reduced Rns-dependent transcription from each promoter indicating that the molecules bound to these sites apparently function independently of one another, with each having an additive effect upon fimbrial promoter activation.

Conclusion: This study demonstrates that the ETEC virulence regulator Rns is required for the expression of all known 5b fimbriae. Since Rns is also known to control the expression of additional ETEC fimbriae, including those within subclasses 5a and 5c, the inactivation or inhibition of Rns could be an effective strategy to prevent ETEC infections.

Figure 1

Rns–dependent transcription start sites of fimbrial promoters. The transcription start sites of CS17, CS19, and PCFO71 fimbrial promoters were mapped by primer extension of RNA isolated from rns+ and rns::kan strains. The first nucleotide of each mRNA is underlined and wavy arrows indicate the direction of transcription. Putative–10 hexamers are shown in boxes. Lanes labeled GA and TC contain Maxam–Gilbert sequencing ladders. These are excision reactions and are therefore offset from the primer extension products by -1 nucleotide. RNA was isolated after cultures reached an optical absorbance of 1.0 at 595 nm.

Figure 2

Identification of Rns binding sites in vitro. DNase I footprints of MBP–Rns bound the noncoding strands of CS17, CS19, and PCFO71 fimbrial promoters. The nucleotide sequence of each promoter is shown below each gel image with predicted -10 and -35 hexamers in boxes. Numbering is relative to the Rns–dependent transcription start site, denoted by wavy arrows, of each promoter. The central 12 nucleotides of each binding site, which are partially conserved, are shown in bold. Lanes labeled TC and GA contain Maxam–Gilbert sequencing ladders.

Figure 3

Gel mobility assays of wild–type and mutagenized Rns binding sites. The sequence of each binding site is shown with numbering relative to the Rns–dependent transcription start site of each promoter. For the gel mobility assays additional flanking sequences were included with DNA fragments ranging in size from 148 to 257 bp. Nucleotides within the conserved core of each binding site are shown in bold. Point mutations within each binding site are shown above each sequence. Mutagenized binding sites are designated with allele numbers at the end of each site's name. Since each DNA fragment was produced by PCR, primer annealing to sequences with partial homology sometimes produced faint secondary bands as evident in lanes without MBP–Rns.

Figure 4

Function of Rns binding sites in vivo. Rns–dependent and Rns–independent expression of β-galactosidase from reporter constructs integrated into the chromosome of K-12 strain MC4100. Cells were harvested from overnight cultures with an optical absorbance between 0.7 and 0.8 at 580 nm. Mutagenized binding sites are designated with allele numbers at the end of each site's name. n ≥ 3