PafR, a novel transcription regulator, is important for pathogenesis in uropathogenic Escherichia coli

Abstract

The metV genomic island in the chromosome of uropathogenic Escherichia coli (UPEC) encodes a putative transcription factor and a sugar permease of the phosphotransferase system (PTS), which are predicted to compose a Bgl-like sensory system. The presence of these two genes, hereby termed pafR and pafP, respectively, has been previously shown to correlate with isolates causing clinical syndromes. We show here that deletion of both genes impairs the ability of the resulting mutant to infect the CBA/J mouse model of ascending urinary tract infection compared to that of the parent strain, CFT073. Expressing the two genes in trans in the two-gene knockout mutant complemented full virulence. Deletion of either gene individually generated the same phenotype as the double knockout, indicating that both pafR and pafP are important to pathogenesis. We screened numerous environmental conditions but failed to detect expression from the promoter that precedes the paf genes in vitro, suggesting that they are in vivo induced (ivi). Although PafR is shown here to be capable of functioning as a transcriptional antiterminator, its targets in the UPEC genome are not known. Using microarray analysis, we have shown that expression of PafR from a heterologous promoter in CFT073 affects expression of genes related to bacterial virulence, biofilm formation, and metabolism. Expression of PafR also inhibits biofilm formation and motility. Taken together, our results suggest that the paf genes are implicated in pathogenesis and that PafR controls virulence genes, in particular biofilm formation genes.

FIG 1

pafR and pafP contribute to pathogenesis of uropathogenic E. coli. Comparative colonization levels, presented as competitive index, in the urine, bladder, and kidneys of CBA/J mice 48 h after transurethral inoculation with a mixture (cochallenge) of wild-type CFT073 with the ΔpafRP strain (n = 19) (A), the ΔpafRP strain carrying a plasmid expressing pafRP (n = 10) (B), the ΔpafR strain (n = 10) (C), or the ΔpafP strain (n = 9) (D). Bars indicate the median competitive index. P values for cochallenge infections were determined using the Wilcoxon matched-pair test.

FIG 2

Monitoring expression of GFP from P_paf, the putative paf promoter, in CFT073 cells. (A) Expression of P_paf::gfp in the presence of the indicated carbon sources was imaged by fluorescence microscopy. (B) Expression of P_paf::gfp in the presence of 190 different carbon sources was monitored in phenotypic microarray (PM) plates. Fluorescence units represent the recorded fluorescence normalized to cell density. Shown are results with representative carbon sources. The results with all carbon sources are presented in Table S1 in the supplemental material.

FIG 3

Regulatory activities of PafR. (A) PafR is capable of preventing premature termination of transcription. Plasmids carrying the E. coli K-12 bglG gene, the CFT073 pafR gene, or the cloning vector only were introduced into MA152, an E. coli K-12 strain which is deleted for the bgl operon and carries a chromosomal fusion of the bgl promoter and terminator to the lacZ gene. The ability of the plasmid-encoded proteins to enable lacZ expression was tested by measuring β-galactosidase levels. (B) PafR is a major regulator. Shown are groups of genes that were differentially expressed in PafR-expressing CFT073 bacteria and in ΔpafRP bacteria, as indicated by oligonucleotide-based microarray analysis. Black bars, upregulated genes; gray bars, downregulated genes. The lists of all upregulated and downregulated genes in PafR-expressing CFT073 compared to those in ΔpafRP cells are presented in Data Sets S1 and S2, respectively, in the supplemental material.

FIG 4

Expression of PafR in UPEC affects biofilm formation and motility. (A) An array of biofilm-related genes is upregulated in PafR-expressing CFT073 cells compared to results for ΔpafRP cells, as indicated by oligonucleotide-based microarray analysis. (B) Biofilm formation by wild-type CFT073 and PafR-expressing cells, assessed by the crystal violet coloration assay. (C) Average diameters of CFT073 and PafR-expressing colonies on soft agar plates. (D) Colonies of CFT073 and PafR-expressing cells grown for 12 h on soft agar plates.

FIG 5

PafR downregulates the level of mal gene transcripts and proteins in UPEC. (A) The entire mal regulon is downregulated in PafR-expressing CFT073 compared to expression in ΔpafRP bacteria, as indicated by oligonucleotide-based microarray analysis. (B) CFT073 but not E. coli K-12 bacteria, which express PafR from a plasmid, cannot grow on minimal medium plates that contain maltose as a sole carbon source. (C and D) Maltose-binding protein (MBP), detected by Western blotting, in ΔpafRP, wild-type, and PafR-expressing CFT073 bacteria cultured in LB (C) or in minimal medium with maltose as a sole carbon source (D). (E) MBP is expressed in comparable levels in E. coli K-12 bacteria that express PafR and in those that do not express it, as indicated by Western blotting with anti-MBP antibodies. Purified MBP was used as a positive control.