The lrp gene and its role in type I fimbriation in Citrobacter rodentium

Abstract

Citrobacter rodentium is a murine pathogen that is now widely used as an in vivo model for gastrointestinal infections due to its similarities with human enteropathogens, such as the possession of a locus for enterocyte effacement (the LEE island). We studied the lrp gene of C. rodentium and found that it encodes a product highly similar to members of the Lrp (leucine-responsive regulatory protein) family of transcriptional regulators, able to recognize leucine as an effector and to repress the expression of its own structural gene. In enterobacteria, Lrp is a global regulator of gene expression, as it controls a large variety of genes, including those coding for cell appendages and other potential virulence factors. Based on the well-established role of Lrp on the expression of pilus genes in Escherichia coli, we also studied the role of Lrp in controlling the formation of the type I pilus in C. rodentium. Type I pili, produced by the fim system, are virulence factors of uropathogens, involved in mediating bacterial adhesion to bladder epithelial cells. Yeast agglutination assays showed that Lrp is needed for type I pilus formation and real-time PCR experiments indicated that Lrp has a strong leucine-mediated effect on the expression of the fimAICDFGH operon. Mutant studies indicated that this positive action is exerted mainly through a positive control of Lrp on the phase variation mechanism that regulates fimAICDFGH expression. A quantitative analysis of its expression suggested that this operon may also be negatively regulated at the level of transcription.

FIG. 1.

(A) Schematic representation of the trdX-lrp-ftsK region on the C. rodentium chromosome. Short arrows indicate the position of annealing of synthetic oligonucleotides. (B) β-Galactosidase assay performed with the E. coli strains CV975 (ilvIH::lacZ), CV1008 (ilvIH::lacZ lrp::Tn10), and AC13 (CV1008 carrying plasmid pAC12), indicated as lrp⁺, lrp⁻, and lrp⁻/pAC12, respectively. Cells were grown in minimal medium (white bars) and leucine-supplemented minimal medium (gray bars).

FIG. 2.

β-Glucuronidase assay performed on C. rodentium strains AC49 (ATCC 51459, wild type carrying plasmid pAC47), AC52 (EM2, lrp null carrying plasmid pAC47), AC62 (ATCC 51459, wild type carrying plasmid pAC61), indicated as wild type/pAC47(gusA), lrp⁻/pAC47 (gusA), and wild type/pAC61 (gusA lrp), respectively. Cells were grown in minimal medium (white bars) and leucine-supplemented minimal medium (gray bars). The activity value obtained for strain AC49 grown in the absence of leucine was considered 100% activity. All values are the average of at least three independent experiments.

FIG. 3.

Schematic representation of the fim region on the C. rodentium chromosome. Arrows indicate the transcription orientation; short arrows indicate the position of annealing of synthetic oligonucleotides. Enlarged is the fimS element in the ON orientation. Three Lrp boxes and two inverted repeats (IRL and IRR) are also indicated. The three Lrp boxes are indicated in the order 2-1-3 in homology to the E. coli model (19).

FIG. 4.

Real-time PCR experiment performed to monitor fimAICDFGH expression in various growth conditions. Cells of the wild-type ATCC 51459 strain were grown in minimal medium (white bars), rich medium in aerated conditions (gray bars), and rich medium in static conditions (black bars) and collected during the exponential growth phase, at entry into stationary growth phase, or after 3 h of stationary growth phase. Total RNA was extracted, and cDNA was synthesized and used in the reactions with an ABI PRISM 7500 sequence detection system (PE Applied Biosystems). The fluorescence signal due to SYBR Green intercalation was monitored to quantify the double-stranded DNA product formed in each PCR cycle. The ΔΔC_t method was used to calculate the relative amount of specific RNA present in each sample, and the transcriptional induction was estimated by comparison to values relative to the wild-type strain grown in minimal medium at exponential phase.

FIG. 5.

Yeast agglutination experiments. We mixed 10 μl of 3% (wt/vol) Saccharomyces cerevisiae yeast cells on a glass slide with the same volume of three bacterial cultures of Citrobacter rodentium EM2 (A), wild-type (wt) (B), and EM3 (C) strains at an optical density of 595 nm.