RegA, an AraC-like protein, is a global transcriptional regulator that controls virulence gene expression in Citrobacter rodentium

Abstract

Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5alpha, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

FIG. 1.

Genetic location of regA: schematic diagram of regA and surrounding ORFs. P1A2 and EMH1 indicate the sites of insertion of the Tn5 transposon and kanamycin resistance gene, respectively. The amplified region containing regA is indicated by a dashed rectangle, and the amplified region containing adcA is indicated by a dotted rectangle.

FIG. 2.

Comparison of the predicted amino acid sequences of the regulatory proteins AggR, Rns, and PerA with the amino acid sequence of RegA. Sequences were aligned by using the ClustalW program. The amino acids in the putative helix-turn-helix regions are underlined. Identical amino acid residues are indicated by asterisks, conserved residues are indicated by a colons, and semiconserved residues are indicated by periods. C. r., C. rodentium.

FIG. 3.

Colonization of C57BL/6 mice by derivatives of C. rodentium. The data are the means and standard errors of the mean for feces from at least five individual mice at selected time points after inoculation. Mice received (via oral gavage) 2 × 10⁹ CFU of C. rodentium wild-type strain ICC169 (▪), regA deletion mutant EMH1 (▿), or trans-complemented regA mutant EMH1(pEH4) (•).

FIG. 4.

Effect of adcA from C. rodentium on cell-cell adherence in E. coli DH5α. (A) Settling of bacterial cells after 90 min, (B) quantitative autoaggregation, and (C) the microscopic appearance of bacteria were investigated using static liquid suspensions of strains DH5α(pAT153) (vector control) (•) and DH5α(pEH32) (adcA cloned from strain ICC169) (○). The data are the means of two independent assays. The aggregation phenotype in panel C was visualized by phase-contrast microscopy (original magnification, ×1,000).

FIG. 5.

Adherence of E. coli DH5α strains to HEp-2 cells. (A) DH5α(pAT153) (nonadherent vector control). (B) DH5α(pEH32) expressing adcA of C. rodentium showing a diffuse pattern of adherence. (C) DH5α(pEH6, pEH32) expressing both adcA and regA of C. rodentium showing an enhanced diffuse adherence pattern. Bar = 50 μm. The arrows indicate adherent bacteria. The preparations were stained with Giemsa stain. The mean ± standard deviation number of adherent bacteria per HEp-2 cell is shown for each strain.

FIG. 6.

Colonization of C57BL/6 mice with derivatives of C. rodentium. The data are the means and standard errors of the means for feces from at least five individual mice at selected time points after inoculation. Mice received (via oral gavage) 2 × 10⁹ CFU of (A) C. rodentium wild-type strain ICC169 (▪) or adcA deletion mutant EMH2 (□) or (B) wild-type strain ICC169 (▪) or kfc deletion mutant EMH3 (○). The limit of detection is indicated by a dotted line. *, P < 0.05; **, P < 0.005 (two-tailed Student's t test).