The CpxRA System in Uropathogenic Escherichia coli Regulates Major Virulence Genes

Background:

CpxRA is a two component system that is found in uropathogenic Escherichia coli (UPEC) and other members of the Enterobacteriaceae. CpxA is both a sensor kinase and a phosphatase that controls the activity of its response regulator CpxR. Major functions of CpxRA are to repair and reduce protein traffic across the envelope; thus, activation of the CpxRA system downregulates expression of multiple virulence determinants. CpxRA activating mutants in Haemophilus ducreyi and Salmonella typhimurium are less virulent in human and murine models of infection, making CpxRA an attractive antivirulence target. In competition experiments, both cpxA (system active) and cpxR (system inactive) deletion mutants are attenuated compared to wildtype (WT) UPEC in a murine model of urinary tract infection. To better understand the mechanisms underlying their attenuation, we determined the genes regulated by the CpxRA system using RNA-Seq.

Methods:

Total RNA was extracted from four independent samples of cpxA mutant, cpxR mutant, and WT strains using TRIzol. mRNA libraries were prepared using Script-Seq complete kit and sequenced using Illumina NextSeq platform. Differential gene expression between the strains was determined using a cutoff of 2-fold change and a false discovery rate of < 0.1. RNA-Seq results were confirmed by quantitative reverse transcription PCR.

Results:

516 genes were differentially regulated between the cpxA and cpxR mutants. Of these, 310 were downregulated in the cpxA mutant compared to the cpxR mutant; these encoded proteins that function in motility, adhesion, and envelope biogenesis. Type 1 fimbriae, which are encoded by the fim genes, were downregulated in the cpxA mutant; fim mutants are unable to colonize the urinary tract in a mouse model of UTI. Similarly, pap genes were downregulated in the cpxA mutant compared to the cpxR mutant; Pap proteins play an essential role in the pathogenesis of pyelonephritis. Compared to the cpxA mutant, the cpxR mutant had a significant downregulation of iron acquisition genes.

Conclusion:

Both activation and inactivation of CpxRA downregulates a number of genes that encode established virulence factors utilized by UPEC to infect its host.