SoxS Increases the Expression of the Zinc Uptake System ZnuACB in an Escherichia coli Murine Pyelonephritis Model

Abstract

Paralogous transcriptional regulators MarA, Rob, and SoxS act individually and together to control expression of more than 80 Escherichia coli genes. Deletion of marA, rob, and soxS from an E. coli clinical isolate prevents persistence beyond 2 days postinfection in a mouse model of pyelonephritis. We used microarray analysis to identify 242 genes differentially expressed between the triple deletion mutant and its parent strain at 2 days postinfection in the kidney. One of these, znuC of the zinc transport system ZnuACB, displayed decreased expression in the triple mutant compared to that in the parental strain, and deletion of znuC from the parental strain reduced persistence. The marA rob soxS triple deletion mutant was less viable in vitro under limited-Zn and Zn-depleted conditions, while disruption of znuC caused a reduction in the growth rates for the parental and triple mutant strains to equally low levels under limited-Zn or Zn-depleted conditions. Complementation of the triple mutant with soxS, but not marA or rob, restored the parental growth rate in Zn-depleted medium, while deletion of only soxS from the parental strain led to low growth in Zn-depleted medium. Both results suggested that SoxS is a major regulator responsible for growth under Zn-depleted conditions. Gel shift experiments failed to show direct binding of SoxS to the znuCB promoter, thus suggesting indirect control of znuCB expression by SoxS. While SoxS expression in the triple mutant fully restored persistence, increased expression of znuACB via a plasmid in this mutant only partially restored wild-type levels of persistence in the kidney. This work implicates SoxS control of znuCB expression as a key factor in persistence of E. coli in murine pyelonephritis.

The contribution of PmrAB to the virulence of a clinical isolate ofEscherichia coli

“…This construct was transformed into DH5α and Ap R transformants were confirmed for the presence of the pPmrAB plasmid by PCR sequencing. Plasmid pPmrAB was digested with restriction enzyme BbsI and ligated to a blunt-ended cat cassette PCR amplified from plasmid pKD3 described previously, 6 creating plasmid pPmrAB::Cm. This plasmid was selected for its Ap R /Cm R phenotype and confirmed with PCR.…”

“…6 Briefly, a total of 5 × 10 7 cfu consisting of a 1:1 mixture of independently grown strains were used to inoculate 7-12 mice. The competitive indices were determined by culturing kidney homogenates on LB-agar with and without Cm.…”

“…[1][2][3][4] The three related transcription factors, MarA, Rob, and SoxS, act in both individual and redundant manners to affect antibiotic resistance and virulence of Escherichia coli, through known and unknown mechanisms. 5,6 These three transcription factors play a key role in the persistence of E. coli in a murine kidney infection model, where deletion of all three genes significantly decreases the bacterial load in the kidney after three days of successful colonization. 5 Previous microarray data show that expression levels of genes associated with the pmrAB regulon 6,7 were decreased in a triple mutant lacking the transcription factors MarA, Rob, and SoxS compared with its parent strain, when extracted from a murine pyelonephritis infection model.…”

“…5,6 These three transcription factors play a key role in the persistence of E. coli in a murine kidney infection model, where deletion of all three genes significantly decreases the bacterial load in the kidney after three days of successful colonization. 5 Previous microarray data show that expression levels of genes associated with the pmrAB regulon 6,7 were decreased in a triple mutant lacking the transcription factors MarA, Rob, and SoxS compared with its parent strain, when extracted from a murine pyelonephritis infection model. 6 The pmrAB operon encodes a two-component regulatory system present in many bacterial species, 8,9 which mediates a wide range of functions, most notably modification of the lipid A moiety of lipopolysaccharide (LPS).…”

“…5 Previous microarray data show that expression levels of genes associated with the pmrAB regulon 6,7 were decreased in a triple mutant lacking the transcription factors MarA, Rob, and SoxS compared with its parent strain, when extracted from a murine pyelonephritis infection model. 6 The pmrAB operon encodes a two-component regulatory system present in many bacterial species, 8,9 which mediates a wide range of functions, most notably modification of the lipid A moiety of lipopolysaccharide (LPS). 10,11 Such modification of LPS alters the host immune response to the bacterial pathogen, and indeed, studies in Salmonella enterica serovar Typhimurium 12 and Francisella novicida 13 show that pmrAB contributes to virulence in animal models of infection.…”