Expression of the soxR gene of Pseudomonas aeruginosa is inducible during infection of burn wounds in mice and is required to cause efficient bacteremia

Abstract

Burn wounds are prone to infection by Pseudomonas aeruginosa, which is an opportunistic pathogen causing various human diseases. During infection, the bacterium senses environmental changes and regulates the expression of genes appropriate for survival. A purine-auxotrophic mutant of P. aeruginosa was unable to replicate efficiently on burn wounds, suggesting that burn wounds are purine-deficient environments. An in vivo expression technology based on purEK gene expression was applied to the burned mouse infection model to isolate P. aeruginosa genes that are specifically induced during infection. Four such in vivo-inducible (ivi) genetic loci were identified, including the gene for a superoxide response regulator (soxR), the gene for a malate synthase G homologue (glcG), an antisense transcript of a putative regulator responding to copper (copR), and an uncharacterized genetic locus. SoxR of Escherichia coli is known to regulate genes involved in protecting the bacterium against oxidative stress. The expression of soxR was proven to be highly inducible during the infection of burned mice and also inducible by treatment with paraquat, which is a redox-cycling reagent generating intracellular superoxide. The SoxR protein functions as an autorepressor in the absence of paraquat, whereas in the presence of paraquat, this autorepression is diminished. Furthermore, a soxR null mutant was shown to be much more sensitive than wild-type P. aeruginosa to macrophage-mediated killing. In support of this observation, a soxR null mutant exhibited a significant delay in causing systemic infections in the burned mice. Since most mortality in burn patients is caused by systemic infection, the defect in the ability to cause efficient bacteremia in burned mice suggests an important role of the soxR gene in the infection of burn wounds.

FIG. 1

Comparison of growth rates of the IVET isolate SF21 and the purEK deletion strain PAK-AR2 in vitro (A) and in vivo (B). For in vitro growth (A), a mixture of the two bacteria was inoculated into MinA and grown at 37°C for 1, 3, and 7 days. Bacterial cell densities were determined by colony counting on appropriate media. For in vivo growth (B), a mixture of the two bacteria was inoculated on the surfaces of mouse burn wounds. Both bacterial strains were recovered from burn wounds after 1, 3, and 7 days of incubation to determine the number of viable cells.

FIG. 2

Overexpression of the SoxR protein. E. coli harboring either the fusion vector pQE32 (lane 2) or His-SoxR fusion construct pHW9808 (lane 3) was induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) for 3 h, and the total cellular proteins were separated on a sodium dodecyl sulfate–12% polyacrylamide gel, followed by Coomassie blue staining. The His-SoxR fusion protein is marked by an arrow. Lane 1, protein molecular mass standards (high range; Gibco BRL).

FIG. 3

Comparison of the SoxR amino acid sequences of E. coli, S. typhimurium, and P. aeruginosa PAK. The helix-turn-helix motif (boldface) in the N terminus (with a possible DNA-binding function) and a region with four cysteines (CX₂CXCX₅C) (dots) in the C terminus (a putative metal binding site) are marked.

FIG. 4

Induction of the soxR gene by treatment with paraquat (PQ). PAK strains containing pDN19lacΩ (vector control), pHW9802 (soxR::lacZ fusion), or pHW9811 (pbpC::lacZ fusion) were grown in L broth at 37°C in the presence of 0 to 1.0 mM PQ. Samples were taken after 3 h of induction to measure β-galactosidase activities. Average values from three repeated tests are shown.

FIG. 5

Expression of the soxR gene in the presence and absence of a functional soxR gene. The soxR::lacZ fusion construct (pHW9802) and a lacZ fusion vector (pDN19lacΩ) were each introduced into wild-type PAK or a soxR null mutant, PAK-soxR::Ω (soxR). Overnight cultures of the bacteria were used to measure β-galactosidase activities. Average values from three repeated tests are shown.

FIG. 6

Effect of soxR on virulence of P. aeruginosa. Burned mice were infected with either PAK or the soxR mutant PAK-soxR::Ω, and the bacterial cells were recovered from burned skin tissues (A), spleens (B), or livers (C) after 1, 3, and 7 days of incubation. Three mice were used for each strain at each time point. This experiment was performed twice. Error bars indicate standard deviations.

FIG. 7

Effect of soxR on survival of P. aeruginosa within activated macrophages. Activated macrophages were isolated by peritoneal lavage 4 days after injection of thioglycolate. Bacterial cells were opsonized with mouse serum and were incubated with macrophages for phagocytosis. At the indicated times, bacterial cells were recovered by lysis of macrophages with deoxycholate solution and plated on L agar containing appropriate antibiotics. This experiment was repeated in triplicate. Wild-type P. aeruginosa PAK, a soxR null mutant strain, and the soxR null mutant strain complemented by a plasmid (pHW9812) containing the intact soxR gene were used. Error bars indicate standard deviations.