Hypochlorous acid and hydrogen peroxide-induced negative regulation of Salmonella enterica serovar Typhimurium ompW by the response regulator ArcA

Abstract

BackgroundHydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by Salmonella enterica serovar Typhimurium (S. Typhimurium) upon internalization by phagocytic cells. In order to survive, bacteria must sense these signals and modulate gene expression. Growing evidence indicates that the ArcAB two component system plays a role in the resistance to reactive oxygen species. We investigated the influx of H2O2 and HOCl through OmpW and the role of ArcAB in modulating its expression after exposure to both toxic compounds in S. Typhimurium.ResultsH2O2 and HOCl influx was determined both in vitro and in vivo. A S. Typhimurium ompW mutant strain (∆ompW) exposed to sub-lethal levels of H2O2 and HOCl showed a decreased influx of both compounds as compared to a wild type strain. Further evidence of H2O2 and HOCl diffusion through OmpW was obtained by using reconstituted proteoliposomes. We hypothesized that ompW expression should be negatively regulated upon exposure to H2O2 and HOCl to better exclude these compounds from the cell. As expected, qRT-PCR showed a negative regulation in a wild type strain treated with sub-lethal concentrations of these compounds. A bioinformatic analysis in search for potential negative regulators predicted the presence of three ArcA binding sites at the ompW promoter region. By electrophoretic mobility shift assay (EMSA) and using transcriptional fusions we demonstrated an interaction between ArcA and one site at the ompW promoter region. Moreover, qRT-PCR showed that the negative regulation observed in the wild type strain was lost in an arcA and in arcB mutant strains.ConclusionsOmpW allows the influx of H2O2 and HOCl and is negatively regulated by ArcA by direct interaction with the ompW promoter region upon exposure to both toxic compounds.

Bacterial Responses to Reactive Chlorine Species

“…Strains lacking ycfR are more sensitive to several stress conditions, including exposure to HOCl (31). HOCl treatment leads to the repression of outer membrane porin ( omp ) genes in S. enterica and P. aeruginosa (78, 95, 102). OmpW increases the HOCl permeability of S. enterica , making a △ ompW mutant more resistant to HOCl treatment (78).…”

“…HOCl treatment leads to the repression of outer membrane porin ( omp ) genes in S. enterica and P. aeruginosa (78, 95, 102). OmpW increases the HOCl permeability of S. enterica , making a △ ompW mutant more resistant to HOCl treatment (78). Consistent with the model that the presence of Omp family members correlates with increased HOCl permeability and sensitivity, acid-adapted S. enterica cells, which have an increased complement of those proteins (66), are more sensitive to HOCl than nonadapted cells (67).…”

ompW is cooperatively upregulated by MarA and SoxS in response to menadione

“…The OmpW protein is an immunogenic 22 kDa (Jalajakumari & Manning, 1990) minor porin and has been related to osmoprotection (Hu et al , 2005), the efflux and resistance towards paraquat (Gil et al , 2009) and the influx of hydrogen peroxide and hypochlorous acid (Morales et al , 2012). It is regulated by diverse environmental conditions including temperature, salinity, nutrient availability, oxygen levels (Nandi et al , 2005), paraquat (Gil et al , 2009) and reactive oxygen species (Morales et al , 2012), among others, and is differentially regulated at the transcriptional level by FNR (anaerobiosis), ArcA (H 2 O 2 and NaOCl) and SoxS (paraquat) (Bouchal et al , 2010; Morales et al , 2012; Gil et al , 2009).…”

“…It is regulated by diverse environmental conditions including temperature, salinity, nutrient availability, oxygen levels (Nandi et al , 2005), paraquat (Gil et al , 2009) and reactive oxygen species (Morales et al , 2012), among others, and is differentially regulated at the transcriptional level by FNR (anaerobiosis), ArcA (H 2 O 2 and NaOCl) and SoxS (paraquat) (Bouchal et al , 2010; Morales et al , 2012; Gil et al , 2009). …”

“…3). These sites are located approximately 100 nt upstream from the ArcA-binding site (from −70 to −55), required for the negative regulation in response to H 2 O 2 and NaOCl (Morales et al , 2012). This suggests that under the assayed conditions, ArcA is not active, since, as observed in the presence of NaOCl, when ArcA, MarA and SoxS are present, ompW is negatively regulated.…”