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

<p>Abstract</p> <p>Background</p> <p>Hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by <it>Salmonella enterica</it> serovar Typhimurium (<it>S</it>. 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 <it>S.</it> Typhimurium.</p> <p>Results</p> <p>H₂O₂ and HOCl influx was determined both <it>in vitro</it> and <it>in vivo</it>. A <it>S</it>. Typhimurium <it>ompW</it> mutant strain (∆<it>ompW</it>) 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 <it>ompW</it> 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 <it>ompW</it> promoter region. By electrophoretic mobility shift assay (EMSA) and using transcriptional fusions we demonstrated an interaction between ArcA and one site at the <it>ompW</it> promoter region. Moreover, qRT-PCR showed that the negative regulation observed in the wild type strain was lost in an <it>arcA</it> and in <it>arcB</it> mutant strains.</p> <p>Conclusions</p> <p>OmpW allows the influx of H₂O₂ and HOCl and is negatively regulated by ArcA by direct interaction with the <it>ompW</it> promoter region upon exposure to both toxic compounds.</p

Probing the ArcA regulon under aerobic/ROS conditions in Salmonella enterica serovar Typhimurium

BackgroundHydrogen peroxide (H2O2) is a reactive oxygen species (ROS), which is part of the oxidative burst encountered upon internalization of Salmonella enterica serovar Typhimurium (S. Typhimurium) by phagocytic cells. It has previously been established that, the ArcAB two-component system plays a critical role in ROS resistance, but the genes regulated by the system remained undetermined to date. We therefore investigated the ArcA regulon in aerobically growing S. Typhimurium before and after exposure to H2O2 by querying gene expression and other physiological changes in wild type and ΔarcA strains.ResultsIn the ΔarcA strain, expression of 292 genes showed direct or indirect regulation by ArcA in response to H2O2, of which 141were also regulated in aerobiosis, but in the opposite direction. Gene set enrichment analysis (GSEA) of the expression data from WT and ΔarcA strains, revealed that, in response to H2O2 challenge in aerobically grown cells, ArcA down regulated multiple PEP-PTS and ABC transporters, while up regulating genes involved in glutathione and glycerolipid metabolism and nucleotide transport. Further biochemical analysis guided by GSEA results showed that deletion of arcA during aerobic growth lead to increased reactive oxygen species (ROS) production which was concomitant with an increased NADH/NAD+ ratio. In absence of ArcA under aerobic conditions, H2O2 exposure resulted in lower levels of glutathione reductase activity, leading to a decreased GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio.ConclusionThe ArcA regulon was defined in 2 conditions, aerobic growth and the combination of peroxide treatment and aerobic growth in S. Typhimurium. ArcA coordinates a response that involves multiple aspects of the carbon flux through central metabolism, which ultimately modulates the reducing potential of the cell

Probing the ArcA regulon under aerobic/ROS conditions in Salmonella enterica serovar Typhimurium

Abstract Background Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS), which is part of the oxidative burst encountered upon internalization of Salmonella enterica serovar Typhimurium (S. Typhimurium) by phagocytic cells. It has previously been established that, the ArcAB two-component system plays a critical role in ROS resistance, but the genes regulated by the system remained undetermined to date. We therefore investigated the ArcA regulon in aerobically growing S. Typhimurium before and after exposure to H2O2 by querying gene expression and other physiological changes in wild type and ΔarcA strains. Results In the ΔarcA strain, expression of 292 genes showed direct or indirect regulation by ArcA in response to H2O2, of which 141were also regulated in aerobiosis, but in the opposite direction. Gene set enrichment analysis (GSEA) of the expression data from WT and ΔarcA strains, revealed that, in response to H2O2 challenge in aerobically grown cells, ArcA down regulated multiple PEP-PTS and ABC transporters, while up regulating genes involved in glutathione and glycerolipid metabolism and nucleotide transport. Further biochemical analysis guided by GSEA results showed that deletion of arcA during aerobic growth lead to increased reactive oxygen species (ROS) production which was concomitant with an increased NADH/NAD+ ratio. In absence of ArcA under aerobic conditions, H2O2 exposure resulted in lower levels of glutathione reductase activity, leading to a decreased GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio. Conclusion The ArcA regulon was defined in 2 conditions, aerobic growth and the combination of peroxide treatment and aerobic growth in S. Typhimurium. ArcA coordinates a response that involves multiple aspects of the carbon flux through central metabolism, which ultimately modulates the reducing potential of the cell

Participation of the <i>Salmonella</i> OmpD Porin in the Infection of RAW264.7 Macrophages and BALB/c Mice

<div><p><i>Salmonella</i> Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, <i>Salmonella</i> must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the <i>Salmonella</i> Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a Δ<i>ompD</i> strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of <i>ompD</i> caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the Δ<i>ompD</i> strain showed increased ability to survive and replicate in target organs of infection. The <i>ompD</i> transcript levels showed a down-regulation when <i>Salmonella</i> resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the Δ<i>ompD</i> strain produced lower levels of reactive oxygen species, suggesting that down-regulation of <i>ompD</i> could favor replication of <i>Salmonella</i> inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.</p></div

Expression of <i>S.</i> Typhimurium 14028 s <i>ompD</i> inside macrophages.

<p>Transcript levels of <i>ompD</i> (A) and <i>ompW</i> (B) from strain 14028 s were quantified by qRT-PCR by recovering total RNA from the infected macrophages at 1 to 12 h post infection, and from bacteria of the inoculating culture (I). Experiments were performed in triplicate. Asterisks represent significant statistical differences between the control and mutants strains (* <i>p</i>≤0.05; ** <i>p</i>≤0.005;*** <i>p</i>≤0.001). Values are mean ± SD.</p