Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress

Abstract

Background: Salmonella Typhimurium is a Gram-negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella-containing vacuole where Salmonella is exposed and survives oxidative stress induced by H₂O₂ through modulation of gene expression. After exposure of Salmonella to H₂O₂, the expression of the porin-encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post-transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA.

Results: In this work we sought to evaluate the transcriptional and post-transcriptional regulation of ompX under H₂O₂ stress. We demonstrate that ompX expression is induced at the transcriptional level in S. Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H₂O₂ does not translate into increased protein levels in the wild-type strain, suggesting that ompX mRNA is also regulated at a post-transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H₂O₂-induced stress in Salmonella during the exponential growth phase in Lennox broth.

Conclusions: Our results demonstrate that ompX mRNA is regulated in response to H₂O₂ by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Keywords: H2O2 stress; Transcriptional regulation; Translational regulation; ompX.

Fig. 1

Analysis of ompX expression. A mRNA level of ompX in the wild-type strain of S. Typhimurium 14028 s. B Fluorescence activity of GFP under the control of the ompX promoter (-353 to -1) in the wild-type S. Typhimurium background. C Immunodetection of the OmpX::3xFlag protein, measured after exposure of the strain to 2 mM H₂O₂ for 20, 45 and 60 min. The control received no treatment. Ten µg of total proteins were loaded. White bars represent the control (no treatment), and red bars represent cells treated with 2 mM H₂O₂. The graph represents the average of 3 independent experiments (mean ± SD or SE?)

Fig. 2

Regulation model of ompX mRNA under H₂O₂ stress. A Binding sites of the oxyS, micA and cyaR sRNAs in the 325 bp-regulatory region of ompX mRNA (the whole transcript is 836 bp long) under oxidative stress (oxyS in light blue, cyaR in orange, and micA in purple, translation start site in pink). B Schematic representation of the proposed interaction of oxyS sRNA with the ompX mRNA. The prediction was made using IntaRNA [4, 49],numbers indicate positions in the ompX mRNA and the oxyS sRNA sequence, respectively

Fig. 3

Role of cyaR, micA and oxyS sRNAs in oxidative stress. A Percentage of survival based on Colony Forming Units (CFU) of the sRNA-mutants under peroxide stress compared to S. Typhimurium 14028 s under control conditions. B ROS accumulation of the single sRNA mutants under peroxide stress. C ompX mRNA levels in wild-type, ΔcyaR, ΔmicA, ΔoxyS, Δhfq and ΔryhB strains of S. Typhimurium 14028 s were measured by qRT-PCR. Strains were exposed to 2 mM H₂O₂ for 20 min (red bars); the control had no treatment (white bars). The graph represents the average of 5 independent experiments (mean ± SD or SE?)

Fig. 4

Effect of rifampicin on ompX mRNA under H₂O₂-induced stress. ompX mRNA levels were measured by qRT-PCR in the following strains: wild-type, ΔmicA, ΔcyaR, ΔoxyS and Δhfq. When cultures reached an OD₆₀₀ ≈ 0.4, a pulse of rifampicin (20 µg/ml) was added or not (+ RIF red circles or –RIF inverted blue triangles) to the culture at time 0 min. At each time point, RNA was isolated and subjected to qRT-PCR as described in Materials and Methods. Post-test Bonferroni: *p < 0.05, ***p < 0.01 and ****p < 0.0001, (mean ± SD or SE?)