Generated by paraquat, we also measured expression of oxyS, a small

Generated by paraquat, we also measured expression of oxyS, a small regulatory RNA in E. coli that has PX-478 structure previously been shown to be upregulated in response to hydrogen peroxide, control expression of several stress response genes, and protect E. coli from peroxide-induced DNA damage[32]. We observed a consistent dose- and time-dependent increase of oxyS mRNA in E. coli Trichostatin A site treated with paraquat (Fig. 3C). Interestingly, the oxyS upregulation slightly precedes ibpABFig 3. E. coli upregulate ibpAB in the presence of the superoxide generator, paraquat. E. coli ibpA (A), ibpB (B), and oxyS (C) mRNA was measured by real-time PCR in mid-log phase bacterial cultures that were stimulated for the indicated times with the indicated concentrations of paraquat. Data are presented as means ?sd (n = 3, *p<0.05 vs. 0mM paraquat). doi:10.1371/journal.pone.0120249.gPLOS ONE | DOI:10.1371/journal.pone.0120249 March 23,6 /IbpAB Protect Commensal E. coli against ROSFig 4. E. coli ibpAB upregulation in response to paraquat treatment and phagocytosis by macrophages is mediated in-part by oxyS. A) E. coli ibpA and ibpB mRNA was measured using real-time PCR in mid-log phase cultures of E. coli NC101 or E. coli NC101oxyS 5 min after exposure to vehicle control (0mM) or 10mM paraquat. B and C) Intracellular ibpA and ibpB mRNA was measured using real-time PCR in wt BMDMs incubated with E. coli NC101 or E. coli NC101oxyS for the indicated times. Data are presented as means ?sd (n = at least 3 wells/timepoint, *p<0.05 vs. E. coli NC101). doi:10.1371/journal.pone.0120249.gupregulation. These data indicate that superoxides transiently induce ibpAB expression in E. coli and suggest the possibility that oxyS mediates the superoxide-induced upregulation of ibpAB.E. coli ibpAB expression is positively controlled by the oxyS small regulatory RNAUsing a reporter-gene screen, others have previously shown that oxyS expression up- or downregulates 20 genes in E. coli, several of which are stress response genes[32]. However, oxyS has not previously been described to regulate expression of the ibpAB operon. Since we determined that superoxides induce oxyS expression shortly before ibpAB expression (Fig. 3), we hypothesized that oxyS may upregulate ibpAB expression. To test this, we measured ibpAB expression in paraquat-treated E. coli NC101 or oxyS-deficient E. coli (NC101oxyS) and found that ibpAB expression was significantly attenuated in unstimulated as well as paraquat-stimulated NC101oxyS (Fig. 4A). To determine whether upregulation of ibpAB in macrophages was also dependent on oxyS, we incubated BMDMs with E. coli NC101 or NC101oxyS for the indicated times and measured ibpAB mRNA levels in intracellular bacteria. At one hour after the addition of bacteria, ibpAB mRNA was significantly lower in NC101oxyS compared with NC101 (Fig. 4B and C). However, this difference was absent by 6 hours. Therefore, oxyS-dependent factors mediate ibpAB expression in intra-macrophage E. coli at early, but not late, stages of intracellular survival. The mechanisms by which the oxyS small regulatory RNA controls ibpAB mRNA levels are still unknown.Expression of ibpAB is associated with enhanced E. coli survival within macrophagesHaving determined that E. coli upregulate ibpAB in response to ROS in culture and in macrophages, we hypothesized that ibpAB expression protects E. coli from killing by ROS in macrophages. In order to address this hypothesis, we incubated BMDMs from wt or gp91phox-/- mice w.Generated by paraquat, we also measured expression of oxyS, a small regulatory RNA in E. coli that has previously been shown to be upregulated in response to hydrogen peroxide, control expression of several stress response genes, and protect E. coli from peroxide-induced DNA damage[32]. We observed a consistent dose- and time-dependent increase of oxyS mRNA in E. coli treated with paraquat (Fig. 3C). Interestingly, the oxyS upregulation slightly precedes ibpABFig 3. E. coli upregulate ibpAB in the presence of the superoxide generator, paraquat. E. coli ibpA (A), ibpB (B), and oxyS (C) mRNA was measured by real-time PCR in mid-log phase bacterial cultures that were stimulated for the indicated times with the indicated concentrations of paraquat. Data are presented as means ?sd (n = 3, *p<0.05 vs. 0mM paraquat). doi:10.1371/journal.pone.0120249.gPLOS ONE | DOI:10.1371/journal.pone.0120249 March 23,6 /IbpAB Protect Commensal E. coli against ROSFig 4. E. coli ibpAB upregulation in response to paraquat treatment and phagocytosis by macrophages is mediated in-part by oxyS. A) E. coli ibpA and ibpB mRNA was measured using real-time PCR in mid-log phase cultures of E. coli NC101 or E. coli NC101oxyS 5 min after exposure to vehicle control (0mM) or 10mM paraquat. B and C) Intracellular ibpA and ibpB mRNA was measured using real-time PCR in wt BMDMs incubated with E. coli NC101 or E. coli NC101oxyS for the indicated times. Data are presented as means ?sd (n = at least 3 wells/timepoint, *p<0.05 vs. E. coli NC101). doi:10.1371/journal.pone.0120249.gupregulation. These data indicate that superoxides transiently induce ibpAB expression in E. coli and suggest the possibility that oxyS mediates the superoxide-induced upregulation of ibpAB.E. coli ibpAB expression is positively controlled by the oxyS small regulatory RNAUsing a reporter-gene screen, others have previously shown that oxyS expression up- or downregulates 20 genes in E. coli, several of which are stress response genes[32]. However, oxyS has not previously been described to regulate expression of the ibpAB operon. Since we determined that superoxides induce oxyS expression shortly before ibpAB expression (Fig. 3), we hypothesized that oxyS may upregulate ibpAB expression. To test this, we measured ibpAB expression in paraquat-treated E. coli NC101 or oxyS-deficient E. coli (NC101oxyS) and found that ibpAB expression was significantly attenuated in unstimulated as well as paraquat-stimulated NC101oxyS (Fig. 4A). To determine whether upregulation of ibpAB in macrophages was also dependent on oxyS, we incubated BMDMs with E. coli NC101 or NC101oxyS for the indicated times and measured ibpAB mRNA levels in intracellular bacteria. At one hour after the addition of bacteria, ibpAB mRNA was significantly lower in NC101oxyS compared with NC101 (Fig. 4B and C). However, this difference was absent by 6 hours. Therefore, oxyS-dependent factors mediate ibpAB expression in intra-macrophage E. coli at early, but not late, stages of intracellular survival. The mechanisms by which the oxyS small regulatory RNA controls ibpAB mRNA levels are still unknown.Expression of ibpAB is associated with enhanced E. coli survival within macrophagesHaving determined that E. coli upregulate ibpAB in response to ROS in culture and in macrophages, we hypothesized that ibpAB expression protects E. coli from killing by ROS in macrophages. In order to address this hypothesis, we incubated BMDMs from wt or gp91phox-/- mice w.