Small heat-shock proteins, IbpAB, protect non-pathogenic Escherichia coli from killing by macrophage-derived reactive oxygen species

Abstract

Many intracellular bacterial pathogens possess virulence factors that prevent detection and killing by macrophages. However, similar virulence factors in non-pathogenic bacteria are less well-characterized and may contribute to the pathogenesis of chronic inflammatory conditions such as Crohn's disease. We hypothesize that the small heat shock proteins IbpAB, which have previously been shown to reduce oxidative damage to proteins in vitro and be upregulated in luminal non-pathogenic Escherichia strain NC101 during experimental colitis in vivo, protect commensal E. coli from killing by macrophage-derived reactive oxygen species (ROS). Using real-time PCR, we measured ibpAB expression in commensal E. coli NC101 within wild-type (wt) and ROS-deficient (gp91phox(-/-)) macrophages and in NC101 treated with the ROS generator paraquat. We also quantified survival of NC101 and isogenic mutants in wt and gp91phox(-/-) macrophages using gentamicin protection assays. Similar assays were performed using a pathogenic E. coli strain O157:H7. We show that non-pathogenic E. coli NC101inside macrophages upregulate ibpAB within 2 hrs of phagocytosis in a ROS-dependent manner and that ibpAB protect E. coli from killing by macrophage-derived ROS. Moreover, we demonstrate that ROS-induced ibpAB expression is mediated by the small E. coli regulatory RNA, oxyS. IbpAB are not upregulated in pathogenic E. coli O157:H7 and do not affect its survival within macrophages. Together, these findings indicate that ibpAB may be novel virulence factors for certain non-pathogenic E. coli strains.

Fig 1. Kinetics of E. coli ibpAB upregulation during phagocytosis by macrophages.

J774 macrophages (A) or BMDMs (B) were incubated with E. coli NC101. At the indicated times, ibpA and ibpB mRNA in gentamicin-resistant (i.e. intracellular) E. coli was quantified by real-time PCR. Data are presented as means ± sd (n = at least 3 wells/timepoint, *p<0.05 vs. time 0).

Fig 2. Upregulation of ibpAB in intracellular E. coli is partially-dependent on reactive oxygen species.

A) Intracellular E. coli ibpA and ibpB mRNA was measured by real-time PCR in J774 macrophages that were incubated with E. coli NC101 for up to 3 hrs in the presence or absence of the vacuolar H⁺-ATPase inhibitor, bafilomycin-A1. B-E) Intracellular E. coli ibpA and ibpB mRNA was measured by real-time PCR in BMDMs from wt, Inos ^-/- (B and C) or gp91phox ^-/- (D and E) mice that were incubated with E. coli NC101for up to 6 hrs. Data are presented as means ± sd (n = at least 3 wells/timepoint, *p<0.05 vs. media control or wt BMDMs).

Fig 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).

Fig 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 NC101ΔoxyS 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 NC101ΔoxyS for the indicated times. Data are presented as means ± sd (n = at least 3 wells/timepoint, *p<0.05 vs. E. coli NC101).

Fig 5. IbpAB protect commensal E. coli NC101, but not pathogenic E. coli O157:H7 from reactive oxygen species-mediated killing within macrophages.

Viable, gentamicin-resistant (i.e. intracellular) E. coli were measured at the indicated timepoints after infection of wt or gp91phox ^-/- BMDMs with E. coli NC101 / E. coli NC101ΔibpAB (A) or E. coli O157:H7 / E. coli O157:H7ΔibpAB (B). Data are presented as means ± sem (n = 3–4 wells/timepoint, *p<0.05 vs. all other conditions in panel A and vs. both E. coli strains in gp91phox ^-/- BMDMs in panel B).

Fig 6. Commensal E. coli NC101, but not pathogenic E. coli O157:H7 upregulate ibpAB after phagocytosis by macrophages.

A and B) IbpA and ibpB mRNA in intracellular E. coli was measured using real-time PCR at the indicated times following infection of wt BMDMs with E. coli NC101 or E. coli O157:H7. Data are presented as means ± sd (n = at least 3 wells/timepoint, *p<0.05 vs. E. coli O157:H7).