The global stress response regulator oxyS in an adherent-invasive Escherichia coli strain attenuates experimental colitis

Abstract

Crohn's disease and ulcerative colitis in humans and experimental immune-mediated colitis in mice are likely due in part to overactive immune responses to resident intestinal bacteria, including certain strains of adherent-invasive Escherichia coli (E. coli) such as E. coli NC101. We have previously shown that specific E. coli NC101 stress responses are upregulated during experimental colitis and attenuate inflammation. However, the roles of broader stress response pathways in E. coli NC101 during experimental colitis are unknown. We hypothesize that the global stress response regulator in E. coli, oxyS, also reduces experimental colitis. We show that intestinal E. coli NC101 upregulate oxyS expression during colitis in monocolonized interleukin-10 deficient mice. Furthermore, we demonstrate that oxyS-sufficient E. coli NC101 have decreased motility and biofilm formation in vitro and attenuated intestinal translocation and colitogenic potential in vivo compared with oxyS-deficient E. coli. These data suggest that activation of a generalized E. coli stress response, oxyS, reduces experimental colitis and may be a potential therapeutic target.

Keywords: Stress response; flagellum; interleukin-10; motility; translocation.

Figure 1.

E. coli oxyS expression levels in luminal bacteria during experimental colitis. E. coli oxyS transcripts relative to bacterial 16S transcripts were quantified in cecal bacterial RNA from wild-type (WT) and interleukin-10 deficient (Il10^−/−) mice monocolonized with E. coli NC101 for the indicated times. Data are expressed as means ± SEM (n=4–5 mice/group, *p<0.05 Il10^−/− v. WT).

Figure 2.

In vitro characterization of E. coli NC101ΔoxyS. a) Aerobic growth in LB liquid media measured by optical density at 600nm. Data presented as the means ± SEM (n=3 cultures/strain). b) Survival in the presence of the indicated concentrations of H₂O₂. Data are expressed as percent survival compared to cultures without added H₂O₂ and are presented as means ± SEM (n=3 cultures per strain per condition, *p<0.05 relative to NC101ΔoxyS). c) Motility in soft agar over 12 hours. Data are expressed as the diameter of the growth ring (mm) and are presented as means ± SEM (n=3 assays per strain per condition, *p<0.05 relative to NC101. d and e) Flagellar gene expression during growth. Data are expressed as the abundance of flhD and fliC transcripts relative to bacterial 16S at the indicated times of growth in liquid LB and are presented as the means ± SEM (n=3 assays per strain per condition, *p<0.05 relative to NC101).

Figure 3.

E. coli oxyS attenuates experimental colitis in monocolonized Il10^−/− mice. a) Colony forming units of E. coli in cecal contents, b) Composite histological inflammation scores (range 0–16), and c) IFNγ secretion by mesenteric lymph node cells stimulated ex vivo with sterile NC101 bacterial lysate. Data are presented as means ± SEM (n=6 mice/group/timepoint, *p<0.05 relative to NC101-colonized mice. d) Representative photomicrographs of cecal tissue from mice monocolonized for 80 days (40x).

Figure 4.

E. coli oxyS decreases biofilm formation in vitro and bacterial translocation in the cecum of monocolonized Il0^−/− mice. a) Quantity of bacterial biofilm on a polystyrene surface as measured by the optical density of crystal violet-stained bacterial biofilms (570nm)/optical density of bacterial cultures prior to biofilm staining (630nm). Data are presented as the means ± SEM (n=16 cultures per strain). b-d) Colony forming units of tissue associated E. coli in the cecum and distal colon from Il10^−/− mice monocolonized with E. coli for 1 week. Data are presented as the means ± SEM (n=5 mice/group, *p<0.05 relative to NC101).