Helicobacter bilis gamma-glutamyltranspeptidase enhances inflammatory stress response via oxidative stress in colon epithelial cells

Abstract

Helicobacter bilis (H. bilis) infection is associated with cases of inflammatory bowel Disease, thyphlocolitis, hepatitis and cholecystitis. However, little is known about the bacterial virulence determinants or the molecular mechanisms involved. Recently, H. bilis γ-glutamyltranspeptidase (HBgGT) was shown to be a virulence factor decreasing host cell viability. Bacterial gGTs play a key role in synthesis and degradation of glutathione and enables the bacteria to utilize extracellular glutamine and glutathione as sources of glutamate. gGT-mediated loss of cell viability has so far been linked to DNA damage via oxidative stress, but the signaling cascades involved herein have not been described. In this study, we identified enhanced ROS production induced by HBgGT as a central factor involved in the activation of the oxidative stress response cascades, which finally activate CREB, AP-1 and NF-κB in H. bilis infected colon cancer cells. IL-8, an important pro-inflammatory chemokine that is a common downstream target of these transcription factors, was up-regulated upon H. bilis infection in an HBgGT dependent manner. Moreover, the induction of these signaling responses and inflammatory cytokine production in host cells could be linked to HBgGT-mediated glutamine deprivation. This study implicates for the first time HBgGT as an important regulator of signaling cascades regulating inflammation in H. bilis infected host epithelial cells that could be responsible for induction of inflammatory disorders by the bacterium.

Figure 1. Superoxide production induced by HBgGT in colon cancer cells.

A) NBT assay visualization of formazan crystal formation in HCT116 and DLD-1 cells in response to wild type and gGT deficient H. bilis infection for 20 hours at MOI 50. Cells were also treated with recombinant HBgGT (5µg/ml) or inactive HBgGT (5µg/ml) for 20 hours. Following treatment, cells were stained for formazan crystals (dark blue) and counterstained with safranin. TNFα (20ng/ml) was used as a positive control. B) Quantification of crystal formation in HCT116 and DLD-1 cell lysates at OD 650. Results are expressed as mean of three independent experiments normalized to the untreated control. *p<0.05, **p<0.005, ***p<0.0005. Asterisks on top of bars indicate significance relative to untreated control; asterisks on bars indicate significance level between indicated conditions.

Figure 2. Activation of oxidative stress-associated signaling pathways upon H. bilis infection.

A) NF-κB, AP-1 and CREB transcriptional activity in gGT proficient and gGT deficient H. bilis infected HCT116 colon cancer cells. Transiently transfected HCT116 cells with corresponding luciferase reporter plasmids were co-cultured with H. bilis and H. bilis ΔgGT at MOI 5 and 50 for 24 hours. Bars represent mean of relative luciferase values to renilla of 3 independent experiments normalized to the untreated control. *p<0.05, **p<0.005. Asterisks on top of bars indicate significance relative to untreated control; asterisks on bars indicate significance level between indicated conditions. B) p-IκBα (40 kDa), c-jun (48kDa) and p-CREB (43 kDa) protein levels detected by western blot. HCT116 cells were lysed after 10 hours of infection at the MOI indicated. β-actin (45 kDa) was used as a loading control. One representative blot of three independent experiments is shown. C) Confocal image of HCT116 cells showing nuclear translocation of NF-κB subunit p65 after 24 hour H. bilis infection at MOI 50 or recombinant HBgGT (5µg/ml) treatment. TNFα (20ng/ml) was used to induce nuclear translocation of p65 and the inactive gGT (5µg/ml) was used as control. Actin was stained with phalloidin to allow visualization of total cell area.

Figure 3. Influence of HBgGT on the activation of the NF-κB, AP-1 and CREB pathways.

NF-κB, AP-1 and CREB transcriptional activity in transiently transfected HCT116 (A), and DLD-1 and LS174T (B) cells. Recombinant HBgGT (5µg/ml) was added to H. bilis ΔgGT-infected cells and transcriptional activity determined after 24 hours of infection. Results are expressed as mean of relative luciferase activity to renilla of three independent experiments, normalized to the untreated control. *p<0.05, **p<0.005, ***p<0.0005. Asterisks on top of bars indicate significance relative to untreated control; asterisks on bars indicate significance level between indicated conditions.

Figure 4. Influence of HBgGT on IL-8 secretion by colon cancer cells.

A) IL-8 secretion determined by ELISA in cell culture supernatants of HCT116 and DLD-1 cells treated with recombinant HBgGT (5µg/ml) or infected with H. bilis or H. bilis ΔgGT (MOI 50) for different time points. Mean values of three independent experiments are shown. B) IL-8 levels after 24 hour of treatment secreted by HCT116 and DLD-1 cells upon HBgGT treatment or H. bilis infection. Results are expressed as mean of three independent experiments. *p<0.05 **p<0.005, ***p<0.0005. Asterisks on top of bars indicate significance relative to untreated control; asterisks on bars indicate significance level between indicated conditions.

Figure 5. Effect of glutamine supplementation on the activation of NF- κB, AP-1 and CREB pathways after H. bilis infection.

A) NF-κB, AP-1 and CREB transcriptional activity. Transiently transfected HCT116 cells were infected with H. bilis (MOI 50). Infected cells were supplemented with 3mM L-glutamine (Supplementary Gln) in addition to the 2mM present in the culture medium (Input Gln) for 24 hours where indicated. Untreated cells cultured in 2mM L-glutamine in culture medium served as the untreated control. L-glutamine free medium was used to starve the cells of glutamine for the same time period. Results are expressed as mean of relative luciferase activity to renilla of three independent experiments, normalized to the untreated control. *p<0.05, **p<0.005, ***p<0.0005. Asterisks on top of bars indicate significance relative to untreated control; asterisks on bars indicate significance level between indicated conditions. B) p-IκBα, c-Jun and p-CREB protein levels analyzed by western blot in HCT116 after 10 hours H. bilis infection (MOI 50). Where indicated regular cell culture medium containing 2mM L-glutamine (Input Gln) was supplemented with additional 3mM L-glutamine (Supplementary Gln). β-actin was used as a loading control. One representative blot from three independent experiments is shown. C) ROS production was determined by NBT assay in HCT116 and DLD-1 cells after L-glutamine supplementation of cells infected with H. bilis (MOI 50) or treated with HBgGT PIM (pre-incubated medium with 5µg of HBgGT/ml of culture medium) for 20 hours. L-glutamine free medium was used to starve the cells of glutamine. TNFα (20µg/ml) was used as a positive control. D) Quantification of superoxide production in similarly treated HCT116 and DLD-1 cell lysates at OD 650. Results are expressed as mean of three independent experiments, normalized to the untreated control. *p<0.05, **p<0.005, ***p<0.0005. Asterisks on top of bars indicate significance relative to untreated control; asterisks on bars indicate significance level between indicated conditions.

Figure 6. Influence of glutamine supplementation on IL-8 secretion by H. bilis co-cultures and HBgGT-treated cells.

A) IL-8 production by HCT116 and DLD-1 cells. H. bilis (MOI 50) infected cells were supplemented with 3mM L-glutamine (Gln). Supernatants of 24 hour treated cells were collected and IL-8 secretion determined by ELISA. Results are expressed as mean of three independent experiments. *p<0.05, **p<0.005, ***p<0.0005. B) IL-8 levels secreted by HCT116 and DLD cells after glutamine supplementation (Gln 2mM, where indicated) of HBgGT PIM (pre-incubated medium, 5µg HBgGT/ml of cell culture medium). Heat-inactivated HBgGT PIM (inactive HBgGT pre-incubated at 5µg/ml of culture medium) was used as an enzymatically inactive control. Results are expressed as mean of three independent experiments. *p<0.05, **p<0.005, ***p<0.0005.

Figure 7. Model of host cell response modulated by HBgGT.

Glutamine deprivation, exhaustive consumption of glutathione, and subsequent generation of free radicals by H. bilis gGT induce several oxidative stress response cascades in host cells, cumulating in IL-8 secretion.