SopB activates the Akt-YAP pathway to promote Salmonella survival within B cells

Abstract

B cells are a target of Salmonella infection, allowing bacteria survival without inducing pyroptosis. This event is due to downregulation of Nlrc4 expression and lack of inflammasome complex activation, which impairs the secretion of IL-1β. YAP phosphorylation is required for downregulation of Nlrc4 in B cells during Salmonella infection; however, the microorganism's mechanisms underlying the inhibition of the NLRC4 inflammasome in B cells are not fully understood. Our findings demonstrate that the Salmonella effector SopB triggers a signaling cascade involving PI3K, PDK1 and mTORC2 that activates Akt with consequent phosphorylation of YAP. When we deleted sopB in Salmonella, infected B cells that lack Rictor, or inhibited the signaling cascade using a pharmacological approach, we were able to restore the function of the NLRC4 inflammasome in B cells and the ability to control the infection. Furthermore, B cells from infected mice exhibited activation of Akt and YAP phosphorylation, suggesting that Salmonella also triggers this pathway in vivo. In summary, our data demonstrate that the Salmonella effector inositide phosphate phosphatase SopB triggers the PI3K-Akt-YAP pathway to inhibit the NLRC4 inflammasome in B cells. This study provides further evidence that Salmonella triggers cellular mechanisms in B lymphocytes to manipulate the host environment by turning it into a survival niche to establish a successful infection.

Keywords: Akt; B cells; IL-1β; Salmonella; SopB; YAP.

Figure 1.

Salmonella effector SopB promotes Akt activation and YAP phosphorylation in B cells. (a) Western blot analysis of pAkt S473, pAkt T308, pYAP S127 and the corresponding total protein levels in B cells from BALB/c mice infected with Salmonella Typhimurium wild-type, Salmonella Typhimurium ΔsopB, Salmonella Typhimurium ΔsopB-pSopB or Salmonella Typhimurium ΔsopB-pSopB C460S at an MOI of 50. (b) Protein levels of pAkt S473, pAkt T308 and pYAP relative to total Akt or total YAP levels were normalized to the control (not infected). (c) Western blot analysis of pAkt S473, pAkt T308, pYAP S127 and the corresponding total protein levels in B cells from BALB/c mice pretreated for 30 minutes with the Akt inhibitor AKT VIII (2,12 µM) or the PDK1 inhibitor GSK23334470 (50 µM) and infected with Salmonella Typhimurium wild-type at an MOI of 50. Inhibitors were present before, during and after infection. (d) Protein levels of pAkt S473, pAkt T308 and pYAP S127 relative to total Akt and total YAP levels were normalized to the control (not infected). (e) Western blot analysis of pAkt S473 and pYAP S127 and the corresponding total protein levels in B cells from Rictor^fl/fl and Cd19Cre/Rictor^fl/fl mice infected with Salmonella Typhimurium wild-type at an MOI of 50. (f) Protein levels of pAkt S473 and pYAP S127 relative to total Akt and total YAP levels were normalized to the control (not infected). Data are representative Western blotting images or are expressed as the mean ± S.D. of at least three different experiments. Data were analyzed by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Activation of Akt by Salmonella in B cells follows a canonical PI3K activation mechanism. (a) Western blot analysis of pAkt S473, pAkt T308 and total Akt in B cells from BALB/c mice pretreated for 60 minutes with the PI3K inhibitor wortmannin at a concentration of 0,2 µM or 1 µM and infected with Salmonella Typhimurium wild-type at an MOI of 50. (b) Protein levels of pAkt T308 and pAkt S473 relative to total Akt levels were normalized to the control (not infected). (c) Western blot analysis of p-p85 Y458 and total p85 in B cells from BALB/c mice infected with Salmonella Typhimurium wild-type at an MOI of 50. (d) Protein levels of p-p85 Y458 relative to total p85 levels were normalized to the control (not infected). (e) Representative confocal immunostaining of PIP3 in B cells from BALB/c mice infected with Salmonella Typhimurium wild-type-GFP at an MOI of 50. Data are representative Western blotting images or are expressed as the mean ± S.D. of three different experiments. Data were analyzed by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 5 µm.

Figure 3.

Salmonella promotes Akt-YAP pathway activation to block IL-1β secretion. (a) IL-1β secretion levels in supernatants of B cells from BALB/c mice infected with Salmonella Typhimurium wild-type or ΔsopB at an MOI of 50. (b) IL-1β secretion levels in supernatants of B cells from BALB/c mice pretreated for 60 minutes with the PI3K inhibitor wortmannin (0.2 µM) or for 30 minutes with the Akt inhibitor AKT VIII (2,12 µM) or the PDK1 inhibitor GSK23334470 (50 µM) and infected with Salmonella Typhimurium wild-type at an MOI of 50. Inhibitors were present before, during and after infection. Viability of infected-B cell not treated with inhibitors was similar to infected-B cell treated with inhibitors (data not shown) (c) IL-1β secretion levels in supernatants of B cells from Rictor^fl/fl and Cd19Cre/Rictor^fl/fl mice infected with Salmonella Typhimurium wild-type at an MOI of 50. Data are expressed as the mean ± S.D. of three different experiments. Data were analyzed by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Salmonella activates the Akt-YAP pathway through SopB to survive in B cells. (a) Bacteria kinetic survival in B cells from BALB/c mice infected with Salmonella Typhimurium wild-type or Salmonella Typhimurium ΔsopB at an MOI of 50. (b) Survival percentage was calculated using the following formula (CFUs 24 h PI/CFUs 1 h PI). (c) Bacteria kinetic survival in B cells from BALB/c mice pretreated for 60 minutes with the PI3K inhibitor wortmannin (0.2 µM) or for 30 minutes with the Akt inhibitor AKT VIII (2,12 µM) or the PDK1 inhibitor GSK23334470 (50 µM) and infected with Salmonella Typhimurium wild-type at an MOI of 50. Inhibitors were present before, during and after infection. Viability of infected-B cell not treated with inhibitors was similar to infected-B cell treated with inhibitors (data not shown) (d) Survival percentage was calculated using the following formula: (CFUs 24 h PI/CFUs 1 h PI). (e) Bacteria kinetic survival in B cells from Rictor^fl/fl and Cd19Cre/Rictor^fl/fl mice infected with Salmonella Typhimurium wild-type at an MOI of 50. (F) Survival percentage was calculated using the following formula: (CFUs 24 h PI/CFUs 1 h PI). Data are expressed as the mean ± S.D. of at least three different experiments. Survival percentage data were analyzed by Student’s t-test. Survival kinetic data were analyzed by two-way ANOVA with a post hoc Bonferroni test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

Salmonella SopB activates the Akt-YAP pathway in B cells infected in vivo. (A) Flow cytometric analysis of pAkt S473, pAkt T308 and pYAP S127 in B cells from BALB/c mice infected intraperitoneally with 50 CFUs of Salmonella Typhimurium wild-type-GFP or Salmonella Typhimurium ∆sopB-GFP. (B) Mean fluorescence intensity of pAkt S473, pAkt T308 and pYAP S127. Data are expressed as the mean ± S.D. of three different experiments. Data were analyzed by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

Salmonella effector SopB activates the Akt-YAP pathway to prevent IL-1β secretion. Through PI3K, SopB promotes the production of PIP3, which recruits Akt. Next, PDK1 and mTORC2 phosphorylate Akt (at threonine 308 and serine 473, respectively) to activate it. Once active, Akt phosphorylates the coactivator YAP at serine 127, leading to YAP sequestration in the cytoplasm and preventing it from heterodimerizing with the transcription factor p73. Without the p73-YAP heterodimer, the transcription of Nlrc4 is reduced, which impacts the processing and secretion of the pro-inflammatory cytokine IL-1β and leads to the survival of Salmonella in B cells.