Research ArticleHost-Microbe Interactions

β-Barrel outer membrane proteins suppress mTORC2 activation and induce autophagic responses

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Science Signaling  27 Nov 2018:
Vol. 11, Issue 558, eaat7493
DOI: 10.1126/scisignal.aat7493
  • Fig. 1 Purified OMPs trigger autophagy in macrophages and intestinal epithelial cells.

    (A) THP1 cells were treated with control buffer or the indicated doses of E. coli OmpA, S. Typhimurium OmpF, E. coli K12FimH, S. Typhimurium FliC, corresponding volume of buffer containing detergent octyl-β-glucopyranoside, or previously boiled E. coli OmpA for 2 hours. In addition, control small interfering RNA (siRNA)– and SlamF8 siRNA–treated THP1 cells were treated with increasing doses of the indicated OMP or buffer. Cells were stained with LysoTracker and analyzed by flow cytometry. Means ± SD of the percentage of cells with OMP-mediated endosomal acidification are shown from three independent experiments. For control siRNA compared to SlamF8 siRNA upon VDAC1 treatment, P < 0.05, Wilcoxon test; for E. coli OmpA compared to boiled E. coli OmpA, P < 0.05, Wilcoxon test. (B) Representative flow cytometry images for (A) showing LysoTracker staining of THP1 cells treated with the indicated doses of E. coli OmpA and previously boiled E. coli OmpA for 2 hours, representative of three independent experiments. The gated population shows the percentage of cells with LysoTracker staining for each condition. (C) THP1 cells were transfected with control or Atg16L1 siRNA, treated with 250 nM P. aeruginosa OprF for 2 hours, and stained with LysoTracker. Means ± SD from three independent experiments are shown. **P < 0.005, t test. Right: Atg16L1 Western blot from a representative experiment showing the efficiency of RNA silencing. (D) Representative flow cytometry images for (C) showing LysoTracker staining of control and Atg16L1 siRNA–transfected THP1 cells treated with P. aeruginosa OprF for 2 hours. The gated population shows the percentage of cells with LysoTracker staining for each condition. (E) Representative Western blot for LC3B and p62 using cell lysates prepared from THP1 cells treated with 100 nM OmpA for 15 min, with or without bafilomycin before treatment for 2 hours. Blots are representative of three independent experiments. (F) Representative Western blots showing p62 degradation and LC3B lipidation in THP1 cells transfected with SlamF8, SlamF2, or control siRNAs and treated with 100 nM VDAC1. Blots are representative of three independent experiments. (G) Cleavage of GFP-LC3 in HEK293T cells treated with 100 nM E. coli OmpA or rapamycin for 15 min. Blots are representative of three independent experiments.

  • Fig. 2 SlamF8 and XRCC6 are important for OMP-mediated autophagy in macrophage and epithelial cells.

    (A and B) Representative immunofluorescence (A) showing LC3B puncta in XRCC6 or control siRNA–transfected Caco2 cells, treated with OmpA and/or chloroquine (CQ) as indicated. Images are representative of three independent experiments. Scale bar, 20 μm. LC3B puncta were counted from a total of 100 cells for each condition from three different experiments (B). ***P < 0.001, t test comparing effect of control and XRCC6 siRNA in OmpA-treated cells. (C) Percentage of LysoTracker-positive cells induced in response to E. coli OmpA treatment of BMDMs from SlamF8−/−, SlamF1,5,6−/−, SlamF4−/−, and SlamF1,4,5,6+/+ mice for 2 hours. Means ± SD of three independent experiments using freshly derived BMDMs are shown. F1.516,4.549 = 92.62, P < 0.005 for overall comparisons of matched data between SlamF8−/− and SlamF8+/+, repeated-measures one-way analysis of variance (ANOVA); F3,12 = 52.57, P <0.0001 for comparisons between SlamF8−/− and SlamF8+/+ BMDMs at individual OMP doses, repeated-measures one-way ANOVA. (D) Representative flow cytometry images for (C) showing LysoTracker staining of SlamF8+/+ and SlamF8−/− BMDMs treated with the indicated doses of E. coli OmpA for 2 hours. (E) Percentage of cells stained with LysoTracker upon rapamycin treatment of SlamF8+/+ and SlamF8−/− BMDMs. Data are shown from three independent experiments using freshly derived BMDMs. (F) Representative Western blot for p62 in control and SlamF8 siRNA–transfected THP1 cells upon OMP treatment. Data are representative of results from three independent experiments. (G) PKC activity from SlamF8+/+ and SlamF8−/− BMDMs treated with 100 nM E. coli OmpA or VDAC1 as indicated. Means ± SD from three independent experiments are shown. ***P < 0.005. (H) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of SlamF8 mRNA levels in THP1 and Caco2 cells after treatment with 10 nM OMP for 16 hours. Means ± SD of three independent experiments performed in technical triplicate are shown. *P < 0.05. (I) Representative Western blot for p62 and LC3B expression from Caco2 cells transfected with XRCC6 or control siRNA and treated with 100 nM OmpA. Data are representative of results from three independent experiments.

  • Fig. 3 OMPs inhibit mTORC2 and Akt activation and inhibit intracellular bacterial replication.

    (A) Representative Western blots probing the phosphorylation of mTOR at Ser2481 and Ser2448 in Caco2 cells in response to 100 nM OmpA treatment for 15 min. Data are representative of results from three independent experiments. (B) Representative Western blots for phosphorylation of Akt at Ser473 in Caco2 cells upon treatment with 0, 5, 10, 50, and 100 nM OmpA for 15 min. Data are representative of results from three independent experiments. (C) Representative Western blot showing time course of OMV-mediated p62 and LC3B degradation in Caco2 cells treated with B. theta OMVs (10 μg/ml). Data are representative of results from three independent experiments. (D) Representative Western blot for GFP showing cleavage of GFP-LC3 in HEK293T cells transfected with GFP-LC3 and treated with B. theta OMVs (0, 1, 5, and 10 μg/ml) for 15 min. Data are representative of results from three independent experiments. (E) Representative Western blot showing LC3B levels in BMDMs treated with B. theta OMVs (10 μg/ml) for the indicated times. Data are representative of results from three independent experiments using freshly derived BMDMs. (F) Representative Western blot showing Akt Ser473 phosphorylation in BMDMs treated with control (C), LPS (L), and OmpA (O) or cotreated with LPS + OmpA (LO) for 15 min or 1 hour after S. Typhimurium (S.Tm) infection at a multiplicity of infection (MOI) of 10. Data are representative of results from three independent experiments using freshly derived BMDMs. (G) Representative Western blot showing the time course of mTOR Ser2481 and Akt Ser473 phosphorylation in total lysates from S. Typhimurium–infected BMDMs at an MOI of 10. Data are representative of results from three independent experiments using freshly derived BMDMs. (H) Means ± SD of colony-forming units (CFUs) in BMDMs infected with S. Typhimurium at an MOI of 10, further treated with OMP or control buffer 1 hour after infection. (I) Ratio of CFUs at 16 hours/2 hours in control or OMP-treated S. Typhimurium–infected BMDMs. Means ± SD from three independent experiments using freshly derived BMDMs are shown. **P < 0.01. (J) Mechanistic model of action of OMPs on SlamF8 in macrophages and XRCC6 in epithelial cells.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/558/eaat7493/DC1

    Fig. S1. Representative purification of an OMP.

    Fig. S2. Purified OMPs trigger autophagy but not cell death in THP1 cells.

    Fig. S3. SlamF8 and XRCC6 are required for OMP-mediated autophagy in macrophages and epithelial cells.

    Fig. S4. Purified OmpA induces surface expression of HLA-DR and facilitates Atg16L1-dependent bacterial clearance in THP1 cells.

  • This PDF file includes:

    • Fig. S1. Representative purification of an OMP.
    • Fig. S2. Purified OMPs trigger autophagy but not cell death in THP1 cells.
    • Fig. S3. SlamF8 and XRCC6 are required for OMP-mediated autophagy in macrophages and epithelial cells.
    • Fig. S4. Purified OmpA induces surface expression of HLA-DR and facilitates Atg16L1-dependent bacterial clearance in THP1 cells.

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