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The Salmonella effector protein SopD targets Rab8 to positively and negatively modulate the inflammatory response

Nature Microbiology volume 6, pages 658–671 (2021)Cite this article

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Abstract

The food-borne bacterial pathogen Salmonella Typhimurium uses a type III protein secretion system to deliver multiple proteins into host cells. These secreted effectors modulate the functions of host cells and activate specific signalling cascades that result in the production of pro-inflammatory cytokines and intestinal inflammation. Some of the Salmonella-encoded effectors counteract this inflammatory response and help to preserve host homeostasis. Here, we demonstrate that the Salmonella effector protein SopD, which is required for pathogenesis, functions to both activate and inhibit the inflammatory response by targeting the Rab8 GTPase, which is a negative regulator of inflammation. We show that SopD has GTPase-activating protein activity for Rab8 and, therefore, inhibits this GTPase and stimulates inflammation. We also show that SopD activates Rab8 by displacing it from its cognate guanosine dissociation inhibitor, resulting in the stimulation of a signalling cascade that suppresses inflammation. We solved the crystal structure of SopD in association with Rab8 to a resolution of 2.3 Å, which reveals a unique contact interface that underlies these complex interactions. These findings show the remarkable evolution of a bacterial effector protein to exert both agonistic and antagonistic activities towards the same host cellular target to modulate the inflammatory response.

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Fig. 1: SopD is a GAP for Rab8.
Fig. 2: SopD enhances pro-inflammatory signalling by antagonizing RAB8 through its GAP activity.
Fig. 3: Crystal structure of the SopD–Rab8 complex and functional analyses of the binding interface.
Fig. 4: SopD activates Rab8 through a functional interface unrelated to its GAP activity.
Fig. 5: The structural basis for GDI displacement by SopD.
Fig. 6: SopD positively and negatively modulates S. Typhimurium-induced inflammatory signalling through its independent Rab8-modulating activities.

Data availability

The atomic coordinates and structure factors generated in this study have been deposited in the PDB under the accession code 7BWT. Source data are provided with this paper.

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Acknowledgements

We thank J. Wang (Tsinghua University, China) for suggestions on structure data processing; the staff of the BL17U1 and BL19U1 beamlines of the National Facility for Protein Science Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility (SSRF) for assistance during data collection; and X. Li (Shandong University, Core facilities for life and environmental sciences) for help with the XRD. Work in X.G.’s laboratory was supported by the National Key R&D Program of China (no. 2018YFE0113000), the National Natural Science Foundation of China (nos. 31770143 and 31901943), the Major Basic Program of Natural Science Foundation of Shandong Province (no. ZR2019ZD21), the Youth Interdisciplinary Innovative Research Group of Shandong University (no. 2020QNQT009) and the Taishan Young Scholars Program (no. tsqn20161005). Work in J.E.G.’s laboratory was supported by NIH grants (nos. R01AI055472 and R01AI079022).

Author information

Author notes

  1. These authors contributed equally: Huan Lian, Kun Jiang, Ming Tong.

Authors and Affiliations

  1. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

    Huan Lian & Jorge E. Galán

  2. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    Kun Jiang, Ming Tong, Zhe Chen, Xiaoyu Liu & Xiang Gao

  3. School of Life Sciences, Shandong University, Qingdao, China

    Xiang Gao

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Contributions

H.L. performed all of the functional analyses, animal and cell biological experiments. K.J., M.T. and Z.C. carried out the biochemical characterization of SopD and solved its crystal structure bound to Rab8. J.E.G. and X.G. supervised the study. J.E.G. and X.G. wrote the paper with comments from all of the authors.

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Correspondence to Jorge E. Galán or Xiang Gao.

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Peer review information: Nature Microbiology thanks Samuel Miller, Laurent Terradot and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Size-exclusion chromatography analyses of Rab8 in the presence of SopD or SopD2.

Purified Rab81–183 preloaded with GTPγS was incubated with purified SopD or SopD2 and subjected to size-exclusion chromatography in a Superdex 75 increase column. Elution profiles along with SDS-PAGE analyses of the elution fractions are shown. This experiment was conducted at least three times with equivalent results.

Source data

Extended Data Fig. 2 Rab8 is recruited to the Salmonella-induced membrane ruffles.

Henle-407 cells were transiently transfected with a plasmid expressing GFP-Rab8A and subsequently infected with wild-type S. Typhimurium, its ∆sopD isogeneic mutant, or left uninfected (mock). Fifteen minutes after infection, cells were fixed and stained with anti-GFP antibody, to visualize Rab8A (green), rhodamine-labelled Phalloidin, to visualize the actin cytoskeleton (red), and 4’,6-diamidino-2-phenylindole (DAPI) to visualize nuclear and bacteria DNA (blue). Scale bar: 10 µm. This experiment was conducted at least three times with equivalent results.

Extended Data Fig. 3 Rab8 negatively modulates Salmonella-induced pro-inflammatory signaling.

Effects of Rab8a (a and b), Rab8b (c), or Rab8ab (d) deficiency on S. Typhimurium-induced AKT activation. Control or deficient Raw264.7 or HT29 (as indicated) cells were infected with wild-type S. Typhimurium with a multiplicity of infection of 2 and 10, respectively, and at the indicated times after infection, the levels of phosphorylated Akt were determined by immunoblotting analyses as indicated in Materials and Methods. Values are normalized to the β-actin signal, which served as a loading control. The western blots for this experiment are shown in Fig. 2.

Source data

Extended Data Fig. 4 SopD enhances pro-inflammatory signaling by antagonizing Rab8 through its GAP activity.

(a) Expression levels of SopD and its catalytic mutant SopDR312A in stable cell lines (Raw264.7). Stable cell lines (Raw264.7) expressing HA-tagged SopD or SopDR312A were lysed before immunoblotting analysis with the antibodies directed to the HA tag and to β-actin (as a loading control). (b) Effect of the expression of SopD or its catalytic mutant SopDR312A on LPS-induced activation of AKT, p70S6K, Erk1/2, and p38 MAP, and NF-κB signaling pathways. Raw264.7 cells stably expressing HA-tagged SopD or its GAP-deficient mutant SopDR312A were treated with LPS (100 ng/ml) for the indicated times, lysed, and analyzed by immunoblotting with antibodies specific for the phosphorylated state of AKT, p70S6K, p38, and Erk1/2, as well as an antibody to I-κBα and β-actin (as a loading control). The quantification of the western blot analyses is shown on Fig. 2e.

Source data

Extended Data Fig. 5 PI3-kinase is required for S. Typhimurium-induced signaling.

(a) Effects of the PI3-kinase inhibitor Wortmannin on S. Typhimurium-induced phosphorylation of AKT. Raw264.7 (MOI=2) or HT29 cells (MOI=10) were pre-treated with increasing concentrations of Wortmannin (10 nM to 1000 nM) for 30 min. Cells were then infected with wild-type S. Typhimurium for 30 min and cell lysates were analyzed by immunoblotting with antibodies directed to the phosphorylated (activated) form of Akt or β-actin (as a loading control). Controls included cells left uninfected or infected with the ∆sopB S. Typhimurium mutant strain, which is defective for the activation of AKT. Quantification of the immunoblots is shown on the right panels. (b) Effects of the PI3-kinase inhibitor Wortmannin on S. Typhimurium-induced transcription of cytokine genes. Raw264.7 cells (MOI=5) and HT29 cells (MOI=20) were pre-treated with Wortmannin (100 nM) for 30 min. Cells were left uninfected or infected with wild-type S. Typhimurium for 4 hs and the cytokine mRNA levels were measured by qPCR assay. Data are the mean ± standard deviation of three independent experiments. ** P < 0.01, *** P < 0.001 (unpaired two-sided t test).

Source data

Extended Data Fig. 6 Size-exclusion chromatography profile of SopD/GDP-bound Rab8 complex.

Rab81–183 preloaded with GDP was incubated with SopD, and subjected to size-exclusion chromatography with Superdex 200 increase column. The elution profile along with the SDS-PAGE analyses of the elution fractions of the SopD/Rab8 complex are shown. This experiment was conducted at least three times with equivalent results.

Source data

Extended Data Fig. 7 SDS-PAGE analyses of the elution fractions of the size-exclusion chromatography analyses of SopD carrying mutations in amino acids defining its interface with Rab8.

Purified Rab8a1–183 preloaded with GDP was incubated with purified SopD or the indicated SopD mutants and subjected to size-exclusion chromatography (see the chromatographic profile in Fig. 3e). Fractions were collected, separated on an SDS-PAGE, and stained with Coomassie blue. This experiment was conducted at least three times with equivalent results.

Source data

Extended Data Fig. 8 The SopDE293A mutation disrupts the ability of SopD to form a complex with Rab8.

FLAG-epitope tagged SopD or SopDE293A were co-expressed with GFP-Rab8 or empty vector in HEK-293T cells. The cell lysates were co-immunoprecipitated with anti-FLAG M2 beads followed by immunoblotting with anti-GFP and anti-Flag antibodies. The quantification of the intensity of the bands is shown in the right panel. This experiment was conducted at least three times with equivalent results.

Source data

Extended Data Fig. 9 SopD lacks guanine-nucleotide exchange activity.

Mant-GDP-loaded Rab8a (2 μM) was incubated with Rabin8 (0.4 μM) (positive control), SopD (2 μM), 2 μM SopDR312A (2 μM), SopDE293A (2 μM) or buffer only (negative control), in the presence of 5 μM GTPγS. The decreased fluorescence as a result of the mant-GDP to GTPγS exchange was monitored over time. Data are expressed as the mean ± SD from three independent experiments.

Source data

Extended Data Fig. 10 Effect of SopD or its mutants on cytokine production in S. Typhimurium-infected cells.

Raw264.7 cells were infected with the indicated S. Typhimurium strains (MOI=5) and 18 hours after infection, the levels of the indicated cytokines in cell supernatants were quantified by ELISA. Values are the mean ± SD of three independent measurements. ** P < 0.01, *** P < 0.001, ns: not significant (unpaired two-sided t test).

Source data

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Lian, H., Jiang, K., Tong, M. et al. The Salmonella effector protein SopD targets Rab8 to positively and negatively modulate the inflammatory response. Nat Microbiol 6, 658–671 (2021). https://doi.org/10.1038/s41564-021-00866-3

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