Host-mediated ubiquitination of a mycobacterial protein suppresses immunity

Abstract

Mycobacterium tuberculosis is an intracellular pathogen that uses several strategies to interfere with the signalling functions of host immune molecules. Many other bacterial pathogens exploit the host ubiquitination system to promote pathogenesis1,2, but whether this same system modulates the ubiquitination of M. tuberculosis proteins is unknown. Here we report that the host E3 ubiquitin ligase ANAPC2—a core subunit of the anaphase-promoting complex/cyclosome—interacts with the mycobacterial protein Rv0222 and promotes the attachment of lysine-11-linked ubiquitin chains to lysine 76 of Rv0222 in order to suppress the expression of proinflammatory cytokines. Inhibition of ANAPC2 by specific short hairpin RNA abolishes the inhibitory effect of Rv0222 on proinflammatory responses. Moreover, mutation of the ubiquitination site on Rv0222 impairs the inhibition of proinflammatory cytokines by Rv0222 and reduces virulence during infection in mice. Mechanistically, lysine-11-linked ubiquitination of Rv0222 by ANAPC2 facilitates the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6, preventing the lysine-63-linked ubiquitination and activation of TRAF6. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.

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Fig. 1: Rv0222 inhibits host inflammatory responses.
Fig. 2: Rv0222 interacts with and inhibits TRAF6 signalling.
Fig. 3: Rv0222 is K11-ubiquitinated at K76 by host ANAPC2.
Fig. 4: K11-linked ubiquitination of Rv0222 by ANAPC2 suppresses anti-tuberculosis immunity.

Data availability

RNA-sequencing data have been deposited in Gene Expression Omnibus (GEO) under accession codes GSM4005162, GSM4005163, GSM4005164, GSM4005165, GSM4005166 and GSM4005167. Other data that support the findings of this study are available within the Source Data and Supplementary Information. Source Data for Figs. 1, 2, 4 and Extended Data Figs. 1, 2, 4, 5 are provided with the paper.

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Acknowledgements

We thank M. Rape (Howard Hughes Medical Institute) and M. Matsumoto (Genentech) for providing antibody against K11/K48-branched ubiquitin chains; L.-D. Lyu (CAS Key Laboratory of Synthetic Biology) for technical assistance in constructing the Rv0222-knockout strain; K. Mi (CAS Key Laboratory of Pathogenic Microbiology and Immunology) for the pMV261 plasmid; X. Cao (Second Military Medical University) for ubiquitin plasmids; G. Meng (Institute Pasteur of Shanghai) for pLKO.1-TRAF6 plasmids; F. Shao (National Institute of Biological Sciences) and C. G. Feng (Sydney Medical School, University of Sydney) for critical reading of the manuscript; and members of the B.G. laboratory for discussions and technical assistance. This project was supported by grants from the Chinese National Program on Key Basic Research Project (2017YFA0505900 to B.G.; 2017YFC0840300 to Z.R.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020200 to Z.R.), the National Natural Science Foundation of China (91842303, 31730025, 91542111, 81330069, 31030028 and 30525012 to B.G.; 81800004 to L.W.; and 81520108019 and 813300237 to Z.R.), Fundamental Research Funds for the Central Universities (22120180024 to L.W.), the clinical key discipline construction project from the Shanghai Municipal Health Commission (2017ZZ02003 to B.G.) and Shanghai Pulmonary Hospital (fkyq1909 to L.W.).

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Contributions

L.W. and B.G. designed the study. L.W. and J. Wu performed most experiments and analysed data. T.T., H. Liang, M.Z. and C.P. performed the qPCR and co-immunoprecipitation experiments and analysed data. H. Liu and F.L. provided technical help and assisted with manuscript preparation. J.C., Z.L., Y.W., X.W. and R.Z. performed mouse infection experiments and analysed data. J. Wang and H.Y. constructed the knockout H37Rv strain. The Rv0222 structure (Protein Data Bank code 6LDZ) was solved and analysed by J.L., Y.R. and Z.R. The H37Rv strain was stored by X.H. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Zihe Rao or Baoxue Ge.

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Extended data figures and tables

Extended Data Fig. 1 Rv0222 inhibits host inflammatory responses.

a, b, Luciferase assay of HEK293T cells transfected with the NF-κB (a) or AP-1 (b) reporter gene with Rv0222 plasmid (+) or vector plasmid (−). c, Immunoblot of lysates from iBMDMs transfected with a plasmid encoding Flag-tagged Rv0222 for 48 h, following infection with H37Rv for the indicated times (MOI = 5). p-p65, phosphorylated p65, an indicator of NF-κB activation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, a loading control. d, Immunoblot of Rv0222 protein in H37Rv, ΔRv0222, H37Rv(ΔRv0222 + Rv0222) and H37Rv(ΔRv0222 + Rv0222(K76A)) cells with anti-Rv0222 antibody. Coomassie blue staining was used as a control. eg, qPCR analysis of Il1b (e), Il6 (f) and Il12 p40 (g) mRNA from peritoneal macrophages infected with H37Rv or ΔRv0222 for the indicated times (MOI = 5). h, i, Lactate dehydrogenase assay for cell death (h) and CFU assay (i) in mice peritoneal macrophages infected with H37Rv or ΔRv0222 for 3 h (i), 12 h or 24 h (MOI = 5). j, C57BL/6 mice were aerosol-infected with roughly 200 CFUs per mouse of H37Rv, H37Rv(ΔRv0222 + GFP) or H37Rv(ΔRv0222 + Rv0222) for 30 days. Histopathology was assessed in lung sections stained with acid-fast (scale bars, 100 μm (top) and 20 μm (bottom)). Two-tailed unpaired Student’s t-test (a, b, ei) was used for statistical analysis. Data are representative of one experiment with at least three independent biological replicates in c, d, j; and show mean ± s.e.m. of n = 3 cultures in a, b, ei. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 2 Rv0222 interacts with and inhibits TRAF6 signalling.

a, Immunoblot of lysates from peritoneal macrophages infected with H37Rv or ΔRv0222 for the indicated times (MOI = 5). b, c, qPCR analysis of Il1b (b) and Il6 (c) mRNA from peritoneal macrophages treated with the p38 inhibitor SB203580, JNK inhibitor SP600125 or NF-κB inhibitor PDTC for 1 h following H37Rv or ΔRv0222 infection for 8 h (MOI = 5). d, Immunoblots and immunoprecipitates of lysates from HEK293T cells transfected with the indicated plasmids. eh, Luciferase assay of the effect of Rv0222 on the activation of TRAF6/TAK1 in HEK293T cells transfected with a plasmid encoding NF-κB (e, g) or AP-1(f, h) luciferase reporter or indicated plasmids for 24 h. ik, qPCR analysis of Il1b (i, right), Il6 (j) and Il12 p40 (k) mRNA from control or TRAF6-knockdown iBMDMs infected with H37Rv or ΔRv0222 for 4 h (MOI = 5). i, Left, immunoblots of TRAF6 in lysates from control (‘Scrambled’) or TRAF6-knockdown (TRAF6-1, TRAF6-2) iBMDMs. ln, qPCR analysis of Il1b (l, right), Il6 (m) and Il12 p40 (n) mRNA from control or TAK1-knockdown iBMDMs infected with H37Rv or ΔRv0222 for 6 h (MOI = 5). l, Left, immunoblots of TAK1 in lysates from control or TAK1-knockdown iBMDMs. o, Immunoblots of lysates from control or TRAF1-knockdown iBMDMs infected with H37Rv for the indicated times (MOI = 5). pr, qPCR analysis of Il1b (p), Il6 (q) and Il12 p40 (r) mRNA from control or TRAF1-knockdown iBMDMs infected with H37Rv for the indicated times (MOI = 5). s, t, Immunoblots and immunoprecipitation of lysates from HEK293T cells transfected with a plasmid encoding HA-tagged Ub (s) or K63-Ub (t), Flag-tagged TRAF6 and increasing amounts of Myc-tagged Rv0222 (0 μg, 1 μg and 2 μg). u, Immunoblots of lysates from control or SHP1- or SHP2-knockdown iBMDMs. vy, qPCR analysis of Il1b and Il6 mRNA from control or SHP1/2-knockdown iBMDMs infected with H37Rv or ΔRv0222 for 6 h (MOI = 5). Two-tailed unpaired Student’s t-tests (b, c, en, vy) were used for statistical analysis. Data are representative of one experiment with at least three independent biological replicates (a, d, o, su) and are mean ± s.e.m in b, c, en, pr, vy. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 3 Rv0222 is K11-ubiquitinated at K76 by host ANAPC2.

a, b, Immunoblots and immunoprecipitates of HEK293T cell lysates transfected with a plasmid encoding Flag-tagged Rv0222 and HA-tagged wild-type ubiquitin (Ub) or HA-tagged Ub with a single lysine residue left unmutated among K6, K11, K27, K29, K33, K48 and K63. c, Immunoblots and immunoprecipitates of lysates from peritoneal macrophages infected with H37Rv(ΔRv0222 + Rv0222) for the indicated times (MOI = 5). d, Immunoblots and immunoprecipitates of HEK293T cell lysates transfected with a plasmid encoding Flag-tagged Rv0222 and Myc-tagged ANAPC2, RNF7 and BIRC2. e, Immunoblots and immunoprecipitates of HEK293T cell lysates transfected with the indicated plasmids. Data are representative of one experiments with at least three independent biological replicates. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 4 K11-linked ubiquitination of Rv0222 by ANAPC2 suppresses TRAF6 signalling.

a, b, Luciferase assay of HEK293T cells transfected with a plasmid encoding NF-κB (a) or AP-1(b) luciferase reporter or the indicated plasmids for 24 h. c, Immunoblots and immunoprecipitates of HEK293T cell lysates transfected with the indicated plasmids. Six-week-old female SCID mice were aerosol-infected with roughly 100 CFUs per mouse of H37Rv, H37Rv(ΔRv0222 + GFP), H37Rv(ΔRv0222 + Rv0222) or H37Rv(ΔRv0222 + Rv0222(K76A)). d, e, qPCR analysis of Il1b (d) and Il6 (e) mRNA in lung tissues from mice infected for 7 days. fh, Immunoblots and immunoprecipitates of HEK293T cell lysates transfected with indicated plasmids. i, Luciferase assay of control or ANAPC2-knockdown HEK293T cells transfected with a plasmid encoding NF-κB luciferase reporter or the indicated plasmids for 24 h. jl, Immunoblots and immunoprecipitates of lysates from control or ANAPC2-knockdown HEK293T cells transfected with the indicated plasmids. mo, qPCR analysis of Il1b (m), Il6 (n) and Il12 p40 (o) mRNA from control or ANAPC2-knockdown THP1 cells infected with H37Rv or ΔRv0222 for the indicated times (MOI = 5). p, Immunoblot of lysates from control or ANAPC2-knockdown iBMDMs infected with H37Rv for the indicated times (MOI = 5). Two-tailed unpaired Student’s t-test (a, b, d, e, i, mo) were used for statistical analysis. Data are representative of one experiment with at least three independent biological replicates (c, fh, jl, p) and are mean ± s.e.m. in a, b, i, mo. d, e, Cumulative data from two independent experiments (n = 6 mice). For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 5 K11-linked ubiquitination of Rv0222 by ANAPC2 suppresses anti- tuberculosis immunity.

a, Survival of six-week-old female SCID mice aerosol-infected with roughly 100 CFUs per mouse of H37Rv, H37Rv(ΔRv0222 + GFP), H37Rv(ΔRv0222 + Rv0222) or H37Rv(ΔRv0222 + Rv0222(K76A)). b, Bacterial titres in lungs from mice infected for 1 day or 14 days. Gehan–Breslow–Wilcoxon test (a) and two-sided Mann–Whitney U-test (b) were used for statistical analysis. Cumulative data from two independent experiments (n = 37 mice in a; n = 4 mice infected for 1 day and n = 8 mice infected for 14 days in b). Source data

Extended Data Fig. 6 Summary diagram.

The K11-linked ubiquitination (K11ub) of M.tuberculosis-secreted protein Rv0222 by host ANAPC2 suppresses the TLR2/TRAF6/NF-κB-mediated expression of proinflammatory cytokine mRNAs (Il1b and Il12 p40), part of innate immunity, by facilitating the recruitment of SHP1/2 to polyubiquitinated (K63ub) TRAF6. Mtb, M. tuberculosis.

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Supplementary Figure 1: Uncropped western blot images with molecular weight markers and indication of how the gels were cropped.

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Supplementary Table 1: Strains, plasmids and primers used in in this study.

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Wang, L., Wu, J., Li, J. et al. Host-mediated ubiquitination of a mycobacterial protein suppresses immunity. Nature 577, 682–688 (2020). https://doi.org/10.1038/s41586-019-1915-7

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