Article
Mice and infection
All animal experiments were reviewed and approved by the Animal
Experiment Administration Committee of Tongji University School of
Medicine and were conducted in accordance with the National Insti-
tutes of Health (NIH) Guidelines for the Care and Use of Laboratory
Animals. Female SPF C57BL/6 and SCID mice were purchased from
Slaccas. Six- to eight-week-old female C57BL/6 mice were used for
macrophage separation. Six-week-old C57BL/6 and SCID female mice
were divided randomly into cages and infected by an aerosol method
with approximately 100–200 CFUs of different H37Rv strains for four
weeks (using an inhalation exposure system from Glas-col)^28 ,^29 at the
Biosafety Level-3 (BSL-3) Laboratory. Mice were killed at 1, 7, 14 or 30
days after infection. Lungs were collected and homogenized in 1 ml of
PBS. Mycobacterial burden was determined by plating tenfold serial
dilutions of each tissue homogenate on Middlebrook 7H10 agar plates.
Colonies were counted after three to four weeks of incubation at 37 °C
in 5% CO 2. For histology, lungs were removed, fixed in 4% paraformal-
dehyde in PBS and embedded in paraffin. Sections were cut and stained
with haematoxylin and eosin (H&E) or Ziehl–Neelsen stain (acid-fast
bacillus) according to standard protocols. Imaging was performed by
microscopy (TCS CP5II, Leica). All mice were age- and sex-matched in
each experiment. Sample size was based on empirical data from pilot
experiments. For in vitro M. tuberculosis infections, macrophages were
infected with a single-cell suspension of bacteria at an MOI of 5.
shRNA-mediated gene interference
Short hairpin RNA in the pLKO.1 vector was introduced into target cells
via lentiviral transduction. pLKO.1-TRAF6-1 and pLKO.1-TRAF6-2 were
gifts from G. Meng^30. The targeting sequences were: human ANAPC
shRNA, 5′-GCAGATTAAAGCAAGTCAGAT-3′; mouse Anapc2 shRNA,
5′-GCGACGTTCTTCAGACATCAT-3′; mouse Tak1 shRNA, 5′-GCGCC
TGAAGTATTTGAAGGT-3′; mouse Shp1 shRNA, 5′-GCTAGACTGTG
ACATTGATAT-3′; mouse Shp2 shRNA, 5′-CGTGTTAGGAACGTCAA
AGAA-3′; mouse Traf1 shRNA, 5′-GCATCCTTTGATGGTACTTTC-3′.
HEK293T cells were transfected with pSPAX2, pMD2.G and PLKO.1 con-
taining shRNAs targeting different genes. Lentiviruses were collected
48 h later and were used to infect iBMDMs (for ANAPC2, TRAF6, TRAF1,
TAK1, SHP1 and SHP2 knockdown) or HEK293T cells and THP1 cells
(for ANAPC2 knockdown). Subsequently, selection with puromycin
(2 μg ml−1) was carried out.
Ubiquitination assay
HA-tagged ubiquitin plasmids were gifts from X. Cao^31. HEK293T cells
were transiently transfected with the indicated plasmids for 48 h.
Rv0222 or TRAF6 was immunoprecipitated with anti-Flag M2 affinity gel
or protein A/G plus anti-TRAF6 antibody, and the obtained complexes
were washed three times with urea (8 M, in PBS) to separate proteins
bound by noncovalent bonds. Ubiquitinated Rv0222 or ubiquitinated
TRAF6 was detected by immunoblot analysis using HA-specific anti-
bodies or specific ubiquitin antibody. For in vitro experiments, His-
Rv0222 was purified from E. coli BL21 and APC/C was purified from
G1 HEK293T cells by anti-CDC27 antibody (Santa Cruz) and protein
A/G (Santa Cruz) at 4 °C. The beads were washed three times with SB
(25 mM HEPES pH 7.5, 1.5 mM MgCl 2 , 5 mM KCl, 1 mM dithiothreitol
(DTT), 1× protease inhibitor, 15 mM creatine phosphate, 2 mM ATP) plus
0.05% Tween-20, and twice with SB before being resuspended in 40 ml
SB. Beads were incubated with 100 nM E1 (Boston Biochem), 125 nM
UbcH10 (Boston Biochem), 1.5 mg ml−1 ubiquitin, 20 mM ATP, 15 mM
creatine phosphate, creatine phosphokinase, 1 mM DTT in UBAB buffer
(25 mM Tris/HCl pH 7.5, 50 mM NaCl, 10 mM MgCl 2 ). The reaction was
started when Rv0222 was added, incubated at 30 °C for 1 h, stopped
when loading buffer was added and analysed by western blot with anti-
ubiquitin antibody.Statistical analysis
Statistical significance between groups was determined by two-tailed
Student’s t-test, two-tailed analysis of variance followed by Bonferroni
post hoc test or two-sided Mann–Whitney U-test. Differences were
significant at P < 0.05. The experiments were not randomized, and the
investigators were not blinded to allocation during experiments and
outcome assessment.Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.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
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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.).Author 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.
Competing interests The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-019-
1915-7.
Correspondence and requests for materials should be addressed to Z.R. or B.G.
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