682 | Nature | Vol 577 | 30 January 2020
Article
Host-mediated ubiquitination of a
mycobacterial protein suppresses immunity
Lin Wang1,8, Juehui Wu1,2,8, Jun Li3,8, Hua Yang^1 , Tianqi Tang^2 , Haijiao Liang1,2, Mianyong Zuo^2 ,
Jie Wang^1 , Haipeng Liu1,4, Feng Liu^1 , Jianxia Chen1,4, Zhonghua Liu^1 , Yang Wang^2 , Cheng Peng^2 ,
Xiangyang Wu^2 , Ruijuan Zheng^1 , Xiaochen Huang^1 , Yajun Ran^3 , Zihe Rao3,5,6,7* & Baoxue Ge1,2,4*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
pathogenesis^1 ,^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.To identify M. tuberculosis proteins that inhibit host inflammatory
responses, we examined the effects of 208 mycobacterial secreted
proteins and lipoproteins on the activation of NF-κB^3 , a transcription
factor that is involved in numerous cellular processes, including inflam-
mation. Of these mycobacterial proteins, Rv0222—a serodiagnostic
target for tuberculosis^4 —inhibited NF-κB activation. Consistent with
this, transient expression of Rv0222 in HEK293T cells markedly inhib-
ited the activation of both NF-κB and AP-1, another transcription factor
(Extended Data Fig. 1a, b). Transfection with Rv0222 also resulted in
reduced activation of NF-κB signalling in immortalized bone-marrow-
derived macrophages (iBMDMs) infected with M. tuberculosis, again sug-
gesting that Rv0222 inhibits NF-κB activation (Extended Data Fig. 1c).
Rv0222 inhibits host immunity
To further investigate the effect of Rv0222 on host immune responses,
we generated a Rv0222-deletion mutant of the M. tuberculosis strain
H37Rv (H37RvΔRv0222) (Extended Data Fig. 1d). Primary peritoneal
macrophages—which serve as both a habitat for, and first line of defence
against, M. tuberculosis—were infected with H37Rv or H37RvΔRv
before gene-expression profiling. Macrophages infected with the
H37RvΔRv0222 strain had much higher mRNA levels of proinflamma-
tory cytokines, including interleukin (IL)-1b, IL-6 and IL-12, than those
infected with wild-type H37Rv (Fig. 1a and Extended Data Fig. 1e–g), but
there was no significant difference in cell death (P = 0.76 when infected
for 12 h and P = 0.91 when infected for 24 h) and bacterial entry (P = 0.67)
(Extended Data Fig. 1h, i). Consistent with this finding, the lung tissue
of mice infected with H37RvΔRv0222 had much higher levels of mRNAs
encoding IL-1b, IL-6 and the IL-12 p40 subunit than did the lung tissue
of mice infected with H37Rv (Fig. 1b–d). Furthermore, complementa-
tion of H37RvΔRv0222 with Rv0222, but not with green-fluorescent
protein (GFP), restored the inhibition of Il1b, Il6 and Il12 p40 expres-
sion upon infection with H37Rv (Fig. 1e–g and Extended Data Fig. 1d).
Together, these results provide further evidence that Rv0222 inhibits
M. tuberculosis-triggered inflammatory responses.
We next investigated the functional relevance of Rv0222 in the patho-
genesis of M. tuberculosis infection. The lung tissues of C57BL/6 mice
infected with H37Rv(ΔRv0222 + GFP) had less immune-cell infiltra-
tion and fewer inflammatory lesions than those infected with H37Rv
(Fig. 1h). As shown above, H37RvΔRv0222 induced a higher level
of Il12 p40 expression than did wild-type H37Rv (Fig. 1d). IL-12 p
has been reported to increase the differentiation of T-helper-1 (TH1)https://doi.org/10.1038/s41586-019-1915-
Received: 8 October 2018
Accepted: 21 November 2019
Published online: 15 January 2020
(^1) Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China. (^2) Department of Microbiology and Immunology, Tongji
University School of Medicine, Shanghai, China.^3 Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai,
China.^4 Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China.^5 Laboratory of Structural Biology, Tsinghua University,
Beijing, China.^6 State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and College of Pharmacy, Nankai University, Tianjin, China.^7 National Laboratory of
Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Beijing, China.^8 These authors contributed equally: Lin Wang, Juehui Wu, Jun Li. *e-mail: raozh@
mail.tsinghua.edu.cn; [email protected]