Nature | Vol 577 | 30 January 2020 | 687complemented with Rv0222, but not with Rv0222(K76A), induced
much lower expression of proinflammatory cytokines in the lungs of
M. tuberculosis-infected mice (Fig. 4d–f and Extended Data Fig. 4d,
e). Together, these results suggest that K76 is essential for Rv0222 to
inhibit host proinflammatory cytokines.
We also found that Rv0222(K76A) did not bind as efficiently as wild-
type Rv0222 to either TRAF6 or SHP1 (Extended Data Fig. 4f, g). Con-
sistent with this, overexpression of Rv0222 rather than Rv0222(K76A)
in HEK293T cells markedly enhanced the association between SHP
and TRAF6 (Extended Data Fig. 4h). Thus, the increased Rv0222-
induced binding of SHP1 to TRAF6 may require K11-linked polyubiq-
uitination on K76 of Rv0222. Although further functional studies are
required to elucidate the mechanism by which K11-linked polyubiq-
uitination of Rv0222 leads to an enhanced interaction between SHP
and TRAF6 and hence to the inhibitory effect of Rv0222, one possible
explanation is that the polyubiquitin chains are required directly
to form a Rv0222–SHP1–TRAF6 complex. Alternatively, K11-linked
polyubiquitination might cause a conformational change in Rv
that renders it more accessible for the binding of TRAF6 and SHP1,
enhancing their interaction and inhibiting the K63-linked polyubiq-
uitin of TRAF6.
Given that ANAPC2 promotes the K11-linked polyubiquitination
of Rv0222 at K76, we next investigated whether ANAPC2 is involved
in suppressing TRAF6 signalling by Rv0222. In ANAPC2-knockdown
HEK293T cells, TRAF6-induced NF-κB luciferase reporter activity
was not substantially inhibited by Rv0222 (Extended Data Fig. 4i).
Moreover, silencing of ANAPC2 with specific shRNA markedly reduced
the binding of Rv0222 with TRAF6 in HEK293T cells (Extended Data
Fig. 4j). ANAPC2 knockdown also impaired the inhibitory effect of
Rv0222 on K63-linked polyubiquitination of TRAF6 in HEK293T
cells (Extended Data Fig. 4k). Furthermore, in ANAPC2-knockdown
HEK293T cells, Rv0222 was considerably less able to enhance the asso-
ciation of SHP1 with TRAF6 (Extended Data Fig. 4l). Finally, we found
that infection with H37RvΔRv0222 induced much higher proinflam-
matory cytokine expression than did infection with H37Rv in THP
cells, but ANAPC2-knockdown THP1 cells infected with H37RvΔRv
showed an even lower level of proinflammatory cytokine expression
than did those cells infected with H37Rv (Extended Data Fig. 4m–o).
Silencing of ANAPC2 by specific shRNA led to a marked reduction in
cytokine levels in both H37Rv- and H37RvΔRv0222-infected cells, sug-
gesting that ANAPC2 may also regulate the expression of cytokines
in a Rv0222-independent manner. We also observed that knock-
down of ANAPC2 by specific shRNA in iBMDMs almost eliminated
the H37Rv-induced activation of MAP kinases and NF-κB (Extended
Data Fig. 4p). ANAPC2 is an important host E3 ligase, but it remains
to be seen whether ANAPC2 is involved in activating the inflamma-
tory signalling pathway through other mechanisms. Together, our
results suggest that the host ubiquitin ligase ANAPC2 regulates the
inhibitory effect of Rv0222 on cytokine expression by promoting its
K11-linked polyubiquitination.
Virulence of Rv0222 requires ubiquitination
To investigate the pathological relevance of the K11-linked poly-
ubiquitination of Rv0222 in M. tuberculosis infection, we challenged
C57BL/6 and SCID^18 mice with wild-type H37Rv, H37Rv(ΔRv0222 + GFP),
H37Rv(ΔRv0222 + Rv0222) or H37Rv(ΔRv0222 + Rv0222(K76A)),
and examined histopathology and bacterial burden in the lungs. For
C57BL/6 mice, infection with H37RvΔRv0222 complemented with
wild-type Rv0222 (rather than with the K76A mutant) led to more-
severe histological damage and more immune-cell infiltration into
the lungs (Fig. 4g). For SCID mice, at eight weeks after infection,
almost all mice infected with H37Rv or H37Rv(ΔRv0222 + Rv0222)
died, but those infected with H37Rv(ΔRv0222 + GFP) or H37Rv
(∆Rv0222 + Rv0222(K76A)) lived for much longer (Extended Data
Fig. 5a). Similarly, the growth defects of H37Rv(ΔRv0222 + GFP) in the
lung tissue of infected C57BL/6 mice or SCID mice were rescued when
the strain was complemented with wild-type Rv0222 but not with the
K76A mutant (Fig. 4h, i and Extended Data Fig. 5b), suggesting that the
virulence of H37Rv bacteria requires the K11-linked ubiquitination of
Rv0222 at K76.
Our findings identify Rv0222 as a previously unrecognized com-
ponent of the immunological signature of M. tuberculosis. Previous
studies have shown the accumulation of ubiquitin on host membra-
nous structures containing M. tuberculosis^19 –^23 , and the interaction of
a bacterial ubiquitin-binding protein with a host signalling molecule^8 ;
however, mycobacteria possess only a pupylation system for pro-
tein post-translational modification^24 , rather than an endogenous
ubiquitination system. Our results reveal a mechanism for subverting
host immunity that depends on the ubiquitination of M. tuberculo-
sis Rv0222 by a host E3 ligase, ANAPC2, highlighting the versatility
of host–M. tuberculosis interactions (Extended Data Fig. 6). Future
research will expand to the role of ubiquitination mediated by other
host E3 ligases^16 ,^25. Notably, the K11-linked ubiquitination of Rv0222 by
ANAPC2 seems to have a different cellular effect to the APC/C-triggered
degradation of many cell-cycle regulators^26 : the Rv0222 ubiquitina-
tion promotes the binding of phosphatases to immune signalling
molecules, rather than substrate recognition by the proteasome^14.
Our findings could pave the way to the development of effective anti-
tuberculosis treatments that target the Rv0222–ANAPC2 interface.Online content
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