684 | Nature | Vol 577 | 30 January 2020
Article
of Il1b and Il6 in macrophages infected with H37RvΔRv0222 (Extended
Data Fig. 2b, c), suggesting that Rv0222 may inhibit the M. tuberculosis-
induced expression of proinflammatory cytokines by downregulating
the activation of the p38, JNK and NF-κB pathways.
M. tuberculosis infection induces the expression of proinflamma-
tory cytokines via the Toll-like receptor (TLR) signalling pathway^8 ,^9 ,
which is composed of several adaptor proteins, namely tumour necro-
sis factor (TNF)-receptor-associated factor 6 (TRAF6), transforming
growth factor-β (TGFβ)-activated kinase 1 (TAK1) and TAK1-binding
protein 1 (TAB1)^10. We found that TRAF6 interacts with Rv0222 (Fig. 2a,
b and Extended Data Fig. 2d). Moreover, through co-immunoprecip-
itation and confocal microscopy analysis of macrophages infected
with H37Rv(ΔRv0222 + Rv0222), we observed an endogenous inter-
action between Rv0222 and TRAF6 (Fig. 2c, d). Among TRAF-family
proteins, Rv0222 associates with TRAF1 and TRAF6 (Extended Data
Fig. 2d). Rv0222 efficiently blocked the TRAF6-triggered, but not the
TAK1-plus-TAB1-triggered, activation of NF-κB and AP-1 (Extended Data
Fig. 2e–h). Knockdown of TRAF6 by specific shRNA markedly attenu-
ated the inhibitory effect of Rv0222 on the expression of Il1b, Il6 and
Il12 p40 (Extended Data Fig. 2i–k). Consistent with this, knockdown of
TAK1 in iBMDMs by specific shRNA also markedly reduced the Rv0222-
mediated inhibitory effect on the expression of Il1b, Il6 and Il12 p
(Extended Data Fig. 2l–n). However, TRAF1 had no marked effect on
the activation of the NF-κB or MAP kinase signalling pathways, or on
cytokine expression in M. tuberculosis-infected macrophages (Extended
Data Fig. 2o–r). Together, these data suggest that Rv0222 may inhibit
the activation of host MAP kinase and NF-κB signalling by targeting
the TRAF6 pathway.
Activation of TRAF6 is a ubiquitination-dependent process^10. We
found that overexpression of Rv0222 markedly inhibited both total
ubiquitination and K63-linked ubiquitination of TRAF6 in HEK293T
cells (Extended Data Fig. 2s, t), whereas deletion of Rv0222 markedly
enhanced K63-linked ubiquitination of TRAF6 in macrophages infected
with H37Rv (Fig. 2e). The protein tyrosine phosphatases SHP1 and SHP
(hereafter SHP1/2) have been reported to interact with TRAF6 and inhibit
its ubiquitination^11. Therefore, we next examined the effect of Rv0222 on
the interaction between SHP1/2 and TRAF6. In HEK293T cells, Rv
promoted the binding of SHP1/2 with TRAF6 (Fig. 2f, g). We generated
iBMDMs in which Shp1 and Shp2 were stably knocked down (Extended
Data Fig. 2u), and found that silencing of Shp1/2 by specific shRNA elimi-
nated the inhibitory effect of Rv0222 on cytokine expression (Extended
Data Fig. 2v–y). Together, these data suggest that Rv0222 facilitates the
association of SHP1/2 with TRAF6, thus inhibiting TRAF6 activation.K11-linked Rv0222 ubiquitination by ANAPC
Given that TRAF6 serves as a classical E3 ligase for the K63-linked
polyubiquitination of multiple signalling molecules^12 , and that Rv0.0.1.1.Co-localization ratiob Flag–TRAF
HA–Rv
IP: Flag
IB: HA
IP: Flag
IB: Flag
Lysate
IB: HA++–- ++
TRAFRvdaecDAPI ΔRv0222 + Rv0222 TRAF6 Flag–Rv0222 MergeRvFlag–Rv
HA–TRAF
IP: Flag
IB: HA
IP: Flag
IB: FlagLysate
IB: HA++–- ++
RvTRAFTRAFTime (h)IB: K63-UbIP: TRAF6 K63-UbIP: TRAF
IB: TRAF6 TRAF013013H37Rv Rv0222 Flag–TRAF
HA–SHP
Myc–Rv+–++
–+++
–––+
IP: Flag
IB: HA
IP: Flag
IB: Flag
Lysate
IB: HA
Lysate
IB: MycSHPTRAFSHPRvIP: Flag
IB: Flag
Lysate
IB: HA
Lysate
IB: MycTRAFSHPRvFlag–TRAF
HA–SHP
Myc–Rv
IP: Flag
IB: HA SHP+–++
–+++
–––+ΔRv0222 + Rv
Time (h)IP: Flag
IB: Rv036
IP: Flag
IB: TRAFLysate
IB: TRAFRvTRAFTRAFf gOverlap
coef cientPearson’s rFig. 2 | Rv0222 interacts with and inhibits TR AF6 signalling.
a, b, Immunoblot (IB) and immunoprecipitation (IP) of lysates of HEK293T cells
transfected with various plasmids as indicated. c, Endogenous interaction of
Rv0222 with TR AF6 in H37Rv(ΔRv0222 + Rv0222)-infected peritoneal
macrophages (MOI = 5). d, Left, images show confocal microscopy of peritoneal
macrophages infected with H37Rv(ΔRv0222 + Rv0222) for 3 h (MOI = 5). Scale
bar, 5 μm. Right, the co-localization ratio from 60 macrophages (mean ± s.e.m.).
e, Peritoneal macrophages were infected with H37Rv or ΔRv0222 for the
indicated times (MOI = 5); cell lysates were immunoprecipitated with an anti-
TRAF6 antibody; and immunoprecipitates were analysed with an anti-K63-Ub
antibody. f, g, Immunoblot and immunoprecipitation of lysates from HEK293T
cells transfected with plasmids as indicated. Data in a–g represent one
experiment with at least three independent biological replicates. For gel
source data, see Supplementary Fig. 1.