Methods
Bacterial strains and cells
Bacterial strains are described in Supplementary Table 1. Mycobacterial
strains were grown in Middlebrook 7H9 broth supplemented with 10%
oleic acid–albumin–dextrose–catalase (OADC) and 0.05% Tween-
(Sigma) or Middlebrook 7H10 agar (BD) supplemented with 10% OADC.
When required, the antibiotic kanamycin or hygromycin was added at a
concentration of 100 μg ml−1 for mycobacterial strain selection. Myco-
bacteria cultures were grown up to mid-log phase (an optical density
at 600 nm of approximately 0.6). Aliquots of bacterial cultures were
prepared in 20% glycerol and Middlebrook 7H9 medium and preserved
at −80 °C. The CFU per ml of the stocks was titred by plating serial dilu-
tions on Middlebrook 7H10 agar plates plus 10% OADC. These stocks
were used for all subsequent infections of macrophages and mice.
HEK293T cells (ATCC CRL-3216) and iBMDMs (provided by F. Shao,
National Institute of Biological Sciences, Beijing) were maintained in
Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented
with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco) and
100 U ml–1 penicillin and streptomycin. Macrophages and THP1 cells
(ATCC TIB-202) were cultured in RPMI-1640 medium supplemented
with 10% (v/v) FBS. Peritoneal macrophages were obtained from mice
three days after injection of thioglycollate (BD). We routinely tested
all cells for mycoplasma contamination, and used only those cells that
tested negative for further study.
Plasmids, reagents and antibodies
Plasmids are described in Supplementary Table 1. The following anti-
bodies were used for western blot or immunoprecipitation: from
Sigma-Aldrich, monoclonal mouse anti-Flag M2 affinity gel (cata-
logue number/clone: A2220/M2), rabbit anti-HA antibody (H6908/
polyclonal), rabbit anti-GAPDH antibody (SAB2701826/polyclonal),
rabbit anti-Flag antibody (F7425/polyclonal), rabbit anti-Myc antibody
(C3956/polyclona) and rabbit anti-K11 antibody (MABS107-I/2A3/2E6);
from Cell Signaling Technology, rabbit anti-APC2 antibody (12301/
polyclonal), rabbit anti-phosphorylated (p)-p65 antibody (3033/93H1),
rabbit anti-p-p38 antibody (9215/3D7), rabbit anti-p-Jnk antibody
(9251/polyclonal), rabbit anti-K63-polyubiquitin antibody (5621/poly-
clonal), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody
(7074) and anti-mouse IgG HRP-linked antibody (7076); from Santa
Cruz Biotechnology, rabbit anti-TRAF6 antibody (sc-7221/polyclonal).
Antibody against K11/K48-branched ubiquitin chains was provided
by M. Rape (Howard Hughes Medical Institute) and M. Matsumoto
(Genentech)^14. The rabbit polyclonal antibody against Rv0222 was
generated by immunization of rabbits with purified histidine-tagged
Rv0222 fusion protein, in collaboration with ABclonal Biotech. For
western blot, all primary antibodies were diluted 1/1,000 and sec-
ondary antibodies were diluted 1/2,000. For immunofluorescence,
monoclonal mouse anti-Flag M2 antibody was used at 1/500 dilution
and rabbit anti-TRAF6 at 1/100 dilution. Corresponding Alexa Fluor
633- or Alexa Fluor 555-labelled anti-rabbit or anti-mouse antibodies
(Invitrogen) were used as secondary antibodies (1/1,000 dilution).
Fluorescein isothiocyanate (FITC) for strain dyeing was from Sangon
Biotech (catalogue number 3326-32-7).
Construction of Mycobacteria strains
We used the pYUB854 system^27 to generate the H37Rv strain with dele-
tion of the gene encoding Rv0222 (H37RvΔRv0222). In brief, upstream
flanking sequence (UFS) and downstream flanking sequence (DFS)
either side of the Rv0222 gene were amplified from H37Rv genomic
DNA. Following purification, the PCR products of UFS and DFS were
ligated into the pGEM-Tamp vector (Promega), and the resulting plas-
mid was transformed into competent E. coli DH5α cells, which were
plated on LB agar plus 100 μg ml−1 ampicillin. Following overnight
incubation of isolated white colonies in LB broth plus ampicillin,
PCR analysis was used as above to identify successful ligations. The
cloned UFS or DFS fragment was then subcloned into pYUB
digested with the appropriate restriction enzyme. The temperature-
sensitive phage pHA159 and the pYUB854 vector, containing both
UFS and DFS fragments for a particular Rv0222 gene disruption,
were digested with PacI; following gel extraction, these fragments
were ligated using T4 DNA ligase to create a shuttle plasmid, which
was then in vitro packaged using the Lambda DNA Packaging Sys-
tem (Promega) and transformed into E. coli HB101 cells. Successful
plasmids were identified by PacI digestion, followed by antibiotic
selection on LB plus hygromycin (100 μg ml−1) plates. The plasmid
was then transduced into M. smegmatis (mc^2 155) at the permissive
temperature of 30 °C, which allows replication and lysis, and a high-
titre lysate was produced (more than 10^10 ml−1). Transduction into
H37Rv was then carried out with the high-titre lysate at an MOI of 10 at
the nonpermissive temperature of 37 °C, which results in the delivery
of the substrate. Cells-only and phage-only controls were included.
Following incubation for two to three weeks on 7H10 plus OADC,
glycerol and hygromycin, colonies were picked and grown in liquid
medium (7H9 plus OADC, glycerol, Tween-80 and hygromycin) for
two weeks; then, gene deletion was confirmed as above via PCR using
primers outside the upstream and downstream flanking regions, with
a shift in size indicating a mutant. Immunoblot analysis was also used
to confirm the deletion of Rv0222 in the H37Rv strain. The shuttle
vector pMV261 (provided by K. Mi, Institute of Microbiology, Bei-
jing, China) was used to complement the strain H37RvΔRv0222 with
wild-type Rv0222 (with a carboxy-terminal Flag-tag) or to create the
strain H37Rv Rv0222(K76A) (with a C-terminal Flag-tag). Expression
of Rv0222 or its mutants (with a C-terminal Flag-tag) in mycobacteria
was examined by immunoblot analysis.Transfection and confocal microscopy
HEK293T cells were transiently transfected using Lipofectamine 2000
(catalogue number 11668; Invitrogen). Confocal microscopy was per-
formed as described^11.Luciferase assay
The dual-luciferase reporter assay system (Promega) was used for lucif-
erase assays. HEK293T cells were transiently transfected with pNF-κB-
luc, p-AP-1-luc, pRL-TK and indicated plasmids for 24 h.Reverse transcription-PCR analysis
RNA preparation and qPCR analysis were performed as described using
gene-specific primers (Supplementary Table 1).Immunoprecipitation and immunoblotting
For immunoprecipitation, HEK293T cells were transiently trans-
fected using Lipofectamine 2000. After 48 h, cells were washed with
phosphate-buffered saline (PBS) and then lysed in cell-lysis buffer for
western blotting and immunoprecipitation (Beyotime), supplemented
with 1% protease-inhibitor cocktail (catalogue number P8340, Sigma).
After 30 min on ice, the lysates were centrifuged at 13,523g for 10 min at
4 °C to remove debris. The cell lysates were incubated with anti-Flag M
affinity gel overnight at 4 °C. For endogenous immunoprecipitation,
primary peritoneal macrophages were stimulated with mycobacteria
for the indicated time periods. The cells were lysed, and the lysates
were incubated with anti-TRAF6 antibody and protein A/G (sephrasose)
overnight at 4 °C. The sepharose samples were centrifuged, washed
five times with cell-lysis buffer and boiled with SDS loading buffer for
10 min. After separation by SDS–PAGE, equivalent amounts of protein
were electroblotted onto nitrocellulose membranes. The membrane
was blocked, incubated with primary antibodies, and washed three
times before incubation with secondary antibody. After a final wash,
analysis was conducted using enhanced chemiluminescence reagent
(Thermo Scientific).