Extracellular fibrinogen-binding protein released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3

Abstract

Many pathogens secrete effectors to hijack intracellular signaling regulators in host immune cells to promote pathogenesis. However, the pathogenesis of Staphylococcus aureus secretory effectors within host cells is unclear. Here, we report that Staphylococcus aureus secretes extracellular fibrinogen-binding protein (Efb) into the cytoplasm of macrophages to suppress host immunity. Mechanistically, RING finger protein 114, a host E3 ligase, mediates K27-linked ubiquitination of Efb at lysine 71, which facilitates the recruitment of tumor necrosis factor receptor associated factor (TRAF) 3. The binding of Efb to TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 (cellular-inhibitor-of-apoptosis-1) complex, which mediates K48-ubiquitination of TRAF3 to promote degradation, resulting in suppression of the inflammatory signaling cascade. Additionally, the Efb K71R mutant loses the ability to inhibit inflammation and exhibits decreased pathogenicity. Therefore, our findings identify an unrecognized mechanism of Staphylococcus aureus to suppress host defense, which may be a promising target for developing effective anti-Staphylococcus aureus immunomodulators.

Full text

Article https://doi.org/10.1038/s41467-022-33205-z
Extracellular fibrinogen-binding protein
released by intracellular Staphylococcus
aureus suppresses host immunity by
targeting TRAF3
Xiaokai Zhang
1,6
, Tingrong Xiong
1,6
,LinGao
1,6
,YuWang
1,2,6
,LuxuanLiu
3
,
Tian Tian
1
,YunShi
4
, Jinyong Zhang
1
, Zhuo Zhao
1
, Dongshui Lu
1
,PingLuo
1
,
Weijun Zhang
1
,PingCheng
1
, Haiming Jing
1
,QiangGou
1
, Hao Zeng
1,7
,
Dapeng Yan
5,7
& Quanming Zou
1,7
Many pathogens secrete effectors to hijack intracellular signaling regulators in
host immune cells to promote pathogenesis. However, the pathogenesis of
Staphylococcus aureus secretory effectors within hostcellsisunclear.Here,we
report that Staphylococcus aureus secretes extracellular fibrinogen-binding
protein (Efb) into the cytoplasm of macrophages to suppress host immunity.
Mechanistically, RING finger protein 114, a host E3 ligase, mediates K27-linked
ubiquitination of Efb at lysine 71, which facilitates the recruitment of tumor
necrosis factor receptor associated factor (TRAF) 3. The binding of Efb to
TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 (cellular-inhibitor-of-
apoptosis-1) complex, which mediates K48-ubiquitination of TRAF3 to pro-
mote degradation, resulting in suppression of the inflammatory signaling
cascade. Additionally, the Efb K71R mutant loses the ability to inhibit inflam-
mation and exhibits decreased pathogenicity. Therefore, our findings identify
an unrecognized mechanism of Staphylococcus aureus to suppress host
defense, which may be a promising target for developing effective anti-Sta-
phylococcus aureus immunomodulators.
S
taphylococcus aureus (S. aureus) is one of the most common bacterial
strains causing hospital- and community-acquired pneumonia asso-
ciated with significant mortality worldwide1,2. It is estimated that ~85%
of the human population carries or previously carried S. aureus3.The
nasal cavity is the main site of S. aureus colonization in the human
body4, providing a pathogen reservoir that significantly increases the
chance of secondary S. aureus pulmonary infection. However, the
morbidity associated with S. aureus pneumonia is far below the bac-
terial carrying rate due to the powerful host immune system. Resident
macrophages, an important class of innate immune cells, are the first
to encounter invading pathogens5. Besides killing the invading
pathogens, macrophages can produce pro-inflammatory cytokines,
Received: 31 January 2022
Accepted: 8 September 2022
Check for updates
1
National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third
Military Medical University, Chongqing 400038, China.
2
Department of Basic Courses, NCO School, Third Military Medical University, Shijiazhuang 050081,
China.
3
Collegeof Medicine,Southwest Jiaotong University,Chengdu 610083, China.
4
Institute of Biopharmaceutical Research, WestChina Hospital, Sichuan
University, Chengdu, Sichuan 610041, China.
5
Department of Immunology, School of Basic Medical Sciences, Shanghai Institute of Infectious Disease and
Biosecurity & Shanghai Public Health Clinical Center, Fudan University, Shanghai 200032, China.
6
These authors contributed equally: Xiaokai Zhang,
Tingrong Xiong, Lin Gao, Yu Wang.
7
These authors jointly supervised this work: Hao Zeng, Dapeng Yan, Quanming Zou. e-mail: zeng1109@163.com;
dapengyan@fudan.edu.cn;qmzou2007@163.com
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chemokines, and lipid mediators that recruit other innate immune
cells, such as neutrophils, monocytes, and dendritic cells, to orches-
trate immune responses and fight infections6. Therefore, resident
macrophages represent a critical defense line that S. aureus must
overcome in order to propagate in the host7.
S. aureus secretes multiple effectors that functionally evade or
inhibit host immune responses8. Previous pathogenic studies on S.
aureus have mainly focused on the effect of its virulent factors on the
cytomembrane and its receptors in innate immune cells. Several
hemolysins, leukocidins, and phenol-soluble modulins have been
identified to lyse cells by forming pores on the membranes of mac-
rophages and other innate immune cells9. Other bacterial factors,
including staphylococcal superantigen-like protein 3 and lipoylated
E2 subunit of the pyruvate dehydrogenase complex, released from S.
aureus can suppress macrophage activation through inhibiting Toll-
like receptors (TLR) activation10,11. Recently, it has been demonstrated
that S. aureus can invade and survive within macrophages and other
host cells12. Even in a hostile environment, such as that in macro-
phages, S. aureus can develop specific countermeasures to evade the
immune response. Numerous intracellular bacteria have been shown
to weaken host immune defenses bysecreting virulent factors capable
of hijacking macrophage signaling pathways13. However, which effec-
tors are secreted by intracellular S. aureus to manipulate the signaling
pathways of macrophages and their underlying mechanisms remains
unclear.
To inhibit the intracellular survival of invading pathogens, the
inflammatory signaling pathway promotes the expression of various
pro-inflammatory cytokines including tumor necrosis factor (TNF),
interleukin 1β(IL-1β), IL-6, and IL-1214,15.Inthepresentstudy,wefound
that extracellular fibrinogen-binding protein (Efb) released by intra-
cellular S. aureus inhibits pro-inflammatory cytokine expression in
macrophages. Previous studies have suggested that Efb can block the
function of C3b, inhibit the formation of platelet-leukocyte complexes,
and bind fibrinogen to prevent neutrophil activation16,17. However, lit-
tle is known about the role of intracellular Efb in modulating host
inflammatory signaling pathways.
In this work, we demonstrate that intracellular S. aureus secretes
Efb into the cytoplasm of macrophages to inhibit expression of pro-
inflammatory cytokines and suppress host immunity by interacting
with tumor necrosis-associated factor 3 (TRAF3). Mechanistically, the
interaction between Efb and TRAF3 requires K27-linked ubiquitination
of Efb mediated by a host E3 ubiquitin ligase, RING finger protein 114
(RNF114).
Results
Efb inhibits host pro-inflammatory responses
To identify the anti-inflammatory components secreted by intracel-
lular S. aureus, we tested mature chains of 78 secretory proteins of S.
aureus on nuclearfactor-κB(NF-κB) activation in HEK293T cells using a
dual-luciferase reporter gene assay (SupplementaryFig. 1a, b). Efb was
one of the proteins shown to inhibit NF-κB activation induced by TNF-α
(Supplementary Fig. 1b). Efb was co-expressed with an NF-κBreporting
gene in HEK293T cells to verify the inhibitory role of intracellularly
expressed Efb on activation of the NF-κB pathway (Supplementary
Fig. 1c). Next, we found that adenoviral vector-mediated intracellular
overexpression of Efb strongly suppressed phosphorylation of p65
(Supplementary Fig. 1d) and mRNA levels of pro-inflammatory cyto-
kines, including Tnf,Il1β,Il6,andIl12p40 (Supplementary Fig. 1e), in
peritoneal macrophages (PMs) stimulated with heat-killed S. aureus.
These results suggest that intracellular Efb inhibits the transcription of
pro-inflammatory cytokines likely through inhibiting the NF-κB
pathway.
To validate the anti-inflammatory activity of physiological Efb in
an infection model, we generated an S. aureus Newman strain with an
Efb-deletional mutant (ΔEfb) and a complementation strain (ΔEfb +
Flag −Efb). As shown in Supplementary Fig. 2a, western blot analysis
with anti-Efb or anti-Flag antibodies and silver staining of poly-
acrylamide gel electrophoresis (PAGE) revealed Efb expression in the
culture supernatant of the corresponding strains. Consistent with a
previous report18, the absence of Efb did not affect S. aureus growth
(Supplementary Fig. 2b).
Next, we found that macrophages infected with the ΔEfb strain
significantly increased the mRNA levels of Tnf,Il1β,Il6,andIl12p40 in
infected macrophages, while the ΔEfb + Flag −Efb strain restored the
inhibitory effect of Efb (Fig. 1a, Supplementary Fig. 3a). However, the
inhibitory effect of Efb on the expression of inflammatory cytokines
was lost after stimulation with only the culture supernatants of S.
aureus (Supplementary Fig. 3b) or non-contact co-culture of mac-
rophages and S. aureus in Transwells (Supplementary Fig. 3c). These
data suggest that Efb released by intracellular S. aureus, not Efb
released by extracellular S. aureus, exerts anti-inflammatory func-
tions. In the control experiments, we found no difference in intra-
cellular S. aureus loading among macrophages infected with
Newman, ΔEfb, or ΔEfb + Flag −Efb strains for 6 h (Supplementary
Fig. 3d, e, GFP did not affect the expression of Efb in S. aureus;
Supplementary Fig. 3f), indicating that the above-observed differ-
ences in mRNA levels of inflammatory cytokines are Efb-dependent.
Compared to the Newman strain, the ΔEfb strain also exhibited
accelerated phosphorylation of p65, p38, JNK, and ERK in macro-
phages (Supplementary Fig. 4a, b), suggesting that Efb may also
inhibit the mitogen-activated protein kinase (MAPK) pathway in
addition to NF-κB pathway. These results provide further evidence
that physiological Efb released by intracellular S. aureus suppresses S.
aureus-triggered inflammatory responses in macrophages.
To investigate the role of Efb in vivo, we established a trachea
cannula infection model by challenging 6-week-old C57BL/6 mice with
Newman, ΔEfb, or ΔEfb + Flag −Efb strains. At 24 h post infection, there
was increased expression of TNF-α,IL-1β, IL-6, and IL-12 in the lungs of
mice infected with the ΔEfb strain compared to those infected with the
Newman and ΔEfb + Flag −Efb strains (Fig. 1b). In agreement, the
bacterial burden in the lungs of Newman and ΔEfb + Flag −Efb infected
mice was significantly higher than that of ΔEfb infected mice (Fig. 1c).
There were also more intact alveolar spaces, along with less infiltration
of neutrophils and lymphocytes, in the lungs of ΔEfb infected mice
(Fig. 1d, e). We also found that ΔEfb directly led to an increase in
survival rates in lethal pneumonia (Supplementary Fig. 5a) and bac-
teremia (Supplementary Fig. 5b) models, as well as alleviated festering
areas inthe skin infection model (Supplementary Fig. 5c, d), compared
to Newmanand ΔEfb + Flag −Efb infected mice. Together, these results
suggest that Efb released by intracellular S. aureus may act as an inhi-
bitor of host inflammatory responses, making Efb an important factor
for S. aureus infection.
Efb interacts with TRAF3
Immunoelectronmicroscopy and immunofluorescent confocal micro-
scopy showed that Efb was secreted by intracellular S. aureus into the
cytoplasm of the macrophages and gradually accumulated over time
during infection (Supplementary Fig. 6a, b). TLR signaling, especially
TLR2, plays an important role in detecting S. aureus and inducing the
expression of pro-inflammatory cytokines during infection10,19. There-
fore, using a co-immunoprecipitation assay, we screened Efb-
interactive proteins from a list of key signaling molecules in the TLR
pathway. TRAF3 appeared to be the only Efb-associated protein
(Supplementary Fig. 7a and Fig. 2a, b). Further experiments in ΔEfb +
Flag −Efb infected macrophages demonstrated that Efb released by
intracellular S. aureus interacted physiologically with TRAF3 (Fig. 2c, d
and Supplementary Fig. 7b). The in vitro GST-pull down assay also
demonstrated a direct interaction between purified Efb and TRAF3
(Supplementary Fig. 7c, d). In addition, the zinc finger domain of
TRAF3 was responsible for the interaction (Fig. 2e, f).
Article https://doi.org/10.1038/s41467-022-33205-z
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Efb inhibits pro-inflammatory responses by stabilizing TRAF3
Next, we examined whether TRAF3 mediated the inhibition effects of
Efb on host pro-inflammatory responses. We generated conditional
knockout mice for TRAF3 (genotype: TRAF3[flox/flox, Lyz2-Cre]) using
CRISPR/Cas9-mediated genome editing and isolating the PMs from
these mice. TRAF3 deficiency effectively abrogated Efb’s inhibitory
effects on the mRNA levels of Tnf,Il1β,Il6,andIl12p40 in PMs (Fig. 3a).
Furthermore, Efb’s inhibitory effects were lost in lungs of TRAF3[flox/flox,
Lyz2-Cre] mice, but notin TRAF3[flox/flox] mice (Fig. 3b).Consistentwiththis
result, there were no significant differences in the pulmonary bacteria
burdens and histopathology between TRAF3[flox/flox, Lyz2-Cre] mice infec-
ted with the Newman and ΔEfb strains (Fig. 3c–e). TRAF3 mediatesNF-
κB, MAPK, and type I interferon pathways20. Therefore, we used NIK
SMI1 (NIK inhibitor), GSK8612 (TBK1 inhibitor), and 5Z-7-oxozeaeno
(TAK1 inhibitor) to test which pathway mainly mediates pro-
inflammatory cytokine production of macrophages infected with S.
aureus. The results show that 5Z-7-oxozeaeno, but not NIK SMI1 and
GSK8612, inhibited pro-inflammatory cytokine production. TAK1
mainly mediated the canonical NF-kB and MAPK pathways21. There-
fore, the canonical NF-kB and MAPK pathways may play a dominant
role in this mechanism. And the anti-inflammatory effects of Efb could
still be observed in macrophages treated with NIK SMI1 and GSK8612,
but not with 5Z-7-oxozeaeno (Supplementary Fig. 8a–c). Collectively,
these in vitro and in vivo results indicate that Efb may suppress host
inflammatory responses via inhibiting TRAF3-mediated canonical NF-
κB and MAPK pathway activation.
Previous studies have demonstrated that K48 and K63 ubiquiti-
nation of TRAF3 is essential for activation of the NF-κBandMAPK
pathways22–25. Here, we found that, compared to the Newman strain,
the ΔEfb strain markedly reduced the amounts of TRAF3 in macro-
phages, suggesting that Efb may stabilize TRAF3 (Fig. 4a and Supple-
mentary Fig. 9a). Consistently, both overexpressed and physiological
Efb significantly reduced the K48-linked ubiquitination of TRAF3 in
HEK293T cells and macrophages (Fig. 4b, c, Supplementary Fig. 9b).
However, we found little evidence of K63-linked ubiquitination of
TRAF3 in macrophages infected with S. aureus (Supplementary
Fig. 9c), indicating that Efb may mainly affect the K48-linked ubiqui-
tination of TRAF3. The degradative ubiquitination of TRAF3 is typically
mediated by cIAP1/2, and the modification effects require TRAF2 to act
as a bridge between TRAF3 and cIAP1/226,27. We found that TRAF2-
mediated K48-linked ubiquitination of TRAF3 in macrophages infected
by S. aureus (Supplementary Fig. 9d), and Efb could prevent TRAF3
from interacting with TRAF2 and cIAP1 (Fig. 4d, e, Supplementary
Fig. 9e), resulting in inhibition of K48-linked ubiquitination of TRAF3 in
both a cell-free ubiquitylation reaction system and HEK293T cells
(Fig. 4f, Supplementary Fig. 9f). We further demonstrated that the zinc
finger domain of TRAF3 was responsible for its binding to TRAF2
(Supplementary Fig. 9g), suggesting that Efb could competitively bind
TRAF3 and affect TRAF2 binding to TRAF3.
Lysines 106 and 155 of TRAF3 were reported as the main K48-
linked ubiquitination sites28, and our results showed that Efb mainly
inhibited the K48-linked ubiquitination of lysine 155 in the zinc finger
Fig. 1 | Efb inhibits host pro-inflammatory responses. a Quantitative polymerase
chain reaction (qPCR) analysis of Tnf,Il1b,Il6,andIl12p40 mRNA from alveolar
macrophage (MH-S) infected with Newman, ΔEfb, or ΔEfb + Flag −Efb for indicated
times (MOI = 25; **P= 0.0014, 0.0052, ****P<0.0001, ***P=0.0003insequence,
Tnf;****P<0.0001, Il1b;****P< 0.0001, <0.0001, <0.0001, ***P=0.0001,
****P< 0.0001, ***P= 0.0002, in sequence, Il6;**P=0.0019,*P=0.0126, 0.0391,
0.0118, **P= 0.0020, 0.0020, in sequence, Il12p40). bELISA quantification of TNF-
α, IL-1β, IL-6, and IL-12 levels in lung tissue homogenized in 1ml PBS 24 h after
infection with Newman, ΔEfb, or ΔEfb + Flag −Efb. C57BL/6 mice were infected by
intratracheal administration with the test strains (2 × 108CFUs per mouse) for the
indicated times (**P= 0.0095, ****P<0.0001, in sequence, TNF-α;*P=0.0109,
****P<0.0001, in sequence, IL-1β;*P= 0.0187, ***P= 0.0008, in sequence, IL-6;
*P= 0.0206, ***P= 0.0002, in sequence, IL-12). cQuantification of the bacterial
CFUs of lung tissue homogenates obtained in b (***P=0.0001, ****P< 0.0001).
d,eHistopathologyof lung tissues was assessed in H&E sections stained from mice
infectedfor 24 h; scale bars, 1,000μm(top)and200μm (bottom), the boxedareas
at the top are enlarged below (**P=0.0036, *P= 0.0110). Student’s two-tailed
unpaired t-test (a,b,d) or two-tailed Mann–Whitney Utest (c) was used for sta-
tistical analysis. Data are representative of three experiments with at least three
independent biological replicates. The bars show the mean and standard deviation
of n=3(a), n=9(b,c), and n=5(d) pergroup. Source dataare provided as a Source
Data file.
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domain of TRAF3 (Fig. 4g and Supplementary Fig. 9h). Next, we
expressed wild-type TRAF3 in TRAF3 knockout macrophages to
restore the function of TRAF3 and expressed TRAF3 K155R in TRAF3
knockout macrophages to mimic the effects of Efb on TRAF3. After
infection, the pro-inflammatory cytokines induced by the ΔEfb stain
remained higher than the Newman str ain in wild-type TRAF3, but not in
TRAF3 K155R-expressing macrophages (Supplementary Fig. 9i). Taken
together, these results suggest that Efb disturbs the TRAF3/TRAF2/
cIAP1 complex, inhibits the K48-linked ubiquitination of TRAF3 at
lysine 155, and prevents TRAF3 degradation in macrophages during S.
aureus infections.
RNF114 mediated K27-linked ubiquitination of Efb
Several studies have suggested that bacteria can utilize the post-
translational modification system of the host to modify their effectors
for pathogenesis29,30. Given that Efb is small in size (mature protein,
15kD) and contains 20 lysine residues that are common sites for ubi-
quitination, we investigated the ubiquitination of intracellular Efb.
Plasmids expressing different ubiquitins (Ub, K6, K11, K27, K29, K33,
K48, and K63) were co-transfected with Efb into HEK293T cells. Co-
immunoprecipitation results revealed multiple types of ubiquitination
of Efb, with K27-linked polyubiquitination being most dominant
(Supplementary Fig. 10a). Furthermore, we detected the endogenous
K27-linked polyubiquitin conjugates of intracellular Efb in macro-
phages infected with the ΔEfb + Flag −Efb strain (Fig. 5a, Supplemen-
tary Fig. 10b), indicating that Efb is polyubiquitinated by K27 in host
cells. We also carried out proteomics analysis on immunoprecipitated
overexpressed Efb in HEK293T cells using tandem mass spectrometry
(MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to UPLC
(Supplementary Table 3). We identified a protein named RNF114 as an
E3 ligase with K27-linked polyubiquitination activity31. Overexpression
co-immunoprecipitation (Fig. 5b, c) and endogenous co-
immunoprecipitation (Fig. 5d, Supplementary Fig. 10c) consistently
demonstrated reproducible Efb interaction with RNF114. Over-
expression of RNF114 also increased the K27-linked (but not other
types), polyubiquitination of Efb in HEK293T cells (Fig. 5e, Supple-
mentary Fig. 11), and RNF114 KD by specific siRNA reduced the K27-
linked polyubiquitination of Efb in macrophages (Fig. 5f). These results
collectively suggest that RNF114 is an important ubiquitinase that
mediates K27-linked polyubiquitination of Efb.
To determine which lysine residue of Efb was modified by K27-
linked polyubiquitination, we constructed 20Efb mutants by replacing
lysine with arginine and co-transfected these mutants with a plasmid
expressing K27 ubiquitin in HEK293T cells. The immunoprecipitation
results revealed that the K71R mutant significantly reduced the K27-
linked polyubiquitination of Efb (Supplementary Fig. 12a). Con-
sistently, RNF114 did not promote the K27-linked ubiquitination of
K71R mutant of Efb in HEK293T cells (Fig. 5g). Next, we replaced all
other Efb lysines with arginines, except lysine 71, and RNF114 still
maintained the K27-linked polyubiquitination of Efb at lysine 71 (Sup-
plementary Fig. 12b, c). Moreover, physiological K27-linked ubiquiti-
nationof the K71R mutant of Efb disappeared in macrophages (Fig. 5h).
These results suggest that RNF114 can mediate K27-linked poly-
ubiquitinion of Efb at lysine 71.
Fig. 2 | Efbinteracts withTRAF3. a,bImmunoblots of wholecell lysates (WCL)and
immunoprecipitation (IP) products of WCL from HEK293T cells transfected with
indicated plasmids. cImmunoblots of WCL and IP products of WCL from MH-S
infected with ΔEfb+ Flag −Efb for indicated times (MOI = 25).
dImmunofluorescence assay of MH-S infected with ΔEfb + Flag−Efb for 4 h
(MOI = 25), scale bars, 2 μm. eTruncation of TRAF3. fImmunoblots of WCL and IP
products of WCL from HEK293T cells transfectedwith indicated plasmids; WT, full-
length TRAF3. Data are representative of three experiments with at least three
independent biological replicates. Source data are provided as a Source Data file.
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Ubiquitination of Efb facilitates the stabilization of TRAF3
To investigate whether RNF114 is involved in suppressing
TRAF3 signalingby Efb, we observed theenhancing effectof RNF114 on
Efb inhibiting K48-linked ubiquitination of TRAF3 (Supplementary
Fig. 13a). When compared to wild-type Efb, the K71R mutant appeared
to reverse the inhibition of Efb on the K48-linked ubiquitination of
TRAF3 (Supplementary Fig. 13b). The results obtained from ΔEfb +
Flag −Efb and ΔEfb + Flag −Efb K71R infected macrophages (Supple-
mentary Fig. 13c–e) showed that the K71R-Efb mutant reduced the
inhibitory effects on TRAF2 and cIAP1 mediated K48-linked poly-
ubiquitination of TRAF3 at physiological levels. Furthermore, we found
that ΔEfb infection in RNF114 KD macrophages resulted in an even
lower level of pro-inflammatory cytokine expression compared to
Newman infection (Supplementary Fig. 13f), and we found that RNF114
affected the TRAF3 expression level only when Efb was present (Sup-
plementary Fig. 13g), suggesting that RNF114 is involved in the reg-
ulation of inflammatory cytokine production during S.aureus infection
in macrophages independent of Efb and TRAF3. Together, these
results suggest that RNF114 promotes K27-linked ubiquitination of Efb
to facilitate TRAF3 stabilization.
Ubiquitinated Efb inhibits host pro-inflammatory responses
Next, the reconstitution experiments using ΔEfb infected macro-
phages demonstrated that wild-type Efb, but not the K71R-mutant
strain, was able to restore inhibition of infection-induced pro-inflam-
matory cytokine production (Fig. 6a, Supplementary Fig. 13c). To
investigate the function of the K71R mutant on the innate immune
response in vivo, we infected 6-week-old mice with the Newman, ΔEfb,
ΔEfb + Flag −Efb, or ΔEfb + Flag −Efb K71R strains and evaluated the
expression levels of pro-inflammatory cytokines in the lungs. Com-
pared with ΔEfb infection, ΔEfb + Flag −Efb, but not ΔEfb + Flag−Efb
(K71R), significantly suppressed pro-inflammatory cytokine produc-
tion in the affected lungs (Fig. 6b). The bacterial load of ΔEfb and
ΔEfb + Flag −Efb (K71R) strains in the lungs was significantly lower than
that of the Newman and ΔEfb + Flag −Efb strains (Fig. 6c). Consistently,
histopathology results revealed more severe lung damage and more
neutrophil and lymphocyte infiltration in the lungs of ΔEfb mice
reconstituted with wild-type but not the K71R-mutant Efb strains
(Fig. 6d, e). These results suggest that the pathogenesis of S. aureus
might rely on the K27-linked ubiquitination of Efb at its lysine 71
residue.
Discussion
Opsonophagocytic killing conducted by macrophages and neu-
trophils is critical for the host to combat S. aureus, a classical
extracellular pathogen7. However, in recent years, accumulating
reports revealed that S. aureus can invade and survive within mac-
rophages, neutrophils, and other host cells12. As a reservoir of S.
aureus, macrophages play an important role in regulating the
inflammatory responses that are critical for eliminating invading
pathogens6,12. To achieve a successful infection, S. aureus must
overcome activation of the inflammatory pathways of macrophages.
Fig. 3 | Efb inhibits host pro-inflammatory responses via TRAF3.a qPCR analysis
of Tnf,Il1b,Il6,andIl12p40 mRNA of PMs infected with Newman, ΔEfb (MOI = 25 )
for 4 h; wild-type PMs (WT) were isolated from TRAF3[flox/flox] mice; TRAF3knockout
(KO) PMs were isolated from TRAF3[flox/flox, Lyz2-Cre] mice (**P= 0.0096, Tnf;
***P=0.0001, Il1b;**P= 0.0011, Il6;**P= 0.0015, Il12p40). bELISA quantification of
TNF-α, IL-1β, IL-6, and IL-12 levels in lung tissues from TRAF3[flox/flox] (Flox) or
TRAF3[flox/flox, Lyz2-Cre] (TRAF3 KO) mice, homogenized in 1 ml PBS and infected with
Newman, ΔEfb for 24 h (2 × 10 8CFUs per mouse; ****P< 0.0001, TNF-α;*P=0.0184,
IL-1β;*P= 0.0427,IL-6; **P= 0.0089,IL-12). cQuantification of the bacterial CFUs of
lung tissue homogenates obtained in b(**P=0.0030).d,eHistopathology of
lung tissues was assessed in H&E sections stained from mice infected for 24 h;
scale bars, 1000 μm(top)and200μm (bottom), the boxed areas at the top are
enlarged below (****P<0.0001).Student’s two-tailed unpaired t-test (a,b,d)or
two-tailed Mann–Whitney Utest (c) was used for statistical analysis. Data are
representative of three experiments with at least three independent biological
replicates. The bars show the mean and standard deviation of n=3(a), n=9
(b,d), and n=5(e) mice per group. Source data are provided as a Source
Data file.
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Secreting multiple effectors is a unique strategy that S. aureus has
adopted to cope with environmental challenges9. However, whether
and how the secreted effectors of intracellular S. aureus interfere
with the inflammatory pathways of macrophages remains unclear. In
the present study, we screened 78 secreted effectors on activation of
the NF-κB pathway. The results showed that more than half of the
secretory proteins decreased NF-kB activation. We conducted sev-
eral bioinformatics and literature searches on these proteins,
especially α-hemolysin (WP-000857483.1), serine protease SplE
(WP-001038759.1), and cysteine protease staphopain A (WP-
000827748.1). We found that the inhibitory effects of these three
proteins were false positives due to the fact that overexpressing
these proteins in HEK293T cells led to cell deformation or death
(Supplementary Fig. 1f). Moreover, this validation work is still in
progress. Efb was the first protein we discovered that could inhibit
both NF-κB and MAPK, as well as inhibit S. aureus-induced expression
of pro-inflammatory cytokines, including TNFα,IL-1β, IL-6, and IL-12,
in vitro and in vivo, respectively. And we determined that the Efb
does not influence the in vitro killing ability of isolated macrophages
(Supplementary Fig. 3d–f) or neutrophils (Supplementary Fig. 14)
in vitro. In the early stages of infection, increased levels of TNFαand
IL-1βare critical for eliminating bacterium in human S. aureus
bacteremia32.IL-1βhas also been shown to be a key cytokine for the
eradication of S. aureus in experimental models33. Our results indi-
cate that decreased bacterial burdens in lung tissues infected with
ΔEfb could be attributed to elevated TNF-αand IL-1βlevels,
which may lead to more neutrophil or other immune cell chemotaxis
or activation. Thus, inhibiting host pro-inflammatory responses may
be one way by which Efb contributes to the pathogenicity of S.
aurues.
Although the post-translational modification system in bacteria is
very simple in comparison to eukaryotes34, accumulating studies have
shown that some bacteria effectors can be modified by the host post-
translational modification system29. Under certain conditions, some
bacteria effectors can disturb host post-translational modification35 to
facilitate infection. Our present study found that a host E3 ligase,
RNF114, ubiquitinates Efb of S. aureus to suppress host immunity. K27-
linkedubiquitination serves a varietyof functions, including enhancing
protein–protein interactions, promoting proteasomal degradation,
and providing binding platforms for DNA repair proteins36. RNF114 has
been reported as a host E3 ligase with K27-linked polyubiquitination
activity with degradation effects in porcine and sea perch37,38.How-
ever, our results showed that K27-linked ubiquitination of Efb by
RNF114 did not lead to degradation of Efb but promoted the interac-
tion between Efb and TRAF3.
Efb has been identified as an immune evasion effector, and its
complement inhibitory effect depends on its C-terminal16. However,
our results demonstrate that replacing lysine 71 with arginine in the
N-terminal of Efb can eliminateits inhibitoryeffects in vivo and in vitro,
suggesting that the inhibitory effects of Efb are independent of its
complement effects in vivo. On the other hand, the immunosuppres-
sive abilities of Efb were shown to be correlated with inhibiting the
Fig. 4 | Efb stabilizes TRAF3. a Immunoblots of WCL from MH-S infected with
Newman andΔEfb (MOI = 25). bImmunoblots of WCL and IP products of WCL from
HEK293T cells transfected with indicated plasmids. Ub, wild-type ubiquitin; K48,
Ub with a single 48 lysine residue left; K63 Ub with a single 63 lysine residue left.
cImmunoblots of IP products of WCL from MH-S infected with Newman and ΔEfb
(MOI = 25). dImmunoblots of WCL and IPproducts from HEK293T cellstransfected
with indicated plasmids. eImmunoblots of IP products from MH-S infected with
Newman and ΔEfb (MOI = 25). fEfb inhibits K48-linked ubiquitin chains on TRAF3
in vitro. gImmunoblots of WCL and IP products from HEK293T cells transfected
with indicated plasmids. K/R, replace all lysine of TRAF3 with arginine; R106K,
replace 106 arginine of K/R with lysine; R155K, replace 155 arginine of K/R with
lysine. R106/155K, replace 106 and 155 arginine of K/R with lysine. Data are repre-
sentative of three experiments withat least three independent biological replicates.
Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-022-33205-z
Nature Communications | (2022) 13:5493 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved

formation of platelet-leukocyte complexes and binding fibrinogen to
prevent neutrophil activation, which depends on the N-terminal of
Efb17. However, lysine 71 of Efb was not the key residue in the inter-
action between Efb and fibrinogen according to a previous report39.
Platelets have been reported to play an important role in
inflammation40. However, there is no detailed information as to which
amino acid of Efb plays a key role in the interaction between the Efb
N-terminal and platelets. Our results demonstrate that the direct
inhibitory effect of Efb on pro-inflammatory cytokines was accom-
plished through interaction with TRAF3 in myeloid cells, including
mature macrophages, monocytes, and granulocytes. The anti-
inflammatory effects of Efb are critical to the in vivo change in S.
aureus load and pathogenicity in mice. However, it is still possible that
our in vivo results were partly due to the interaction between Efb and
platelets or other unknown mechanisms.
TRAF3 is a tri-faced immune regulator that has distinct roles
depending on its targeted receptors, even within the same cell, and is
also highly cell-type-dependent20. TRAF3 has been reported to posi-
tively regulate type I interferon production while negatively regulating
NF-κB and MAPK pathways22. Our results demonstrate that TRAF3
regulates S. aureus-induced activation of NF-κBandMAPKpathwaysin
macrophages. S. aureus infection induces the degradation of TRAF3 in
macrophages. However, Efb can inhibit degradation by disturbing the
K48-linked ubiquitination of TRAF3 in HEK293T cells, PMs, and
alveolar macrophages (MH-S). K48-linked ubiquitination of TRAF3 for
degradation is accomplished by cIAP1/2, which requires the bridging
effects of TRAF2. Our dataindicate thatthe interaction between TRAF3
and TRAF2 was disturbed by Efb, and the inhibition on K48-linked
ubiquitination of TRAF3 by Efb was accomplished by disrupting the
TRAF3-TRAF2-cIAP1/2 complex.
As stated in previous reports, TLR2 and MyD88 in innate
immune cells play a key role in detecting S. aureus41–43. We demon-
strated that TLR2 and MyD88 were critical in promoting pro-
inflammatory responses of macrophages infected by S. aureus
(Supplementary Fig. 15a). As stated in a report by Perkins et al.44,
macrophages stimulated by P3C, a TLR2 ligand, overnight (≥24 h)
upregulated TRAF3 protein expression. However, their results also
showed that the TRAF3 mRNA levels were not significantly increased
until 8 h post treatment. In the present study, we measured TRAF3
protein expression immediately after S. aureus infection using wild-
type, TLR2−/−, and MyD88−/−macrophages. We found that the
decrease in TRAF3 protein expression was related to the TLR2-
MyD88 pathway (Supplementary Fig. 15b).
The emergence of multi-drug resistant strains and the lack of a
vaccines has made S. aureus a global concern. Immune evasion has
been proven to be the main pathogeneses for S. aureus infection9,
and is also one of the main challenges for vaccine development45.We
identified that K27-linked ubiquitination of Efb by RNF114 binding to
TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 complex
and prevents K48-ubiquitination-mediated TRAF3 degradation,
resulting in suppressed pro-inflammatory cytokine production
in vivo and in vitro. The present findings identify a previously
unrecognized mechanism that S. aureus uses to suppress host
immunity (Supplementary Fig. 16). In addition, the Efb-RNF114
interface may be a promising target for the development of effec-
tive anti-S. aureus treatments.
Fig. 5 | Efb is K27-ubiquitinated at K71 by host RNF114. a Immunoblots of IP
products of WCL from MH-S infected with ΔEfb + Flag −Efb for indicated times
(MOI = 2 5). b,cImmunoblots of WCLand IP products from WCL of HEK 293 T cells
transfected with indicated plasmids. dImmunoblots of IP products of WCL from
MH-S infected with ΔEfb + Flag −Efb for indicated times (MOI = 25). eImmunoblots
of WCL and IP products from HEK293T cells transfected with indicated plasmids.
fImmunoblots of WCL and IP products from MH-S or RNF114 knockdown (KD)
MH-S infected with ΔEfb + Flag −Efb for indicated times (MOI = 25).
gImmunoblots of WCL and IP products from HEK293T cells transfected with
indicated plasmids. K71R, replace 71 lysine of Efb wi th arginine. hImmunoblots
of WCL and IP products from MH-S infected with ΔEfb + Flag −Efb or ΔEfb +
Flag −Efb K71R for indicated times (MOI = 25). Data are representative of three
experiments with at least three independent biological replicates. Source data
are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-022-33205-z
Nature Communications | (2022) 13:5493 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Methods
Bacterial strains and cells
Bacterial strains adopted in the present study are described in Sup-
plementary Table 1. The Escherichia coli strains DH5αand BL21 (Tian-
gen Biotech, China) were grown in LB medium. When required, the
antibiotics ampicillin (100 μg/ml) (Sanggon Biotech) or kanamycin
(50 μg/ml) (Sanggon Biotech) were used for the E. coli strain selection.
The S. aureus strains were grown in trypticase soy broth (OXOID,
CM0129B). Chloramphenicol (25 μg/ml) (Sanggon Biotech) was used
for the selection of S. aureus strains when required. Unless noted, all
bacteria were grown at 37°C in a shaking incubator at 200 rpm in
tubes kept at a 45° angle.
HEK293T cells (CRL-3216), obtained from the American Type
Culture Collection, were maintained in Dulbecco’smodified Eagle’s
medium (DMEM; HyClone) supplemented with 10% (v/v) fetal bovine
serum (FBS, HyClone) and 100 U/ml penicillin and streptomycin
(HyClone). MH-S cells (CRL-2019), obtained from the American Type
CultureCollection, were maintained in Roswell Park MemorialInstitute
(RPMI)−1640 medium (HyClone) supplemented with 10% FBS. Perito-
neal macrophages (PMs) were obtained from 6-week-old wild-type or
mutant C57BL/6J mice as follows: mutant mice and their wild-type
littermates were injected with 2 ml Thioglycolate Broth (4%) intraper-
itoneally. Three days later, the peritoneal lavage fluid was collected
from the mice and washed with PBS three times. PMs were grown in
DMEM supplemented with 10% FBS. Neutrophils were isolated from
mice blood using anti-Ly6G MicroBeads (Miltenyi, 130-120-337) and
cultured in RPMI-1640 medium (HyClone) containing 10% FBS.
Plasmids, reagents, and antibodies
Expressing plasmids were constructed by inserting a synthetic gene
segment in the vector, the names of which are listed in Supple-
mentary Table 1. The following antibodies were used for western
blot, immunoprecipitation, or immunofluorescence assays: rabbit
anti-TRAF3 (PA5-20165, Invitrogen; ab36988, Abcam), mouse anti-
TRAF3 (sc6933, Santa Cruz), rabbit anti-TRAF2 (4724, Cell signaling
technology, CST), mouse anti-Flag (F1804, Sigma-Aldrich), rabbit
anti-HA (3724, CST), rabbit anti-Myc (2040, CST), rabbit anti-
phospho-p65 (3033, CST), rabbit anti-phospho-p38 (9215, CST),
rabbit anti-phospho-Erk1/2 (9101,CST),rabbitanti-phospho-JNK
(4668, CST), rabbit anti-GFP (2956, CST), rabbit anti-K27 (ab181537,
Abcam), rabbit anti-K48 (8081, CST), rabbit anti-K63 (5621, CST),
rabbit anti-cIAP1 (ab2399, Abcam), rabbit anti-RNF114 (ab97303,
Fig. 6 | Efb inhibits host immunity depending on K27-ubiquitination. a qPCR
analysis of Tnf,Il1b,Il6,andIl12p40 mRNA from PMs infected with Newman,
ΔEfb, ΔEfb + Flag −Efb, or ΔEfb + Flag −Efb K71R for indicated time s (MOI = 2 5;
***P= 0.0001, ****P< 0.0001, ***P= 0.0004, in sequence, Tnf;
*P=0.0128,**P= 0.0034, 0.0034, in sequence, Il1b;**P=0.0024,***P= 0.0010,
0.0010, in sequence, Il6;***P= 0.0010, 0.0001, 0.0002, in sequence, Il12p40).
bELISA quantification of TNF-α, IL-1β, IL-6, and IL-12 levels in lung tissues
homogenized in 1 ml PBS, infected with Newman, ΔEfb, ΔEfb + Flag −Efb, or
ΔEfb + Flag −Efb K71R (2 × 108CFUs per mouse) for 24 h (****P<0.0001,
***P =0.0001, 0.0003, in sequence, TNF-α;****P< 0.0001, <0.0001,
*P= 0.0163, in sequence, IL-1β;**P= 0.0065, ****P< 0.0 001, *P= 0.0111, in
sequence, IL-6; ***P=0.0008, **P=0.0019, *P= 0.0105, in sequence, IL-12).
cQuantification of the bacterial CFUs in lung tissue homogenates obtained in
b(**P= 0.0079, 0.0013, 0.0065). d,eHistopathology of lung tissues was
assessed in H&E sections stained from mice infected for 24 h; scale bars, 1,000
μm(top)and200μm (bottom), the boxed areas at the top are enlarged below
(***P= 0.0001, 0.0007, **P= 0.0020). Student’s two-tailed unpaired t-test
(a,b) or two-tailed Mann–Whitney Utest (c) was used for statistical analysis.
Data are representative of three experiments with at least three independent
biological replicates. The bars show the mean and standard deviation of n=3
(a), n=9(b,c), and n=5(d) per group. Source data are provided as a Source
Data file.
Article https://doi.org/10.1038/s41467-022-33205-z
Nature Communications | (2022) 13:5493 8
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Abcam), Alexa Fluor Plus 488 conjugated goat anti-mouse IgG
(A32723, Invitrogen), Alexa Fluor Plus 555 conjugated goat anti-
mouse IgG (A32727, Invitrogen), Alexa Fluor Plus 647 conjugated
goat anti-rabbit IgG (A32733, Invitrogen); Anti-Efb was generatedby
immunization of rabbits with the protein of Efb, rabbit anti-GST
(CW0085M, Cwbio), rabbit anti-His (CW0083M Cwbio); rabbit anti-
GAPDH (G9545, Sigma-Aldrich), mouse anti-Flag M2 Affinity Gel
(A2220, Sigma-Aldrich), mouse anti-HA Magnetic Beads (88836,
Thermo Fisher), mouse anti-TRAF3 agarose beads (sc6933 AC,
Santa Cruz), and goat anti-rabbit IgG (5127, CST), goat anti-mouse
IgG (96714, CST). For western blot assays, all primary antibodies
weredilutedat1:1000andsecondaryantibodiesweredilutedata
1:5000. For immunofluorescence, monoclonal mouse anti-Flag M2
antibody was used at 1:500 dilution and rabbit anti-TRAF3 at a 1:200
dilution. Corresponding Alexa Fluor Plus 488 labeled goat anti-
mouse IgG, Alexa Fluor Plus 555 labeled goat anti-mouse IgG or
Alexa Fluor Plus 647 labeled goat anti-rabbit IgG were used as at a
1:200 dilution. DAPI Stain Solution for nuclear strain was from
Sangon Biotech (E607303). For flow cytometry, rat anti-mouse
Ly6G PE (551461, BD), rat anti-mouse CD11b-FITC (557396, BD) were
used at a 1:100 dilution.
Construction of S. aureus strains
The pBT2 vector (provided by X. Rao, Army Medical University,
Chongqing) was used to generate the S. aureus Newman strain with a
deletion of the gene encoding Efb (ΔEfb) using an allelic replacement
strategy as previously described46. The primersfor the construction of
the Efb knockout vector are listed in Supplementary Table 2. The
deletion of Efb was confirmed by PCR and Sanger sequencing. The
pLI50 vector (provided by X. Rao, Army Medical University, Chongq-
ing) was used to complement the ΔEfb strain with the wild-type Efb or
Efb K71R gene driven by the original promotor of Efb. The expression
of Efb or its mutants in the supernatant of S. aureus was examined by
immunoblot assays.
Transfection and confocal microscopy
HEK293T cells were transiently transfected using PEI (23966-2; Poly-
sciences) according to instructions of the manufacturer. PMs were
transduced or transfected with adenovirus (Hanbio) or jetMSSENGER
(150-07, Polyplus). MH-S cells were transiently transfected using the
INVI DNA RNA Transfection Reagent (IV1216100, Invigentech). Con-
focal microscopy was performed as described previously46.MH-SCells
were fixed with4% formaldehyde for 10 minat 25 °C, permeabilized for
30 min in PBScontaining 0.3% Triton X-100, and then blocked for 1 h at
4 °C in a blocking buffer (1% BSA in PBS). Next, the cells were incubated
with the indicated antibodies at 4 °C overnight and secondary anti-
bodies at room temperature for 1 h. After staining with DAPI, images
were obtained using a Zeiss LSM 780 confocal laser microscopy
system.
Immunoelectronmicroscopy
After 4h infection with Newman or ΔEfb strains, MH-S cells were fixed
in immunoelectronmicroscopy fixative (Wanwu; G1124) for 2 h at 4 °C.
Ultrathin cryosections (70 nm) were prepared as previously
described47, and sections were sequentially labeled with rabbit anti-
Flag antibody, followed by sheep anti-rabbit antibody coupled with
10 nm gold particles. The stained sections were observed under a
Hitachi electron microscope HT7800.
Luciferase assay
HEK293T cells were transiently transfected with pNF-κB–luc, pRL–TK,
and the indicated plasmids for 24 h. After TNF-α(210-TA, R&D) sti-
mulation for 6 h, the dual-luciferase reporter assay system (RG028,
Beyotime) was used to detect luciferase activity according to the
instruction of the manufacturer.
Immunoprecipitation and immunoblot assays
Immunoprecipitation and immunoblot assays were performed as
previously described. Briefly, HEK293T cells were transiently trans-
fected with plasmids using PEI. After 48 h, culture supernatants were
removed at corresponding times, and the cells were washed three
times with PBS. Cells were lysed in cell lysis buffer (Beyotime) sup-
plemented with 1% protease inhibitor cocktail (4693116001, Roche).
After centrifugation, the supernatants of cell lysates were incubated
with indicated gels at 4 °C overnight. For endogenous immunopreci-
pitations, PMs and MH-S were infected with S. aureus for the indicated
times.The cell lysates were subsequently incubated with indicated gels
at 4 °C overnight. After being centrifuged, the gels were then washed
three times with cell lysis buffer and boiled with 1× SDS loading buffer.
Equivalent amounts of total proteins were separated by SDS-PAGE and
electro-blotted onto PVDF membranes. The membranes were then
probed with antibodies, followed by exposure conducted using an ECL
reagent (32209 or 34095, Thermo Fisher).
Ubiquitination assay
HA-TRAF3 was purified from HEK293T cells using an anti-Flag M2
Affinity Gel. The beads were washed three times with cell lysis buffer.
Beads were incubated in E3 ligase buffer, which contained Flag-TRAF2
and Flag-cIAP1 overexpressing cell lysate, ubiquitin, Mg-ATP, with or
without E1, E2, or Flag-Efb overexpressing cell lysate. All samples were
incubated at 37 °C for 1h in a metal bath by gently shaking. After
removing the supernatants, 50 μl2×SDS-PAGEgelloadingbufferwas
added to the obtained beads followed by heating to 95 °C for 5 min
prior to western blot analysis.
Real-time PCR analysis
RNA preparation and qPCR analysis were performed as described
previously30 using gene-specific primers (Supplementary Table 2).
Total RNA of cells was isolated using RNAiso Plus (9109; Takara). Next,
RNA (1 µg) was reverse-transcribed using the PrimeScript™RT Reagent
Kit (RR037; Takara) to generate cDNA. SYBR RT-PCR Kit (QPK-212;
Toyobo) were used for the quantitative real-time RT-PCR analysis.
Gene amplification was performed using the ΔΔCt method, and gene
expression was normalized to that of GAPDH.
Mice infection model
Six-week-old female specific-pathogen-free C57BL/6 mice were pur-
chased from Beijing HFK Bioscience CO., LTD. Traf3[flox/flox] and
Traf3[flox/flox, Lyz2-Cre] mice were purchased from Cyagen Biosciences.
They were housed under 12 light/12 dark cycles, ~18–23 °C, 40–60%
humidity and specific-pathogen-free (SPF) conditions at the National
Engineering Research Center of Immunological Products. All animal
experiments were reviewed and approved by the Animal Experiment
Administration Committee of Army Medical University and were
conducted in accordance with governmental guidelines and institu-
tional policies for the Care and Use of Laboratory Animals. Six-week-
old C57BL/6 mice were divided randomly into cages and infected by
intratracheal administration with 2 × 108CFUs or 6 × 108CFUs of dif-
ferent S. aureus strains in 20 μl PBS for 24 h or a few days. After the
mice were sacrificed at the indicated times, the lungs were collected
and homogenized in 1 ml of PBS for ELISA and CFU assays. S. aureus
burden was determined by plating serial dilutions of each tissue
homogenate on tryptic soy agar (TSA) plates, which were incubated
at 37 °C. Colonies were counted after 12 h of incubation. For histo-
logical analysis, lungs were removed and fixed in 4% paraformalde-
hyde in PBS and embedded in paraffin. Sections were cut and stained
with hematoxylin and eosin (H&E) according to standard protocols.
Imaging was performed using microscopy (ECLIPSE 80i, Nikon). For
skin and blood infection models, the doses of S. aureus were 2 × 108
and 1 × 108CFUs. All mice were age- and sex-matched in each
experiment. The sample size was determined based on data from
Article https://doi.org/10.1038/s41467-022-33205-z
Nature Communications | (2022) 13:5493 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved

pilot experiments. For in vitro S. aureus infections, macrophages
were infected with a single-cell suspension of bacteria at an moieties
of infection (MOI) of 25.
Statistics and reproducibility
Data are expressed as mean ± s tandard deviation (SD). GraphPad Prism
8 was used for statistical analysis. The sample sizes, reproducibility of
experiments and the statistical tests used are presented in the figure
legends. ZEN 2.1 on Zeiss LSM 780 confocal laser microscopy system
was used for immunohistochemistry data collection and analysis. CFX
Manager Software v3.0 on BioRad CFX96Touch was used for qRT-PCR
data collection and analysis. Bio-Rad ChemDoc Touch was used for
western blot data collection and analysis. BD biosciences FACSDiva
software on FACSCanto was used for Flow cytometry data collection
and analysis.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
All data are available within the present article and Supplementary
Information, or in the Source Data files. Source data are provided with
this paper.
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Acknowledgements
We thank Xiancai Rao (Third Military Medical University, China) for pro-
viding the pBT2 and pLI50 vectors and S. aureus Newman strain; Lixin
Zheng (LISB/NIAID/NIH, USA) for critical reading of the manuscript; and
Weilong Shang (Third Military Medical University, China), Yi Yang (Third
Military Medical University, China), and Xianzhi Meng (Southwest Uni-
versity, China) for technical assistance. This work was supported by
grants from the National Natural Science Foundation of China (Nos.
81902036, 31970138) and the National Natural Science Foundation of
Chongqing (cstc2019jcyj-msxmX0377).
Author contributions
Q.Z., D.Y., H.Z., and X.Z. designed the experiments. X.Z. and T.X. wrote
the manuscript. X.Z., T.X., L.G., Y.S., J.Z., and Z.Z. analyzed experimental
results. X.Z., T.X., L.G., Y.W., L.L., and T.T. carried out the experiments.
D.L., P.L., W.Z., P.C., H.J., and Q.G. provided technical help. All authors
discussed the results and commented on the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-022-33205-z.
Correspondence and requests for materials should be addressed to
Hao Zeng, Dapeng Yan or Quanming Zou.
Peer review information Nature Communications thanks Friedrich Götz
and the other anonymous reviewer(s) for their contribution to the peer
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