Yersinia YopJ negatively regulates IRF3-mediated antibacterial response through disruption of STING-mediated cytosolic DNA signaling

    Yersinia YopJ Negatively Regulates IRF3-mediated antibacterial response through Disruption of STING-Mediated Cytosolic DNA Signaling Ye Cao, Kai Guan, Xiang He, Congwen Wei, Zirui Zheng, Yanhong Zhang, Shengli Ma, Hui Zhong, Wei Shi PII: DOI: Reference:

S0167-4889(16)30258-0 doi:10.1016/j.bbamcr.2016.10.004 BBAMCR 17952

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

7 June 2016 30 September 2016 7 October 2016

Please cite this article as: Ye Cao, Kai Guan, Xiang He, Congwen Wei, Zirui Zheng, Yanhong Zhang, Shengli Ma, Hui Zhong, Wei Shi, Yersinia YopJ Negatively Regulates IRF3-mediated antibacterial response through Disruption of STING-Mediated Cytosolic DNA Signaling, BBA - Molecular Cell Research (2016), doi:10.1016/j.bbamcr.2016.10.004

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ACCEPTED MANUSCRIPT Yersinia

YopJ

response

Negatively

Regulates

through Disruption

of

IRF3-mediated

STING-Mediated

DNA

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a, b, c

Kai Guan, c Xiang He, c Congwen Wei, c Zirui Zheng, c Yanhong

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Ye Cao,

Cytosolic

T

Signaling

antibacterial

Zhang, c Shengli Ma, a, b, c Hui Zhong, c* and Wei Shi a, b* a

Key Laboratory for Molecular Enzymology & Engineering, the Ministry of

c

College of Life Science, Jilin University, Changchun 130012, China.

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b

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Education, Jilin University, Changchun 130012, China.

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of

Email

addresses:

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Authors

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Biotechnology, Beijing 100850, P.R. China. [email protected]

(Ye

Cao);

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[email protected] (Kai Guan);[email protected] (Xiang He); [email protected] (Congwen Wei); [email protected] (Zirui Zheng); (Yanhong

Zhang);

[email protected]

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[email protected] (Shengli Ma). *

Corresponding authors at: Key Laboratory for Molecular Enzymology &

Engineering, the Ministry of Education, Jilin University, Changchun 130012, China, Tel.: 0431-85155216 (Wei Shi); State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Beijing 100850, P.R. China Tel.: 010-66931821 (Hui Zhong). Email address: [email protected] (Wei Shi); [email protected](Hui Zhong) . Running title: YopJ inhibits IRF3 activity by targeting STING

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ACCEPTED MANUSCRIPT Abbreviations: BMDMs, bone marrow-derived macrophages; c-di-GMP, cyclic diguanylate monophosphate; DMEM, Dulbecco’s modified Eagle’s medium;

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ER, endoplasmic reticulum; FBS, fetal bovine serum; GST, Glutathione

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S-transferase; HEK293T, human embryonic kidney; IB, immunoblotting; IHC, immunohistochemical; K63, lysine 63; MAPK, mitogen-activated protein kinase; MAP2Ks, mitogen-activated protein kinase kinases; MAVS, mitochondrial

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antiviral signaling protein; M-CSF, macrophage colony-stimulating factor;

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NF-B, nuclear factor B; PAMPs, the pathogen associated molecule patterns; PBS, phosphate-buffered saline; PRRs, pattern recognition receptors; RIG-I,

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retinoic acid-induced gene I; SD, standard deviations; sictrl, siRNA control;

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siSTING, STING siRNA oligos; STING, stimulator of interferon genes; SUMO,

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small ubiquitin related modifiers; TAK1, TGFβ-activated kinase 1; TBK1, TANK-binding kinase 1; TBST, Tris-buffered saline and tween 20; TMAs, tissue

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microarrays; TNFα, tumor necrosis factor α; T3SS, type III secretion system; WT Y. pestis, wild type Yersinia pestis; YopE, Yersinia outer protein E; YopM, Yersinia outer protein M (YopM); YopJ, Yersinia outer protein J; YopJ C172A, the 172th cysteine residue of YopJ was replaced by alanine; ΔYopJ Y. pestis, Y. pestis lacking YopJ; Yops, Yersinia outer proteins.

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ACCEPTED MANUSCRIPT Highlights: Our studies demonstrate that YopJ targets STING to inhibit IRF3 signaling.

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Our studies show YopJ interacts with STING to block its ER-to-Golgi traffic and

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remove its K63-linked ubiquitination chains. Deubiquited STING perturbs the formation of STING-TBK1 complex and the activation of IRF3. Our studies show the 172th cysteine of YopJ mediates STING deubiquitination

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and IRF3 signaling inhibition.

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Consequently, mice infected with WT and ΔYopJ/YopJ bacteria induced lower levels of IRF3 and IFN-β, decreased inflammation and reduced staining of

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STING as compared to ΔYopJ and ΔYopJ/YopJ C172A strains infection.

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The data herein reveal a previously unrecognized mechanism by which YopJ

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modulates innate immune signaling.

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ACCEPTED MANUSCRIPT Abstract The Yersinia outer protein J (YopJ) plays a pivotal role in evading the host

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immune response and establishes a persistent infection in host cells after

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bacterial infection. YopJ is a cysteine protease and can act as a deubiquitinating enzyme that deubiquitinates several targets in multiple signaling pathways. Stimulator of interferon genes (STING) is a critical adapter

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for the induction of interferon regulatory factor 3 (IRF3) phosphorylation and

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subsequent production of the cytokines in response to nucleic acids in the cytoplasm. Our studies demonstrate that YopJ targets STING to inhibit IRF3

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signaling. Specially, YopJ interacts with STING to block its ER-to-Golgi traffic

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and remove its K63-linked ubiquitination chains. Deubiquited STING perturbs

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the formation of STING-TBK1 complex and the activation of IRF3. The 172th cysteine of YopJ mediated STING deubiquitination and IRF3 signaling

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inhibition. Consequently, mice infected with WT and ΔYopJ/YopJ bacteria induced lower levels of IRF3 and IFN-β, decreased inflammation and reduced staining of STING as compared to ΔYopJ and ΔYopJ/YopJ C172A strains infection. The data herein reveal a previously unrecognized mechanism by which YopJ modulates innate immune signaling. Keywords: Yop J, STING, Deubiquitination, IRF3, IFN-β.

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ACCEPTED MANUSCRIPT Introduction The host innate immune system provides a critical first line of defense against

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infectious agents, including pathogenic bacteria [1]. When pathogenic bacteria

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invade hosts, the pathogen associated molecule patterns (PAMPs) can be recognized by pattern recognition receptors (PRRs) and then trigger the rapid induction of cytokines to eliminate the pathogens [2].

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Several independent groups have identified STING as a central and

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multifaceted mediator of the innate immune response [3] . Later studies show that STING acts as not only an adaptor, but also a direct sensor of cytosolic

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DNA and cyclic diguanylate monophosphate (c-di-GMP) [4-6]. Cytosolic

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detection of pathogen-derived DNA and c-di-GMP are the major mechanisms

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of bacterial-induced interferon production. STING is ubiquitinated during the invasion of certain nucleic acids and then separated from the endoplasmic

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reticulum (ER) to form punctate complexes with TBK1 at the Golgi apparatus [7-10]. The STING-TBK1 complexes further activate the phosphorylation of IRF3, and the phosphorylated IRF3 forms dimers to activate transcription of downstream genes in the nucleus [11-14]. Therefore, STING functions as both an adaptor and a sensor, and has been demonstrated to facilitate downstream signal transmission to IRF3 and nuclear factor B (NF-B). Many Gram-negative bacterial pathogenic agents have acquired strategies to escape host immune system by injecting effector proteins into host cells by using a type III secretion system (T3SS). Y. pestis injects at least 6 kinds of

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ACCEPTED MANUSCRIPT Yersinia outer proteins (Yops) into the infected macrophages, resulting in the inhibition of the inherent immune signaling, which ultimately allows the bacteria

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to establish a systemic infection [15, 16]. Through subverting the innate

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immune response, YopJ nullifies the activation of pro-inflammatory cytokines, and subsequently induces macrophage apoptosis to prevent CD8+ T-cell responses and contributes to successful infection [17]. YopJ, but not the

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catalytically inactive YopJ C172A, inhibits the mitogen-activated protein kinase

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(MAPK) and the NF-B signaling pathways by acting as both an acetyltransferase and deubiquitinase [18-20]. Although NF-B is found to be

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one of the host target molecule of the YopJ, Y. pestis could affect host immune

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system at multiple levels. In this study, we show that T3SS effector YopJ in Y.

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pestis targets STING and inhibits cytosolic DNA antibacterial signaling, revealing a previously unrecognized mechanism by which YopJ modulates

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innate immune signaling.

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ACCEPTED MANUSCRIPT Materials and methods Strains, plasmids and siRNA

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Y. pestis biovar Microtus str. 91001 was used as wild type in this study and

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ΔYopJ Y. pestis was constructed based on the wild one. The pACYC184 vector was used to construct the YopJ or YopJ C172A (the 172th cysteine residue was replaced by alanine) overexpressing strains (ΔYopJ/YopJ or

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ΔYopJ/YopJ C172A Y. pestis). The mammalian expression plasmids of retinoic

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acid-induced gene I (RIG-I), mitochondrial antiviral signaling protein (MAVS), STING, TBK1, Yersinia outer protein E (YopE), Yersinia outer protein M

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(YopM), YopJ, ubiquitin, K48-ubiquitin, K63-ubiquitin encoding Flag-, Myc-,

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HA-fusion proteins were constructed by inserting PCR-amplified fragments

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into pcDNA3-Flag, pCMV-Myc, pCMV-HA vacant vectors respectively and the 172th nucleotide of the YopJ gene was mutated from cysteine to alanine by

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using a Quik-Change Mutagenesis Kit (Stratagene) to generate the Myc-YopJ C172A plasmids (Supplementary Table S1). pCDH-EF1-Flag-STING was constructed

by

inserting

pCDH-EF1-MCS-T2A-puro

PCR-amplified vacant

vector.

fragments The

of

reporter

STING

into

constructs

pGL3-IRF3-luc/UAS-luc (IRF-luciferase reporter plasmids), pGL3-IFN-β-luc have been described earlier [21-23]. Glutathione S-transferase (GST) fusion proteins were generated by expression in pGEX4T-1-based vectors (Amersham Biosciences Biotech) in Escherichia coli BL21 (DE3). Enzyme analysis and DNA sequencing were performed to verify all kinds of plasmids.

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ACCEPTED MANUSCRIPT The fluctuation of A260/A280 of all plasmids solution was between 1.75 and 1.85. siRNA (20nM, GenePharma) was transfected by jetPRIME® reagent

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(114-01, Polyplus) following the manufactory’ s instructions and the sequences

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of siRNA were showed in Supplementary Table S2. Bacterial infection was performed 48 hrs later after the transfection of siRNA. Cell culture and transfections

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Human embryonic kidney (HEK293T) and the murine macrophage cell line

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RAW264.7 were grown in Dulbecco’s modified Eagle’s medium (DMEM, 10-013, Corning cellgro) supplemented with 10% heat-inactivated fetal bovine

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serum (FBS, 10100-147, Gibco), 2 mM L-glutamine, 100 U/ml penicillin, and

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100 mg/ml streptomycin. Thp-1 cells were grown in RPMI Medium 1640

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(31800-022, Gibco) with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin and treated by TPA (0.1 μg/ml,

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723134, Sigma) for 48 hrs before using. Transient transfections were performed with jetPRIME® reagent following the manufactory’s instructions. Bone marrow-derived macrophages (BMDMs) isolation BMDMs were isolated from C57BL/6 mice, cultured in DMEM containing 20 ng/ml macrophage colony-stimulating factor (M-CSF, eBiosciences, CA, USA) for 6 days, and then seeded into 6-well plates 24 hrs before infection. Bacterial genome DNA extracting and bacterial infection WT Y. pestis was cultured in Hiss Agar (CM1102, LAND BRIDGE) and the genome DNA was extracted using the TIANamp Bacteria DNA Kit (DP302,

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ACCEPTED MANUSCRIPT TIANGEN) with the A260/A280 of DNA solution between 1.75 and 1.85. Both the stimulation of Y. pestis genome DNA (Y. pestis DNA) and poly (dA: dT)

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(P0883, Sigma–Aldrich) were at the concentration of 0.2 μg/ml for 6 hrs.

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RAW264.7 macrophages were infected with Y. pestis at a multiplicity of infection (MOI) of 20. The infection was initiated by centrifuging the plate at 700g for 5 min. After incubation for 1 hour at 37 °C, the plates were washed

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three times with 1 phosphate-buffered saline (PBS), transferred into fresh

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medium containing gentamicin (100 μg/ml) to kill the extracellular bacteria. Samples were subjected to immunoblotting (IB). Normalization of the levels of

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of each band.

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-Tubulin was used to measure the mean intensity for quantifying the density

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Dual luciferase reporter assays HEK293T cells were transfected with reporter plasmids (1 μg per plate,

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pGL3-IRF3-luc/UAS-luc or pGL3-IFN-β-luc), Renilla luciferase constructs (pRL-TK vector, 0.02 μg per plate, E2241, Promega), and various combinations of expression plasmids using jetPRIME® reagent after being seeded in 12-well plates for 24 hrs. Indicated empty vectors were used to equalize the total amounts of plasmids. The reporter activity of prepared cell extracts was detected using the Dual-Luciferase® Reporter Assay System (E1910, Promega). Results were presented as fold relative to the activity of uninfected or unstimulated cells. Data were representative of three independent experiments.

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ACCEPTED MANUSCRIPT Immunoprecipitation and IB analysis Cell lysates were prepared in lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM

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phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 mM sodium fluoride,

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protease inhibitors cocktail, 5% Nonidet P-40). Anti-Flag M2 Affinity Gel (A2220, Sigma–Aldrich) or anti-mouse IgG conjugated beads (42472, Sigma-Aldrich) were used for immunoprecipitation of soluble proteins. An

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aliquot of the total lysates (5%, v/v) was included as a control. IB was

anti-HA

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performed with anti-Acetylated-Lysine (9441; Cell Signaling Technology), (H9658;

Sigma-Aldrich),

HRP-conjugated

anti-Flag

(A8592;

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Sigma-Aldrich), anti-IRF3 (2241-1; Epitomics), anti-IRF3 Phospho pS386

(A5598;

Sigma-Aldrich),

anti-RIGI

(sc-98911;

Santa

Cruz

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anti-Myc

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(2562-1; Epitomics), anti-MAVS (sc-166583; Santa Cruz biotechnology),

biotechnology), anti-TBK1 (3296-1; Epitomics), anti-ubiquitin (sc-166553,

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Santa Cruz Biotechnology), anti-ubiquitin K63-linkage specific (05-1308; HERCK MILIIPORE), anti-ubiquitin K48-linkage specific (6919-s; Epitomics), anti-STING (ab92605; abcam) or anti-α-Tubulin (T6074; Sigma-Aldrich) antibody, respectively. Films (X-OMAT BT film, Carestream) were used to visualize

the

antigen-antibody

complexes

by

chemiluminescence

(NEL104001EA, PerkinElmer Life Sciences). GST pull-down assays DE3 was used to express the GST and GST fusion proteins. The subsequent purification of both kinds of proteins was according to the protocol (28-9523-61,

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ACCEPTED MANUSCRIPT Amersham Biosciences Biotech). Cell lysates were incubated with 5 μg purified GST or GST fusion proteins bound to glutathione beads at 4 ºC for 2

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hrs. The absorbates were washed by lysis buffer and IB was performed with

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the total lysates (5%, v/v) as a control. Immunofluorescence

Cells were washed three times with 1 PBS. For Flag-STING, Myc-YopJ, or

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Golgin 97 visualization, cells were fixed with 2% paraformaldehyde for 15 min

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at 37°C and permeabilized for 5 min with 0.2% Triton X-100. Samples were blocked with 5% skim milk in Tris-buffered saline and tween 20 (TBST) for 1

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hour. Anti-Flag (1:150, SAB4301135, Sigma-Aldrich), anti-Myc (1:150, A5598,

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Sigma-Aldrich), and anti-Golgin 97 (1:150, sc-74632, Santa Cruz) antibodies

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were used to detect the Flag-STING, Myc-YopJ and Golgin 97 proteins, respectively. Staining was visualized with secondary antibodies conjugated

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with Alexa Fluor 405 (ab175649, abcam), 594 (SA00006-7, Proteintech) or 488 (SA00003-3, Proteintech) and the images were captured with a digital camera under a confocal microscope (Zeiss LSM510). To see the colocation of STING and Golgi more intuitively, we interchanged the colors of STING and YopJ in the analysis software. Cycloheximide chase assay HEK293T cells were cotransfected with Flag-STING and Myc-vector or Myc-YopJ, treated with cycloheximide (50 μM) for the indicated time points. Samples were analyzed by IB with anti-Flag and anti-Myc antibody. The

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ACCEPTED MANUSCRIPT half-life of the STING protein was calculated as the time required for degradation of 50% of the protein.

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RNA analysis

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Total RNA was extracted from samples using TRNzol-A+ Regent (DP421, TIANGEN), and first-strand cDNA was generated from total RNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311,

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TRANSGENE). Quantitative RT-PCR was performed using AceQ qPCR SYBR

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Green Master Mix (Q121-02, VazymeTM) in triplicate and analyzed on an ABI Prism 7700 analyzer (Applied Biosystems, Foster, CA, USA). All real-time

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values were normalized to GAPDH. The primer sequences were showed in

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Supplementary Table S3. [24].

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Ubiquitination assays

HEK293T cells were cotransfected with Flag-STING, Myc-YopJ or Myc-vector

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together with HA-ubiquitin, HA-K48 ubiquitin or HA-K63 ubiquitin. For detection of endogenous ubiquitinated-STING following WT, ΔYopJ, ΔYopJ/YopJ or ΔYopJ/YopJ C172A Y. pestis infection, cells were infected at MOI=20. Cells were treated with MG132 for 8 hrs before harvesting cells. Immunoprecipitated were performed with Anti-Flag M2 Affinity Gel or Protein A/G plus-Agarose together with anti-STING antibody. The ubiquitination signal was detected using anti-HA, anti-ubiquitin, anti-ubiquitin K63-linkage specific or anti-ubiquitin K48-linkage specific antibody, respectively. In situ PLA assays

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ACCEPTED MANUSCRIPT RAW264.7 macrophages were transiently transfected with Flag-STING, cells were infected with different strains of Y. pestis after 24 hrs. The infected cells

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were treated according to the protocol of Duolink In Situ PLA® Probe

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Anti-Rabbit MINUS (DUO92005, OLINK BIOSCIENCE). The interaction between exogenous STING and endogenous TBK1 was determined by the intensity and distribution of the red spots under fluorescent microscopy.

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Histopathological scoring

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Liver and spleen tissues were fixed in 10% phosphate-buffered formalin, dehydrated by a tissue processor and embedded in paraffin. Microtome (5 μm)

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was used to section tissues and then hematoxylin and eosin (HE) were stained

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by an autostainer. Histological scoring of coded slides was assessed by a

system.

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single pathologist, blinded to the source of the slides, based on a modified

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Immunohistochemical (IHC) staining IHC staining for STING was performed on the paraffin-embedded tissue microarray. The tissue microarrays (TMAs) were performed on 5 μm thick sections. A graded alcohol series was used for tissue slides de-paraffinizing and rehydrating. Antigen retrieval was performed by placing the slides in 10 mM sodium citrate buffer (pH 6.0) and maintained at a sub-boiling temperature for 10 min. The slides were immersed in 10% normal goat serum in 1 PBS for 30 min to block non-specific staining, followed by primary antibody overnight at 4°C in a humidified chamber. 1 PBS was used for slides washing. Then,

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ACCEPTED MANUSCRIPT secondary antibody was used for 30 min at room temperature. Statistical Analysis

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Two-tailed Student’s t test was used to statistically analyze the difference

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between two groups. All data points were the average of triplicates, with error bars representing standard deviations (SD). All data were representative of

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considered statistically significant.

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results from at least three independent experiments. **P<0.01 or ##P<0.01 was

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ACCEPTED MANUSCRIPT Results YopJ inhibits IRF3 activity induced by cytosolic DNA pathway

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Due to the importance of IRF3 in host defense against bacterial infection [25],

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we initiated our study by examining the extent of cytosolic DNA activated IRF3 activation in THP-1 cells. c-di-GMP can be directly recognized by STING to activate TBK1-IRF3 signaling. To this end, cells carrying pGL3-IRF3–

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luc/UAS-luc and pRL-TK vector were transfected together with Y. pestis DNA

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or c-di-GMP. As was shown, the IRF3 activity was increased by cytosolic Y. pestis DNA and c-di-GMP (Fig. 1A). Similar enhancement of IFN-β luciferase

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activity by Y. pestis DNA or c-di-GMP was also observed in THP-1 cells (Fig.

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1A). Since STING expression is very low in HEK293T cells (Data not shown),

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we reconstitute responsiveness of HEK293T cells to c-di-GMP by Flag-STING overexpression. As we can see, Y. pestis DNA or c-di-GMP transfection also

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increased IRF3 and IFN-β reporters in HEK293T cells (Fig.1B). These data suggested that Y. pestis infection can trigger cytosolic DNA sensing pathway by its genome DNA or its second messenger. We next would like to determine if Yops were capable of blocking DNA-mediated induction of IRF3 activation. Expression constructs for YopE, YopJ and YopM were generated and cotransfected into THP-1 cells respectively together with Y. pestis genome or c-di-GMP. As shown in Fig. 1C, the induction of IRF3 activation by Y. pestis DNA or c-di-GMP was only inhibited by YopJ. YopJ inhibited IRF3 induction by Y. pestis DNA or c-di-GMP

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ACCEPTED MANUSCRIPT in a dose-dependent manner (Fig. 1D). Similar repression of Y. pestis DNA-induced activation of IRF3 reporters by YopJ was also observed in

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HEK293T cells (Fig. 1E and F), suggesting that YopJ is able to inhibit IRF3

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activity induced by cytosolic DNA pathway. YopJ associates with STING

Due to the importance of STING in cytosolic DNA innate immune signaling, a

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possible link between YopJ and STING were proposed. To test this possibility,

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Flag-STING was cotransfected together with Myc-YopJ. IB analysis of anti-Flag immunoprecipitates with an anti-Myc antibody showed a significant

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association between Myc-YopJ and Flag-STING (Fig. 2A). Specially, YopE and

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YopM failed to associate with STING (Fig. 2B). To demonstrate the interaction

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of YopJ and STING in vitro, lysates from HEK293T cells expressing Flag-STING were incubated with GST or GST-YopJ fusion protein. Analysis of

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the absorbates by IB analysis with anti-Flag antibody showed that STING bound GST-YopJ, but not GST (Fig. 2C). It has been known that STING relocalizes from ER to Golgi after activation [9, 10, 13]. We thus wanted to explore the effect of YopJ on STING translocation in response to c-di-GMP treatment. Immunofluorescence results showed that YopJ colocalized with STING even in the absence of c-di-GMP transfection (Fig. 2D). As expected, more colocalization of STING and Golgin97 were identified in response to c-di-GMP stimulation. However, in cells with overexpressed YopJ, the colocalization of STING with Golgin97 were

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ACCEPTED MANUSCRIPT significantly reduced (Fig. 2D). These results suggest that YopJ blocks STING translocation from ER to Golgi after activation through their physical

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association.

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YopJ blocks IRF3 signaling by inducing STING for degradation To further explore the mechanism for the inhibitory effect of YopJ on IRF3 antiviral signaling, we examined the effect of YopJ on STING abundance.

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Immunofluorescence results showed that the staining intensity of STING was

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much weaker in cells with overexpressed YopJ (Fig. 2D). In addition, a striking reduction in STING expression with overexpressed YopJ was found in a dose

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dependent manner (Fig. 3A). A study in 2007 reported that YopJ influenced the

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expression of CMV-based plasmids [26]. We then constructed STING into an

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EF1 promoter plasmid pCDH-EF1-MCS-T2A-Puro and found YopJ could still reduce STING expression (Supplementary Fig. S1A and B). Specially, YopE or

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YopM expression did not change STING level (Fig. 3B). Quantitative RT-PCR revealed no change in endogenous STING mRNA level with increased YopJ expression,

suggesting

that

YopJ

down-regulates

STING

by

posttranscriptional modification (Supplementary Fig. S1C). We then assessed whether YopJ was able to mediate the degradation of endogenous STING under physiological conditions. RAW264.7 macrophages infected with WT Y. pestis or ΔYopJ Y. pestis showed that STING level was higher in cells infected with ΔYopJ Y. pestis as compared with that of WT Y. pestis, whereas the protein levels of RIG-I, MAVS and TBK1 was not affected

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ACCEPTED MANUSCRIPT by YopJ deletion (Fig. 3C). To further delineate the mechanism for the YopJ-mediated STING degradation, IRF3 phosphorylation level was analyzed

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following Y. pestis infection. As illustrated in Fig. 3D, ΔYopJ Y. pestis infection

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strongly stimulated the signals for IRF3 activation than WT Y. pestis infection. We then investigated the half-life of STING in cells with overexpressed YopJ. As was shown in Fig. 3E, the approximated half-life of STING was

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considerably reduced by YopJ introduction. In reporter assays, YopJ, but not

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YopE or YopM, dramatically suppressed STING-induced activation of the IRF3 and IFN-β reporters activation (Fig. 3F and G). Taken together, these results

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indicate that YopJ targets STING for degradation to block the phosphorylation

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and activation of IRF3 signaling.

signaling

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YopJ 172th cysteine is the active site for blocking STING-mediated

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Previous reports showed that YopJ functions by deubiquitinating various signal transduction proteins [20, 26, 27]. A catalytic cysteine mutant of YopJ (YopJ C172A) is unable to inhibit signaling pathways, which prompted us to evaluate the contribution of YopJ protease activity in blocking STING activity [20]. Transfection of HEK293T cells with plasmids encoding Flag-STING together with WT YopJ or YopJ C172A revealed that the levels both of STING protein abundance and IRF3 phosphorylation were diminished considerably by WT YopJ, but not YopJ C172A (Fig. 4A). Consistently, ΔYopJ and ΔYopJ/YopJ C172A Y. pestis also had a compromised activity on inhibiting STING-induced

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ACCEPTED MANUSCRIPT IRF3 promoter activation compared to WT and ΔYopJ/YopJ Y. pestis (Fig. 4B). As YopJ targeted STING for degradation, protein levels of STING were

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checked after infection with different strains of Y. pestis at the indicated time

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points. Consistently, the protein level of STING was reduced significantly by WT and ΔYopJ/YopJ Y. pestis, while ΔYopJ and ΔYopJ/YopJ C172A Y. pestis had a compromised activity on STING down-regulation (Fig. 4C). To

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demonstrate that the STING signaling pathway was a critical way for YopJ

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induced interferon response, we infected RAW264.7 macrophages with different strains of Y. pestis. The results showed that ΔYopJ and ΔYopJ/YopJ

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C172A Y. pestis could activate the IFN-β expression more strikingly than WT

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and ΔYopJ/YopJ Y. pestis did (Fig. 4D).

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Previous studies indicated that the activated STING forms dimers to assemble with TBK1, leading to IRF3 activation and the final induction of type I interferon

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[28]. To further examine the mechanism of the inhibitory effect of YopJ in innate immune signaling under physiological conditions, we explored the effect of YopJ on STING-TBK1 interaction. Immunoprecipitation experiment indicated that STING-TBK1 interaction was reduced strikingly by YopJ (Supplementary Fig. S2). Then STING-TBK1 complexes were visualized by an in situ proximity ligation assay (PLA). Small spots of STING-TBK1 complex in RAW264.7 macrophages infected with different strains of Y. pestis were observed. Results showed that ΔYopJ and ΔYopJ/YopJ C172A Y. pestis-infected RAW264.7 macrophages displayed significantly increasing in abundance of STING-TBK1

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ACCEPTED MANUSCRIPT complexes as compared to those from WT and ΔYopJ/YopJ Y. pestis-infected RAW264.7 macrophages (Fig. 4E and F). These results indicate the

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importance of YopJ deubiquitinating protease activity in down-regulating

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STING activity. YopJ deubiquitinates STING

It has been reported that the removal of K63-linked ubiquitin chains from

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TRAF6 by YopJ inactivates the NF-B signaling pathway [20]. Since STING

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ubiquitination is indispensable for its function in IFN-β signaling, we then checked the effect of YopJ on STING ubiquitination modification. As was

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shown in Fig. 5A, ΔYopJ Y. pestis induced much more obvious ubiquitination of

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STING than WT Y. pestis did, indicating that YopJ might act as a

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deubiquitinating enzyme. Furthermore, the ubiquitination modification of STING was decreased by ectopic YopJ expression in response to c-di-GMP or

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poly (dA: dT) stimulation (Fig. 5B and C). Actually, basal STING ubiquitination level was sharply decreased by YopJ introduction (Fig. 5B and C). Furthermore, STING ubiquitination induced by c-di-GMP or poly (dA: dT) was nearly unchanged by YopJ C172A (Fig. 5B and C). Since YopJ is both a deubiquitinating enzyme and an acetyltransferase, we next explored whether the acetyltransferase activity of YopJ had any effect on STING acetylation. Flag-STING were cotransfected with Myc-YopJ or Myc-YopJ C172A plasmids and immunoprecipitation were performed to check if STING was been acetylated. The validity of Acetylated-Lysine antibody was shown in

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ACCEPTED MANUSCRIPT Supplementary Fig.S3A. As was shown in Supplementary Fig. S3B, neither YopJ nor YopJ C172A could acetylate STING. Therefore, YopJ triggers STING

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deubiquitination by its Cys-172 protease activity.

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By using K48 and K63 ubiquitin antibodies in ubiquitination assay, only anti-K63 ubiquitin antibody could react with immunoprecipitated STING in macrophages after Y. pestis infection (Fig. 5D and Supplementary Fig. S3C). endogenous

ubiquitination

assay

showed

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The

that

the

K63-linked

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ubiquitination modification of STING was significantly decreased in WT and ΔYopJ/YopJ Y. pestis-infected macrophages compared to ΔYopJ/YopJ C172A

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Y. pestis-infected macrophages, while the K48-linked ubiquitination was barely

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detected (Fig. 5D and Supplementary Fig. S3C). Using an ubiquitin mutant

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with only one lysine at position 48 or 63 available for conjugation (HA-K48 ubiquitin or HA-K63 ubiquitin), we found that c-di-GMP or poly (dA: dT)

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transfection led to increased K63-linked but not K48-linked ubiquitination of STING, which was significantly abrogated by YopJ (Fig. 5E and F), indicating YopJ mediates K63-linked deubiquitination of STING. Since YopJ-mediated degradation of STING was partially inhibited by the proteasome inhibitor MG132, but not lysosome inhibitor NH4Cl (Fig. 5G), we conclude that the YopJ-induced

K63-linked

deubiquitination

may

direct

STING

to

proteasome-mediated degradation. YopJ contributes to bacterial virulence by regulating STING-mediated antibacterial response

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ACCEPTED MANUSCRIPT To further delineate the role of YopJ mediated STING down-regulation in Y. pestis virulence, BMDMs were infected with different strains of Y. pestis at the

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indicated time points. The mean numbers of bacteria recovered from WT and

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ΔYopJ/YopJ Y. pestis-infected BMDMs were considerably higher than those from ΔYopJ and ΔYopJ/YopJ C172A Y. pestis-infected BMDMs (Fig. 6A). After down-regulation of STING, the mean number of bacteria recovered from

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ΔYopJ Y. pestis-infected siSTING cells was much higher than ΔYopJ Y.

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pestis-infected siCtrl cells, while the mean number of bacteria recovered from WT Y. pestis-infected siSTING cells were barely increased (Fig. 6B), indicating

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a major function of YopJ in STING degradation is required for virulence. To

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establish the role of YopJ in Y. pestis infection, mice were challenged by

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intravenous injection of WT, ΔYopJ, ΔYopJ/YopJ or ΔYopJ/YopJ C172A Y. pestis. Firstly, HE staining was performed to examine disease pathology of

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formalin-fixed liver and spleen. Total pathological severity scoring indicated a significantly increased inflammation, tissue injury and necrosis in ΔYopJ or ΔYopJ/YopJ C172A bacteria infected tissues, while WT and ΔYopJ/YopJ strains generally caused lower levels of inflammation and necrosis (Fig. 6C). These observations suggest that YopJ is essential for Y. pestis virulence. IHC staining was then performed to examine the STING staining in situ. Consistently, significantly increased staining of STING in the inflammatory lesions of the liver and spleen from ΔYopJ and ΔYopJ/YopJ C172A-infected mice was identified as compared to which of WT and ΔYopJ/YopJ-infected

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ACCEPTED MANUSCRIPT mice (Fig. 6D). These results demonstrate that YopJ contributes to bacterial

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virulence by negatively regulating STING-mediated antibacterial response.

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ACCEPTED MANUSCRIPT Discussion YopJ has been reported to be a deubiquitinating enzyme that negatively

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regulates NF-B signaling via removing K63-linked ubiquitination conjugates

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from critical proteins, such as TRAF2, TRAF6, and IKK [20]. In this study, we investigated the mechanisms of the IRF3 antagonism imposed by YopJ in a deubiquitinase catalytic dependent manner. We showed that YopJ, but not

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YopJ C172A, blocked the assembly of STING-TBK1 complexes and IRF3

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activation. Importantly, we have demonstrated that K63-linked ubiquitination signals of STING were dramatically reduced in the presence of YopJ, which

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might contribute to destabilizing STING. We propose that the deubiquitination

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and degradation of STING during Y. pestis infection is a new bacterial strategy

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to modulate the host antibacterial responses. Recent studies identify that pathogenic bacteria can evade the host immune

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system during the course of infection through targeting at various proteins in multiple signaling pathways. Different groups have already demonstrated that YopJ blocks the MAPK and NF-B signaling pathways by acting as both deubiquitinase and acetyltransferase. In 2000, it was reported that YopJ is an ubiquitin-like protein protease by which small ubiquitin related modifiers (SUMO) of unidentified conjugated proteins were cleaved [27]. Subsequently, YopJ was proved to act as a deubiquitinase targeting at TRAF proteins to inhibit NF-B signaling pathways [20, 26]. In 2006 and 2012, YopJ was regarded

as

a

serine/threonine

acetyltransferase

24

targeting

both

at

ACCEPTED MANUSCRIPT mitogen-activated protein kinase kinases (MAP2Ks) and TGFβ-activated kinase 1 (TAK1) [18, 19]. Our data show that the deubiquitinase activity of

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YopJ also targets at a scaffolding protein, STING. Therefore, YopJ modulates

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various inflammatory signaling pathways at multiple levels with different mechanisms.

There are various mechanisms for mammalian cells to limit bacterial growth

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inside endosome and lysosome compartments [29]. Under physiological

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surroundings, most bacterial colonization in host cells can be restrained by innate immune system [2, 30]. Previous studies indicated that STING plays a

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pivotal role both in the process of ubiquitin colocalization of specific bacterial

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subpopulation and the initiating of autophagy targeting [31]. The limited

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bacterial killing by macrophages relies on the delivery of this population to lysosome. In the process of keeping the host cytosol from bacterial colonizing,

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TBK1 is also necessary for activating autophagy by recognizing the ubiquitin-coated bacteria. Because YopJ associates with STING, inhibits STING translocation from ER to Golgi and suppresses STING-TBK1 interaction, it is reasonable to propose that this bacterial protein interferes with cell signaling initiated from TLRs, STING, TBK1, or autophagy pathways during Y. pestis infection. We also demonstrated that YopJ, but not YopJ C172A, could inhibit the activation of IFN-β production both in macrophages and under physiological conditions in mice. The K48-linked ubiquitination of STING couldn’t be detected after infection with different strains of Y. pestis.

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ACCEPTED MANUSCRIPT K48-linked ubiquitination of STING has been identified in response to viral infection in several papers [32, 33]. Failure to detect K48-linked ubiquitination

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in our experiments may due to its low level in response to bacterial infection.

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As YopJ induced STING degradation can be partially rescued by MG132 treatment, we propose that YopJ-induced K63-linked deubiquitination may direct STING to proteasome-mediated degradation, which needs further

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investigation. It should be noted that the protein stability was increased via

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K63-linked ubiquitination [34]. In conclusion, we show that YopJ could inhibit the activation of IFN-β production in macrophages by binding to STING and

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removing its K63-linked ubiquitination.

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YopP in Y. enterocolitica, a 94% identical of YopJ in Y. pseudotuberculosis and

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Y. pestis, shows greater ability for secretion, translocation, and consequent cytotoxicity than YopJ [35, 36]. But both expression of YopP and complete

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absence of YopJ in Y. pseudotuberculosis resulted in attenuation of virulence, which shows that reasonable levels of YopJ-mediated cytotoxicity are indispensable for optimized virulence for specific pathogenic species [37]. Other studies also showed that this virulence characteristic of YopJ is essential for the establishment of a systemic infection in mice [38, 39].Here we found that the mean numbers of bacteria recovered from ΔYopJ and ΔYopJ/YopJ C172A Y. pestis-infected cells were dramatically reduced compared to those from WT and ΔYopJ/YopJ Y. pestis-infected cells, indicating the importance of YopJ for Y. pestis to establish a systemic infection. Together, our work show

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ACCEPTED MANUSCRIPT that the interaction between YopJ and STING not only helps us to understand the mechanism of pestis to evade the host immunity, but also suggests that

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YopJ may form a possible target for pestis treatment.

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Funding

This work was supported in part by the Basic Research Program of China (2012CB518900), the National Natural Science Foundation of China

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(31170029, 31207911 and 31270800), the China Postdoctoral Science

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Foundation Grant (2013M542507). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the

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Competing interests

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manuscript.

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The authors do not have any potential conflicts of interest to declare.

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The authors declare that they have no competing interests.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. YopJ inhibits IRF3 activity induced by cytosolic DNA pathway THP-1

(A)

or

HEK293T

cells

(B)

were

transfected

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B)

with

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(A,

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pGL3-IRF3-luc/UAS-luc or pGL3-IFN-β-luc together with pRL-TK vector and stimulated by Y. pestis genome or c-di-GMP for 6 hrs before harvesting. Luciferase activity was measured and normalized for transfection efficiency. E)

THP-1

(C)

or

HEK293T

cells

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(C,

(E)

were

transfected

with

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pGL3-IRF3-luc/UAS-luc together with indicated plasmids and stimulated by Y. pestis genome or c-di-GMP for 6 hrs before harvesting. Luciferase activity was

F)

THP-1

(D)

or

HEK293T

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(D,

D

measured and normalized for transfection efficiency. cells

(F)

were

transfected

with

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pGL3-IRF3-luc/UAS-luc, pRL-TK vector together with increasing amount of Myc-YopJ and stimulated by Y. pestis genome or c-di-GMP for 6 hrs before

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harvesting. Luciferase activity was measured and normalized for transfection efficiency.

Whole cell lysates were analyzed by IB with the indicated antibodies. α-Tubulin was used as equal loading control. Cell-based studies were performed at least three independent times with comparable results. Data represent means ± the standard errors of the means. Student’s t test was used for statistical analysis: **P<0.01; ##P<0.01, versus different control respectively.

Figure 2. YopJ associates with STING

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ACCEPTED MANUSCRIPT (A) HEK293T cells were cotransfected with Myc-YopJ and Flag-vector or Flag-STING

expression

plasmids

respectively.

anti-Flag

or

IgG

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immunoprecipitates and whole cell lysates were analyzed by IB with anti-Myc

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or anti-Flag antibody.

(B) Myc-STING expression plasmids were transfected into HEK293T cells together with Flag-vector, Flag-YopE Flag-YopJ or YopM. Anti-Flag or IgG

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immunoprecipitates and whole cell lysates were analyzed by IB with anti-Myc

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or anti-Flag antibody.

(C) HEK293T cells were transfected with Flag-STING expressing plasmids.

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The GST-YopJ fusion protein absorbates from cell lysates were analyzed by IB

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with anti-Flag antibody (top panel). Loading of the GST proteins was assessed

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by coomassie blue staining (bottom panel). (D) Representative confocal immunofluorescent images in HEK293T cells

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transfected with the indicated plasmids in the presence or absence of c-di-GMP. Original magnification ×100. Scale bars, 10 μm. Cell-based studies were performed at least three independent times with comparable results.

Figure 3. YopJ blocks IRF3 signaling by inducing STING for degradation (A) HEK293T cells were cotransfected with increasing amount of Myc-YopJ expression plasmids together with Flag-STING, and whole cell lysates were analyzed by IB with the indicated antibodies. α-Tubulin was used as equal

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ACCEPTED MANUSCRIPT loading control. (B) Myc-STING were cotransfected with Flag-vector, Flag-YopE, Flag-YopJ or

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Flag-YopM expression plasmids respectively, and whole cell lysates were

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analyzed by IB with the indicated antibodies. α-Tubulin was used as equal loading control.

(C) RAW264.7 macrophages were infected with WT or ΔYopJ Y. pestis

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(MOI=20). Whole cell lysates were analyzed by IB with the indicated

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antibodies. α-Tubulin was used as equal loading control. (D) RAW264.7 macrophages were infected with WT or ΔYopJ Y. pestis

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(MOI=20) for 0, 2, 4, 6, 8 hrs respectively, and whole cell lysates were

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loading control.

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analyzed by IB with the indicated antibodies. α-Tubulin was used as equal

(E) HEK293T cells were cotransfected with Flag-STING and Myc-vector or

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Myc-YopJ respectively. After 24 hrs, cells were treated with cycloheximide (50 μM) for 0, 2, 4, 6, 8 hrs respectively, and whole cell lysates were analyzed by IB with the indicated antibodies. α-Tubulin was used as equal loading control. (F, G) HEK293T cells were cotransfected with pGL3-IRF3-luc/UAS-luc (F) or pGL3-IFN-β-luc (G) together with pRL-TK vector and the indicated plasmids. Luciferase activity was measured 24 hrs later and normalized for transfection efficiency. Whole cell lysates were analyzed by IB with the indicated antibodies. α-Tubulin was used as equal loading control. Cell-based studies were performed at least three independent times with

30

ACCEPTED MANUSCRIPT comparable results. Data represent means ± the standard errors of the means.

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Student’s t test was used for statistical analysis: **P<0.01.

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Figure 4. YopJ 172th cysteine is the active site for blocking STING mediated signaling

(A) Flag-STING was cotransfected with Myc-vector, Myc-YopJ or Myc-YopJ

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C172A expression plasmids into HEK293T cells respectively, and whole cell

as equal loading control. pGL3-IRF3-luc/UAS-luc,

pRL-TK

vector

were

cotransfected

with

D

(B)

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lysates were analyzed by IB with the indicated antibodies. α-Tubulin was used

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Myc-vector, Myc-YopJ or Myc-YopJ C172A expression plasmids respectively.

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Luciferase activity was measured 24 hrs later and normalized for transfection efficiency. Whole cell lysates were analyzed by IB with the indicated antibodies.

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α-Tubulin was used as equal loading control. (C) RAW264.7 macrophages were infected with WT, ΔYopJ, ΔYopJ/YopJ, ΔYopJ/YopJ C172A Y. pestis (MOI=20) for 0, 2, 4, 6, 8 hrs respectively. Whole cell lysates were analyzed by IB with the indicated antibodies. α-Tubulin was used as equal loading control. (D) RAW264.7 macrophages were infected with WT, ΔYopJ, ΔYopJ/YopJ or ΔYopJ/YopJ C172A Y. pestis (MOI=20) respectively. RNA was extracted and mRNA level of IFN-β was analyzed by quantitative RT-PCR. The data were normalized to the expression of the GAPDH reference gene.

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ACCEPTED MANUSCRIPT (E) Quantification of PLA signals per cell in (F), presented relative to which of control cells treated with solvent.

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(F) In situ PLA of STING-TBK1 complexes in RAW264.7 macrophages

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infected with different strains of Y. pestis (MOI=20). STING-TBK1 complexes were red spots. Original magnification ×100. Scale bars, 10 μm. Cell-based studies were performed at least three independent times with

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comparable results. Data represent means ± the standard errors of the means.

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Student’s t test was used for statistical analysis. **P<0.01.

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Figure 5. YopJ deubiquitinates STING

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(A) THP-1 cells were transfected with Flag-STING expression plasmids. After

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infecting with WT or ΔYopJ Y. pestis (MOI=20). Cells were treated with MG132 for 8 hrs before harvesting cells. Anti-Flag immunoprecipitates and whole cell

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lysates were analyzed by IB with the indicated antibodies. (B-C) HEK293T cells were cotransfected with Flag-STING, HA-ubiquitin together with Myc-vector, Myc-YopJ or Myc-YopJ C172A expression plasmids respectively in the presence or absence of c-di-GMP (B) or poly (dA: dT) (C) stimulation. Cells were treated with MG132 for 8 hrs before harvesting cells. Anti-Flag immunoprecipitates and whole cell lysates were analyzed by IB with the indicated antibodies. (D) RAW264.7 macrophages were infected with WT, ΔYopJ, ΔYopJ/YopJ or ΔYopJ/YopJ C172A Y. pestis (MOI=20) respectively. Cells were treated with

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ACCEPTED MANUSCRIPT MG132 for 8 hrs before harvesting cells. Anti-STING immunoprecipitates were analyzed by IB with the indicated antibodies.

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(E, F) HEK293T cells were cotransfected with Flag-STING and Myc-vector or

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Myc-YopJ together with HA-ubiquitin, HA-K48 or HA-K63 ubiquitin expression plasmids respectively in the presence or absence of c-di-GMP (E) or poly (dA: dT) (F) stimulation. Cells were treated with MG132 for 8 hrs before harvesting

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cells. Anti-Flag immunoprecipitates whole cell lysates were analyzed by IB with

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indicated antibodies.

(G) HEK293T cells were cotransfected with Flag-STING and Myc-vector or

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Myc-YopJ and treated with DMSO, MG132 or NH4Cl respectively. Whole cell

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lysates were analyzed by IB with the indicated antibodies. α-Tubulin was used

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as equal loading control.

Cell-based studies were performed at least three independent times with

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comparable results.

Figure 6. YopJ contributes to bacterial virulence by regulating STING-mediated antibacterial response (A) BMDMs were infected with WT, ΔYopJ, ΔYopJ/YopJ or ΔYopJ/YopJ C172A Y. pestis (MOI=20) respectively for the indicated time poins. Then BMDMs were collected using 5% deoxysodium cholate, smeared to solid Hiss Agar. The quantity of Y. pestis clones were estimated after 36 hrs. (B) BMDMs were transfected with siCtrl or siSTING. After 48 hrs, they were

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ACCEPTED MANUSCRIPT infected with WT or ΔYopJ Y. pestis (MOI=20) respectively for the indicated time points. Then BMDMs were collected using 5% deoxysodium cholate,

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smeared to solid Hiss Agar. The quantity of Y. pestis clones were estimated

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after 36 hrs.

(C) Liver or spleen from mice infected with WT, ΔYopJ, ΔYopJ/YopJ or ΔYopJ/YopJ C172A Y. pestis (MOI=20) respectively for 3 days were subjected

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to immunohistochemical staining of STING. All images are representative and

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taken at magnification ×200. Data shown are representative from two independent experiments (n = 5 mice per strain).

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(D) Liver, or spleen from mice infected with WT, ΔYopJ, ΔYopJ/YopJ or

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ΔYopJ/YopJ C172A Y. pestis respectively for 3 days were subjected to

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histopathological analysis by HE staining (MOI=20). Each micrograph shows an example of the pathology caused by each strain. All images are

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representative and were taken at magnification ×100. Data shown are representative from two independent experiments (n = 5 mice per strain).

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