Article

A Single Bacterial Immune Evasion Strategy

Dismantles Both MyD88 and TRIF SignalingPathways Downstream of TLR4

Graphical Abstract

Highlights

d The Yersinia effector YopJ blocks TRIF signaling and IFNb

production in DCs

d YopJ does not block myddosome formation, NF-kB, or IRF3

activation induced by TLR4

d Blocking MAPKs is sufficient to prevent MyD88- and TRIF-

dependent gene expression

d YopJ interference with TRIF signaling prevents DC-mediated

activation of NK cells

Rosadini et al., 2015, Cell Host & Microbe 18, 682–693December 9, 2015 ª2015 Elsevier Inc.http://dx.doi.org/10.1016/j.chom.2015.11.006

Authors

Charles V. Rosadini, Ivan Zanoni,

Charlotte Odendall, ..., Igor E. Brodsky,

Joan Mecsas, Jonathan C. Kagan

Correspondencejonathan.kagan@childrens.harvard.edu

In Brief

The mechanisms by which pathogens

block TLR4 signaling are unclear.

Rosadini et al. demonstrate that Yersinia

pseudotuberculosis utilizes the effector

YopJ to interfere with MAPK pathways

downstream of TRIF and MyD88 in

dendritic cells. This singular target of

YopJ is sufficient to dismantle the entire

TLR4-dependent transcriptional

response.

Cell Host & Microbe

Article

A Single Bacterial Immune Evasion StrategyDismantles Both MyD88 and TRIF SignalingPathways Downstream of TLR4Charles V. Rosadini,1 Ivan Zanoni,1,2,3 Charlotte Odendall,1 Erin R. Green,4 Michelle K. Paczosa,5 Naomi H. Philip,6

Igor E. Brodsky,6 Joan Mecsas,4,5 and Jonathan C. Kagan1,*1Harvard Medical School and Division of Gastroenterology, Boston Children’s Hospital, Boston, MA 02115, USA2Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan 20126, Italy3Unit of Cell Signalling and Innate Immunity, Humanitas Clinical and Research Center, Rozzano 20089, Italy4Graduate Program in Molecular Microbiology, Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston,

MA 02111, USA5Graduate Program in Immunology, Sackler School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA6Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

*Correspondence: jonathan.kagan@childrens.harvard.edu

http://dx.doi.org/10.1016/j.chom.2015.11.006

SUMMARY

During bacterial infections, Toll-like receptor 4 (TLR4)signals through the MyD88- and TRIF-dependentpathways topromotepro-inflammatory and interferon(IFN) responses, respectively. Bacteria can inhibit theMyD88 pathway, but if the TRIF pathway is also tar-geted is unclear. We demonstrate that, in addition toMyD88, Yersinia pseudotuberculosis inhibits TRIFsignaling through the type III secretionsystemeffectorYopJ. Suppression of TRIF signaling occurs duringdendritic cell (DC) andmacrophage infection and pre-vents expression of type I IFN and pro-inflammatorycytokines. YopJ-mediated inhibition of TRIF preventsDCs from inducingnatural killer (NK) cell productionofantibacterial IFNg. During infection of DCs, YopJpotently inhibits MAPK pathways but does not pre-vent activation of IKK- or TBK1-dependent pathways.This singular YopJ activity efficiently inhibits TLR4transcription-inducing activities, thus illustrating asimple means by which pathogens impede innateimmunity.

INTRODUCTION

Innate sensing of bacteria is mediated by proteins called Toll-like

receptors (TLRs). TLRs detect common microbial features

known as pathogen-associated molecular patterns (PAMPs)

and activate signaling networks to promote inflammatory gene

expression leading to clearance of the invadingmicrobe (Iwasaki

and Medzhitov, 2004). Pathogenic bacteria, however, have

evolved mechanisms to inhibit TLR signaling pathways (Baxt

et al., 2013). In particular, bacteria often target signaling down-

stream of TLR4, a receptor for bacterial lipopolysaccharide

(LPS) (Poltorak et al., 1998). TLR4 activates two distinct signaling

networks: the MyD88-dependent and TRIF-dependent path-

682 Cell Host & Microbe 18, 682–693, December 9, 2015 ª2015 Else

ways (Oda and Kitano, 2006). LPS sensing by TLR4 at the

plasma membrane promotes the MyD88-dependent rapid acti-

vation of the transcription factors AP-1 and NF-kB (Kawai

et al., 1999). TLR4 is then delivered to endosomes, where it sig-

nals through the TRIF pathway to induce late-phase AP-1 and

NF-kB activation. TRIF also induces the phosphorylation and nu-

clear translocation of IFN regulatory factor 3 (IRF3), an important

regulator of type I IFN (IFNa and b) production (Fitzgerald et al.,

2003b; Yamamoto et al., 2003). TRIF-dependent signaling acts

with the MyD88-dependent pathway to promote inflammatory

cytokine production (Hirotani et al., 2005), but can be differenti-

ated from MyD88-dependent signaling by this latter activity of

IRF3 activation and type I IFN production.

Most investigations into bacterial strategies to disrupt TLR4

have focusedon their ability to blockMyD88-dependent signaling

(Rosadini and Kagan, 2015). In contrast, the extent to which bac-

teria interfere with TRIF-dependent responses is unclear. Recent

work indicates thatTRIF regulates immunity tobacterial infections

in mice (Broz et al., 2012; Gurung et al., 2012; Jeyaseelan et al.,

2007; Power et al., 2007; Rathinam et al., 2012). TRIF-induced

type I IFN also promotes the ability of dendritic cells (DCs) to acti-

vate natural killer (NK) cells to release IFNg, a cytokine with anti-

bacterial activity (Fernandez et al., 1999; Zanoni et al., 2013).

Together, these findings indicate that TRIF regulates antibacterial

processes, which prompted us to examine if pathogenic bacteria

have developed strategies to inhibit TRIF signaling.

Pathogenic Yersinia spp., including Y. pestis, Y. enterocolitica,

and Y. pseudotuberculosis (Yptb), inhibit innate immune re-

sponses. Examples of Yersinia-encoded immune evasion strate-

gies include modifying their LPS structure to be less detectable

by TLR4 (Montminy et al., 2006) and secreting effector proteins

into host cells via a type III secretion system (Trosky et al.,

2008). Notably, the type III effector YopJ acetylates proteins

within the MyD88 signaling network, leading to blockade of

NF-kB and AP-1 activation and also phagocyte death (Mittal

et al., 2006; Monack et al., 1997; Mukherjee et al., 2006; Palmer

et al., 1998; Paquette et al., 2012; Schesser et al., 1998). It is

unclear whether the actions of YopJ or any other Yersinia

effector influence the TRIF pathway induced by TLR4.

vier Inc.

Interestingly, TRIF is required for the protection of mice from

Y. enterocolitica infection (Sotolongo et al., 2011), suggesting a

possible need to evade TRIF-mediated signaling events. Thus,

we hypothesized that Yersinia spp. may harbor mechanisms

that would inhibit the TRIF-dependent pathway and investigated

the ability of one of the Yersinia spp. to suppress TRIF signaling

and type I IFN production in immune cells.

In this study, we report that Yptb inhibits TRIF-dependent re-

sponses, including type I IFN expression, in DCs and macro-

phages via a YopJ-dependent mechanism. Surprisingly, we find

that YopJ does not inactivate proteins within the TRIF-IRF3 axis

or IkBkinase (IKK)-dependentprocesses inDCs,but ratherspecif-

ically inhibits mitogen-activated protein kinase (MAPK) pathways

and AP-1 activation. We show that inhibition of MAPK pathways

is sufficient to inhibit expression of a broad range of cytokines

induced by TLR4. Further, YopJ-dependent inhibition of TRIF

signaling impairs the ability of DCs to induce IFNg production by

NK cells. These data reveal that Yptb specifically targets MAPKs

during infection and that this singular activity is sufficient to pre-

vent induction of the TLR4-dependent transcriptional program.

RESULTS

Yptb Suppresses TRIF-Dependent Gene Expression inMacrophages and DCsTo determine if Yptb suppresses TRIF-dependent signaling, mu-

rine bonemarrow-derived DCs (BMDCs) were infected with Yptb

strain Yp2666 and a Yp2666 strain lacking its virulence plasmid.

The strain lacking the virulence plasmid, which encodes the type

III secretion system and Yop effector proteins, is herein referred

to as plasmid�. Infected cells were examined for MyD88 or

TRIF-dependent gene expression (Figure 1A). As expected,

Yp2666-infected cells induced minimal expression of Il6, a

gene that requires MyD88 signaling for full induction. In contrast,

cells infected with the plasmid� strain elicited robust expression

of this gene. Compared to the plasmid� strain, Yp2666 also

repressed expression of three genes considered to be activated

downstream of TRIF, Ifnb1 (IFNb), Cxcl10, and Rsad2 (Fitzgerald

et al., 2003a; Yamamoto et al., 2003) (Figure 1A). This observa-

tion suggested that virulence plasmid-encoded genes were

important for blocking TRIF responses. Because YopJ sup-

presses innate immune responses, we examined a yopJ null

mutant generated in the Yp2666 background and found that

this mutant phenocopied the plasmid� strain for its inability to

block Ifnb1, Cxcl10, and Rsad2 expression (Figure 1A). YopJ-

dependent inhibition of Ifnb1, Cxcl10, and Rsad2 was observed

at both high (100) and low (20) MOIs (Figures 1A and S1A). These

data suggest that, in addition to blocking cytokine expression,

YopJ has the ability to block IFN expression.

We also determined if YopJ was required for blocking TRIF

signaling in macrophages. Immortal bone marrow-derived mac-

rophages (iBMDMs) were infected with Yp2666 or the yopJ

mutant and analyzed for gene expression. YopJ-dependent inhi-

bition of Il6, Ifnb1, Cxcl10, and Rsad2 was observed in these

cells (Figure 1B) and in primary BMDMs (Figure S1B). Thus,

Yptb suppresses TRIF-dependent responses in macrophages

in a YopJ-dependent manner.

To verify the role for YopJ in blocking expression of the genes

examined, BMDCs were infected with yopJ mutants harboring

Cell Host &

an empty plasmid vector or a vector expressing yopJ or yopP

(the yopJ homolog from Y. enterocolitica). Expression of yopJ

or yopP complemented the yopJ mutant for the ability to block

Il6 and Ifnb1 expression in BMDCs, while the strain containing

the empty vector could not (Figure 1C). YopJ is an acetyltransfer-

ase whose enzymatic activity is required for its immune-evasion

activities (Mittal et al., 2006; Mukherjee et al., 2006). Therefore,

we examined the requirement for YopJ catalytic activity in block-

ing gene expression. BMDCs were infected with Yptb parent

strain, Yp32777, and a strain harboring a yopJ mutant deficient

in a critical active site residue, C172A (yopJC172A). Whereas

Yp32777 suppressed Il6, Ifnb1,CXCL10, and Rsad2 expression,

the strain expressing yopJC172A was unable to block expression

of these genes (Figure 1D). Collectively, these data establish that

YopJ and its catalytic activity are required for blocking the

expression of all genes examined.

Potentially, Yptb interferes with all signaling pathways exam-

ined by inducing YopJ-dependent cell death. To address this,

we performed experiments where bacteria-induced gene

expression was examined side-by-side with cell viability. Cell

viability was examined by flow cytometry using the stains an-

nexin V (AV) (which labels apoptotic cells) and propidium iodide

(PI) (which labels necrotic cells). Cells that stain negative for both

stains are considered viable. Infection of BMDCs with the parent

strain Yp2666 or the yopJ null mutant resulted in only a slight in-

crease in AV/PI staining over uninfected control cells at 1 and 2 hr

post-infection (Figures 1E and S1C). Importantly, throughout the

assay the majority of cells (>85%) were negative for AV/PI stain-

ing (i.e., healthy cells), and the observed staining pattern was not

different between Yp2666 and yopJ mutant infections (Figures

1E and S1C). Thus, loss of cell viability is not likely to be respon-

sible for the YopJ-dependent differences in gene expression

observed in BMDCs at these time points.

Cell viability was also assessed for Yptb-infected macro-

phages. A time-dependent increase in cell death was observed,

with YopJ-dependent death being most apparent 2 hr post-

infection (Figures 1F and S1D). Similar results were also

observed for infection of primary BMDMs (Figure S1E). These

data indicate YopJ-dependent effects on cell viability are most

apparent in macrophages, as opposed to DCs, which is consis-

tent with prior work (Brodsky and Medzhitov, 2008).

Inhibition of Gene Expression By YopJ Is Independent ofYopJ-Induced Cell DeathYopJ-induced cytotoxicity proceeds via a RIPK1/caspase 8- or

RIPK3-dependent pathway leading to caspase-1 activation

and subsequent cell death (Philip et al., 2014; Weng et al.,

2014). To examine further if YopJ-induced activation of

caspases and death-related pathways plays a role in blocking

innate immune signaling, we infected RIPK3-deficient BMDCs

treated with a pan-caspase inhibitor (zVAD-fmk). These

conditions render cells resistant to YopJ-induced death (Philip

et al., 2014; Weng et al., 2014). RIPK3-deficient cells

treated with zVAD-fmk were protected from death induced by

YopJ, compared with these cells treated with vehicle only or

wild-type (WT) BMDCs treated with either vehicle or zVAD-fmk

(Figures 2A and S2A). Despite the inability of Yp2666 to induce

any detectable cytotoxicity in RIPK3-deficient cells treated

with zVAD-fmk, YopJ-dependent inhibition of Il6 and Cxcl10

Microbe 18, 682–693, December 9, 2015 ª2015 Elsevier Inc. 683

Figure 1. Yptb Inhibits TRIF-Dependent Gene Expression via a YopJ-Dependent Mechanism

(A–F) BMDCs (A, C, and D) and iBMDMs (B) were infected with strains indicated at an MOI of 100. Gene expression relative to Gapdh was analyzed by qPCR at

times indicated or 2 hr post-infection (C). Uninfected cells (Con) were included in all experiments. Analysis of cell viability was performed by flow cytometry using

propidium iodide and annexin V stains at times indicated for BMDCs (E) or iBMDMs (F) and was performed in parallel with (A) or (B), respectively. For gene

expression analysis, bars represent the average and error bars represent the standard deviation of triplicate readings from one representative experiment of

three. Relative statistics were calculated using ANOVA with Bonferroni’s multiple comparison test. See also Figure S1.

684 Cell Host & Microbe 18, 682–693, December 9, 2015 ª2015 Elsevier Inc.

Figure 2. YopJ Inhibits TRIF-Dependent Gene Expression Independent of Cytotoxicity

(A–F) Cell viability (A, C, and F) or gene expression (B, D, and E) were determined forWT andRipk3�/�BMDCs (A and B),Ripk3/Caspase 8�/�BMDCs (C and D),

or Ripk3�/� BMDMs (E and F). Cells were treated with either vehicle (DMSO) or pan-caspase inhibitor (zVAD-fmk) prior to infection at an MOI of 100. Gene

expression relative toGapdhwas analyzed by qPCR at 4 hr (B and D) or 3 hr (E) after infection. Uninfected cells (Con) were used as a negative control. Analysis of

cell viability was performed by flow cytometry using propidium iodide and annexin V staining. For gene expression analysis, bars represent the average and error

bars represent the standard deviation of triplicate readings from one representative experiment of two (B and E) or three (D). Relative statistics were calculated

using ANOVA with Bonferroni’s multiple comparison test. See also Figure S2.

expression was observed (Figure 2B). To complement these

studies, we examined RIPK3/caspase 8 double mutant BMDCs,

which are resistant to YopJ-mediated cell death (Philip et al.,

2014; Weng et al., 2014), as confirmed by AV/PI staining at

4 hr post-infection (Figures 2C and S2B). Under these condi-

tions, YopJ-dependent inhibition of gene expression remained

(Figure 2D). These collective observations establish that YopJ-

dependent cell death is not required for the ability of Yptb to pre-

vent TRIF-dependent gene expression in BMDCs.

The contribution of YopJ-induced cytotoxicity toward gene

expression in macrophages was also examined in RIPK3-defi-

cient BMDMs treated with vehicle control or with zVAD-fmk.

Yp2666 retained the ability to block Il6 or Rsad2 expression in

these cells by a mechanism dependent on YopJ (Figure 2E).

Cell Host &

Notably, the cytotoxic actions of YopJ were readily apparent in

macrophages, as vehicle-treated RIPK3-deficient BMDMs in-

fectedwithYp2666exhibitedadecrease in viability, ascompared

with yopJ mutants at 3 hr post-infection (Figures 2F and S2C).

Treatment of these cells with zVAD-fmk was protective against

YopJ-induced cell death compared to cells treated with vehicle

control (Figures 2F and S2C). Together, these data demonstrate

that the ability of YopJ to block gene expression occurs indepen-

dently of any cell death programs activated during infection.

YopJ Suppresses MyD88- and TRIF-DependentTranscriptional Responses Downstream of TLR4Genes, such as Cxcl10 or Il6, can be induced by several recep-

tors. To determine the mechanism by which YopJ inhibits these

Microbe 18, 682–693, December 9, 2015 ª2015 Elsevier Inc. 685

Figure 3. YopJ Suppresses MyD88 and TRIF

Transcriptional Responses Downstream

of TLR4

(A–C) Gene expression induced during Yp2666

infection of WT versus TLR4-deficient (Tlr4) (A),

MyD88-deficient (Myd88) (B), or TRIF-deficient

(Ticam1) BMDCs (C). BMDCs were infected with

strains indicated at an MOI of 100. Gene expres-

sion relative to Gapdh was analyzed by qPCR at

times indicated. Uninfected cells (Con) were used

as a negative control. Bars represent the average

and error bars represent the standard deviation of

triplicate readings from one representative experi-

ment of two (B) or three (A and C). Relevant

statistics were calculated using ANOVA with Bon-

ferroni’s multiple comparison test.

genes, we defined the receptor(s) required for gene expression in

BMDCs, with a focus on TLR4.We comparedWT and TLR4-defi-

cient BMDCs for responses to infection with Yp2666 or the yopJ

mutant. Only WT cells were capable of responding to the yopJ

mutant whereas TLR4-deficient cells could not (Figure 3A).

Neither WT nor TLR4-deficient cells induced Il6 or Cxcl10

expression in response to Yp2666 infection. These data demon-

strate that the TLR4 pathway is responsible for induction of gene

expression in response to Yptb in BMDCs.

Il6 and Ifnb1 require contribution of MyD88 and TRIF signaling

pathways downstream of TLR4 for full induction (Yamamoto

et al., 2003). Inhibition of MyD88-dependent signaling by YopJ

may therefore affect expression of TRIF-dependent genes indi-

rectly. To address this possibility, we performed infections in

MyD88-deficient cells, which only permit signaling via the TRIF

pathway. YopJ retained the ability to block Il6 and Cxcl10 tran-

scription in MyD88-deficient BMDCs (Figure 3B). As expected,

TRIF (Ticam1)-deficient BMDCs were unable to induce Cxcl10

expression in response to Yp2666 or yopJmutant infection (Fig-

ure 3C). These collective data indicate YopJ can target the TRIF

pathway downstream of TLR4 directly, and not as an indirect

consequence of inhibiting MyD88 signaling.

YopJ Does Not Interfere with TRIF-Dependent IRF3ActivationTo determine if components of the TRIF pathway are inactivated

by YopJ, we interrogated stages of the pathway starting with

TLR4 activation. Normally, upon stimulation with LPS, TLR4 ini-

tiates the assembly of a protein complex called the myddosome,

which is a supramolecular organizing center (SMOC) that coordi-

nates all signals necessary for inflammatory cytokine expres-

686 Cell Host & Microbe 18, 682–693, December 9, 2015 ª2015 Elsevier Inc.

sion. This complex consists of the adap-

tors TIRAP and MyD88, and IRAK family

kinases (Kagan et al., 2014; Lin et al.,

2010; Motshwene et al., 2009). To assess

effects of YopJ on TLR4 activation, we

monitored myddosome formation during

Yp2666 or yopJ mutant infection in

iBMDMs. Equal levels of IRAK4 were co-

immunoprecipitated with MyD88 from

iBMDMs infected with Yp2666 or the

yopJ mutant (Figure 4A), suggesting that

YopJ does not interfere with TLR4 activation. Next, to signal

via the TRIF pathway, TLR4 must be internalized where it can

activate TRIF from endosomes (Kagan et al., 2008; Zanoni

et al., 2011). To examine if TLR4 endocytosis was affected by

YopJ, we monitored loss of TLR4 surface expression. TLR4

was endocytosed equally in iBMDMs infected with Yp2666 or

a yopJ mutant (Figure 4B).

Endosomal TLR4 promotes the TRIF-dependent activation of

the IKK family member TBK1, a kinase that activates IRF3 and

induces IFNb expression (Fitzgerald et al., 2003a). As YopJ inac-

tivates IKKs (Mittal et al., 2006; Mukherjee et al., 2006; Zhou

et al., 2005), it was possible YopJ interferes with TRIF signaling

by preventing TBK1 activation. To address this possibility,

BMDCs were infected with Yp2666 or the yopJ mutant and

examined for TBK1 phosphorylation and phosphorylation and

nuclear translocation of IRF3. No YopJ-dependent effects on

TBK1 phosphorylation, IRF3 phosphorylation, or p-IRF3 nuclear

translocation were observed in WT or MyD88-deficient BMDCs

(Figures 4C and 4D). Thus, despite the ability to block TRIF-

dependent gene expression, YopJ cannot interfere with TLR4

activation, endocytosis, or the activation of IRF3.

YopJ Inhibits Gene Expression by Blocking MAP KinasePathways in BMDCsWe considered the possibility that YopJ inhibits TRIF-dependent

gene expression by blocking IKKs, which are necessary for the

activation of NF-kB. We therefore assessed degradation of

IkBa during infection of BMDCs as a readout for IKK activity.

No YopJ-dependent effects on the rate or extent of IkBa degra-

dation were observed, even when IkBa resynthesis was blocked

through the use of cycloheximide (Figure 5A). This finding was

Figure 4. YopJ Does Not Block TRIF-Depen-

dent IRF3 Activation

(A–D) iBMDMs were infected with strains indicated

and monitored for myddosome formation (A) or

TLR4 endocytosis (B). Myddosome formation was

assessed 1 hr after infection. TLR4 endocytosis

was examined by flow cytometry. WT or Myd88-

deficient BMDCs (Myd88) were infected with

strains indicated and monitored for TBK1 phos-

phorylation (C) and IRF3 phosphorylation and p-

IRF3 nuclear translocation (D). Phosphorylation of

TBK1 was assessed by immunoblot of cell lysates

with a p-S172-specific antibody. Total TBK1 was

monitored by immunoblot with an antibody specific

for total TBK1. IRF3 activation was assessed after

infection of BMDCs by isolating nuclear and cyto-

plasmic fractions and immunoblotting with anti-

bodies specific for p-IRF3-IRF3 or total IRF3.

Immunoblotting for Lamin and GAPDH served as

controls for nuclear and cytoplasmic fractions,

respectively. Uninfected cells (Con) served as a

negative control. Data are one representative of

two independent experiments.

surprising, as previous studies in BMDMs reported that YopJ

prevents IkBa degradation (Zhang et al., 2005; Zhou et al.,

2005). Complementary assays to assess nuclear translocation

of the NF-kB subunit p65 also revealed no YopJ-dependent

effects in WT or MyD88-deficient BMDCs (Figure 5B). Collec-

tively, these data suggest IKK activity is not affected by YopJ

during the first hr of infection of BMDCs.

In addition to IKK and TBK1, TRIF signaling activates MAPK

pathways and the downstream transcription factor AP-1. To

examine the influence of YopJ on MAPK pathways, we as-

sessed phosphorylation of ERK, JNK, and p38, and their up-

stream MAP kinases kinases (MKKs), MEK1/2, MKK3/6,

MKK4, and MKK7, during infection. YopJ-dependent inhibition

of all MKKs and MAPKs was observed in either WT or MyD88-

deficient BMDCs (Figures 5C and 5D). These data establish

that YopJ blocks TRIF-dependent MAPK activation. Inhibition

of MAPK pathways was dependent on the catalytic activity of

YopJ, as a strain expressing yopJC172A was incapable of sup-

pressing MAPK activation (Figure S3A). To examine the func-

tional significance of YopJ inhibition of MAPK pathways, we

examined activation of AP-1. The parental strain Yp32777 sup-

pressed phosphorylation of the AP-1 subunit c-Jun, whereas

the strain expressing yopJC172A could not (Figure S3B). As

Cell Host & Microbe 18, 682–693,

was observed for MAPK activation,

YopJ-dependent AP-1 inhibition was

also observed in MyD88-deficient

BMDCs (Figure S3B). Importantly, AP-1

inhibition by Yp32777 occurred at times

where no difference in p65 nuclear trans-

location could be observed between

Yp32777 and yopJC172A mutant infec-

tions (Figure S3B). Similar results were

obtained with MOI of 20 (Figure S3C).

Collectively, these data demonstrate

that the enzymatic activity of YopJ pre-

vents MAPK-mediated activation of AP-

1 induced by both MyD88 and TRIF during times when neither

IKKs nor TBK1 are inactivated.

Potentially, Yptb requires no activity other than inhibiting

MAPKs to dismantle TLR4-induced gene expression. Inhibiting

MAPK activation by alternative means should therefore abolish

TLR4-induced gene expression. We treated WT and MyD88-

deficient BMDCswithMAPK-specific pharmacological inhibitors

and assessed gene expression in response to infection.

Compared with the vehicle control, inhibition of the p38 (but

not ERK) pathway suppressed expression of all genes examined

in response to the yopJ mutant to levels similar to those elicited

by Yp2666 infection (Figure 5E). Although inhibition of the JNK

pathway suppressed Ifnb1 expression, it had no effect on

Cxcl10 or Il6 expression (Figure 5E). Because YopJ inhibits all

three MAPK pathways, the effects of combinations of inhibitors

on gene expression were examined. Combinations including

ERK+p38, p38+JNK, or all three inhibitors combined sup-

pressed Ifnb1, Cxcl10, and Il6 expression in response to the

yopJmutant (Figure 5F). The combination of ERK and JNK inhib-

itors also suppressed Ifnb1 expression, but to a lesser degree

than combinations that inhibited p38 (Figure 5F). Large-scale

transcriptional analysis of BMDCs treated with vehicle or a com-

bination of three MAPK inhibitors revealed that YopJ blocked

December 9, 2015 ª2015 Elsevier Inc. 687

Figure 5. YopJ Inhibits Cytokine Production in BMDCs by Blocking MAPKs

(A–F) WT BMDCs (A) or WT and MyD88-deficient BMDCs (Myd88) (B and C) were infected with strains indicated at MOI 100 and monitored for IkBa degradation

(A), p65 nuclear translocation (B), MKK phosphorylation (C), and MAPK phosphorylation (D) at times indicated. Nuclear and cytoplasmic fractions were isolated,

and immunoblotting for Lamin and GAPDH verified nuclear and cytoplasmic fractions, respectively (B). Whole-cell lysates were collected, and immunoblotting for

GAPDH or total MAPKs served as loading controls (A, C, and D). BMDCs were pretreated with DMSO (vehicle control), CHX (50 ng/ml), or specific inhibitors for

ERK, p38, or JNK or a combination of inhibitors at the time of infection (A) or prior to infection with strains indicated (E and F). At 90 min post-infection, gene

expression relative toGapdhwas analyzed by qPCR (E and F). Uninfected cells (Con) served as a negative control in all experiments. Bars represent the average

and error bars represent the standard deviation of triplicate readings from a one representative experiment. Data are representative of three independent

experiments. Relevant statistics were calculated using ANOVA with Bonferroni’s multiple comparison test. See also Figure S3 and Table S1.

688 Cell Host & Microbe 18, 682–693, December 9, 2015 ª2015 Elsevier Inc.

Figure 6. Inhibition of TRIF-Dependent Re-

sponses by Yptb Disrupts DC-Mediated NK

Cell Activation

(A–G) Experimental layout: NK cells and DCs were

mixed in 0.7:1 ratio and infected with strains indi-

cated at an MOI of 100 (A). At 2 hr post-infection,

cells were treated with antibiotics to kill bacteria

(A). Gene expression, cytokine production, and cell

viability weremonitored at times indicated (A). Four

hours post-infection, gene expression relative to

cell-specific housekeeping genes (Control: Gapdh

for DCs or Ncr1 for NK cells) was monitored by

qPCR (B and C), and DC cell viability was analyzed

by flow cytometry with propidum iodide and an-

nexin V staining (D). IFNb production was assessed

by bioassay at times indicated (E). At 18 hr post-

infection, IFNg production was assessed by ELISA

(F). At time 0, exogenous IFNb was added at

200 U/ml where indicated. Viability of NK cells,

infected in parallel with co-cultures in (F), was

analyzed by flow cytometry using propidum iodide

and annexin V staining (G). Uninfected cells (Con)

were used as a negative control in all experiments.

For gene expression analysis, bars represent the

average and error bars represent the standard

deviation of triplicate readings from one repre-

sentative experiment of two. Statistics were

calculated using ANOVAwith Bonferroni’s multiple

comparison test. See also Figure S4.

expression of all genes examined (Table S1 and Figure S5D).

Likewise, inhibition of MAPK pathways suppressed TLR4-induc-

ible genes in response to the yopJ mutant to levels similar to

those observed with Yp2666 infection of vehicle-treated BMDCs

(Table S1 and Figure S3D). To complement these studies, we

compared gene expression in WT and p38a-deficient BMDMs

(Kim et al., 2008). p38a deficiency reduced expression of

Ifnb1, Cxcl10, and Rsad2 in response to the yopJ mutant to

levels similar to those observed with Yp2666 infection (Fig-

ure S3E). p38a-deficient BMDMs were not impaired for Il6

expression in response to the yopJmutant infection (Figure S3E),

possibly as a result of the remaining activity of other p38 iso-

forms in these cells (Figure S3F). These data support the idea

that inhibition of MAPKs by YopJ is sufficient to inhibit MyD88

and TRIF-dependent gene expression in BMDCs.

YopJ Inhibits DC Mediated Activation of NK Cells, butHas a Minor Overall Influence on Infection In VivoWe considered the physiological significance of TRIF in DC

biology. Upon encountering LPS, IFNb production by DCs pro-

motes NK cell activation and release of IFNg, a cytokine with

antibacterial activities (Zanoni et al., 2013). To determine if

Cell Host & Microbe 18, 682–693,

YopJ inhibition of TRIF signaling prevents

DC-mediated NK cell activation, amixture

of BMDCs and NK cells were infected

with Yp2666 or the yopJ mutant. After

2 hr, antibiotics were added to kill bacteria

(Figure 6A). Gene expression, IFN pro-

duction, and cell viability were assessed

4 and 18 hr later (Figure 6A). At 4 hr

post-infection, expression of Rsad2, an

IFN-stimulated gene (ISG) that can be used to monitor IFN ac-

tivity, was suppressed in a YopJ-dependent manner (Figure 6B).

NK cells did not produce any IFN when infected with Yptb, as NK

cells cultured alone with bacteria did not express Rsad2 or Ifng

(Figures 6B and 6C). However, the co-cultures of NK+BMDC

induced Ifng expression in response to yopJ mutant infections

(Figure 6C). Notably, this response was blocked by Yp2666 (Fig-

ure 6C). No YopJ-dependent cytotoxicity was observed

throughout this experiment (Figures 6D and S4A). In addition to

blocking mRNA production, IFNb and IFNg secretion was sup-

pressed in a YopJ-dependent manner, as assessed via bioassay

and ELISA, respectively (Figures 6E and 6F). Only NK+BMDC

mixtures were capable of producing IFNg, which illustrates the

requirement of DCs for stimulating NK cells to produce IFNg (Fig-

ure 6F). Interestingly, addition of exogenous IFNb restored the

ability of Yp2666-infected NK+BMDC mixtures to produce

IFNg to levels similar to that elicited by yopJ mutant infection

(Figure 6F). This restoration of IFNg by exogenous IFNb indicates

that the NK cells are not intrinsically incapable of producing

IFNg. NK cell viability was assessed in parallel with the IFNg pro-

duction at 18 hr, and no YopJ-dependent effects on AV/PI stain-

ing were observed (Figures 6G and S4B).

December 9, 2015 ª2015 Elsevier Inc. 689

As an alternative approach, rather than co-infecting BMDCs

and NK cells, we performed assays where NK cells were added

to infected BMDCs after addition of antibiotics to kill the bacteria

(Figure S4C). Under these conditions, NK cells are never

exposed to living bacteria, yet YopJ-dependent inhibition of

Rsad2 expression, Ifng expression, and IFNg production was still

observed (Figures S4D–S4F). This result indicates that the inac-

tivation of BMDCs by YopJ is sufficient to prevent NK cell activa-

tion. Furthermore, we verified that TRIF-dependent signaling is

required for activation of NK cells by BMDCs, as TRIF-deficient

BMDCs could not stimulate IFNg production from NK cells dur-

ing any infection (Figures S4D–S4F). Collectively, these data

establish that at least one consequence of YopJ-mediated inhi-

bition of TRIF signaling in BMDCs is to abrogate IFNg production

by NK cells.

Lastly, the relative roles of MyD88 and TRIF in a murine model

of infection were examined. WT, TRIF-deficient, and MyD88-

deficient mice were infected by gavage with either Yp2666 or

the yopJ mutant and assessed for survival and bacterial burden

in the spleens and livers. We did not observe a difference in sur-

vival between WT mice infected with either bacterial strain (Fig-

ure S4G). However, compared to WT mice, Yp2666 infection

appeared more lethal for mice deficient in MyD88 or TRIF, and

those infected with Yp2666 fared worse than those infected

with yopJ mutants (Figures S4G–S4I). To address this further,

we analyzed the effect of mouse genotype on resistance to

Yptb infection and found MyD88-deficient mice were signifi-

cantly reduced for their ability to survive infections compared

to wild-type mice (Figure S4H). TRIF-deficient mice exhibited a

similar trend, although not significant, from infections of wild-

type mice (p = 0.1597). One possible explanation for this differ-

ence between MyD88 and TRIF deficiencies is that MyD88 plays

a broader role in host defense than simply mediating TLR4

signaling, as this adaptor is also important for IL-1R and TLR2-

dependent activities crucial for controlling Yptb in vivo (Brodsky

et al., 2010; Dessein et al., 2009; Philip et al., 2014; Weng et al.,

2014). Lastly, MyD88 mutants displayed higher bacterial bur-

dens in the spleen and liver (Figures S4J and S4K). However,

bacterial burden was not influenced by the lack of YopJ (Figures

S4J and S4K). While additional work is necessary to clarify the

relative roles of TRIF and MyD88 during Yersinia infections

in vivo, these findings support the idea that both adaptor path-

ways activated by TLR4 play a role in host defense against these

enteric pathogens.

DISCUSSION

In this study, we demonstrate that Yptb suppresses TRIF-depen-

dent responses during infection of primary phagocytic cells,

including DCs and macrophages. The strongest evidence sup-

porting this conclusion comes from analysis of MyD88-deficient

cells, where the only operable TLR4-dependent pathway is TRIF.

TRIF signaling can therefore be included in the growing list

of innate immune pathways that are targeted by virulent

pathogens.

Our mechanistic analysis of the stages of TLR4 signaling re-

vealed that YopJ does not interfere with any TLR4-dependent

activities that specifically promote TRIF signaling, such as

TLR4 endocytosis, TBK1 phosphorylation, or IRF3 activation.

690 Cell Host & Microbe 18, 682–693, December 9, 2015 ª2015 Else

In addition, we observed no YopJ-dependent effects on myddo-

some formation or downstream NF-kB activation. Rather, the

only proteins inactivated by YopJ in primary cells were MKKs

and their downstream effectors. Direct evidence supporting

this conclusion derives from our finding that YopJ inhibits the

phosphorylation of MKKs, MAPKs, and c-Jun at times where

IkBadegradation, p65 phosphorylation, and p65 nuclear translo-

cation were unabated. These data reveal that YopJ has a mini-

mal ability to interfere with any pathway other than those

activated by MKKs during infections and indicate this effector

is a highly specific map kinase toxin, as suggested by Bliska

and colleagues (Palmer et al., 1999).

Despite this specificity for inactivating MKKs in vivo, YopJ is

well known to be a promiscuous enzyme in vitro, acetylatingmul-

tiple kinases. The specificity of YopJ toward its substrates during

infection is therefore not intrinsic, but must result from some fea-

ture(s) within the cytosol of infected cells. In this regard, we note

that much of the previous work describing inhibition of the IKK

pathway by YopJ was performed in immortal or non-immune

cells (Schesser et al., 1998; Zhou et al., 2004, 2005), which

may contain kinetically slow TLR4 pathways, as compared

with primary phagocytes. Thus, in cell types that contain an in-

efficient TLR4 pathway, injected YopJ may have more time to

acetylate substrates and may therefore inactivate a broader

spectrum of pathways. One prediction of this idea is that

increasing the time that YopJ is present in the host cytosol

should broaden the spectrum of substrates it can functionally

inactivate. Indeed, acetylation studies are commonly performed

by overexpression of YopJ, conditions where the timeline for

substrate identification and pathway inhibition is on the order

of hours to days (Mittal et al., 2006; Mukherjee et al., 2006;

Paquette et al., 2012). In contrast, our data indicate that during

infections of phagocytes, YopJ has less than 30 min to acetylate

enough substrates to block inflammatory gene expression.

While this short time frame is not sufficient for YopJ to physiolog-

ically inactivate the IKK pathways, theMAPK pathway is strongly

inhibited. Thus, we propose that kinases in the MAPK pathway

are the first and preferred substrates of YopJ. Interestingly, a

YopJ-like effector, VopA of Vibrio parahaemolyticus, does not

block IKKs but only MAPKs (Trosky et al., 2004). We propose

that YopJ-like proteins evolved to target the evolutionarily

earliest innate immune pathways, the MAPKs. Indeed, YopJ be-

longs to a family of effector proteins that are also found in plant

pathogens (Lewis et al., 2011), suggesting that this is an evolu-

tionarily ancient bacterial virulence strategy. Substrates within

MAPK pathways may have been the first targets that YopJ

evolved to inactivate and remain the first targets during any

infection. Secondary substrates (e.g., IKKs and TAK1) may

only be targeted functionally at later points during infection, or

not at all. YopJmay therefore be considered an unusual enzyme,

whose substrate specificity is not hard-wired, but is determined

by the speed of the signaling pathways in the infected cell.

The ability of YopJ to target MAPK pathways highlights a

remarkably simple means by which bacteria can inactivate

TLR4 signal transduction pathways. Rather than evolving

multiple strategies to inactivate the MyD88 and TRIF pathways,

YopJ has directed its actions toward the most common and

ancient node in the TLR network, the MAPKs. The effectiveness

of blocking TLR4-dependent transcription by targeting MAPKs

vier Inc.

may have bypassed the need for the evolution of additional ef-

fectors that inactivate TLR signal transduction. In this regard,

pathogenic Yersinia can use a single effector protein to

completely dismantle the transcription-inducing activity of the

dominant innate immune receptor activated during infection,

TLR4.

Despite the activities of YopJ to dismantle the TLR4 signaling

network, and despite the ability of Yptb to disrupt DC-mediated

NK cell activation, we did not observe a strong YopJ-dependent

phenotype during infection of mice. We also did not observe a

significant phenotype during infections of TRIF-deficient mice.

This disconnect between striking mechanistic phenotypes and

modest phenotypes during infections has been observed in

several pathogenic bacterial effectors. Examples include effec-

tors with elegant means of manipulating host physiology, such

as substrates of the Legionella type IV secretion system (Isaac

and Isberg, 2014) and effectors used by Salmonella spp. to

invade mammalian cells (Zhou et al., 2001). These virulence

factors modulate specific host functions, yet deficiencies in indi-

vidual Legionella or Salmonella effectors are not sufficient to

prevent intracellular replication or invasion, respectively. An

extreme example of this disconnect between in vitro and in vivo

analyses comes from the bacterium used in this study, where

even Yptb lacking their entire type III secretion system remain

capable of establishing infections in certain tissues in vivo

(Balada-Llasat and Mecsas, 2006). Overall, our knowledge of

the requirement for specific virulence activities during actual in-

fections remains incomplete, and this study provides a mandate

for future examination of this issue.

EXPERIMENTAL PROCEDURES

Cell Culture

C57BL/6J (Jax 000664), MyD88-deficient (Jax 009088), TRIF-deficient (Jax

005037), TLR4-deficient (C3H/HeJ, Jax 000659), and WT control for TLR4-

deficient (C3H/HeSnJ, Jax 000661) mice were purchased from Jackson

Labs. RIPK3-deficient and RIPK3/Caspase-8 double deficient mice were

described (Philip et al., 2014). All experiments involving animals were carried

out in accordance with the relevant laws and institutional guidelines. BMDCs

or BMDMs were differentiated from bone marrow in IMDM (GIBCO), 10%

GM-CSF, and 10% FBS, or DMEM (GIBCO), 15% L929 supernatant, and

20% FBS, respectively. Immortal macrophages were cultured in DMEM com-

plete media supplemented with 10% FBS. For experiments using inhibitors,

BMDCs were pretreated with DMSO (vehicle control), zVAD-fmk (100 mM),

specific inhibitors for ERK (PD98059, 50 nM), p38 (SB202190, 20 nM), or

JNK (SP600125, 50 nM) or a combination of these inhibitors for 2 hr prior to

infections. Concentrations of DMSO were normalized across samples. For

IkBa degradation assays, BMDCs were treated with DMSO (vehicle) or cyclo-

heximide (CHX) (50 mg/ml) at the time of infection. NK cells were isolated from

red blood cell-lysed splenocytes using biotin-conjugated anti-mouse CD49b

antibody (Biolegend Cat# 108903, clone DX5) and magnetic-activated cell

sorting (MACs) beads (Miltenyi Biotec). NK cell purity was 93%–96%.

Bacterial Strains and Infections

Yptb strains IP2666 (Yp2666), IP2666 plasmid-deficient (plasmid�), IP2666

yopJ-deficient, and IP2666 yopJ-deficient complemented with yopJ or yopP

were described (Brodsky and Medzhitov, 2008). Yersinia strains 32777

(Yp32777) and 32777 yopJC172A mutant were gifts from Dr. James Bliska

(SUNY Stonybrook). To induce type III secretion of Yop effectors, bacteria

were cultured as described (Brodsky and Medzhitov, 2008). Bacteria were

added to cell cultures at the MOI indicated and centrifuged at 3003 g for

3 min. Infected cells were incubated at 37�C with 5% CO2 for times indicated

in individual experiments.

Cell Host &

Gene Expression Analysis, Bioassay, and ELISA

RNA was isolated from mammalian cell cultures using Qiashedder (QIAGEN)

and GeneJET RNA Purification Kit (Life Technologies). Purified RNA was

analyzed for gene expression on a CFX384 real-time cycler (Bio-Rad) using

TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems) with probes purchased

from Life Technologies (see Supplemental Experimental Procedures). The

type I IFN bioassay was performed as described (Dixit et al., 2010). ELISA

for IFNg was performed using Mouse IFNg ELISA Ready-SET-Go ELISA kit

(eBioscience). Statistics were performed as indicated using Prism 6

(GraphPad).

Cell Staining and Flow Cytometry

AV/PI staining was performed via the manufacturer’s protocol using the

following Biolegend products: cell staining buffer, Annexin V binding buffer

propidium iodide, and anti-annexin V Alexa Fluor 647 conjugate. Populations

were gated based on cells individually stained with either propidium iodide

or annexin V. TLR4 endocytosis assays were performed as described (Zanoni

et al., 2011). For all flow cytometry-based experiments, data were acquired on

a BD FACSCanto II (Becton Dickinson) and analyzed using FlowJo v10

(FlowJo, LLC).

Western Blotting, Cell Fractionation, and Immunoprecipitation

Whole-cell lysates were prepared in RIPA buffer and cleared by centrifugation

at 17,0003 g for 5 min prior to boiling in SDS-loading buffer. For preparation of

cytoplasmic and nuclear fractions, cells were lysed in L1 buffer (50mMTris [pH

8.0], 2mMEDTA, 0.1%NP40, 10%glycerol, and protease/phosphatase inhib-

itors [Roche]) and lysates were centrifuged at 5,000 RPM for 5 min at 4�C to

pellet nuclei. Cytoplasm was isolated and boiled in SDS-loading buffer. Nuclei

were washed once in L1 buffer, boiled in SDS-loading buffer, and homoge-

nized by four passages through a 26G needle. Protein for myddosome analysis

was prepared as described (Bonham et al., 2014). Immunoblotting was per-

formed using standard molecular biology techniques (Supplemental Experi-

mental Procedures).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

four figures, and one table and can be found with this article online at http://

dx.doi.org/10.1016/j.chom.2015.11.006.

ACKNOWLEDGMENTS

We thank James Bliska (SUNY Stonybrook) for Yptb stains 32777 and 32777

yopJ-C172A, Jin Mo Park (Harvard) for p38a F/F-LysMCre marrow, and mem-

bers of the Kagan lab for helpful discussions. J.C.K. is supported byNIH grants

AI113141, and P30 DK34854 and an unrestricted gift from Mead Johnson &

Company. J.C.K. holds an Investigators in the Pathogenesis of Infectious Dis-

ease Award from the Burroughs Wellcome Fund. C.V.R. is supported by NIH

grant DK102317-01. E.R.G., M.K.P., and J.M. were supported by NIH grants

AI113141 and AI113166.

Received: May 15, 2015

Revised: September 9, 2015

Accepted: November 17, 2015

Published: December 9, 2015

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