LETTER. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus

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1 doi: /nture10510 The NLRC4 inflmmsome receptors for cteril flgellin nd type III secretion pprtus Yue Zho 1,2 *, Jieling Yng 1,2 *, Jinjin Shi 2, Yi-Nn Gong 2, Qiuhe Lu 2,HoXu 2, Liping Liu 2 & Feng Sho 2 Inflmmsomes re lrge cytoplsmic complexes tht sense microil infections/dnger molecules nd induce cspse-1 ctivtiondependent cytokine production nd mcrophge inflmmtory deth 1,2. The inflmmsome ssemled y the NOD-like receptor (NLR) protein NLRC4 responds to cteril flgellin nd conserved type III secretion system (TTSS) rod component 35. How the NLRC4 inflmmsome detects the two cteril products nd the moleculr mechnism of NLRC4 inflmmsome ctivtion re not understood. Here we show tht NAIP5, BIR-domin NLR protein required for Legionell pneumophil repliction in mouse mcrophges 6, is universl component of the flgellinnlrc4 pthwy. NAIP5 directly nd specificlly intercted with flgellin, which determined the inflmmsome-stimultion ctivities of different cteril flgellins. NAIP5 enggement y flgellin promoted physicl NAIP5NLRC4 ssocition, rendering full reconstitution of flgellin-responsive NLRC4 inflmmsome in non-mcrophge cells. The relted NAIP2 functioned nlogously to NAIP5, serving s specific inflmmsome receptor for TTSS rod proteins such s Slmonell PrgJ nd Burkholderi BsK. Genetic nlysis of Chromocterium violceum infection reveled tht the TTSS needle protein CprI cn stimulte NLRC4 inflmmsome ctivtion in humn mcrophges. Similrly, CprI is specificlly recognized y humn NAIP, the sole NAIP fmily memer in humn. The finding tht NAIP proteins re inflmmsome receptors for cteril flgellin nd TTSS pprtus components further predicts tht the remining NAIP fmily memers my recognize other unidentified microil products to ctivte NLRC4 inflmmsomemedited innte immunity. The NLR protein NLRC4 (lso known s IPAF) in mcrophges ctivtes cspse-1 nd downstrem inflmmtory response upon sensing cytosolic presence of flgellin during cteril infection 3,4,7,8. To study the mechnism of the NLRC4 inflmmsome, defined iochemicl ssy ws developed y fusing recominnt flgellin croxy-terminl to the mino-terminl domin of nthrx lethl fctor. This domin (designted s LFn here), through inding to nother nthrx protein clled protective ntigen (PA), cn efficiently trnslocte heterologous fusion proteins into mmmlin cytosol through endocytosis-medited entry 9. Using this system, purified flgellin from L. pneumophil (LFn-FlA Lp ) ws found to trigger roust cspse-1 clevge (Fig. 1), IL-1 relese nd pyroptotic deth (Supplementry Fig. 1, ) in primry one-mrrow-derived mcrophges (BMMs). These ctivtions were completely diminished in Nlrc4 2/2 nd cspse-1 2/2 mcrophges (Fig. 1 nd Supplementry Fig. 1, ); Asc 2/2 (Asc lso known s Pycrd) BMMs lso showed little cspse-1 mturtion nd IL-1 relese ut with prtilly ffected pyroptosis due to ASC-independent NLRC4 inflmmsome ctivtion 10. Full ctivtion of NLRC4 inflmmsome requires L470, L472 nd L473 in Legionell flgellin 6. Accordingly, lnine sustitutions of the three leucine residues (3A) generted lrgely inctive LFn-FlA Lp protein (Fig. 1 nd Supplementry Fig. 1, ). LFn-FlA Lp induced similr NLRC4-dependent cspse-1 ctivtion nd pyroptosis in Tlr4 2/2 mcrophges (Supplementry Figs 2, nd 3), excluding possile contriution from residul endotoxin contminnts present in the recominnt protein. Other cteri such s Slmonell typhimurium lso trigger flgellin-dependent NLRC4 inflmmsome ctivtion 3,4,8. Delivery of S. typhimurium (LFn-FliC St )oryersini enterocolitic (LFn-FliC2 Ye ) flgellin into BMMs induced roust cspse-1 ctivtion nd extensive pyroptosis in n NLRC4-dependent mnner (Supplementry Fig. 4, ). Thus, LFn-medited delivery of recominnt flgellin recpitultes ll known genetic properties of flgellin ctivtion of the NLRC4 inflmmsome. For L. pneumophil infection, flgellin-induced cspse-1 ctivtion requires NAIP5 (lso known s BIRC1E), BIR-domin-contining NLR protein 6. A nturl vrint of NAIP5 renders mcrophges from the A/J mouse permissive to L. pneumophil intrcellulr repliction The role of NAIP5 for other cteril flgellins is not cler 6,17.RNA interference (RNAi) knockdown of Nip5 (Supplementry Fig. 3) severely locked LFn-FlA Lp -triggered cspse-1 ctivtion nd pyroptosis (Supplementry Fig. 2, ). Notly, ctivtion of the NLRC4 inflmmsome y LFn-FliC St nd LFn-FliC2 Ye, ut not tht y Slmonell TTSS rod protein (LFn-PrgJ), ws lso drsticlly reduced y short hirpin RNA (shrna)-medited stle knockdown of Nip5 (Fig. 1, c nd Supplementry Fig. 3c). Consistently, flgellintriggered cspse-1 ctivtion during Slmonell nd Legionell infection ws significntly ttenuted in Nip5 knockdown mcrophges (Fig. 1d). The finding tht NAIP5 is possile integrl component of the flgellinnlrc4 pthwy inspired us to investigte whether NAIP5 directly recognizes flgellin. Legionell flgellin ws found to show n evident yest two-hyrid interction with NAIP5, ut not NLRC4, wheres the 3A mutnt showed no interction (Fig. 2). Nip5 is locted within genomic locus contining seven highly homologous Nip genes (Ni) nd four of them (Nip1, Nip2, Nip5 nd Nip6) hve trnscripts in C57BL/6 mice 11. Legionell flgellin lso showed two-hyrid interction with NAIP6, ut not with NAIP1 nd NAIP2 (Fig. 2). Supporting the two-hyrid results, Legionell flgellin expressed in 293T cells redily co-precipitted NAIP5 nd NAIP6, ut not NAIP1, NAIP2 nd NLRC4, wheres the 3A mutnt filed to do so (Fig. 2). The TLR5-inding-deficient mutnt (I391A), which is fully functionl in inflmmsome ctivtion 6,ehvedsimilrly to wild-type flgellin in the co-immunoprecipittion ssy. Flgellin lso co-precipitted NAIP5 encoded y the A/J llele (NAIP5 A/J ) (Fig. 2), which explins the norml or nerly norml cspse-1 ctivtion in L. pneumophil-infected A/J mcrophges 15,18,19. A pnel of nine dditionl flgellins from different cteri ws further profiled (Fig. 2c). In the two-hyrid ssy, flgellins from S. typhimurium, Y. enterocolitic, Photorhdus luminescens nd Pseudomons eruginos showed positive result wheres those from enteropthogenic Escherichi coli (EPEC), enterohemorrhgic E. coli (EHEC), Shigell flexneri, Chromocterium violceum nd Burkholderi thilndensis did not interct with NAIP5 (Fig. 2c nd Supplementry Fig. 5). NAIP5 interction with S. typhimurium flgellin 1 Grdute Progrm in Chinese Acdemy of Medicl Sciences nd Peking Union Medicl College, Beijing , Chin. 2 Ntionl Institute of Biologicl Sciences, Beijing, , Chin. *These uthors contriuted eqully to this work. 596 NATURE VOL SEPTEMBER 2011 Mcmilln Pulishers Limited. All rights reserved 2011

2 RESEARCH BMMs WT Nlrc4 / Asc / Csp1 / LFn-FlA Lp WT 3A WT 3A WT 3A WT 3A LFn-FlA Lp LFn-FliC St LFn-FliC Ye LFn-PrgJ shrna C N5 C N5 C N5 C N5 c Cell deth (%) LFn-FlA Lp LFn-FliC St Control shrna Nip5 shrna LFn-FliC Ye LFn-PrgJ d S. typhimurium WT ΔfliCΔfljB WT ΔfliCΔfljB C C N5 N5 L. pneumophil WT ΔflA WT ΔflA C C N5 N5 Figure 1 A defined iochemicl ssy revels universl role of NAIP5 in flgellin-triggered NLRC4 inflmmsome ctivtion in mouse mcrophges., Effects of nthrx lethl fctor N-terminl-domin-medited intrcellulr delivery of Legionell flgellin (LFn-FlA Lp ) on cspse-1 ctivtion in lipopolyscchride (LPS)-primed BMMs derived from wild-type (WT, C57BL/6) or indicted knockout mice. 3A denotes triple mutnt flgellin (L470A/L472A/L473A). Shown re nti-cspse-1 nd nti-ctin immunolots of culture superntnts (top) nd totl cell lystes (ottom). denotes the processed mture form of cspse-1., c, Effects of Nip5 knockdown on flgellin-induced cspse-1 ctivtion () nd cell deth (c). A Nip5-trgeting (N5) (Supplementry Tle 1) or control (C) shrna ws stly expressed in immortlized BMMs. LFn-FlA Lp, FliC St nd FliC2 Ye re recominnt LFn-tgged flgellins from L. pneumophil, S. typhimurium nd Y. enterocolitic, respectively. LFn-PrgJ is LFn-tgged S. typhimurium TTSS rod protein. c, LDH releses re shown s men vlues 6 stndrd devition (s.d.) from three independent determintions. d, Effects of Nip5 knockdown on flgellin-induced cspse-1 ctivtion during Slmonell nd Legionell infection. DfliCDfljB nd DflA denote flgellin-deficient strins of S. typhimurium nd L. pneumophil, respectively. YC-ULW YC-ULWKH Two-hyrid interction 1 FlA Lp NAIP1 2 FlA Lp NAIP2 3 FlA Lp NAIP5 4 FlA Lp NAIP6 5 FlA Lp mnlrc4 6 FlA Lp (3A) NAIP5 7 hrip3 NAIP5 8 LuX IcmW c Two-hyrid with NAIP5 1: FlA_WT (L. pneumophil) 1 m : FlA_3A (L. pneumophil) 2: FliC_WT (S. typhimurium) 2 m : FliC_3A (S. typhimurium) 3: FliC2 (Y. enterocolitic) 4: FliC (EPEC) 5: FliC (EHEC) 6: FliC (S. flexneri) 7: FliD (C. violceum) 8: Flgellin (Photorhdus luminescens) 9: FliC (B. thilndensis) 10: FliC (type ) (P. eruginos) FlgHA FlA Lp WT WT WT 3A I391A WT WT WT WT IP: Flg Input d HA Flg NAIP1 NAIP2 NAIP5 NAIP5 NAIP5 LFn-flgellin NAIP5 A/J NAIP6 mnlrc4 1 1 m NLRC4 Figure 2 Flgellin intercts specificlly with NAIP5 nd the interction correltes with the ctivity of flgellins from different cteri., Yest twohyrid interction ssys of Legionell flgellin (FlA Lp ) nd different NAIP proteins (or mouse (m)nlrc4). The chrt in the lower right corner summrizes the interction results. The known interction etween Legionell effector LuX nd its secretion chperon IcmW ws included s positive control., Co-immunoprecipittion ssys of Legionell flgellin (FlA Lp ) nd different NAIP proteins (or NLRC4). Shown re immunolots of nti-flg immunoprecipittes (IP: Flg) nd totl cell lystes (Input). I391A is TLR5 inding-deficient flgellin mutnt. c, Summry of yest two-hyrid interction of NAIP5 with different cteril flgellins. The rw dt re shown in Supplementry Fig. 5. d, Cspse-1 ctivtion ssys of LFn-medited delivery of different cteril flgellins into primry BMMs. Numer denottions of different flgellins follow those in c. 29 SEPTEMBER 2011 VOL 477 NATURE Mcmilln Pulishers Limited. All rights reserved

3 RESEARCH LETTER required three leucine residues equivlent to those in Legionell flgellin. When delivered into BMMs, flgellins from S. typhimurium, Y. enterocolitic, P. luminescens nd P. eruginos, ut not those from the other five cteri species, stimulted cspse-1 ctivtion, mcrophge deth nd IL-1 relese (Fig. 2d nd Supplementry Fig. 5, c). The positive NAIP5-inding nd inflmmsome-stimulting ctivities of S. typhimurium nd P. eruginos flgellins gree with their genetic requirements for infection-induced cspse-1 ctivtion 3,4,8,2022. Among those inctive ones, S. flexneri flgellin is not expressed nd dispensle for host innte immune detection of S. flexneri infection 23. Genetic ltions of flgellins from EPEC nd B. thilndensis lso did not ffect infection-induced cspse-1 ctivtion (Supplementry Fig. 6). Thus, the ility of the ten different flgellins to interct with NAIP5 correltes well with their differentil inflmmsome-stimulting ctivity, which further supports the ide tht flgellin is generlly recognized y NAIP5 in triggering NLRC4 inflmmsome ctivtion. NAIP5 nd NLRC4 were then co-expressed in 293T cells nd their possile interctions were investigted. Co-immunoprecipittion of NAIP5 nd NLRC4 ws rely detectle in the sence of flgellin. However, co-expression of Legionell flgellin, ut not the 3A mutnt, significntly incresed the mount of NAIP5 precipitted y NLRC4 (Fig. 3, ). Flgellin ws lso detected in the NLRC4 immunoprecipittes due to the ridging effect of NAIP5. Deletion of the nucleotideinding P-loop in NLRC4 nucleotide-inding nd oligomeriztion domin (NOD) olished flgellin-simulted NLRC4NAIP5 interction (Fig. 3), which grees with the reported interction etween NOD domins from NLRC4 nd NAIP5 (ref. 24). Flgellin lso promoted the ssocition of NLRC4 with NAIP5 A/J, ut neither NAIP1 nor NAIP2 ws precipitted y NLRC4 despite the presence of flgellin (Fig. 3). To test whether the flgellin-stimulted NAIP5NLRC4 complex cn ctivte downstrem signlling, NAIP5 nd NLRC4, together with pro-cspse-1 nd pro-il-1, were co-expressed in 293T cells. Delivery of LFn-FlA Lp, ut not the 3A mutnt, into the trnsfected cells resulted in n evident production of mture IL-1 (Fig. 3c nd Supplementry Fig. 7). Omission of NAIP5, NLRC4 or cspse-1 in this reconstitution olished the response to flgellin FlgNLRC4 WT WT HANAIP5 FlA Lp HA Input IP: Flg Flg HA Δploop c NAIP5 NLRC4 LFn-FlA Lp FlgNLRC4 d mnlrc4pro-il-1 HANAIP A/J 6 NAIP1 NAIP2 NAIP5 NAIP5 A/J NAIP6 FlA Lp WT WT WT 3A WT WT LFn-FlA Lp WT 3A WT 3A WT 3A WT 3A WT 3A Input IP: Flg Flg HA HA WT 3A WT 3A WT 3A WT 3A Figure 3 Flgellin stimultes the NAIP5NLRC4 ssocition nd reconstitution of flgellin ctivtion of the NLRC4 inflmmsome in nonmcrophge cells.,, Co-immunoprecipittion ssys of NAIP5 nd NLRC4 interction in the presence or sence of flgellin. Dploop in denotes n NLRC4 mutnt with deletion of the nucleotide-inding P-loop. c, Reconstitution of flgellin ctivtion of the NLRC4 inflmmsome in non-mcrophge cells. s from 293T cells trnsfected with indicted plsmid comintions nd stimulted with LFn-FlA Lp were nlysed for mture IL-1 () y immunolotting. Expression of trnsfected inflmmsome components for c nd d is in Supplementry Fig. 7. d, Assy of different NAIP proteins in supporting reconstitution of flgellin ctivtion of the NLRC4 inflmmsome in 293T cells. stimultion. NAIP5 A/J lso supported the reconstitution wheres NAIP1 nd NAIP2 filed to do so (Fig. 3d nd Supplementry Fig. 7), consistent with their differentil ssocition with NLRC4 upon flgellin stimultion (Fig. 3). Moreover, the reconstituted NLRC4 inflmmsome exhiited roust responses to flgellins from Slmonell nd Yersini (Supplementry Fig. 8). Ech of the three domins in oth NLR proteins (CARD, NOD nd LRR in NLRC4; BIR, NOD nd LRR in NAIP5) ws essentil for ssemling flgellinresponsive inflmmsome complex (Supplementry Fig. 8). These results indicte tht flgellin recognition y NAIP5 stimultes the physicl ssocition etween NAIP5 nd NLRC4, therey signlling downstrem cspse-1 ctivtion. NAIP6 intercted with flgellin in mnner similr to NAIP5 (Fig. 2 nd Supplementry Fig. 9) nd supported the reconstitution in 293T cells (Fig. 3, d). In fct, NAIP6, mong ll the NAIP proteins, shres the highest sequence identity with NAIP5, of 94.7% (Supplementry Fig. 10). NAIP6 proly hs similr function to NAIP5 in mcrophge detection of flgellin, ut its role might e reltively minor given its much lower expression in primry mcrophges compred with tht of NAIP5 (ref. 12). The NLRC4 inflmmsome lso responds to conserved TTSS rod protein such s PrgJ in S. typhimurium, BsK in B. thilndensis nd EscI in EPEC 5. Delivery of recominnt BsK (LFn-BsK) into BMMs recpitulted such effects nd induced NLRC4-dependent cspse-1 ctivtion nd pyroptosis (Supplementry Fig. 11). Given tht NAIP5 recognizes flgellin nd tht PrgJ ctivtion of the NLRC4 inflmmsome is independent of NAIP5 (ref. 17), we proposed tht other NAIP proteins could recognize the TTSS rod protein. BsK ws found to interct with NAIP2, ut not NAIP1, NAIP5, NAIP6 nd NLRC4, in the two-hyrid ssy (Fig. 4). Co-immunoprecipittion ssy confirmed this NAIP2-specific interction (Fig. 4). This oservtion grees with the ide tht NAIP2 is the most distntly relted to the other NAIP proteins (Supplementry Fig. 10). Reconstitution in nonmcrophge cells further showed tht only NAIP2, ut not ny other NAIP, effected roust IL-1 mturtion upon LFn-BsK stimultion (Fig. 4c). These findings indicte tht NAIP2 is the specific receptor for the TTSS rod protein. To test the requirement of NAIP2 for detecting the TTSS rod protein in mcrophges, Nip2 stle knockdown BMMs were generted. Among the four different shrnas (Nip2-1, 2, 3 nd 4), Nip2-1 nd Nip2-2 considerly reduced Nip2 messenger RNA level wheres Nip2-3 nd Nip2-4 showed intermedite nd negligile efficiency, respectively (Supplementry Fig. 12). Nip2-1 nd Nip2-2 knockdown mcrophges exhiited significnt resistnce in cspse-1 ctivtion nd pyroptosis to LFn-PrgJ or LFn-BsK stimultion (Supplementry Fig. 12d). In contrst, Nip2-3 knockdown mcrophges showed mild resistnce nd Nip2-4 knockdown mcrophges hd norml sensitivity to rod protein stimultions. In Nip2-2 knockdown mcrophges, in which mrna levels of other Nip genes were not ffected (Supplementry Fig. 13), ttenuted cspse-1 ctivtion ws only oserved with the rod protein stimultions, ut not with flgellin stimultions (Fig. 4d). Furthermore, deletion of genes encoding the rod proteins from flgellin-deficient EPEC nd S. typhimurium olished cteril infection-induced cspse-1 ctivtion, nd this effect did not occur in Nip2-2 knockdown mcrophges (Fig. 4e). These results demonstrte the criticl nd specific role of NAIP2 in recognizing the TTSS rod protein for NLRC4 inflmmsome ctivtion. In contrst to mouse mcrophges, humn U937 monocyte-derived mcrophges were unresponsive to intrcellulr delivery of flgellin nd BsK/PrgJ-like rod protein (Supplementry Fig. 14). When profiling our genetic collection of vrious pthogenic cteri, C. violceum strin (deficient in secretion of TTSS effectors) ws identified to e cple of inducing cspse-1 ctivtion in humn U937 monocytes (Supplementry Fig. 15). Notly, further ltion of five possile flgellin genes (DF) cused no reduction in this ctivtion. Stle knockdown of NLRC4 (Supplementry Fig. 16) significntly ttenuted C. 598 NATURE VOL SEPTEMBER 2011 Mcmilln Pulishers Limited. All rights reserved 2011

4 RESEARCH YC-ULW YC-ULWKH Two-hyrid mnlrc4 interction c 1: BsK NAIP1 NAIP1 NAIP2 NAIP5 NAIP5 A/J NAIP6 2: BsK NAIP2 LFn-BsK 3: BsK NAIP5 4: BsK NAIP6 5: BsK mnlrc4 6: hrip3 NAIP2 7: LuX IcmW 8: FlA Lp _3A NAIP2 FlgRFPBsK HANAIP HA Flg HA EPEC S. typhimurium e d LFn-BsK LFn-PrgJ LFn-FlA Lp LFn-FliC P shrna C N2-2 C N2-2 C N2-2 C N2-2 IP: Flg Input ΔfliC ΔfliCΔescl ΔfliC ΔfliCΔescl ΔfliCΔfljB ΔfliCΔfljB ΔfliCΔfljBΔprgJ ΔfliCΔfljBΔprgJ shrna C C N2-2 N2-2 C C N2-2 N2-2 Figure 4 NAIP2 intercts with the TTSS rod protein nd is required for the rod protein to trigger mouse NLRC4 inflmmsome ctivtion.,, Yest two-hyrid () nd co-immunoprecipittion () ssys of interctions etween B. thilndensis rod protein BsK nd different NAIP proteins. c, Reconstitution of BsK ctivtion of the NLRC4 inflmmsome in nonmcrophge cells. s from HeL cells trnsfected with indicted plsmid comintions nd stimulted with LFn-BsK were nlysed for mture IL-1 () y immunolotting. Expression of trnsfected inflmmsome components is in Supplementry Fig. 7. d, Effects of Nip2 knockdown on violceum DF-triggered cspse-1 ctivtion nd pyroptosis (Fig. 5 nd Supplementry Fig. 17). The PrgJ homologue in the C. violceum Cpi-1 TTSS system, CprJ, is encoded in seprte Cpi-1 locus tht hrours severl dditionl TTSS pprtus genes 25 (Fig. 5). Although NLRC4 shrna c f hnaip NLRC4 LFn-CprI C cor 2A WT Cpi-1A locus ΔCp1-1A C C cpr C B A K J I H ΔcprK-J d ΔCpi-1A ΔcorA-C ΔcprK-J ΔcprI ΔcprH 2A WT 2A WT 2A WT ΔCpi-1A Rescue V V CprJ CprI V g e LFn- shrna C C C C NLRC4 ASC hnaip hnlrc4 NAIP2 NAIP5 FlgHACprI WT 2A WT 2A WT 2A WT 2A Input IP: Flg Flg FlA LP Cprl_2A Δcprl V Cprl_WT Δcprl Δcprl CprI 2A cspse-1 ctivtion induced y TTSS rod proteins nd flgellins. Control (C) or Nip2-2 (N2-2) stle knockdown mcrophges (Supplementry Fig. 12) were stimulted with purified LFn-tgged BsK, PrgJ, FlA Lp or Flic P proteins s indicted. e, Effects of Nip2 knockdown on rod-protein-induced cspse-1 ctivtion during EPEC nd Slmonell infection. EPEC E2348/69 DfliCDescI nd S. typhimurium DfliCDfljBDprgJ denote the rod-protein-deficient EPEC nd S. typhimurium strins, respectively, which were constructed on the flgellin-deletion ckground. cprj ws not required for infection-induced cspse-1 ctivtion nd pyroptosis, deletion of the entire Cpi-1 locus lrgely diminished C. violceum-induced NLRC4 inflmmsome ctivtion (Fig. 5, Supplementry Fig. 15 nd Supplementry Fig. 17). Further genetic nlysis of the entire Cpi-1 locus identified cpri, which ws essentil for inducing cspse-1 ctivtion nd pyroptosis (Fig. 5c nd Supplementry Fig. 17). A CprI-expressing plsmid could rescue the deficiencies of inflmmsome ctivtion for oth cpri nd Cpi-1 deletion strins (Fig. 5d). Thus, C. violceum requires cpri to stimulte NLRC4 inflmmsome ctivtion in humn mcrophges. cpri encodes the conserved TTSS needle suunit tht is sequence prlogue of the rod protein 26, rising hypothesis tht the needle protein is the cteril lignd recognized y the humn NLRC4 inflmmsome. Consistent with the ove genetic nlyses, LFnmedited delivery of CprI, ut not other Cpi-1-encoded proteins, triggered roust cspse-1 ctivtion nd pyroptosis in U937 cells Figure 5 C. violceum infection studies revel tht the humn NLRC4 inflmmsome responds to the TTSS needle suunit through specific recognition y humn NAIP. c, Cspse-1 ctivtion ssys of C. violceum infections of humn U937 monocyte-derive mcrophges. DF hs deletions of five possile flgellin genes in C. violceum. Control (C) or NLRC4 (1) stle knockdown cells were used in. DFDCpi-1A mens deletion of the entire TTSS Cpi-1A locus illustrted in the schemtic drwing shown in. Detiled informtion for ll the mutnt strins re listed in Supplementry Tle 3. d, Complementtion of Cpi-1 locus or cpri deletion C. violceum strins y CprI-expressing plsmid. PMA-differentited U937 cells were infected with indicted C. violceum mutnt or rescue strin. 2A is doule mutnt of CprI (V69A/I79A). e, Cspse-1 ctivtion ssys of delivery of CprI into humn U937 mcrophges nd effects of NLRC4 nd ASC knockdown. Control (C) or NLRC4 or ASC stle knockdown cells were stimulted with LFn-CprI or other indicted LFn fusion proteins. f, Reconstitution of CprI ctivtion of the humn NLRC4 inflmmsome in 293T cells. h, humn. g, Co-immunoprecipittion ssy of CprI nd different NAIP proteins (or NLRC4). 29 SEPTEMBER 2011 VOL 477 NATURE Mcmilln Pulishers Limited. All rights reserved

5 RESEARCH LETTER (Supplementry Fig. 18, ), which were lrgely decresed in NLRC4 nd ASC knockdown cells (Fig. 5e nd Supplementry Fig. 18c). Muttion of two hydrophoic residues (V69A/I79A, 2A) in helicl hirpin region in CprI diminished its ctivity of stimulting inflmmsome ctivtion (Fig. 5d, e nd Supplementry Fig. 18). CprI ctivtion of the NLRC4 inflmmsome could lso e roustly reconstituted in 293T cells nd the 2A mutnt remined inctive in this ssy (Fig. 5f). Most importntly, this reconstitution required humn NAIP, the sole NAIP fmily memer in humn. Humn NAIP-sed reconstitution specificlly responded to LFn-CprI, ut not to LFn- FlA Lp nd LFn-BsK; LFn-CprI did not ctivte NAIP5- nd NAIP2-sed reconstitution (Supplementry Fig. 19). Furthermore, CprI redily co-precipitted humn NAIP, ut not ny of NAIP2, NAIP5 nd NLRC4 from 293T cells, nd the nonfunctionl 2A mutnt filed to interct with humn NAIP (Fig. 5g). Homologous needle suunits from EHEC, B. thilndensis, P. eruginos, S. flexneri nd S. typhimurium, ut not those from EPEC nd V. prphemolyticus, lso stimulted NLRC4 inflmmsome ctivtion in U937 cells (Supplementry Fig. 20). Thus, humn NAIP functions nlogously to mouse NAIP5/2, ut specificlly recognizes the TTSS needle suunit to trigger humn NLRC4 inflmmsome ctivtion. In summry, murine NLR proteins NAIP5 nd NAIP2 directly recognize cteril flgellin nd TTSS rod protein, respectively, wheres humn NAIP serves s specific receptor for the TTSS needle protein. Enggement of NAIP receptors y corresponding cteril lignds promotes their physicl ssocition with NLRC4, resulting in ctivtion of the NLRC4 inflmmsome nd mcrophge innte immunity. The inflmmsome-stimulting ctivities of flgellin, TTSS rod nd needle proteins lie in their C-terminl leucine-rich helicl hirpin regions tht shre structurl commonlities 5,27. Thus, other homologous NAIP proteins might recognize dditionl cteril products of similr iochemicl fetures for countercting diverse cteril infections. Our results lso indicte tht NLRC4 cts s n dptor through which inflmmsome ctivtion signls generted from different NAIP receptors re trnsduced to cspse-1. Involvement of n dditionl cytosolic pttern recognition receptor (PRR) protein for sensing one microil product hs previously een noted with NLPR3 nd NALP1-medited inflmmsome ctivtion 28,29. Future studies will proly identify more PRR proteins tht ct sequentilly within single inflmmsome complex in response to microil products or dnger signls. METHODS SUMMARY LFn-medited intrcellulr delivery nd RNAi. For delivery into mcrophges, purified recominnt proteins were wshed with 60% isopropnol to remove the mjority of endotoxin contminnts. LFn-flgellin, LFn-BsK/PrgJ/CprJ, LFn-CprI or other indicted control proteins together with PA proteins were dded into culture medium (serum-free) t finl concentrtion of 1 mgml 21 for ech protein. Cells were further incuted for 1 h (primry BMMs) or 3 h (immortlized BMMs) efore eing sujected to the indicted inflmmsome ctivtion ssys. Trnsient smll interfering RNA (sirna) knockdown in mcrophges ws performed using the INTERFERin regent (Polyplus Trnsfection) y following the mnufcturer s instruction. To chieve stle knockdown in mcrophges, modified plko.1- GFP plsmid hrouring specific shrna (Supplementry Tle 1) ws trnsduced into BMMs or U937 cells y lentivirl infection nd GFP-positive knockdown cells were sorted out y flow cytometry for further functionl nlysis. Full Methods nd ny ssocited references re ville in the online version of the pper t Received 30 My; ccepted 30 August Pulished online 14 Septemer Lmknfi, M. & Dixit, V. M. Inflmmsomes: gurdins of cytosolic snctity. Immunol. Rev. 227, (2009). 2. Schroder, K. & Tschopp, J. The inflmmsomes. Cell 140, (2010). 3. Frnchi, L. et l. Cytosolic flgellin requires Ipf for ctivtion of cspse-1 nd interleukin 1 in slmonell-infected mcrophges. Nture Immunol. 7, (2006). 4. Mio, E. A. et l. Cytoplsmic flgellin ctivtes cspse-1 nd secretion of interleukin 1 vi Ipf. Nture Immunol. 7, (2006). 5. Mio, E. A. et l. Innte immune detection of the type III secretion pprtus through the NLRC4 inflmmsome. Proc. Ntl Acd. Sci. USA 107, (2010). 6. Lightfield, K. L. et l. Criticl function for Nip5 in inflmmsome ctivtion y conserved croxy-terminl domin of flgellin. Nture Immunol. 9, (2008). 7. Amer, A. et l. Regultion of Legionell phgosome mturtion nd infection through flgellin nd host Ipf. J. Biol. Chem. 281, (2006). 8. Broz, P. et l. Redundnt roles for inflmmsome receptors NLRP3 nd NLRC4 in host defense ginst Slmonell. J. Exp. Med. 207, (2010). 9. Milne, J. C., Blnke, S. R., Hnn, P. C. & Collier, R. J. Protective ntigen-inding domin of nthrx lethl fctor medites trnsloction of heterologous protein fused to its mino- or croxy-terminus. Mol. Microiol. 15, (1995). 10. Broz, P., von Moltke, J., Jones, J. W., Vnce, R. E. & Monck, D. M. Differentil requirement for Cspse-1 utoproteolysis in pthogen-induced cell deth nd cytokine processing. Cell Host Microe 8, (2010). 11. Diez, E. et l. Birc1e is the gene within the Lgn1 locus ssocited with resistnce to Legionell pneumophil. Nture Genet. 33, 5560 (2003). 12. Wright, E. K. et l. Nip5 ffects host susceptiility to the intrcellulr pthogen Legionell pneumophil. Curr. Biol. 13, 2736 (2003). 13. Molofsky, A. B. et l. Cytosolic recognition of flgellin y mouse mcrophges restricts Legionell pneumophil infection. J. Exp. Med. 203, (2006). 14. Ren, T., Zmoni, D. S., Roy, C. R., Dietrich, W. F. & Vnce, R. E. Flgellin-deficient Legionell mutnts evde cspse-1- nd Nip5-medited mcrophge immunity. PLoS Pthog. 2, e18 (2006). 15. Zmoni, D. S. et l. The Birc1e cytosolic pttern-recognition receptor contriutes to the detection nd control of Legionell pneumophil infection. Nture Immunol. 7, (2006). 16. Fortier, A., de Chstellier, C., Blor, S. & Gros, P. Birc1e/Nip5 rpidly ntgonizes modultion of phgosome mturtion y Legionell pneumophil. Cell. Microiol. 9, (2007). 17. Lightfield, K. L. et l. Differentil requirements for NAIP5 in ctivtion of the NLRC4 inflmmsome. Infect. Immun. 79, (2011). 18. Lmknfi, M. et l. The Nod-like receptor fmily memer Nip5/Birc1e restricts Legionell pneumophil growth independently of cspse-1 ctivtion. J. Immunol. 178, (2007). 19. Akhter, A. et l. Cspse-7 ctivtion y the Nlrc4/Ipf inflmmsome restricts Legionell pneumophil infection. PLoS Pthog. 5, e (2009). 20. Frnchi, L. et l. Criticl role for Ipf in Pseudomons eruginos-induced cspse-1 ctivtion. Eur. J. Immunol. 37, (2007). 21. Sutterwl, F. S. et l. Immune recognition of Pseudomons eruginos medited y the IPAF/NLRC4 inflmmsome. J. Exp. Med. 204, (2007). 22. Mio, E. A., Ernst, R. K., Dors, M., Mo, D. P. & Aderem, A. Pseudomons eruginos ctivtes cspse1 throughipf. Proc. NtlAcd. Sci. USA 105, (2008). 23. Suzuki, T. et l. Differentil regultion of cspse-1 ctivtion, pyroptosis, nd utophgy vi Ipf nd ASC in Shigell-infected mcrophges. PLoS Pthog. 3, e111 (2007). 24. Dmino, J. S., Oliveir, V., Welsh, K. & Reed, J. C. Heterotypic interctions mong NACHT domins: implictions for regultion of innte immune responses. Biochem. J. 381, (2004). 25. Miki, T. et l. Chromocterium pthogenicity islnd 1 type III secretion system is mjor virulencedeterminnt for Chromocterium violceum-inducedcelldeth in heptocytes. Mol. Microiol. 77, (2010). 26. Worrll, L. J., Lmeignere, E. & Stryndk, N. C. Structurl overview of the cteril injectisome. Curr. Opin. Microiol. 14, 38 (2011). 27. Poyrz, O. et l. Protein refolding is required for ssemly of the type three secretion needle. Nture Struct. Mol. Biol. 17, (2010). 28. Hsu, L. C. et l. A NOD2NALP1 complex medites cspse-1-dependent IL-1 secretion in response to Bcillus nthrcis infection nd murmyl dipeptide. Proc. Ntl Acd. Sci. USA 105, (2008). 29. Poeck, H. et l. Recognition of RNA virusy RIG-I results in ctivtion of CARD9 nd inflmmsome signling for interleukin 1 production. Nture Immunol. 11, 6369 (2010). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We thnk V. Dixit for providing Nlrc4 nd Asc knockout mice, K. Fitzgerld, D. Rdzioch nd A. Ding for immortlized mcrophges, R. Vnce for Nip5 A/J cdna, M. Donnenerg nd J. Girón for EPEC strins, E. Mio for flgellin-deficient S. typhimurium strin, D. Milton nd T. Hong for cteril vectorsnd T. Miki for C. violceum strins. We re grteful to C. Yo for helping with flow cytometry, nd Y. Xu nd the NIBS niml fcility for hndling mouse lines. We thnk memers of the F.S. lortory for helpful discussions nd technicl ssistnce. This work ws supported y the Ntionl Bsic Reserch Progrm of Chin (973 Progrms, 2010CB nd 2012CB518700). Author Contriutions Y.Z. ndj.y. performedexperiments, ssistedy J.S., Y.-N.G., Q.L., H.X. nd L.L. Y.Z., J.Y. nd F.S. nlysed the dt nd wrote the mnuscript. All uthors discussed the results nd commented on the mnuscript. Author Informtion Reprints nd permissions informtion is ville t The uthors declre no competing finncil interests. Reders re welcome to comment on the online version of this rticle t Correspondence nd requests for mterils should e ddressed to F.S. (shofeng@nis.c.cn). 600 NATURE VOL SEPTEMBER 2011 Mcmilln Pulishers Limited. All rights reserved 2011

6 RESEARCH METHODS Plsmids, ntiodies nd regents. DNAs for flgellin were mplified from the corresponding cteril genomic DNA, nd cloned into pet28-lfn vector (Addgene) for recominnt expression in E. coli s descried previously 30,31. BsK nd PrgJ DNAs were mplified from B. thilndensis E264 nd S. typhimurium LT2 strins, respectively, nd inserted into the sme pet28-lfn vector. DNAs for CprI, CprJ, CorB nd CorC were mplified from C. violceum strin (ATCC ccession 12472) nd lso cloned into pet28-lfn vector to prepre recominnt LFn fusion protein. PA expression plsmid ws lso otined from Addgene. To construct the complementtion plsmid for the C. violceum mutnt, CprI or CprJ DNAs with riosome inding site (RBS) sequence were cloned into the pbbr1mcs2 vector. Expression plsmids for pro-cspse-1 nd pro-il-1 were provided y X. Wng (University of Texs Southwestern Medicl Center). cdnas for mouse NAIP1, NAIP2, NAIP5 C57BL/6, NAIP6, humn NAIP nd NLRC4 were mplified from IMAGE EST clones ( , , , , nd , respectively) nd mouse NLRC4 ws mplified from reverse-trnscried mouse cdna. For mmmlin expression, cdnas for ll NLR proteins were cloned into modified pcs2 vectors with n N-terminl, HA or Flg epitope tg. All trunctions nd point muttions were generted y stndrd moleculr iology procedures. All plsmids were verified y DNA sequencing. Antiodies for cspse-1 nd epitopes were otined from Snt Cruz Biotechnology. Other ntiodies used in this study include IL-1 (3ZD; Biologicl Resources Brnch, Ntionl Cncer Institute), HA epitope (Covnce) nd Flg M2 (Sigm). 293T nd HeL cells otined from ATCC were grown in Dulecco s modified Egle s medium contining 10% fetl ovine serum nd 2mM L-glutmine t 37 uc in 5% CO 2 incutor. Cell culture products were from Invitrogen nd ll other chemicls were Sigm-Aldrich products unless noted. Mouse BMMs nd humn monocyte-derived mcrophges. C57BL/6 wild-type mice were from Vitl River Lortory Animl Technology Co. nd cspse-1 2/2 mice 32 were otined from the Jckson Lortory. Nlrc4 2/2 nd Asc 2/2 mice 33 were provided y V. Dixit (Genentech). All knockout lleles hve een crossed onto the C57BL/6 ckground. All niml experiments were conducted following the Ministry of Helth ntionl guidelines for housing nd cre of lortory nimls nd performed in ccordnce with institutionl regultions fter review nd pprovl y the Institutionl Animl Cre nd Use Committee t Ntionl Institute of Biologicl Sciences. Primry BMMs were prepred y following stndrd procedure s previously descried 34. An immortlized mcrophge line derived from C57BL/6 mice ws provided y K. A. Fitzgerld (University of Msschusetts Medicl School) nd TLR4-deficient immortlized BMMs ws gift from A. Ding (Cornell University). Humn U937 monocytes otined from ATCC were cultured in RPMI-1640 contining 10% FBS nd 2 mm L-glutmine nd grown t 37 uc with 5% CO 2.50ngml 21 PMA ws used to induce U937 differentition for 48 h. Differentited U937 cells were digested with 2 mm EDTA in PBS nd sucultured in 24-well pltes for further experiment. Yest two-hyrid nd co-immunoprecipittion ssys. Indicted flgellin, sk nd prgj genes were cloned into the it vector plexade, nd mouse Nip1, Nip2, Nip5, Nip6 cdnas nd Nlrc4 cdnas were cloned into the prey vector pvp16. The it nd prey plsmids were co-trnsformed into the reporter Scchromyces cerevisie strin L40 y using the lithium cette method. Two-hyrid ssys were performed y following clssicl procedure 35. For immunoprecipittion, 293T cells were trnsfected with indicted plsmids. Cells were hrvested nd lysed in uffer contining 50 mm Tris-HCl (ph 7.6), 150 mm NCl nd 1% Triton X-100 supplemented with protese inhiitor mixture (Roche Moleculr Biochemicls). Preclered lystes were sujected to nti-flg M2 immunoprecipittion y following the mnufcturer s instructions. The eds were wshed three times with the lysis uffer nd the immunoprecipittes were eluted in the SDS smple uffer followed y immunolotting nlysis. All the immunoprecipittion ssys were performed more thn three times nd representtive results re shown in the figures. Purifiction of recominnt proteins. E. coli BL21 (DE3) strins hrouring the expression plsmids were grown in LuriBertni medium (tryptone, 10 g l 21, yest extrct, 5 g l 21, NCl, 10.0 g l 21 ) supplemented with pproprite ntiiotics. Protein expression ws induced overnight t 22 uc with 0.4 mm isopropyl-b-dthioglctopyrnoside (IPTG) fter OD 600 nm reched 0.8. Bcteri were hrvested nd lysed in uffer contining 50 mm Tris-HCl (ph 7.6), 300 mm NCl nd 25 mm imidzole. His-tgged proteins were purified y ffinity chromtogrphy using Ni-NTA eds (Qigen). To remove the mjority of endotoxin contminnts, proteins ound onto the Ni-NTA column were sujected to n dditionl wsh with 60% isopropnol in the wsh uffer (.303 column volume). Proteins were then eluted with 250 mm imidzole in 50 mm Tris-HCl (ph 7.6) nd 300 mm NCl. Eluted smples were further dilysed ginst uffer contining 50 mm Tris-HCl (ph 7.6) nd 150 mm NCl to remove the imidzole. Protein concentrtions were estimted y Coomssie lue stining of SDSPAGE gels using BSA s the stndrds. NLRC4 inflmmsome reconstitution in HeL nd 293T cells. For reconstitution in 293T or HeL cells, cells were seeded into 6-well plte 12 h efore trnsfection with indicted comintions of plsmids using the Vigofect regents (Vigorous). The mounts of plsmids used re 2 mg for pro-humn IL-1, 100 ng (HeL cells) or 50 ng (293T cells) for cspse-1, 100 ng for NLRC4 nd 100 ng for NAIP proteins. Twenty-four hours lter, LFn-flgellin or LFn-BsK/PrgJ/CprJ or LFn-CprI together with PA proteins ws dded into the culture medium t the finl concentrtion of 10 mgml 21 for HeL cells (2 mgml 21 for 293T cells) nd incuted for nother 12 h. Cells were hrvested nd lysed in uffer contining 50 mm Tris-HCl (ph 7.6), 150 mm NCl nd 1% Triton X-100. s were resolved onto SDSPAGE gels followed y nti-il-1 immunolotting nlysis. All the reconstitution experiments were performed more thn three times nd representtive results re shown in the figures. RNAi knockdown. For sirna knockdown, immortlized BMMs were cultured in 24-well pltes t density of per well, nd sirna trnsfection ws performed using the INTERFERin regent (Polyplus Trnsfection) y following the mnufcturer s instruction. 2 ml of 20 mm sirna (finl concentrtion, 100 nm) nd 2 ml of INTERFERin regents were used for ech well. Sixty hours fter trnsfection, knockdown efficiency nd cspse-1 ctivtion were monitored y quntittive rel-time PCR (qrtpcr) nd nti-cspse-1 immunolotting nlysis, respectively. To chieve stle knockdown in immortlized BMMs or U937 cells, shrnas trgeting NAIP5, NAIP2, humn NLRC4 or humn ASC (listed in Supplementry Tle 1) were cloned into modified lentivirl vector plko.1, in which puromycin resistnce gene ws replced y GFP coding sequence. plko.1-gfp shrna plsmids were trnsfected together with two pcking plsmids (pcmv-dr8.2 dvpr nd pcmv-vsv-g, oth from Addgene) into 293T cells. Lentivirus expressing shrna ws collected from the superntnt 48 h fter trnsfection nd ws used to infect BMMs for nother 48 h or U937 cells for 12 h. GFP-positive cells were sorted out y flow cytometry. The pool of sorted cells were either directly used in susequent functionl ssys or diluted into 96-well pltes to otin single clones. Knockdown efficiency ws exmined y qrtpcr nlysis or immunolotting nlysis (for ASC knockdown in U937 cells). Cspse-1-medited inflmmsome ctivtion ssys. To ssy cspse-1 ctivtion, culture superntnts of mcrophges treted with indicted stimuli were sujected to TCA precipittion nd the precipittes were nlysed y nticspse-1 immunolotting to detect oth pro-csp1 nd processed mture frgment; cell lystes were lotted with Csp1 nd ctin ntiodies to show the level of pro-csp1 in cell lystes nd ctin loding, respectively. All cspse-1 ctivtion ssys in response to LFn-medited protein delivery nd cteril infection were repeted t lest three times nd the representtive results re shown in the figures. Mture IL-1 relesed into the culture superntnts ws mesured y using the IL-1 ELISA kit (Neoioscience Technology Compny). Pyroptotic cell deth ws mesured y the lctte dehydrogense (LDH) ssy using CytoTox 96 Non-Rdioctive Cytotoxicity Assy kit (Promeg). Cell viility ws determined y the CellTiter-Glo Luminescent Cell Viility Assy (Promeg). qrtpcr nlysis. For qrtpcr nlysis, totl RNA ws extrcted y TRIzol (Invitrogen) nd digested with DNse I (Invitrogen). One microgrm of totl RNA ws reverse-trnscried into cdna using M-MLV reverse trnscriptse (Promeg). qrtpcr nlysis ws performed using the SYBR Premix Ex Tq (TKR) on Applied Biosystems 7500 Fst Rel-Time PCR System. Primers used for qrtpcr nlysis re listed in Supplementry Tle 2. The mrna level of trgeted genes ws normlized to tht of Gpdh for mouse BMMs or to tht of ctin for U937 cells. Bcteril mnipultion nd mcrophge infection. L. pneumophil strins were cultured on uffered chrcol yest extrct gr supplemented with 0.1 mg ml 21 thymidine (BCYET). For infection, cteri were scrped, diluted in sterile wter nd dded to cells. EPEC strins (E2348/69) were grown overnight in 23 YT (tryptone, 16.0 g l 21, yest extrct, 10.0 g l 21, NCl, 5.0 g l 21 ) medium without shking, nd then diluted 1:40 in DMEM medium for 4 h to induce the expression of type III secretion system efore infection. For S. typhimurium infection, overnight 23 YT culture ws diluted 1:100 nd grown for 3 h to induce SPI-1 expression. B. thilndensis E264 ws otined from ATCC nd cultured s descried 31. Wild-type C. violceum strin (ATCC 12472) ws provided y N. Okd nd cultured s previously descried 25. To infect U937 cells, the indicted C. violceum strin cultured overnight t 37 uc in LB roth under conditions of vigorous shking ws diluted 1:100 in fresh LB roth, nd further grown for 3 h to otin n opticl density t A600 of 2.0 to 2.5. The cteri were diluted in serum-free RPMI medium to chieve multiplicity of infection (MOI) of 10. All infection experiments were performed with centrifugtion of 1,000g for 10 min t 22 uc Mcmilln Pulishers Limited. 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7 RESEARCH LETTER The flgellin-deficient L. pneumophil strin (Lp02DflA) ws generted y stndrd homologous recomintion using the suicide plsmid psr47 s. Deletion of genes encoding the type III rod protein in EPEC (DescI) nd S. typhimurium (DprgJ) strins ws chieved y using the suicide vector pcvd442 s descried previously 36.ForgenedeletioninB. thilndensis, modifiedsuicidevector pdm4-phes expressing mutnt phenyllnine synthetse (PheS) for counterselection 37 ws constructed first. Briefly, the scb gene in the commonly used suicide vector pdm4 (provided y D. Milton nd L. Gong) ws replced with 1.1-k PS12- phes frgment (PS12, the promoter of the B. pseudomllei rpsl gene) mplified from pbbr1mcs-km-phes (provided y T. T. Hong). A PCR frgment contining flnking sequences of the trget gene ws then cloned into pdm4-phes. The resulting trgeting vector ws trnsferred into B. thilndensis through E. coli SM10 (lpir)-medited conjugtionl mting. The trnsconjugnts were selected in LB gr medium contining chlormphenicol (50 mgml 21 ) nd streptomycin (100 mgml 21 ). The integrnts were further screened for mrkerless in-frme deletion y growth on M9 gr pltes supplemented with 20 mm glucose nd 0.1% p-chlorophenyllnine. All the mutnts were verified y PCR nd DNA sequencing. Both flgellin genes in B. thilndensis E264, flic (open reding frme (ORF), BTH_I3196) nd flic2 (ORF, BTH_II0151), were deleted to otin the flgellindeficient strin. For gene deletion in C. violceum, the originl pdm4-scb suicide vector ws used. Briefly, PCR frgment contining flnking sequence of the trgeted gene ws cloned into pdm4-scb. The resulting trgeting vector ws trnsferred into C. violceum through E. coli SM10 (lpir)-medited conjugtionl mting. The trnsconjugnts were selected in LB gr medium contining chlormphenicol (17 mgml 21 ) nd nlidixic cid (25 mgml 21 ). The integrnts were further screened for mrkerless in-frme deletion y growth on LB gr pltes contining 16% sucrose without NCl. Detiled informtion for ll deletion strins re listed in Supplementry Tle 3. All the mutnts were verified y PCR nd DNA sequencing. To exmine the role of flgellin in stimulting cspse-1 ctivtion during mouse mcrophge infection, wild-type, type III secretion-deficient DescN (CVD452, provided y M. Donnenerg) nd flgellin-deficient DfliC (AGT01, provided y J. A. Girón) strins of EPEC E2348/69 were used to infect immortlized BMMs t MOI of 10 for 2 h. Wild-type, type III-deficient DipB nd flgellin-deficient DfliC/ flic2 strins of B. thilndensis were used to infect J774 mouse mcrophges t MOI of 10 for 2 h. To ssy the physiologicl function of NAIP5 in detecting cteril flgellin, control or Nip5 stle knockdown immortlized BMMs were infected with S. typhimurium strin (wild type, ATCC s or DfliCDfljB mutnt, flic::tn10 fljb5001::mud-cm; oth strins were provided y E. A. Mio) for 15 min, or L. pneumophil (Lp02 or Lp02DflA) for 40 min t MOI of 50. To ssy the function of NAIP2 in detecting the type III rod protein during infection, control or Nip2 stle knockdown immortlized BMMs were infected with S. typhimurium (DfliCDfljB or DfliCDfljBDprgJ) for 30 min or EPEC E2348/69 strin (DfliC or DfliCDescI) for 2 h t MOI of 50. s nd cell lystes of infected mcrophges were collected nd sujected to cspse-1 ctivtion ssys descried ove. 30. Yo, Q. et l. A cteril type III effector fmily uses the ppin-like hydrolytic ctivity to rrest the host cell cycle. Proc. Ntl Acd. Sci. USA 106, (2009). 31. Cui, J. et l. Glutmine demidtion nd dysfunction of uiquitin/nedd8 induced y cteril effector fmily. Science 329, (2010). 32. Li, P. et l. Mice deficient in IL-1-converting enzyme re defective in production of mture IL-1 nd resistnt to endotoxic shock. Cell 80, (1995). 33. Mrithsn, S. et l. Differentil ctivtion of the inflmmsome y cspse-1 dptors ASC nd Ipf. Nture 430, (2004). 34. Boyden, E. D. & Dietrich, W. F. Nlp1 controls mouse mcrophge susceptiility to nthrx lethl toxin. Nture Genet. 38, (2006). 35. Vojtek, A. B. & Cooper, J. A. Rho fmily memers: ctivtors of MAP kinse cscdes. Cell 82, (1995). 36. Dong, N., Liu, L. & Sho, F. A cteril effectortrgets hostdh-ph domin RhoGEFs nd ntgonizes mcrophge phgocytosis. EMBO J. 29, (2010). 37. Brrett, A. R. etl. Genetic tools for llelicreplcementinburkholderi species. Appl. Environ. Microiol. 74, (2008) Mcmilln Pulishers Limited. All rights reserved