1、Letterhttps:/doi.org/10.1038/s41586-018-0433-3Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose Ping Zhou1,8,Yang She1,2,3,8,Na Dong4,Peng Li1,Huabin He1,Alessio Borio5,Qingcui Wu1,Shan Lu1,Xiaojun Ding6,Yong Cao1,Yue Xu1,Wenqing Gao1,Mengqiu Dong1,Jingjin Ding1,2,Da-Che
2、ng Wang2,Alla Zamyatina5&Feng Shao1,2,7*Immune recognition of pathogen-associated molecular patterns(PAMPs)by pattern recognition receptors often activates proinflammatory NF-B signalling1.Recent studies indicate that the bacterial metabolite d-glycero-d-manno-heptose 1,7-bisphosphate(HBP)can activa
3、te NF-B signalling in host cytosol24,but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified.Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-d-manno-heptose(ADP-Hep),which me
4、diates type III secretion system-dependent NF-B activation and cytokine expression.ADP-Hep,but not other heptose metabolites,could enter host cytosol to activate NF-B.A CRISPRCas9 screen showed that activation of NF-B by ADP-Hep involves an ALPK1(alpha-kinase 1)TIFA(TRAF-interacting protein with for
5、khead-associated domain)axis.ADP-Hep directly binds the N-terminal domain of ALPK1,stimulating its kinase domain to phosphorylate and activate TIFA.The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligandreceptor recognition process.
6、HBP was transformed by host adenylyltransferases into ADP-heptose 7-P,which could activate ALPK1 to a lesser extent than ADP-Hep.ADP-Hep(but not HBP)alone or during bacterial infection induced Alpk1-dependent inflammation in mice.Our findings identify ALPK1 and ADP-Hep as a pattern recognition recep
7、tor and an effective immunomodulator,respectively.Gram-negative bacteria such as Yersinia5,Salmonella6,Burkholderia7 and enteropathogenic Escherichia coli8 induce NF-B-mediated cytokine expression in a type III secretion system(T3SS)-dependent manner.Consistently,infection of 293T cells with Y.pseud
8、otuber-culosis 6(lacking the six T3SS effectors;6 is omitted hereafter)robustly activated NF-B-driven luciferase and eGFP reporters5(Fig.1a,b).From 21,000 Y.pseudotuberculosis transposon mutants,we identified 37 defective in activating both reporters.Most mutations were in T3SS-encoding genes.One mu
9、tant that was more impaired than the T3SS-deficient yopB strain had a functional T3SS with its transposon inserted in hldE(Fig.1ac and Extended Data Fig.1a).Expression of HldE in the transposon or the hldE mutant restored infection-induced NF-B activation.HldE,together with GmhA and GmhB,synthesizes
10、 ADP-d-glycero-d-manno-heptose(ADP-DD-Hep;is omitted hereafter)from d-sedoheptulose 7-phosphate(S7P)through d-glycero-d-manno-heptose 7-phosphate(H7P),HBP and d-glycero-d-manno-heptose 1-phosphate(H1P)(Extended Data Fig.1b).ADP-DD-Hep and ADP-LD-Hep(ADP-l-glycero-d-manno-heptose)undergo interconvers
11、ion,catalysed by HldD.Deletion of gmhB or hldD did not affect Y.pseudotuberculosis-induced activation of NF-B,whereas deletion of gmhA phenocopied the hldE strain(Fig.1b).This seems to suggest that HBP,but not H1P or ADP-Hep,determines Y.pseudotuberculosis-dependent activation of NF-B,echoing the an
12、alyses in Neisseria meningitidis2.However,Y.pseudotuberculosis gmhB,unlike the hldE and gmhA strains,still supported ADP-Hep-dependent autotransporter heptosylation9,10(Extended Data Fig.1c),because of an unknown redundancy to gmhB in ADP-Hep biosynthesis11.When electroporated into 293T cells,synthe
13、tic HBP and ADP-DD-Hep or ADP-LD-Hepbut not S7Pstimulated NF-B activation,with ADP-Hep being the most potent(Fig.1d).H1P was even less active than HBP2(Extended Data Fig.1d).When added directly to 293T cells,only ADP-DD-Hep and ADP-LD-Hep induced activation of NF-B and production of interleukin(IL)-
14、8(Fig.1d,e).This explains the use of transfection in recent reports on HBP2,4.Thus,ADP-Hep is a potent and versatile PAMP.Activation of NF-BeGFP reporter by extracellular ADP-Hep ena-bled us to carry out a fluorescence-activated cell sorting(FACS)-based genome-wide CRISPRCas9 screen(Extended Data Fi
15、g.2a).Following a counterscreen against TNF stimulation,we identified ALPK1,TIFA and TRAF6(each hit by more than one guideRNA(gRNA)that were required for ADP-Hep-induced NF-BeGFP expression(Fig.2a and Supplementary Table1).Upon phosphorylation at T9,TIFA forms foci to activate TRAF6-dependent NF-B s
16、ignalling1214.During the preparation of this manuscript,ALPK1 was identified as contributing to activation of NF-B in Shigella flexneri and Helicobacter pylori3,4.Deletion of ALPK1 or TIFA(Supplementary Table2)abolished ADP-LD-Hep-induced activation of NF-B and expression of IL-8(Fig.2b,c and Extend
17、ed Data Fig.2b,c).Defective NF-B activation in ALPK1/cells was restored by wild-type ALPK1 but not by its kinase-inactive K1067M mutant(Fig.2b);TIFA/was rescued by wild-type TIFA but not by a T9A mutant(Extended Data Fig.2c).ADP-LD-Hep induced phosphorylation of TIFA at T9 and formation of eGFPTIFA
18、foci dependent upon ALPK1 kinase activity(Fig.2d and Extended Data Fig.2d).ADP-LD-Hep did not affect the cytoplas-mic localization of ALPK1 and induced no myosin phosphorylation15(Extended Data Fig.2e,f).ALPK1 and TIFA were also required for the activation of NF-B by electroporation of ADP-Hep(Exten
19、ded Data Fig.2g).ADP-LD-Hep triggered co-immunoprecipitation of TIFA with ALPK1 and TRAF6(Extended Data Fig.2h).Deletion of ALPK1 did not affect activation of NF-B by TNF,NOD1 or NOD2(mediates Salmonella-induced NF-B activation16,17)or MYD88 overexpression(Extended Data Fig.1e).Cells lacking NOD1 an
20、d NOD2 showed intact NF-B responses and TIFA foci following treatment with ADP-LD-Hep(Extended Data Fig.1f,g).Thus,ADP-Hep activates NF-B specifically through the ALPK1TIFATRAF6 axis.Induction of IL-8 expression by Y.pseudotuberculosis,which required hldE and yopB,was blocked by deletion of ALPK1(Fi
21、g.2e).Infection-induced activation of NF-B and formation of eGFPTIFA foci required ALPK1-dependent phosphorylation of TIFA at T9(Extended Data Fig.3ae).Other bacteria,such as diffuse-adhering E.coli(DAEC),enterotoxigenic E.coli(ETEC)and Burkholderia 1National Institute of Biological Sciences,Beijing
22、,China.2National Laboratory of Biomacromolecules,Institute of Biophysics,Chinese Academy of Sciences,Beijing,China.3College of Life Sciences,University of Chinese Academy of Sciences,Beijing,China.4College of Animal Science and Technology,China Agricultural University,Beijing,China.5Department of Ch
23、emistry,University of Natural Resources and Life Sciences,Vienna,Austria.6Beijing Mingde Zhengkang Technologies Co.,Ltd.,Beijing,China.7Tsinghua Institute of Multidisciplinary Biomedical Research,Tsinghua University,Beijing,China.8These authors contributed equally:Ping Zhou,Yang She.*e-mail:1 2 2|N
24、A t U r e|V O L 5 6 1|6 S e P t e M B e r 2 0 1 8 2018 Springer Nature Limited.All rights reserved.Letter reSeArCHcenocepacia also triggered NF-B activation mediated by the ALPK1TIFA axis in an hldE-dependent manner(Extended Data Fig.3f,g).Thus,sensing of ADP-Hep by ALPK1 is not limited to T3SS-cont
25、aining bacteria.ALPK1 contains an-helical domain and a kinase domain linked by an extensive unstructured region(Fig.2f).ALPK1-N492(res-idues 1492)and ALPK1-N492(lacking these residues)were co-immunoprecipitated,independently of ADP-LD-Hep(Fig.2g).Co-expression of ALPK1-N492 and ALPK1-N492,or minimal
26、lythe N-terminal and kinase domains of ALPK1(ALPK1-NTD(1473)and ALPK1-KD(9591244),respectively),was sufficient to allow ADP-LD-Hep or Y.pseudotuberculosis to induce activation of NF-B and phosphorylation of TIFA(Fig.2h and Extended Data Fig.4ae).Unexpectedly,a purified complex of ALPK1-NTD and ALPK1
27、-KD(ALPK1-(N+K);Extended Data Fig.5a)could directly phosphoryl-ate T9 of TIFA(Fig.3a).By contrast,activation of ALPK1-(N+K)in mammalian cells required infection or stimulation by ADP-Hep.Notably,ALPK1-(N+K)from E.coli hldE did not phosphoryl-ate TIFA(Fig.3a).Small-molecule extracts from wild-type bu
28、t not hldE mutant E.coli-derived ALPK1-NTD,when added to 293T cells,potently stimulated ALPK1-dependent phosphorylation of TIFA and activation of NF-B,the latter of which correlated with the amount of ALPK1-NTD(Fig.3b,c).High-performance liquid chromatography(HPLC)of the small-molecule extracts iden
29、tified one active fraction(no.6,the only one with high ultraviolet absorption)(Extended Data Fig.5bd).Mass spectrometry of fraction 6 uncovered three dominant ions with mass-to-charge ratios(m/z)of 619.8,347.9 and 427.8,matching those of ADP-Hep,AMP and ADP,respectively(Extended Data Fig.5e).The pre
30、sumed ADP-Hep ion showed a similar retention time and fragmen-tation pattern to synthetic ADP-Hep(Extended Data Fig.5f,g).The mass of native ALPK1-NTD exceeded that of denatured ALPK1-NTD by 619.32Da(one ADP-Hep)(Fig.3d).Direct binding of E.coli hldE-derived apo-ALPK1-(N+K)to ADP-LD-Hep(but not S7P)
31、was readily detected(Extended Data Fig.5h).We determined the 2.59 crystal structure of ALPK1-NTD puri-fied from wild-type E.coli(Extended Data Table1a).Each asymmetric ahldEGlutamine-synthetaseadenylyltransferaseAmino acid transporterHimar1 transposon1,0002,0003,0004,0005,000(bp)Y.pseudotuberculosis
32、 6/hldE:Himar1bdHoechstEGFPNon infectionyopBhldE:Himar1hldEY.pseudotuberculosis 6hldE:Himar1+pHldEhldE+pHldEehldE/yopB:KangmhAgmhBhldDY.pseudotuberculosis 6HoechsteGFPc100806040200Y.pseudotuberculosis 6Rel.NF-B luciferase activity*Electroporation100806040200Rel.NF-B luciferase activityPBSHepADP-LD-H
33、ep1 M10 M100 M*100806040200Extracellular addition*250200150100500300Electroporation*543210IL-8 secretion(ng ml1)THP-1 cell6*IL8 mRNA/18S rRNATHP-1 cell1501209060300*ADP-DD-HBPS7PNon infectionyopBhldE:Himar1hldE:Himar1+pHldEhldEhldE+pHldEhldE/yopB:KanPBSH1PADP-LD-HepPBSHBPPBSHBPH1PADP-DD-HepADP-LD-He
34、pADP-LD-HepPBSHBPADP-LD-HepFig.1|Transposon screen of Yersinia T3SS-dependent NF-B activation identifies ADP-Hep as a PAMP.a,b,293T cells were infected with indicated Yersinia strains.pHldE is a plasmid expressing HldE.c,Transposon insertion in hldE.d,e,293T(d)or PMA-differentiated THP-1 cells(e)wer
35、e electroporated(d)or extracellularly treated(d,e)with indicated sugars.IL8 mRNA(also known as CXCL8,shown relative to 18S rRNA)and IL-8 secretion were measured by quantitative real-time PCR(qPCR)and enzyme-linked immunosorbent assay(ELISA),respectively.NF-B activation was measured by luciferase act
36、ivity(a,d)or eGFP reporter expression(b)(scale bar,50m).Data shown as mean s.d.from three technical replicates(a,d,e),and representative of three(a,b,d)and two(e)independent experiments.a,d,e,Two-tailed unpaired Students t-test(*P 0.01,*P 0.001).abdcGenegRNAALPK1 ALPK1_3_A120.7 132.2 ALPK1 ALPK1_1_B
37、111.3219.2 ALPK1 ALPK1_2_A57.1 73.6 ALPK1 ALPK1_3_B54.3 92.7 TIFATIFA_3_A75.0109.6 TIFATIFA_3_B68.5 4TIFATIFA_1_A62.4 99.5 TIFATIFA_2_A54.6 93.6 ghWTK/MADP-LD-Hep293T WT293T ALPK1/FlagALPK1FlagTIFApT9-TIFA+FlagALPK1ALPK1-N492Anti-FlagALPK1-N492InputFlagALPK1MycALPK1-N492Flag IPMockADP-LD-HepNTDKDLin
38、kerCN14929811244ALPK1feIL8 mRNA/18S rRNAMockHBP ADP-LD-Hep150200100500250Extracellular addition293T ALPK1/293T WT*IL8 mRNA/18S rRNA150200100500250Y.pseudotuberculosis 6293T ALPK1/293T WT*FLN492N492 N492+N492FlagALPK1Extracellular addition608040200Rel.NF-B luciferase activity100MockADP-LD-Hep*293T AL
39、PK1/FLNTDKD(N+K)FlagALPK1293T WT608040200Rel.NF-B luciferase activity100120Non infectionY.pseudotuberculosis 6*293T ALPK1 KO-1KO-2293T WTFlagALPK1 WTK/MWTK/M456030150Rel.NF-B luciferase activity75Extracellular additionMockADP-LD-Hep*N492N492Non infectionyopBhldEhldE+pHldEN492N492109.0 1268357Ranking
40、FC_exp2FC_exp1Fig.2|CRISPRCas9 screens identify an ALPK1TIFA axis that mediates activation of NF-B induced by ADP-Hep or Yersinia.a,Top eight gRNA hits from CRISPRCas9 screen of ADP-LD-Hep-induced NF-B activation.be,h,Wild-type or ALPK1/293T cells expressing the indicated ALPK1 mutants were treated
41、with ADP-LD-Hep(bd,h)or HBP(c),or infected with Y.pseudotuberculosis(e,h).d,Anti-Flag and anti-pT9-TIFA immunoblots of 293T cells.f,Domain organization of ALPK1.g,Co-immunoprecipitation of ALPK1 N-and C-terminal regions from 293T cells treated with or without ADP-LD-Hep.NF-B activation and IL8 mRNA
42、were assessed by luciferase reporter activity(b,h)or qRTPCR(c,e),respectively.KO-1/2,two ALPK1/clones.Data shown as mean s.d.from three technical replicates(b,c,e,h)and representative of three(a,b,d,h,g)or two(c,e)independent experiments.b,c,e,h,Two-tailed unpaired Students t-test(*P 0.01,*P 0.001).
43、6 S e P t e M B e r 2 0 1 8|V O L 5 6 1|N A t U r e|1 2 3 2018 Springer Nature Limited.All rights reserved.LetterreSeArCHunit contains nine molecules(AI).Molecule A has the highest-quality density map and was used to build the final model,which lacks only the first methionine.The structure bears 18
44、helices(1to18),forming seven antiparallel pairs(14,56,78,910,1113,1415,and 1617)(Fig.3e).The 14 pair features an inser-tion containing 2,3 and loop L1.18,which precedes the L2 tail,flanks the outer surface of the 1617 pair.The one-by-one-packed seven helix pairs form a right-hand solenoid(Fig.3e),re
45、sembling a 12345MBPALPK1-NTDA280nm(mAU)1,5002,0001,00050002,5005060697989Elution volume(ml)ADP-LD-Hep213Mock9012060300Rel.NF-B luciferase activity150*abdfcTIFAHis6ADP-LD-HeppT9-TIFAALPK1-NTDTIFAHis6+ALPK1-(N+K)-E.colihldEWT1234570 kDa100 kDa130 kDaTubulinMycTIFApT9-TIFA21353,988.7653,958.4853,887.82
46、49,832.4343,188.5154,607.8154,874.7764,749.2971,982.0454,608.0843,685.8946,807.0454,563.38 54,651.3954,732.41Native-ESI mass spectrum of His6ALPK1-NTD(E.coli WT)NativeDenaturedMass:54,608.08 53,988.76=619.32 DaMass(kDa)Relative intensityRelative intensity404550556065707580608040200100608040200100N12
47、356789101112C131415L1L2161718D231S236Q67K233F61R116R150R153F29541454101197egSmall-molecule extracts:1.MBPALPK1-NTD(E.coli WT)2.MBPALPK1-NTD(E.coli hldE)3.MBPTIFA(E.coli WT)3004002001000Rel.NF-B luciferase activity500600700293T ALPK1/293T TIFA/293T WTVecALPK1VecTIFA12345*pAD6Fig.3|ADP-Hep binding to
48、ALPK1-NTD and crystal structure of the binding complex.a,ALPK1-(N+K)purified from indicated E.coli strains was incubated with histidine-tagged TIFA(TIFAHis6)in the presence or absence of ADP-LD-Hep.b,Gel-filtration chromatography of MBPALPK1-NTD purified from wild-type E.coli.b,c,293T cells were tre
49、ated with small-molecule extracts from fractions 15(b)or from TIFA or MBPALPK1-NTD(c).Activation of NF-B was assessed by luciferase reporter assay(mean s.d.from three technical replicates);two-tailed unpaired Students t-test,*P 0.001.d,Electrospray ionization mass spectrometry of native and denature
50、d ALPK1-NTD purified from wild-type E.coli.e,Structure of the ALPK1-NTDADP-Hep complex.f,Stereo diagram of the simulated annealing FoFc omit map of ADP-LD-Hep contoured at 3.g,Surface representation of ADP-Hep-bound ALPK1-NTD and overview of the binding pocket.Black dashed lines,hydrogen bonds.a,c,A
