The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy.

The immunogenic po en ial o bac e ial lagella o Salmonella-media ed umo he apy Sebas ian Felgne 1,2,#, Imke Spö ing1, Vinay Pawa 2,4, Dino Kocijancic5, Ma hias P eusse2, Ch is ine Falk3, Man ed Rohde4, Susanne Häussle 2, Sieg ied Weiss5,&, Ma c E ha d 1,6,& 1 In ec ion Biology o Salmonella, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many 2 Depa men o Molecula Bac e iology, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many 3 Ins i u e o T ansplan Immunology, Medical School Hanno e , Hanno e , Ge many 4 Cen al Facili ies o Mic oscopy, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many 5 Ins i u e o Immunology, Medical School Hanno e , Hanno e , Ge many 6 Ins i u e o Bac e ial Physiology, Humbold Uni e si y, Be lin, Ge many NOVELTY AND IMPACT: Balancing sa e y and he apeu ic e icacy emains a majo challenge o he a ional design o bac e ia o immuno he apy applica ions. We demons a ed ha manipula ing he spa io empo al egula ion o lagella syn hesis can con e his balance in Salmonella. Unexpec edly, ab oga ing lagella syn hesis a an ea ly s age esul ed in ou e memb ane esicle o ma ion, which enhanced he immunogenic p ope ies. These enginee ed Salmonella s ains migh ha e he po en ial o se e as ec o pla o m o a ious he apies anging om immuniza ion o cance he apy. KEYWORDS: Salmonella Typhimu ium, hos -pa hogen in e ac ion, lagella, luminex, bac e ia- media ed umo he apy ARTICLE CATEGORY: Cance he apy and p e en ion Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. This a icle has been accep ed o publica ion and unde gone ull pee e iew bu has no been h ough he copyedi ing, ypese ing, pagina ion and p oo eading p ocess which may lead o di e ences be ween his e sion and he Ve sion o Reco d. Please ci e his a icle as doi: 10.1002/ijc.32807 2 # Add ess co espondence o Sebas ian Felgne , Sebas ian. elgne @helmhol z-hzi.de, Helmhol z- Cen e o In ec ion Resea ch, Inho ens aße 7, 38124 B aunschweig, Ge many, Phone: + 49 531 6181 3133 & Con ibu ed equally o his wo k ABBREVIATIONS: PAMP = pa hogen-associa ed molecula pa e n, LPS = lipopolysaccha ide, TLR = oll-like ecep o , PPR = pa e n- ecogni ion ecep o , UK-1 = Uni e sal Kille 1, W = wild- ype, MOI = mul iplici y o in ec ion, LDH = lac a e dehyd ogenase, IL = In e leukin, TNF-α = umo nec osis ac o α, hpi = hou s pos in ec ion, dpi = days pos in ec ion, OMV = ou e memb ane esicle, CT26 = mu ine colon ca cinoma, RenCa = enal adenoca cinoma, ELISA = enzyme-linked immunoso ben assay, BMDM = bone-mo ow de i ed mac ophages Abs ac : 150 wo ds Wo ds: 5152 Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 3 ABSTRACT: Gene ically enginee ed Salmonella Typhimu ium a e po en ec o s o p ophylac ic and he apeu ic measu es agains pa hogens as well as cance . This is based on he po en adju an ici y ha suppo s s ong immune esponses. The physiology o Salmonella is well unde s ood. I simpli ies enginee ing o bo h enhanced immune-s imula o y p ope ies as well as sa e y ea u es, hus, esul ing in an app op ia e balance be ween a enua ion and e icacy o clinical applica ions. A majo i ulence ac o o Salmonella is he lagellum. I is also a s ong pa hogen-associa ed molecula pa e n ecognized by ex a- and in acellula ecep o s o immune cells o he hos . A he same ime, i ep esen s a se ious me abolic bu den. Acco dingly, he bac e ia e ol ed igh egula o y mechanisms ha con ol lagella syn hesis in i o. He e, we sys ema ically in es iga ed he immunogenici y and adju an p ope ies o a ious lagella mu an s o Salmonella in i o and in a mouse cance model in i o. We ound ha mu an s lacking he lagellum-speci ic ATPase FliHIJ o he inne memb ane ing FliF displayed he g ea es s imula o y capaci y and s onges an i- umo e ec s, while emaining sa e in i o. Scanning elec on mic oscopy e ealed he p esence o ou e memb ane esicles in he Δ liF and Δ liHIJ mu an s. Finally, he combina ion o he Δ liF and Δ liHIJ mu a ions wi h ou p e iously desc ibed a enua ed and immunogenic backg ound s ain SF102 displayed s ong e icacy agains he highly esis an cance cell line RenCa. We hus conclude ha manipula ing lagella biosyn hesis has g ea po en ial o he cons uc ion o highly e icacious and e sa ile Salmonella ec o s ains. Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 4 INTRODUCTION Despi e he exponen ial g ow h o biomedical knowledge o e he las decades, we a e s ill acing heal h condi ions ha a e no con ollable. Thus, p esen biomedical esea ch is called upon o p o ide solu ions o his dilemma. In ace o he demog aphic changes wi hin he wo ld popula ion hese p oblems become e en mo e acu e wi h cance being one o he mos p essing p oblems. Consequen ly, scien is s need o de elop no el and/o mo e e ec i e s a egies o accines and immuno he apies. Sea ch o p ope p o ec i e an igens is equi ed o he success o such immune in e en ions bu a he same ime e icacious and sa e adju an s a e equi ed o such s a egies. Employmen o pa hogens as pla o ms o many o such app oaches is he e o e a s aigh o wa d possibili y. These mic oo ganisms a e able o ac as po en ca ie s because hey elici s ong immune eac ions i.e. hey exhibi s ong adju an p ope ies. Vi uses a e o en employed in his con ex 1. Howe e , bac e ia ha e also success ully been es ed. Fo ins ance, Salmonella spp. a e known o elici s ong cellula and humo al immune ac i i ies which unde sco e hei po en ial as an e ec i e li e ca ie 2–4. Salmonella spp. is a pa hogen. I may elici li e h ea ening disease in he hos . Hence, hei pa hogenic p ope ies ha e o be a enua ed o ensu e sa e applica ion. In his case, a enua ion and immune-s imula ion needs o be well in balance o gua an ee sa e y and e icacy 5,6. This ep esen s he basic p oblem o li e bac e ial ca ie s. Immune- ecogni ion, immune-s imula ion as well as immune-e asion o Salmonella a e closely connec ed o he a ailabili y o pa hogen associa ed molecula pa e ns (PAMPs), such as lipopolysaccha ide (LPS) o lagella 7. Responses o LPS, as an agonis o he Toll-like ecep o 4 (TLR-4) and causa i e agen o sepsis a e in ensi ely in es iga ed 8. LPS has been es ablished as one o d i ing o ces o adju an ici y 5. These expe imen s included gene a ion o condi ionally a enua ed Salmonella ca ie s ains. A egula o y connec ion be ween LPS modi ica ions and Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 5 lagella syn hesis ia he R lP/ClpXP pa hway has been sugges ed 9. In addi ion, he impai men o bo h hese wo impo an PAMPs - LPS and lagellum - di ec ly led o a loss o he apeu ic po ency. Appa en ly, such molecules a e essen ial asse s o he adju an ici y o he Salmonella ec o . Howe e , he con ibu ion o he lagellum o he es ablishmen o a success ul in ec ion and i s in ol emen in an e icacious immune s imula ion is less well explo ed. On he one hand, lagella mo ili y con ibu es o pa hogenesis by p omo ing bac e ia-hos in e ac ions, adhe ence and in asion o hos cells 10,11. On he o he hand, as soon as Salmonella eaches i s ana omical des ina ion in he body, lagella syn hesis is a se ious bu den o he mic oo ganism. Down- egula ion o a oid immune ecogni ion by Pa e n-Recogni ion-Recep o s (PPR), like ex acellula TLR-5 o in acellula caspase-1 is essen ial o bac e ial su i al in i o 12,13. This e asion mechanism al eady indica es ha bac e ia cons i u i ely exp essing lagella migh elici s ong immune ac i a ion. The lagellum is a sophis ica ed mac omolecula appa a us composed o se e al housand copies o app oxima ely 25 di e en p o eins. I can be classi ied in o h ee main pa s: i) a basal body embedded in he cy oplasmic memb ane ha a e ses he pe iplasm and cell wall up o he ou e memb ane ( he engine), ii) a long ex e nal ilamen ( he p opelle ) and iii) a lexible, cu ed s uc u e known as he “hook”, which connec s he basal body wi h he igid ilamen 14. S. Typhimu ium usually encodes wo an igenically dis inc ilamen p o eins ( he lagellins FliC o FljB), whose mu ually exclusi e exp ession is egula ed by phase swi ching 15. O no e, s ains cons i u i ely exp essing he phase-2 lagellin p o ein FljB we e ound o be mo e po en in a ge ing umo s in a mu ine umo model compa ed o s ains exp essing phase-1 FliC 16. In line wi h hese obse a ions, Eom and colleagues obse ed an enhanced adju an ici y o Salmonella ha co-exp essed bo h lagellin p o eins FliC and FljB 17. These esul s demons a e ha a modula ion o syn hesis and assembly o lagella migh allow o enginee app op ia ely modi ied bac e ial Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 6 ec o s ains o he apeu ic applica ions. Recen esul s con i m he impo ance o FlaB lagella o success ul Salmonella based cance he apy 18. Fu he mo e, mo ili y, chemo axis and he p esence o lagella as an igen ha e been shown o be impo an o umo he apy as well 18–21. Thus, manipula ions o he a ious lagellum sub-s uc u es may posi i ely in luence he pe o mance o he he apeu ic s ains. In he p esen s udy, we aimed o sys ema ically un a el he connec ion be ween he p esence o lagella componen s and immune s imula o y po ency. We hypo hesized ha manipula ing he spa io empo al onse o lagella syn hesis o o a ious s eps in lagella assembly migh ep esen a alid s a egy o inc ease he adju an powe o Salmonella ec o s ains wi hou inc easing hei pa hogenici y. Thus, we in es iga ed h ee g oups o lagella mu an s (Figu e 1): i) lagellin phase locked mu an s (FliC-ON, FljB-ON, FliC-ON & FljB-ON), ii) non- ilamen ous mu an s (∆ lgK, ∆ liF, ∆ liHIJ) and lagella o e p oduc ion mu an s (∆ lP, ∆ lP ∆ lgM and ∆ lP ∆ lgM ∆ lM). In summa y, ou esul s highligh he impo ance o a con olled spa io empo al egula ion o lagella syn hesis du ing hos -pa hogen in e ac ions. Salmonella mu an s lacking he inne memb ane ing and basal body componen FliF o he lagellum-speci ic ATPase complex FliHIJ displayed he highes he apeu ic e icacy in mu ine umo models despi e he ac ha hey a e unable o assemble lagella. We hus belie e ha he lagellum o Salmonella ep esen s an ideal a ge o immunomodula o y modi ica ions and i migh be possible o gene a e op imized sa e ec o s ains wi h imp o ed adju an p ope ies o p ophylaxis and he apy. Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 7 MATERIALS AND METHODS E hics s a emen : All animal expe imen s we e pe o med acco ding o guidelines o he Ge man Law o Animal P o ec ion and wi h pe mission o he local e hics commi ee and he local au ho i y LAVES (Niede sächsisches Landesam ü Ve b auche schu z und Lebensmi elsiche hei ) unde pe mission numbe 33.9-42502-04-12/0713, 33.9-42502-04-13/1122 and 33.9-42502-04-13/1191. S ains and p epa a ion o inoculum: Bac e ial s ains a e shown in Table S1. S ain cons uc ion was done by P22 phage ansduc ion o λ- ed ecombina ion 22. Salmonella s ains we e g own o e nigh and sub-cul u ed o mid-log phase in LB media a 37 °C. The bac e ia we e washed wice and adjus ed o he desi ed OD600 in py ogen ee PBS. Pla ing se ed as con ol. Cell lines and p ima y cells: Immo alized RAW264.7 mac ophages (RRID:CVCL_0493) we e used o in asion assays and ob ained om Raschke e al. 23 (Salk Ins i u e, San Diego, USA). CT26 umo cells (RRID:CVCL_7524, ob ained om: B a ain e al. 24, Comp ehensi e Cance Cen e , Uni e si y o Alabama, USA) and RenCa umo cells (RRID:CVCL_2174, ob ained om: Wells e al. 25, Tumo Biology Cen e , F eibu g, Ge many) we e used o he subcu aneous mu ine umo model. All cell cul u es used in his s udy we e es ed and con i med as mycoplasma- ee. Bone-ma ow de i ed mac ophages (BMDMs) we e isola ed om he emu o BALB/c mice and di e en ia ed using 20% ( / ) L929 (RRID:CVCL_0462, ob ained om: Monne e al. 26, Na ional Resea ch Cen e o Bio echnology, B aunschweig, Ge many) condi ioned medium in RPMI con aining 10% FCS. All cells we e main ained a 37 °C, 5% CO2 and 90% el. humidi y. Mo ili y assay: The mo ili y o he Salmonella s ains was assessed on semi-solid aga pla es con aining 0.3% (w / ol) aga by inocula ing 2 µl o a bac e ial o e nigh cul u e in o he aga and incuba ed a 37 °C. The swa m diame e was measu ed a e 4 h incuba ion. Flagella immunos aining: Salmonella s ains g own o mid-log g ow h phase we e ixed on L- lysine coa ed mic oscopy slides using o maldehyde (c = 2%, / ) and glu a aldehyde (c = 0.2%, Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 8 / ). Flagellum s aining was accomplished using polyclonal abbi an i-FliC (Di co) as p ima y and an i- abbi Alexa Fluo -488 as seconda y an ibody and he bac e ia we e s ained wi h DAPI (Sigma- Ald ich). Images we e aken using an Axio Obse e mic oscope equipped wi h an Axiocam HR came a (Zeiss) a 100x magni ica ion and analyzed wi h ImageJ. In asion assays: RAW 264.7 and BMDM mycoplasma- ee cells we e used o he phagocy ic up ake and in acellula eplica ion. The assay was pe o med as desc ibed be o e 27 using MOIs o 1 and 10. CFUs we e de e mined by pla ing o se ial dilu ions and compa ed o he co esponding pa en al s ains. Mu ine umo model: Six-week old BALB/c mice (Jan ie ) we e in ade mally inocula ed wi h 5*105 syngeneic CT26 o 2*106 RenCa umo cells in he igh lank. Tumo de elopmen was moni o ed using calipe measu emen s. Upon eaching a umo olume o app ox. 150 mm³, he mice we e injec ed in a enously in o he ail ein wi h 5*106 Salmonella. The apeu ic e icacy: Tumo de elopmen was moni o ed using calipe measu emen s o as long as umo s pe sis ed o un il con on ed wi h a humane endpoin in e ms o exceedingly la ge umo size (~ 1 cm³) o mo bidi y. Body weigh as gene al heal h indica o was moni o ed using a scale. A loss o body weigh below 80% o he o iginal body weigh was incen i e o eu hanize a subjec . TNF-α ELISA measu emen : Supe na an samples o cul i a ed mac ophages we e aken 6 h pos in ec ion. The TNF-α ELISA MaxTM S anda d Ki (Biolegend) was used o de e mine he TNF-α le el acco ding o he manu ac u e ’s manual. Th ee di e en biological eplica es we e analyzed and a PBS ea ed g oup se ed as nega i e con ol. Cy okine, chemokine and g ow h ac o de ec ion in supe na an s and se a: Cy okine, chemokine and g ow h ac o concen a ions in supe na an s o 264.7 RAW mac ophages cells (6 hpi) o se a (1.5 hpi, 6 hpi and 24 hpi) we e quan i ied by he Luminex-based mul iplex echnique acco ding o he manu ac u e ’s ins uc ions (Bio-Rad, USA). S anda d cu es and concen a ions we e Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 9 calcula ed wi h Bio-Plex Manage 6.0, he de ec ion sensi i i y o all p o eins was be ween 1 pg/ml and 40 µg/ml. RNA isola ion and sequencing: RNA isola ion was pe o med as desc ibed p e iously 16. Lib a y p epa a ion o plank onic cul u es g own o mid-log phase was done using he Sc ip SeqTM 2 RNA-Seq Lib a y P ep Ki (Illumina) and he endo ’s p o ocol. Sequence eads we e mapped o he genome sequence o he e e ence s ain Salmonella en e ica subsp. en e ica se o a Typhimu ium UK-1 (Genbank, CP002614.1) using bow ie2 28. Di e en ial gene exp ession was calcula ed using obus gene alized linea models and he quasi-likelihood F- es o he R package edgeR 29. Genes we e conside ed di e en ially exp essed i he old change exp ession was signi ican ly g ea e han 2 (edgeR unc ion glmT ea ) wi h a alse-disco e y a e (FDR) cu o o 0.05. Mul idimensional scaling plo s (MDS) we e isualized using ggplo 2 30. Genome sequencing and SNP calling: Genomic DNA was ex ac ed om plank onic o e nigh cul u es using he DNeasy Blood & Tissue ki (Qiagen) and sequenced using Illumina HiSeq. Single nucleo ide polymo phism (SNP) analysis o DNA sequencing da a was ca ied ou using sam ools mpileup and py hon 31. S a is ics: Signi icance be ween wo g oups was de e mined using he nonpa ame ic Mann- Whi ney es , while one-way analysis o a iance (ANOVA) wi h Bon e oni pos es was used o compa e wo o mo e g oups. Signi icance le els o p < 0.05, p < 0.01, o p < 0.001 we e deno ed wi h as e isks: *, **, and ***, espec i ely. Da a a ailabili y: All aw and p ocessed sequencing da a ha e been submi ed o GEO (GSE116623; h ps://www.ncbi.nlm.nih.go /geo/que y/acc.cgi?acc=GSE116623). All aw and p ocessed genome sequencing da a ha e been submi ed o SRA (SRP153811, h ps://www.ncbi.nlm.nih.go /s a/SRP153811). Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 16 ha may be esponsible o his pa icula pheno ype. We employed scanning elec on mic oscopy o sea ch o al e a ions a he cell su ace o bac e ial shape (Figu e 6). In e es ingly, his analysis e ealed he p esence o ou e memb ane esicles (OMV) o SF102 + Δ liF and SF102 + Δ liHIJ mu an s while all he o he mu an s did no exhibi such s uc u es. Impo an ly, he combina ion o all ea u es (e.g. ΔlpxR ΔpagP ΔpagL - Δa oA - Δ liF o Δ liHIJ) was equi ed o p omo e OMV o ma ion (Supplemen a y Figu e S6). Salmonella de i a i es ha lack a leas one o hese p ope ies did no display any OMVs unde ou condi ions. As OMVs include high amoun s o LPS and a e known o con ain lagella p o eins 44, hus, he inc eased immunogenic po en ial may e y well be a ibu ed o hese s uc u es. Ano he possibili y o he obse ed OMV p oduc ion was due o an independen , unin en ional mu a ion ha may ha e accumula ed du ing he s ain cons uc ions. The e o e, we sequenced he genome o he Δ liF mu an and i s pa en al s ains as i exhibi ed he mos p onounced OMV p oduc ion (Table S3). No appa en SNPs o addi ional dele ions we e de ec ed in he SF102 + Δ liF s ain ha may ha e con ibu ed o he OMV pheno ype. The e o e, he genomic o ansc ip ional o igin o he enhanced OMV o ma ion emains unanswe ed. Δ liHIJ and Δ liF mu an s display ad anced an i- umo p ope ies: Nex , we e alua ed whe he he s ongly induced cy okine pa e n and p oduc ion o OMVs co ela ed wi h he he apeu ic e icacy o he lagella mu an s ains in a mu ine umo model. O no e, umo coloniza ion i sel appea s no o be a ec ed by he modi ica ions o he bac e ial lagella as shown p e iously 33 and con i med by pla ing (da a no shown). CT26 umo -bea ing mice we e in ec ed in a enously wi h 5*106 bac e ia and he body weigh changes as indica o o he heal h bu den o he apy as well as he he apeu ic e icacy was assessed (Figu e 7). Upon bac e ial applica ion, all in ec ed mice su i ed he he apy and weigh loss was a ound 10% a mos . This can be conside ed as mino Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 17 (Figu e 7A). The egula o y mu an s SF102 + Δ lgK and SF102 + Δ lP Δ lgM Δ lM appea ed o be highly a enua ed as he body weigh eco e ed e y as upon an ini ial d op. In con as , he s uc u al lagella mu an s induced a pe sis en weigh loss ha only slowly eco e ed a e 6 days. Howe e , hese s uc u al mu an s SF102 + Δ liF and SF102 + Δ liHIJ displayed he mos p onounced an i- umo e ec . The bac e ia clea ed all he CT26 umo s wi hin 8 o 4 days, espec i ely (Figu e 7B and Supplemen a y Figu e S7 o indi idual umo de elopmen ). In addi ion, he pa en al s ain SF102 and i s Δ lhDC de i a i e we e able o clea 80% o he analyzed umo s (5/6). In con as , he hype - lagella ed s ain SF102 + Δ lP Δ lgM Δ lM and he assembly mu an SF102 + Δ lgK appea ed o be o e -a enua ed. The umo -clea ing capaci y was e en lowe han ha o he pa en al SF102 s ain. In summa y, he Δ liHIJ mu an s ain exhibi ed he s onges po en ial o clea CT26 umo s. Howe e , he Δ liF s ain exhibi ed he bes balance o sa e y and e icacy. The mice eco e ed as es om e ec s o he in ec ion when exposed o hese bac e ia. Combina ion o SF102+Δ liF o SF102+Δ liHIJ s ongly a ec RenCa umo s: Al hough he cause o he s ong p oduc ion o OMVs in he SF102 + Δ liF o SF102 + Δ liHIJ s ains emains unknown, hese s uc u es likely con ibu e o he imp o ed immunogenici y. We he e o e wonde ed how s ongly he he apeu ic po ency o hese s ains was imp o ed. Thus, we es ed hese mu an s agains he highly esis an cance cell line RenCa (Figu e 7C). As shown be o e, he LPS mu an s only e a ded he g ow h RenCa sho ly 5. In con as , employing SF102 + Δ liF and SF102 + Δ liHIJ o he apy d ama ically imp o ed he an i- umo esponse and signi ican ly p olonged he su i al o he RenCa bea ing mice. Thus, he unique combina ion o mu a ions and speci ically he in oduc ion o he gene dele ions Δ liF o Δ liHIJ a ec ing lagella assembly and egula ion esul ed in an i-cance s ains o excep ional he apeu ic po ency. Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 18 DISCUSSION Salmonella exe s unique di ec in e ac ions wi h he hos cell ia specialized sec e ion sys ems, e ec o p o eins o PAMPs. Thus, Salmonella displays g ea po en ial as a highly e sa ile a ge ed deli e y sys em o accina ion and cance immune he apies 45–48. Flagella ep esen one o he majo PAMPs o hese bac e ia. Hence, in he p esen s udy we aimed o in es iga e he e ec s o he s uc u e, spa io- empo al egula ion and syn hesis o he bac e ial lagellum du ing hos - pa hogen in e ac ions and in he apy. To co e a b oad ange o lagella pheno ypes, we in es iga ed he beha io o ele en di e en Salmonella a ian s mu a ed in a ious egula o y and s uc u al componen s o he lagellum. Fo ins ance, dele ing he hin ecombinase esponsible o lagellin phase a ia ion in Salmonella allowed o enginee lagellin phase-locked mu an s ha exp essed ei he FliC o FljB 49. In o de o gene a e a mu an ha co-exp essed bo h ilamen p o eins, we addi ionally dele ed he nega i e egula o o liC ansla ion, FljA, in a FljB-ON backg ound 50. Unde hese condi ions, e e y indi idual lagellum p esumably consis s o a mix u e o bo h p o eins. The a ian exhibi ed educed mo ili y al hough he numbe o lagella pe cell was no mal. I migh be possible ha he s uc u al di e ences be ween bo h lagellin ypes dec ease he e iciency o lagella unc ion, e.g. by impai ing ilamen bundle o ma ion. In compa ison o he W , he FliC-ON phase-locked mu an did no exhibi any supe io immunogenic beha io in i o and in i o. In con as , immune ac i a ion by he FljB-locked s ain was inc eased du ing he ea ly s ages o in ec ion. This co ela ed wi h p e ious obse a ions ha Salmonella exp essing FljB exhibi highe adju an po en ial 17,51. This migh also explain why he s ain SF102, published p e iously, ep esen s a highly immunogenic he apeu ic ec o . I p edomina ely exp esses FljB 16. The bene icial e ec o FljB-only exp ession anishes a he la e s ages o in ec ion. Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 19 Salmonella in he mammalian hos is known o down- egula e lagella exp ession o a oid ecogni ion by TLR-5 o caspase-1 13,52. To coun e ac his escape mechanism, we in es iga ed he ole o egula o s o lagella syn hesis: lP, lgM and lM 38,53,54. As expec ed, s ains bea ing dele ions o ei he gene esul ed in a signi ican ly inc eased lagella ion s a us and enhanced lagellin p oduc ion. In e es ingly, his o e p oduc ion a ec ed bac e ial g ow h in i o. Fu he mo e, when exposing he o e p oducing iple mu an Δ lP Δ lgM Δ lM o mac ophages, we de ec ed inc eased le els o LDH in he supe na an s. This sugges ed ha he hype - lagella ion migh induce in acellula caspase-1 esul ing in py op osis and mac ophage cell dea h 55. Bac e ial dea h would consequen ly be he esul om exposu e o gen amycin in he medium. In i o, such lagella-o e p oducing s ains would no be able o hide om immune ecogni ion o es ablish hei in acellula niche. This is consis en wi h in i o e icacy o his s ain. I appea ed o e -a enua ed. The mice eco e ed al eady 4 dpi and he umo clea ing e icacy was only sligh ly abo e 50%. Thus, hype - lagella ion o he Salmonella ec o could ep esen a p omising s a egy o inc ease accine adju an ici y bu may no be a po en s a egy o umo he apeu ic applica ions. As hype - lagella ion did no appea o be bene icial due o he me abolic bu den and/o he ex ensi e ac i a ion o py op osis, we nex in es iga ed he Δ lgK mu an , which is able o sec e e monome ic lagellin, bu is unable o assemble lagella ilamen s 56. In i o, he Δ lgK mu an induced a s ong IL-6 and TNF-α esponse. This may indica e a s ong s imula ion o he TLR-5 ecep o due o he enhanced le els o monome ic lagellin 57. Howe e , his s ong pheno ype disappea ed in i o. Possibly, soluble lagellin was dilu ed o diges ed in he blood o he issue and did no each concen a ions su icien o s imula e TLR-5 o in acellula ecep o s like NOD-like ecep o s o NLRP3. This also co ela ed wi h he low e icacy in he an i- umo esponse. The e o e, a Δ lgK dele ion may no ep esen a good choice when a emp ing o op imize a ec o s ain o he apy. Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 20 Finally, we es ed s ains wi hou ex acellula lagella. We dele ed he gene coding o FliF, which o ms he MS- ing o he lagella basal body in he inne memb ane 37, o FliHIJ, which is an ATPase complex in ol ed in he expo o lagella building blocks 58. Bo h s ains nei he exp essed no sec e ed lagellin and hus we e non-mo ile. In e es ingly, bo h s ains we e highly immunogenic in i o and in i o. In an o he wise W backg ound, he Δ liF mu a ion induced sec e ion o high le els o p o-in lamma o y cy okines like TNF-α o IL-6. Once he mu a ion was ans e ed on o ou immunogenic backg ound s ain UK-1, he Δ liHIJ mu a ion displayed he mos p onounced cy okine esponse. As FliC is an impo an PAMP ha con e s immunogenici y, his inding was su p ising since hese mu an s a e no able o exp ess lagellin and he e o e lack he lagella ilamen . Impo an ly, in ou he apeu ic model he SF102 + Δ liF and SF102 + Δ liHIJ s ains we e able o clea all CT26 umo s wi hin 8 o 4 days, espec i ely. All mice su i ed he he apy. The e o e, hese s ains exhibi an op imal balance o sa e y and e icacy. As pu a i e eason o hei supe io i y, we made he se endipi ous disco e y ha a subs an ial amoun o OMVs was p oduced by hese mu an s. Al hough he mechanism o OMV p oduc ion s ill emains elusi e, we belie e ha hey signi ican ly con ibu e o he supe io he apeu ic e icacy o hese s ains. The inc eased le els o p o-in lamma o y cy okines may de i e om he LPS ha p edomina ely o ms he esicles o he pu a i e ca go o he OMVs ha migh include bac e ial RNA and DNA 59. In addi ion, ecen p o eome s udies ha e shown ha OMVs can ca y lagella p o eins 44. The MS- ing made o FliF and he ATPase complex FliHIJ a e impo an componen s o he lagella expo appa a us ha sec e es lagella building blocks om he in acellula o he ex acellula space. The dele ion o ei he he Class 2 genes liF o liHIJ esul s in de ec i e lagella basal bodies and he e o e accumula e lagella building blocks in he cy oplasm. Thus, he enhanced OMV p oduc ion migh be a s a egy o he cell o emo e excessi e lagella p o eins. This Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 21 hypo hesis is suppo ed by he ac ha no nega i e egula o con olling lagella syn hesis a he le el o Class 2 gene exp ession was ound o be signi ican ly egula ed. Besides hei immune-s imula o y capaci y, he p esence o OMVs could be u he exploi ed as deli e y sys em in u u e s udies 60,61. The e o e, using dele ions o he Class 2 genes liF and liHIJ in combina ion wi h o he immunomodula o y mu a ions like he SF102 backg ound could u n Salmonella in o a e y e ec i e deli e y ec o pla o m. Va ious immunogenic ca gos o he apeu ic agen s may be anspo ed di ec ly in o umo cells o immune cells o he hos . Taken oge he , he co ec spa io empo al egula ion o lagella syn hesis du ing hos -pa hogen in e ac ions exe s di ec impac on he he apeu ic e icacy o Salmonella ec o s ains. While mu an s o e -exp essing lagella we e oo sensi i e o he immune sys em o he hos , he mu a ions ∆ liF and ∆ liHIJ appea ed o be highly e icacious in ou mu ine umo models. Inc eased immunogenic p ope ies we e con e ed by hese mu a ions. Especially he unique p oduc ion o high numbe s o OMVs induced by mu a ions o liF and liHIJ migh allow o enhance in insic he apeu ic ea u es o he bac e ia and ende Salmonella in o an e icacious deli e y pla o m. Such Salmonella-based ec o s migh ep esen po en s ains o cance he apy, which is e idenced by he subs an ial g ow h e a da ion o he e y esilien RenCa umo s, which ha e been highly esis an o his ype o he apy hus a . Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 22 ACKNOWLEDGEMENTS Ou g a i ude is ex ended o Regina Lesch and Nadine Kö ne o hei expe echnical assis ance. Mo eo e , we hank Roy Cu iss III o p o iding pa en al s ains o Salmonella, along wi h expe ad ice conce ning s ain design. The s udy was suppo ed in pa by he Niede sächsische K ebsgesellscha , he Deu sche K ebshil e, he Bundesminis e ium ü Bildung und Fo schung (BMBF) ia he INDIGO p og am, Hanno e Biomedical Resea ch School (HBRS) (all o SW), a Lich enbe g Fellowship om he Niede sächsiche Minis e ium ü Wissenscha und Kul u (MWK) ( o SF), an Explo a ion G an o he Boeh inge Ingelheim Founda ion (BIS) ( o SF and ME) and he Helmhol z Associa ion Young In es iga o g an no. VH-NG-932 ( o ME). AUTHOR CONTRIBUTIONS S.F., I.S., V.P., D.K., C.F. and M.R. pe o med and analyzed he expe imen s. M.P. and S.H. we e esponsible o he sequencing analyses. S.H., S.W. and M.E. p o ided ideas and c i ical commen s. S.F., S.W. and M.E. w o e he manusc ip . S.W. and M.E. di ec ed he p ojec . CONFLICT OF INTEREST The au ho s decla e ha he e is no con lic o in e es . Accep ed A icle This a icle is p o ec ed by copy igh . All igh s ese ed. 23 REFERENCES 1. Bou nazos S, Ra e ch J V. A enua ed Vaccines o Augmen ed Immuni y. 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