N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence through a type IV pili/cAMP-mediated morphology transition

Domain: earth_science
Published: 2025-10-24
Editorial outcome: minor_revision

Abstract
Amicrobeispathogenicwhenitmanagestosurviveinitshostand,often,isabletoproliferate.Thus,virulenceentailscopingwithhostdefensesinparalleltodisseminationand/orattackofthehost.Howthepathogenendurestheattackbyeffectorsoftheimmuneresponseremainsinsufficientlyunderstood.Here,wereportthatplanktonicPseudomonasaeruginosaisnotimmediatelyvirulentinaDrosophilamodelofacuteinfection.Bacteriaundergoamaturationstepcalledpriming,whichisrequiredfortransitiontovirulence.Primedbacteriaswitchtoabacillusshape,onlyinvivo,proliferateandresisttheactionofaspecificcombinationofantimicrobialpeptides.ThisprimingmechanismrequiresaninterplaybetweentwomajoreffectorsofthetypeIVpili(T4P),FimVandVfr,whichenhancelateralcellwallpeptidoglycansynthesis.Interestingly,N-acetyl-muramicacid(NAM)abolishesthevirulenceoftheinjectedbacteria,whichbecomeround,andpreventsthelocalizationoftheT4PhubproteinFimVatbacterialpoles,andcAMPsignaling.Incontrast,N-acetylglucosamine(NAG)counteractstheactionofNAMbypromotingFimVpolarplacement.Infact,NAGaloneacceleratesthespeedofP.aeruginosapriminginaPilJ-dependentmanner.ThissuggeststhattheNAGsensedbymicroorganismsisacommonsignalthatpromotesvirulencethroughamorphologicalswitch,bothinbacteriaandpathogenicdimorphicyeasts.

Introduction
Pseudomonasaeruginosaisaubiquitousopportunisticpathogenabletoinfectavarietyofhostsfromplantstoanimals1andisasignificantthreatintermsofpublichealth2,3.Besidesthemechanismsofantibioticresistance,itisessentialtounderstandallofthefacetsthatmakethisbacteriumahigh-riskpathogen.AnessentialaspectofP.aeruginosa biologyisitsadaptabilitytoavarietyofnaturalandhostenvironments,allowed by some 60 two component sensing systems, four chemosensorysystems,includingtheChpsystemoftypeIVpili(T4P),variousquorum-sensingsystemsandmultiplesecretionsystemsthatallowtherelease of various secreted virulence factors, from siderophores to toxins1,4,5. A prevailing view of P. aeruginosa pathogenicity is thatit functions according to two distinct, antagonistic modes.Planktonic cellsarecytotoxic,inducestronginflammatoryreactions,andcause acute infections. In contrast, following a lifestyle switch, biofilm- formingbacteriaareattheoriginofchronicrefractoryinfectionssuch asthosefoundincysticfibrosispatients6,7.Thisviewoftwopathogenic lifestylesmayneedtobeamendedasstudiesinvestigateP.aeruginosa pathogenicityatthewholeorganismlevelingeneticmodelorganisms. AnillustrationofitsflexibilityintermsofvirulenceprogramsisprovidedbythreetypesofinfectionmodelsinDrosophilamelanogaster8,9.

Inaninjectionparadigminwhichaccesstothebodycavityisprovided bybypassingnaturalphysicalbarriers,P.aeruginosakillsitshostwithin about two days through uncontrolled bacteremia10–12. In contrast, continuouslyingestedP.aeruginosaleadtothedemiseofthefliesin aboutaweek,againmostlycausedbysepticemiaoncethebacteria haveescapedfromthedigestivetract12,13.Finally,inalatentinfection modelinwhichfliesfeedonP.aeruginosaforalimitedperiodoftime, thebacteriumishardlypathogeniceventhoughitmanagestosilently colonize the internal tissues of the host in the absence of biofilm formation9.

Onepotentialcluetoaccountforthisstrikingdifferenceinvirulence,according to the infection model,may be the toxin-antitoxin (TA)systemsidentifiedsofarinP.aeruginosa14.Indeed,TAsystems maybeinvolvedinpersistence,abacterialtolerancestrategyduring antibioticstreatment15,16.Interestingly,wehaverecentlyreportedthat the latent infection model evokes someof the features of persister cells9.Thehostinhibitionofgrowth(higBA)operonisthebeststudied TAsysteminP.aeruginosasofar17.TheRNaseactivityoftheHigBtoxin isinhibitedbythedirectbindingoftheHigAantitoxin.HigA,inaddition,isalsoabletobindtoDNAandfunctionsasarepressor,notonly ofhigBAitselfbutalsointhepromotersofgenesinvolvedinvirulence suchasmvfRortype3and6secretionsystems18–20.higAexpressionis alsodeterminedindependentlyofthatofthehigBAoperonthroughan internal promoter in the higB locus18. Even though the HigB toxin reducestheproductionofseveralvirulencefactorssuchaspyochelin, pyocyanin,swarmingabilityandbiofilmformation,higAmutantshave beenreportedtobemorevirulentthanwild-typePA14wheninjected intoGalleriamellonellalarvae17,20,21.

Here,wereportthatP.aeruginosaPAO1isnotimmediatelyvirulent uponinjectionintheDrosophilahostandactuallyfailstotriggeraspecificsystemicinnateimmuneresponsefor24h.Virulenceisacquired throughaprimingprocesswithinthehostthatcanbeelicitedbyco- exposurewithN-acetyl-glucosamine(NAG).Theresultingpathogenicity correlateswithaswitchfromanellipsoidtoabacillusshapeandthe abilitytoenduretheactionofaspecificsetofhostantimicrobialpeptides(AMPs).Wedocumentonthebacterialsideaninvolvementofan interplay between higBA, T4P and its chemosensor PilJ, FimV, cyclic adenosine monophosphate (cAMP) signaling, and peptidoglycan synthesisforcellwallelongationthatisrequiredforthemorphologic transitionandswitchtovirulenceofprimedbacteria.

Results
ThehigAantitoxinmutantissusceptibletoacombinationofspecificAMPfamilies
Because PAO1 is hardly virulent in a recently developed oral latent infection model9, we tested the possibility that this phenomenon mightbemediatedbytheHigBtoxin.Weindeedobservedastrong induction of the operon in the gut as well as a relatively modest decreaseinlifespaninducedbytheΔhigBmutantandamoresizable one by the ΔhigBA deletion mutant (Supplementary Fig. 1a–c). Nevertheless,thecontributionofthisTAsystemismuchlessimportant than that of melanization, a major insect host defense9. Unexpectedly,wenoticedthattheΔhigAmutantwaslessvirulentthanwild-typeorotherhigBAmutantswheningestedbymelanization-deficient flies(SupplementaryFig.1d).Wethereforetestedoursetofmutantsin asepticinjurymodelat18°C,aconditionakintothatusedfortesting melanization mutants in the latent infection model. Strikingly, the ΔhigAmutantwasmuchlessvirulentthanwild-typeorothermutants anddisplayedadecreasedbacterialburdenat48h(Fig.1a,b).This phenotypewasalsoobtainedbydirectlyoverexpressinghigB(Fig.1c, d),whichsuggeststhattheΔhigAphenotypeiscausedbytheunrestrainedtoxin,anexplanationsupportedbytheenhancedvirulenceof theΔhigBAmutant(Fig.1a,b).Importantly,theΔhigBAmutantstrains at18°Cgrewatthesamespeedaswild-typePAO1invitro(SupplementaryFig.1e).

As Gram-negative bacteria infections are counteracted by the ImmuneDeficiency(IMD)armofthesystemicimmuneresponse,we testedwhetherthedecreasedvirulenceofΔhigAresultedfromasensitivitytothehostdefense.Indeed,ΔhigAbacteriaexhibitedanalmost fully recovered virulence in the IMD pathway mutant kenny (key) (Fig.1e,f),aphenomenonalsoobservedtoalesserextentinthelatent infectionmodel(comparesurvivalofΔhigAinSupplementaryFig.1d vs.f).TheinvivoexpressionlevelofhigBinΔhigAmutantbacteriawas similarinwild-typeorkeyflies(SupplementaryFig.2a).Ofnote,even thoughtheTollpathwayhasbeenreportedtoparticipateinthehost defenseagainstP.aeruginosa11,ΔhigAbacteriadidnotrecovertheir virulenceintheTollpathwaymutantMyD88(SupplementaryFig.2b). In keeping with a major roleof the IMD pathway inrestraining the virulence of ΔhigA bacteria, we found that flies missing three categoriesofAMPgenefamilies22,Diptericins,Drosocin/Buletin,andAttacins,whichareregulatedmainlybytheIMDpathway(withthe exceptionofAttacinsthatarealsoco-regulatedbytheTollpathway23), werealsohighlysusceptibletothismutantbacterium(Fig.1g).This was not the case for other combinations of AMP gene families still havingthesethreeAMPgenefamiliesnorfordeletionsofsingleAMP gene families tested (Fig. 1h, Supplementary Fig. 2c–g). These data suggest that the stabilization of the HigB toxin makes the bacteria vulnerabletoacombinationofAMPsbelongingtotwo/threefamilies.

InjectedP.aeruginosaisnotimmediatelyvirulentnordoesittriggerthesystemicimmuneresponse
HavingcheckedthatΔhigAbacteriainducetheexpressionofseveral AMP genes at least as well as wild-type PAO1 (Supplementary Fig.3a–d),wenextmonitoredthekineticsofIMDpathwayinduction usingDiptericinsteady-statetranscriptlevels.WhereastheIMDpathwayisknowntobeinducedwithafastkineticswithin3-6hours,we foundasignificantinductionofDiptericinat24hbutnotat12h,as compared toa controlinjection ofphosphatebuffered saline(PBS) (Fig. 2a), in keeping with a lack of proliferation until at least 18h (Fig. 1b). Interestingly, PGRP-LE senses small peptidoglycan (PGN) fragments released by growing and proliferating Gram-negative bacteria24.ThefindingthatΔhigArecoversitsvirulenceinfliesmissing either IMD pathway receptors PGRP-LC or PGRP-LE that senses bacterialgrowthfurthersupportsalackofbacterialgrowthuntilat least 18h (Supplementary Fig. 3e), although we cannot formally excludeanactiveinhibitionoftheIMDpathwaybyinjectedPAO110.We therefore monitored when the ΔhigA bacteria would become more susceptible to the hostdefense by co-injecting an equalamount of wild-type and ΔhigA bacteria. Whereas the mixture killed the flies slightlyslowerthanPAO1alone(Fig.2b),thestrikingobservationwas thattheΔhigAbacteriagrewatthesamerateaswild-typebacteriauntil 36h, before being killed mostlikely through the action of the IMD pathwaybythe48htimepoint(Fig.2c).Weconcludethatthedelayed proliferationofinjectedPAO1isnotcausedbytheactivationofthe IMDpathwaysinceitbecomeseffectiveonlyfrom36honwards.

PriminginthehostpromotesthevirulenceofP.aeruginosa
Wenextaskedwhethernaïvebacteriaaresusceptibletotheactionofa pre-activatedIMDpathway.Whereasbothwild-typeandΔhigAbacteria displayed a similarly strongly decreased virulence when injected at 29°Cintofliesthatoverexpresstheimdgeneattheadultstage(astrong activationoftheimdpathway;SupplementaryFig.4a),thedecreaseof virulence at 18°C was less pronounced in flies that had been pre- challengedbytheinjectionofheat-killedPAO124hoursearlier,with bacterial proliferation observed at 48 but not 24h (Supplementary Fig. 4b, c). These data taken together with those described in the paragraphaboveimplythatwild-typePAO1injectedintowild-typenaïve fliesbecomeresistanttotheactionoftheIMDpathwayaftersome24- 36hours. Next, we pre-challenged wild-type flies by the injection of some100greenfluorescentprotein(GFP)-labeledPAO1followedbythe injection of some 1000 red fluorescent protein (RFP)-labeled PAO1 24h thereafter,aquantitysimilartothatmeasuredinflies24hafterinjection (Fig.2d;correspondingsurvivalexperimentshowninSupplementary Fig.4d).Strikingly,thetiterofRFPbacteriawassignificantlyreduced within1h(25htimepoint)andthosebacteriawereclearedby6h(30h timepoint)bywild-typebutnotkeyflies(Fig.2e),suggestingthatGFP- PAO1hadadaptedtothehostanditsimmuneresponse,whichwasnot thecaseofthenaïveRFP-bacteria.Next,weretrievedbacteriafroman injected host and injected them into flies pre-challenged with heat- killedPAO124hearlier.Remarkably,theprimedbacteriawereimmediatelyvirulentascomparedtonaïvebacteria,whetherthebacteriahad beenretrievedfromwild-typeorkeyflies(Fig.2f;notethattheabsolute numberofinjectedbacteriadoesnothaveastrongeffectonsurvival curves(SupplementaryFig.4e–f)).Thisexperimentfurtherestablishes that priming is not initiated by an exposure to the IMD immune response.Moreover,retrievedbacteriawerealsomorevirulentwhen injectedinimd-overexpressingflies(Fig.2g).Importantly,theretrieved bacteriastartedtoproliferateshortlyaftertheirinjectioninwild-type flies(Fig.2h).Moreover,theΔhigAretrievedfromwildtypefliesat24hpi couldnotkillnaivefliesasfastasthoseretrievedfromkeyflies(SupplementaryFig.4gincontrasttoFig.2f).

Weconcludethatprimingfirstallowsthebacteriatoproliferate activelyandsecondallowsthemtowithstandtheactionoftheIMD pathway,likelybyresistingthecombinedantimicrobialactionofthe two/threeAMPfamilies.

Primingcorrelateswithamorphologicalswitch
WehadnotedinanearlierstudythatPAO1harboranellipsoidshape whengrowninvitro(inLuria-Bertani(LB)orbrain-heartinfusion(BHI) medium)andthatbacteriaretrievedinitiallyfromthehemolymphin the latent infection model display instead a bacillus conformation9. PAO1alsoadoptsarodshapeinwild-typeorkeyhosts48hoursafter injection. Interestingly, we found that ΔhigA mutants displayed a roundedmorphologyinwild-typeandarodoneinkeyflies(Fig.3a-b’). Thetransitionfromellipsoidtorodoccursduringtheprimingperiod: whereasbacteriastillpresentmostlyanellipsoidmorphologyat13h,a mixture of both forms was observed 16h after injection whereas mostlyrodswereobserved24hoursafterinfection,atimeatwhich bacteriastartproliferatingandhavebeenprimed(Fig.3c-c’,Supplementary Fig. 5a-a’). Thus, virulence correlates with a bacterial morphologytransition.

N-acetyl-glucosaminepromotestheprimingofP.aeruginosa andamorphologicalswitch
SinceprimingdoesnotdependonexposuretotheIMDhostdefense, wereasonedthatitmightresultfromanattackbyenzymescirculating inthehemolymphsuchasbacterialorhostlysozymesorchitinases. Pre-exposure of bacteria to lysozyme in vitro for 30minutes in the presenceorabsenceofEthylenediaminetetraaceticacid(EDTA)was sufficienttoincreasethevirulenceofeitherwild-typeorΔhigAbacteria injectedintowild-typeorkeyflies(Fig.3d,SupplementaryFig.5b).The pre-injectionofachitinaseintoflies30minutespriortoachallenge withPAO1similarlyledtoenhancedvirulenceoftheinjectedbacteria (Fig.3e).Ofnote,P.aeruginosaalsoencodesachitinasegenethatis howevernotrequiredforitsvirulenceinPA14bacteriaandwhichis actuallydown-regulatedwhenPAO1bacteriawereinjectedintowild- typeorkeyflies(SupplementaryFig.5c,d)25.Thehydrolaseactivityof bothenzymesreleasesN-acetyl-glucosamine(NAG).Wethereforeco- injected PAO1 and NAG and observed again an enhanced virulence (Fig.3f),whichcorrelateswithanearliertransitiontorodmorphology as most bacteria exhibited it 13h post co-injection (Supplementary Fig.5a-a’). Asexpectedfromaswitchoccurringearlier,theinjectionof NAG24haftertheinitialPAO1challengedidnotalterthevirulenceof PAO1(SupplementaryFig.5e).

AnalternatepossibilityisthatNAGisreleaseddirectlythroughthe metabolismofPGN.Insuchcase,thedigestionofPGNwillinitiallyyield bothNAGandN-acetylmuramicacid(NAM).Whenweco-injectedPAO1 andanequimolarmixtureofNAGandNAM,wedetectedadecreased virulence instead (Fig. 3g), suggesting that PGN hydrolysis may not mediatepriming.Unexpectedly,theco-injectionofPAO1andNAMalone almostabolished thevirulenceofthe bacteriainwild-typehostsand severely decreased it in key flies (Fig. 3h); this striking result further supports the virulence-promoting properties of NAG (compare Fig.3h–g).Again,aninjectionofNAM24haftertheinitialPAO1challengehadnoimpactonthecourseoftheinfection(Supplementary Fig.5f),inkeepingwithalackofmorphologicaltransition24hafterthe co-injection of PAO1 and NAM, which might however, start at 48h (SupplementaryFig.5a-a).Ofnote,thecultureofPAO1witheitherNAM orNAGdidnotaltermuchthegrowthofPAO1invitro,andinanycase, wasclearlydistinctfrominvivogrowth(SupplementaryFig.5g)notdid itimpactthevirulenceofthepurifiedinjectedbacteria(Supplementary Fig.5h-i).NAMandNAGdidalsonotinhibitthelysozymeantimicrobial activityagainstMicrococcusluteusinvitro(SupplementaryFig.5j).

WeconcludethatNAGlikelyoriginatingfromthehostpromotes the transition to a bacillus morphology, which correlates with virulence.

AproteomicanalysisrevealsaninvolvementofthetypeIVpili systeminmediatingtheeffectsofthehigBAoperon
WhereasΔhigAbacteriaretrievedfromkeymutantsrapidlykillwild- typefliespre-challengedwithheat-killedPAO1withinadayandahalf (Fig.2f),thesebacteriaretrievedfromwild-typehostsandinjectedin wild-typeflieskilledthemonlyslightlyfasterthannaïveΔhigAbacteria (SupplementaryFig.4g).ThisobservationsuggeststhatΔhigAbacteria maybepoorlyprimedwhenexposedtotheactionoftheIMDpathway, likelybyresistingthecombinedantimicrobialactionofthe two/threeAMPfamilies.

Inkeepingwiththeirellipsoidshape.Tobetterunderstandtheroleof the higBA operon, we assessed the quantitative expression of PAO1 proteinsinΔhigA,ΔhigB,andΔhigBAbacteriarelativetothosefoundin wild-typebacteriaculturedinvitro(Fig.4a,SupplementaryFig.6aand SupplementaryData1).Proteinsbelongingtothetypethreesecretion system (T3SS) and to the phenazine biosynthesis operon were significantlyrepressedinΔhigAmutantsandrespectivelyappearedtobe eithersomewhatinducedorunalteredinΔhigBorΔhigBAmutants. Thus,the HigA antitoxin appearsto promote the expression of the T3SS.TheectopicexpressionofexsA,whichencodesamajorregulator of T3SS operon expression26, did not rescue the lessened virulence phenotypeofinjectedΔhigA,suggestingthattheT3SSmaynotbea majorcontributortothesusceptibilityofΔhigAmutantstotheIMD response (Supplementary Fig. 6b)12. Indeed, ΔhigB and ΔhigBA are equallyvirulent in the injection model despite the inductionof the T3SSintheformerandnotthelatter(Fig.1a).Likewise,phenazinesare alsounlikelytocontributetopathogenesisinthesepticinjurymodel since ΔhigB and ΔhigBA mutants are more virulent than wild-type bacteriaintheinjectionmodeldespitetheirdecreasedexpressionof phenazinegenesobservedinvitro(Fig.1a).

Incontrast,mostgenesrequiredfortheformationoftypeIVpili (T4P)areexpressedinΔhigBandΔhigBAmutantsatlevelssimilarto thoseencounteredinwild-typebacteriabutareclearlyrepressedin theΔhigAmutants,especiallythepilQandfimVgenewhichrespec- tivelyencodethetypeIVpilinsecretinchannelembeddedintheouter membrane of the bacteria and a PGN-binding protein required for assemblyofthesecretinchannel27.Unexpectedly,thedecreasedpro- teinexpressionofT4Pcomponentswasnotmirroredatthetranscript level(SupplementaryFig.6c),eventhoughtheHigBtoxinisthoughtto destabilizemRNAs28–30.

TheT4PisrequiredfortwitchingmotilityandhigAisalsorequired fortwitchingmotility(SupplementaryFig.6d),furthersupportinga linkbetweenthehigBAoperonandtheT4P.pilJencodesthechemo- sensorofthePil/Chpchemosensingsystem4,31.D’Argenioetal.showed that it is chemo-sensation and not twitching motility per se that is importantforP.aeruginosavirulenceinDrosophilasince24mutants affectingtwitchingmotilitystilldisplayedanormalvirulenceinflies32. WeconfirmedthattheΔpilJmutantwaslessvirulentthanwild-type PAO1(Fig.4b).However,whereasD’Argenioetal.32hadnotobserveda difference in the bacterial burden between wild-type and mutants affectingthepilGHIJKLchpABCDEoperonatthetimepointsexamined, upto18hours,weobservedaprogressivelydecreasingΔpilJbacterial loadfrom24hours onward in the collected hemolymph and from 36hoursonwardinwholeflies.Theseobservationssuggestthatlike ΔhigA mutants, ΔpilJ mutants are sensitive to the fly’s immune response(SupplementaryFig.6e,f).Indeed,ΔpilJmutantsrecovered fullvirulencewhen injected into key and group B mutants (Fig. 4b, SupplementaryFig.6g).WenextoverexpressedpilJinaΔhigAmutant background and significantly rescued the lessened virulence ΔhigA phenotypeinwild-typeflies(Fig.4c).However,thevirulenceofthepilJ- rescued ΔhigA bacteria was somewhat reduced relative to that of ΔhigAbacteriainkeymutantflies,revealingamodestlyimpairedfitnessofthesebacteria,atleastinvivo.

ThecAMP-VfrpathwaybutnottheT4PitselfisimportantforP. aeruginosavirulence
PilJ,togetherwithotherChpproteins,activatesasignalingpathway that regulates the activity of the CyaB cAMP cyclase. cAMP levels controlbacterialcellmorphologyandalsoactivatetheVfrtranscription factor, which regulates the expression of several virulence genes4,31,33–36.Theoverexpressionoftheadenylate-cyclasecodinggene cyaBpartiallyrescuedtheimpairedvirulenceofΔhigAmutants,even though this overexpressionslightlyimpairsthefitnessofthesebacteria (in key flies) (Fig. 4d), as was the case for pilJ overexpression. cAMP levels activate the Vfr transcription factor. Interestingly, we foundvfrexpressiontobeinducedinvivosome40hafterinjection (SupplementaryFig.7a).TofurtherassesstheroleofVfr,wegenerated a deletion mutant and found it to display a reduced virulence equivalent to that exhibited by ΔpilJ mutants with respect to both survivalandbacterialload(Fig.4e,f).However,itsoverexpressiondid not appear to rescue the ΔpilJ mutant phenotype. Conversely, pilJ overexpressionalsodidnotrescuetheΔvfrmutantphenotype(SupplementaryFig.7b).ThemorphologyofΔvfrmutantsresembledthat ofΔhigAmutantinjectedintowild-typeflies(Fig.4g-g),albeitincontrasttothelatter(Fig.3b-b),itdidnotbecomerod-likeinkeymutants. ThismayperhapsreflectarequirementforVfrfunctioninlateralcell wallelongation(thereisstillsomePilJmadeinΔhigAmutants).

TheT4Pisinvolvedinsurfacesensingandretainstheinformation that it has been in contact with a surface over several bacterial generations37–42.WethereforeaskedwhetherexposureofPAO1toa surfaceinvitrowouldmimictheeffectofprimingasinjectedbacteria may temporarily become associated to the surface of host tissues. Eventhoughthecontactwith1.5%agarosedidinducetheexpression ofthexphAgene(SupplementaryFig.7c),thisgenedidnotappearto beinducedinvivo(SupplementaryFig.7d).Importantly,bacteriathat had been pre-exposed to this surface did not display an enhanced virulence when compared to naïve in vitro-grown bacteria (SupplementaryFig.7e).Thus,primingisnotrelatedtothesensingofsurfaces, as further underscored by the near normal virulence of mutant bacteriawithadefectiveT4P(ΔpilA,ΔpilC),asreportedpreviously32 (Fig.4eandSupplementaryFig.7f).

ThePilJchemosensorisrequiredfortheprimingofP. aeruginosabyNAG
Whereasthevirulenceofnaïvewild-typebacteriaisenhancedcloseto thelevelofprimedbacteriauponco-injectionwithNAGintowild-type flies(Fig.3f),wedidnotdetectanychangeinthesurvivalrateofwild- typefliesinjectedwithΔpilJbacteriainthepresenceorabsenceofNAG (Fig.5a).Ofnote,thevirulenceofΔhigAbacteriawasstillsomewhat increased when co-injected with NAG (Fig. 3f), in keeping with a decreased but not abolished expression of PilJ in these mutants (Fig.4a).TheshapeofΔpilJbacteriaremainedellipsoid20hoursafter infectionofeitherwild-typeorkeyflies.Theco-injectionofNAGalso didnotalterthemorphologyoftheinjectedΔpilJbacteria(Fig.5b-b).

Previously,PilJhasbeendocumentedtoberequiredforsensing phenol soluble modulins emitted by Staphylococcus aureus, albeit directbindingofthesepeptidestoPilJhasnotbeenyetdocumented43. A thermal shift assay has been employed to demonstrate a direct bindingofthePilJdomainoftheMcpNchemoreceptortonitrate44.To directly test the hypothesis of a direct binding of PilJ to NAG, we expressedeitherrecombinantfulllengthPilJorafragmentcontaining thetandemligand-bindingdomains(LBD)45(SupplementaryFig.8a,b). As the former is an integral membrane protein, it was found to be unstablewhenthetemperaturewasincreasedto32°Canditsbehavior wasnotchangedbyco-incubationwitheitherNAGorNAM(Supplementary Fig. 8c). As shown in Fig. 5c, the addition of NAG did not influence the thermal shift of the LBD domains. Finally, we used a geneticapproachtoassesstheimportanceoftheLBDdomainsofPilJ. Theoverexpressionoffull-lengthpilJrescuedtheΔpilJmutantphenotype (Fig. 5d). The overexpression of the sole PilJ LBD domains mildly but nevertheless significantly rescued the virulence of ΔpilJ mutants.Unexpectedly,theoverexpressionofapilJgenelackingthe sequencescodingthetandemLBDdomainswasabletofullyrescuethe virulenceofΔpilJmutants,suggestingthattheLBDdomainsarenot essentialforthefunctionofPilJintheprimingprocess(Fig.5d).Thus,NAGmayinteractdirectlyorindirectlythroughotherdomainsofthe PilJprotein,whichdoesnotfunctionasacanonicalchemosensorof this metabolite. To confirmthis finding, we co-injected in wild-type andkeyfliesthepilJmutantscarryingaplasmidthatoverexpressesthe truncated pilJ gene lacking the tandem LBD coding sequences and NAG.Thistreatmentrestoredvirulence(Fig.5e),incontrasttotheΔpilJ mutants exposed to NAG in vivo (Fig. 5a). We next measured the transcriptionlevelofpilA,akeyreadoutgeneforcAMP-Vfrsignaling46, under thesedifferentin vivoconditions.Whereasthe cAMP-Vfrsignaling pathway was activated in wild-type PAO1 by 4hours post- injection,itremainedinactiveinpilJmutants(Fig.5f).Ofnote,NAG supplementationdidnotinfluencetheinductionofpilAexpression, suggestingthatNAGmayinfluenceaPilJ-dependentmechanismthatis distinctfromtheactivationofthecAMP-Vfrpathway.

Incontrast,theco-injectionofNAMstillpotentlydecreasedthe virulenceofpilJmutants(Fig.5g).Unfortunately,theco-incubationof the LBD domains with NAM led to the denaturation of the LBD domains(Fig.5h),foreclosinganyinterpretation.

Interestingly,theNAMtreatment,withorwithouttheadditionof NAG,preventedtheactivationofthecAMP-VfrpathwayinbothPAO1 and pilJ mutants, as monitored by pilA transcript levels 10h after injection thatwereexpressedonlyatverylowlevels(Fig.5i).Nevertheless,theco-injectionofNAGwithNAMdidrescuethevirulenceof PAO1 after 48h (Fig. 5j; Supplementary Fig8d).By monitoring pilA expressionat48hpost-challenge,wefoundthatthecAMP-VfrpathwaydidultimatelygetactivateddespitethepresenceofNAM,inapilJ- dependentmanner(Fig.5k).WeconcludethatNAMtreatmentinhibits orpreventstheactivationofthecAMP-Vfrpathwayduringtheearly stages of the infection, when priming occurs, by an as yet uncharacterizedmechanism.WhileNAMtreatmentalonepreventsaswitchto virulenceforatleastfivedays(Fig.5g),itis,however,abletocounteracttheeffectofNAGonPilJonlyforaperiodlimitedtotheearly stagesoftheinfection.Altogether,thesedatasuggestthatNAGpromotestheswitchtovirulenceindependentlyoftheactivationofthe cAMP-Vfrpathway.

fimVfunctionsdownstreamofhigBA
Asnotedearlier,FimVlevelsarestronglyreducedinΔhigA.TheFimV PGN-bindingproteinisassociatedwiththeT4PandfimVmutantsare moresusceptibletoinjectedPAO132.Incontrasttovfr(Supplementary Fig. 9a), the overexpression of fimV did markedlyrescue the ΔhigA mutantphenotype,actuallybetterthancyaBoverexpression(Fig.4d), even though the higA bacteria overexpressing fimV displayed a somewhatreducedfitnessasobservedinvivowheninjectedintokey mutants(Fig.6a).fimVhasbeenreportedtobegeneticallyupstreamof cyaB;thus,thestrongerintensityofitsrescueofΔhigA,ascomparedto cyaBoverexpression,maymirroracyaB-independentfunctionoffimV, inkeepingwithitsproposedcAMP-independentfunction47.However, fimVoverexpressionfailedtorescueeithertheΔpilJortheΔvfrmutant phenotypes(SupplementaryFig.9b)whereasitdidrescuetheΔhigA phenotype(Fig.6a).Thismayreflectagainthefactthatthereisstill somePilJmadeintheΔhigAmutantsandthatthecAMPpathwaymay still be partially activated in these mutants, as displayed schematicallyinSupplementaryFig.9c.

ThevirulenceofPAO1dependsonFimVpolarlocalization
The restored virulence of ΔhigA by fimV overexpression correlated withabacillusshape(Fig.6b-b’).AsFimVplaysaroleinpositioningthe T4Ptothesiteoffuturecelldivision48,whichwillbecomethepolesof the two new bacterial cells, we investigated its subcellular location withinthebacterialcellbyoverexpressingaGFP-taggedform,whichis functional sincebacteriaweremore elongated (mean of4µm) than wild-type PAO1 (mean of 3µm; compare Fig. 3b-b’ to Fig. 6c, see below).Strikingly,uponitsoverexpression,whereastaggedFimVwas founddistributedratherevenlythroughoutthecellinternalsurfacein 60%ofcellsgrowninBHBfor16hours,itwasfoundtobeenrichedat thepolesofallbacteriainjectedintowild-typeflies24hafterchallenge (Fig.6c),whichsuggeststhatbacterialpoleorganizationchangesasa resultofpriming.Actually,thesebacteriawerehomogenouslylonger in vivo than in vitro, for which size varied extensively (Fig. 6c, see below). The FimV protein contains several identified domains, includingaLysM,acoiled-coil(CC),andacytoplasmicdomain(CD) (Fig.6d)27.TheoverexpressionofconstructsencodingtruncatedFimV proteinsinΔhigAmutantsrevealedthattheLysMdomainislargely dispensable for the rescue, in contrast to the CC and CD domains (Fig.6e).TheexpressionofGFP-taggedFimVmissingeithertheCC(N- terminaltag)orCD(C-terminaltag)domainsrevealedthatthereisstill anenrichmentatthepolarpoleswhentheCDdomainismissingbuta verylimitedonewhentheCCdomainismissing(Fig.6f,SupplementaryFig.9d),suggestingthatthelatterisrequiredforpolarlocalization ofFimV.Indeed,themorphologyofbacteriaoverexpressingtheCC deletedfimVconstructremainedellipsoid(SupplementaryFig.9d-d”’). TheCDdomainissolelyrequiredforextensivelateralcellwallpeptidoglycan synthesis since the bacteria are shorter than when overexpressing full fimV yet retain a rod shape (Fig. 6f’, Supplementary Fig.9d-d”’). Ofnote,apolardistributionofFimVisalsolikelyrequired fortheelongationofthebacteriaasthebacteriaoverexpressingFimV lackingtheCCdomainappearedtobemoreroundishthanelongated (Supplementary Fig. 9d-d”’). The overexpression of the CC or CD deletionmutantgenesimpairedthevirulenceoftheoverexpressing bacteriawheninjectedintowild-typeflies,albeitthetaggeddeletion protein mutants weresomewhat deleterious asthe virulenceof the correspondingbacteriainkeymutantswasmildlyimpaired(SupplementaryFig.9e).

Since both NAM and NAG influence the virulence of injected PAO1 in a cAMP/Vfr independent manner, we next asked whether theymightimpactthelocalizationofFimVtothepolesofbacteria. WefirstinvestigatedthisissueinfimV-overexpressingbacteriaculturedinvitro.TheexposuretoNAGsignificantlyincreasedthelength of bacteria as monitored six hours after culture (Supplementary Fig.9f-f’). Strikingly,theinitialpolarlocalizationofFimVobservedat sixhourswaslostat48hours(SupplementaryFig.9g-g’),inkeeping withtheintermediatesituationwhenweinjectwild-typePAO1after overnightculture(60%ofbacteriahavelostthepolarlocalizationof FimV).Furthermore,whereasuntreatedcellshadkeptarodshape, theNAM-exposedbacteriaappearedtoberoundish(Supplementary Fig. 9g). In vivo, there was no difference between control-injected fimV-overexpressingbacteriaandthoseco-injectedwithNAGat10h (Fig. 6g-g”). However, the examination of bacteria retrieved from hemolymphtwohourspost-injectionrevealedthatFimVenrichment atthebacterialpoleswassignificantlyincreasedinbacteriathathad beenco-injectedwithNAGascomparedtocontrols(Supplementary Fig.9h-h’).Importantly,thisfindingmayaccountforthefasterswitch tovirulenceobservedinPAO1co-injectedwithNAG.Strikingly,those co-injectedwithNAMhadnoenrichedlocalizationandwereactually roundishwithlittlepolarityleft(Fig.6g-g”).

ThesedatademonstratethatNAMinterfereswiththelocalization ofFimVtotheprospectivepolesandtherebyaffectsthemorphology ofPAO1.

ThevirulenceofPAO1correlateswithanelongatedmorphology
Theresultsaboveunderscoretheimportanceofbacterialshapeand length in virulence. The passage from ellipsoid to bacillus shape involvesbothcellelongationandcelldivisionsincethereisacompetitionforPGNsynthesisforlateralcellwallandseptumsynthesis49–51. Wethereforetestedwhethertheoverexpressionofgenesrequiredfor celldivisionorPGNsynthesismightrescuetheΔhigAimpairedvirulencephenotype.Theoverexpressionofgenesencodingsomecomponentsofthedivisome(zipA,ftsQ,andftsW)orgenesrequiredforcell wall elongation (rodA,mreD,and ftsW) didnotalter the ΔhigAphenotype(Fig.6h).Themorphologyofinvitro-culturedΔhigAbacteria overexpressingzipAwaschangedfromellipsoidtorodorfilaments, suggestinganimpactoncelldivision/septationandlateralwallelongation whereas this change was not observed upon ftsW overexpression (Supplementary Fig. 10a). However, both ΔhigA bacteria overexpressingeitherzipAorftsWacquiredarounded,apolar,rather thananellipsoidmorphologywheninjected(SupplementaryFig.10a), inkeepingwiththeirlowvirulence(Table1).Incontrast,thevirulence of ΔhigA bacteria was somewhatincreased when someother genes involvedinPGNsynthesislinkedtocelldivisionwereoverexpressedin this background (ftsI, ftsL, and ftsE/ftsX) (Supplementary Fig. 10b). However,thevirulenceofΔhigAbacteriawasfurtherdecreasedwhen thegenesencodingdivisomecomponentsFtsZ(themajorstructural componentoftheZ-ringthatdrivesdivisionandalimitingfactorfor thisprocess),FtsA,orFtsKwereoverexpressed,almosttoavirulencein the case of ftsZ overexpression, even though their fitness was not impaired,asdocumentedbytheirfullvirulenceinkeymutants(SupplementaryFig.10c).Thus,thisobservationestablishesthatinterfering with cell division through ftsZ overexpression52 makes the ΔhigA mutantsmoresusceptibletotheactionofIMDpathwayeffectors.This correlatesagainwitharoundedmorphologyofthesebacteriainwild- typeflies(SupplementaryFig.10a,Table1).

AllofthegenestestedbyoverexpressioninΔhigAmutantsthat either do not alter its virulence or further decrease it displayed a reduced virulence when overexpressed in wild-type PAO1 (Fig. 6i, SupplementaryFig.10d,eandTable1).Ontheonehand,theoverexpression of ftsZ and ftsA in wild-type bacteria led to only a mild reductioninvirulenceanddidnotimpairtheirfitnessinvivo,asthey wereabletoefficientlykillkeyimmunodeficientflies(Supplementary Fig. 10d). Of note, the strong overexpression of ftsZ in wild-type Escherichiacolibacteriahasbeenreportedtoblockcelldivisionandto leadtofilamentation52,inkeepingwithourinvitrodata(SupplementaryFig.10a,Table1).Amilderyetsimilarfilamentationphenotypewas observeduponftsAoverexpression.Interestingly,thesebacteriadisplayed variable morphologies in vivo, fromellipsoid to filamentous (Supplementary Fig. 10a, Table 1). On the other hand, the overexpression of zipA, rodA, mreD, ftsQ, ftsK, or ftsW led to a reduced virulenceinwild-typeflies,whichwasonlypartiallyaccountedforby theirreducedfitness,asmonitored inkeymutants(Fig.6h,SupplementaryFig.10e).Thereducedvirulencewasmoremarkedinthecase ofzipAandmreDoverexpression.Incontrast,theoverexpressionin wild-typePAO1oftheftsE,ftsI,ftsL,orftsXgenes,whichslightlyrescuedthevirulencedefectofΔhigA(SupplementaryFig.10b),hardly alteredthevirulenceofthesebacteria(SupplementaryFig.10f)even though the genes were indeed overexpressed (Supplementary Fig.10g).Strikingly,bacteriaoverexpressingzipAorftsWformedfilaments in vitro, displayed in contrast a rather rounded to ellipsoid morphologywheninjectedintowild-typeflies,yetwererod-shaped wheninjectedintokeyflies(Fig.6j-j’).Thus,themorphologystrongly differsbetweenbacteriainvitroandinvivoandanellipsoid/rounded shape correlates with a moderate or strongly attenuated virulence. Importantly,theoverexpressionofthesedivisomecomponentsmakes them susceptible to the action of the IMD-dependent AMPs that actuallypreventtheassociatedelongationobservedinvitroorinkey mutants.Ofnote,theoverexpressionofeitherftsZ,rodA,zipA,mreD, ftsAorftsIdoesnotappeartoimpairtheproliferation/growthofbacteriaculturedinvitro(SupplementaryFig.10h).

Weconcludethatbacterialshapecorrelateswithvirulenceinits ability to resist the action of the IMD pathway and that AMPs may interferewithcomponentsofthedivisome.

Methods
AlistofstrainsandresourcesisavailableinSupplementaryData2.

Ethicalstatement
Mice experiments were performed under the guidance of the local South China Agricultural University ethics committee (agreement number2019C013).

Drosophilaandmousemodels
MatedfemaleflieshavebeenusedforallDrosophilaexperiments.The wild type fly strain used throughout the experiments is w[A5001], which was generated from re-isogenized w1118 flies77. key, PGRP-LE, PGRP-LC,MyD88fliesarekeptinourlaboratory78,79.w[A5001]isthe isogenic control flies for the key, MyD88,and PGRP-LC mutant flies. Someoftheflymutantstocksusedinthisstudyhavebeengenerated inaw1118fliesbackground80.PPO1Δ-PPO2ΔandtheseriesofAMPgene- deficientflieswerekindgiftsfromBrunoLemaitre22,81.pubi-Gal4-ptub- Gal80tswereconstructedfromstocksobtainedfromtheBloomington centerstock.UpstreamActivatingSequence(UAS)-imdflieshavebeen describedbyGeorgeletal.82Seven-eight-week-oldfemaleSPFC57BL/ 6JmicewereboughtfromJacksonLaboratories(BarHarbor,ME)and raisedintheGuangdongMedicalLaboratoryAnimalCenterunderthe following conditions: Specific Pathogen-Free (SPF) conditions; mice werehousedat4~5percagewitha12-hourlight/darkcycle,andat 25±1°Cwith40~70%relativehumidity.

Bacteriastrainsandcultureconditions
ThewildtypeP.aeruginosastrainusedinthisstudyisthelaboratory referencestrainPAO1.Mutantsusedherehavebeenconstructedusing thesuicidevectorpEX18AP18basedonhomologousrecombination83. DetailsforeachmutantareshowninSupplementaryData2.Plasmids foroverexpressionofcyaB,exsA,fimV,ftsZ,ftsA,ftsE,ftsI,ftsK,ftsL,ftsQ, ftsW,ftsX,higB,higA,higBA,mreD,pilJ,rodA,sulA,vfr,andzipAwere constructedbycloningthecodingsequencefromPCR-amplifiedPAO1 genomicDNA(primerssynthesizedbyTsingke,China;Supplementary Data2)intoplasmidpMQ70(ClonExpressMultiSOneStepCloningkit, Vazyme,Nanjing,China)andtransformedintheE.coliDH5αstrainfor selection on LB agar plates with 50μg carbenicillin. Gene overexpressioninP.aeruginosawasinducedby5mML-arabinoseinvitro. For inducing bacterial gene overexpressioninvivo, flies were fed a solutionof10mg/mLarabinoseinsucrosesolutiononedaypriorto theinjectionofbacteriaandkeptonthissolutionforthedurationof theexperiment.Allthebacterialstockswerekeptat−80°C;thestocks were retrieved by smearing on LB agar plate and cultured at 37°C overnight.SinglefreshcolonieswerepickedtoinoculatetheBHIbroth overnightandexponentialphasebacterialcultureswereharvestedby centrifugation.Thebacterialpelletwasthenresuspendedandwashed inphosphate-bufferedsalinetwice.

Acuteinfection
TheharvestedP.aeruginosapelletsweresuspendedinPBStomeasure their optical densities and adjusted to OD600 1.0 in PBS and then diluted 100-fold for injection. 3-7-day-old female adult flies were pickedatrandomforinjection,andthen13.8nLofthebacterialsuspension(about100bacteria,unlessotherwiseindicated)wasinjected intothethoraxofeachflyusingtheNanojectIII(Drummond,3-000- 032,USA).Theinfectedflieswereincubatedat18°Cwith60%humidity inaclimatechamberandcountedevery12hoursuntilfliesdiedout.

Latentinfection
Before infection, 3-7-day-old female adult flies were picked for pre- treatmentbyfeedingthemona100mMsucrosesolutiondepositedon aMilliporepadfor2daysat25°C.TheharvestedP.aeruginosapellets weresuspendedinto100mMsucrosesolutionandtheopticaldensity of the bacterial suspension was adjusted to OD 10.0 in sucrose 600 solutionforthenextinfectionexperiment.Eachtubewith20flieswas exposedto600µLbacterialsolutiononaMilliporefilterandplacedat 18°Cfor2days.Then,theinfectedfliesbyingestionweretransferred toanewtubewith600µLof100mMsucrosesolutionsupplemented with100µggentamicinonaMilliporefilterandkeptfor4daysat18°C inthisvialtokillP.aeruginosacellsinthegutlumen.Next,theflies were transferred to new tubes with a filter soaked with 600µL of 100mM sucrose solution only. The flies were counted at regular intervalsuntilfliesdiedout.

Acutelunginfectioninmice
P.aeruginosastrainsweregrownonBHBforwithshaking(220r.p.m.)at 37°Cfor16~18h,thenwashedandsuspendedinPBS,anddilutedto OD6000.1inavolumeof50μL.C57BL/6J(7-to8-week-old)females wereanesthetizedbydiethyletherandwereinfectedbynon-invasive intratrachealinstillationofPAO1attheindicateddose.Groupsofabout 10miceweremonitoredover7daysforsurvivalwhereasbacterialload wasmeasuredasdescribedfurtherbelowinblood,lung,liver,andkidneys36hpost-infection.Animalswerehumanelyeuthanizedwhenthey appearedtobeunderacutedistressandcountedasdiseased.

Bacterialtiterinhemolymph
The empty capillary needle was fixed into the Nanoject III machine (Drummond,3-000-032,USA),thenwasprickedonthethoraxofeach flytocollecthemolymphonthebasisofacapillarityextractioneffect. The collected hemolymph fromeachfly was diluted into 10µL pre- prepared PBS in 1.5mLEppendorf tube. Samples collected abovewere dilutedbyaseriesof10-folddilutionandthenplatedonLBagar.These plateswereputinanincubatorat37°Covernighttocountthecolonyformingunits.

Bacterialtiterinwholeflies
Eachanesthetizedflywasputinto1.5mlEppendorftubewith50ulPBS insideandthencrushedthefliesusingtheMixerMillMM400(Retsch, Germany) at 30Hertz for 10sec. The samples prepared above were dilutedbyaseriesof10-folddilutionandthenplatedonLBagarand incubatedat37°Covernighttocountthecolonyformingunits.

Bacterialtiterinmiceorgans
Blood from anesthetized infected micewascollected by the orbital sinusmethodpriortoeuthanasiaat36hpost-infection;theirorgans were then collected aseptically, weighed, and homogenized in 1mL of PBS. Tissue homogenates were serially diluted and plated on 100µg/mLampicillinLB-agarplatesandcolonyformingunits(CFU) countingwasmade16–18hlater.

RNAextractionandreversetranscriptionforRT-qPCR
RNAextractionandreversetranscriptionwasperformedasdescribed earlier9.GenetranscriptionlevelinfliesandbacterialcellswasmeasuredbyreversequantitativePCRusingaSYBRGreenfluorescentdye. PrimersusedforquantificationweresynthesizedbyTsingke(China) areshown inSupplementary Data 3. The 5x-diluted RTsamples for detectionweremixedwithChamQSYBRqPCRMasterMixaccording tosupplierinstructionsandthenrunonCFX96orCFX384(Biorad, USA).Theexpressionleveloftargetgeneswasnormalizedbythe2-ΔΔCt method against Rpl32 (encoding ribosomal protein 49) transcripts (flies)oroprL/16s(P.aeruginosa)afterhavingcheckedthattheefficienciesoftheprimercouplesweresimilar.ForSupplementaryFig.3, the AMP genes steady-state transcription levels normalized to bacterialloadweremeasuredusingthecarcassofsinglefliesfromwhich hemolymph had been collected to measure the bacterial load. To measurebacterialgenetranscriptionatearlyinfectionstages,upto 10h,ahigherbacterialdose(10^5CFU/fly)hadbeeninoculatedinto flies (Fig. 5f, 5i, 5k).In other cases,bacterialgene transcriptionwas measuredunderstandardizedinfectionconditions.

Co-injectionofPAO1andΔhigAcells
TheovernightculturedPAO1andΔhigAbacterialcellswereharvested, washedanddilutedasforacuteinfection.Thesetwodilutedbacterial cellsweremixed1:1forinjectiontomakesurethatthismixturehasthe sameoverallnumberofbacterialcellsasthePAO1orΔhigAcontrol groups.

Acuteinfectioninimd-overexpressionflies
VirginUAS-imdflieswerecrossedtopubi-Gal4-ptub-Gal80tsmales.The crossedflieswereputat18°Candtheoffspringdevelopedat18°Cinto adultflies.Theimdgenewasoverexpressedbyplacingfliesat29°Cfor at least 5 days before injection procedure. The survival analysis of injectedfliesbyP.aeruginosawasperformedat29°C.

HeatkilledP.aeruginosainjection
TheculturedP.aeruginosawereharvestedandsuspendedinPBStobe adjustedintoOD 10andboiledat95°Cfor30min.Theheat-killed 600 bacteriasolutionwasplatedonLBagartoverifytherenolivebacteria remained. 13.8nL of heat-killed bacteria solution was injected into adultfemaleflies.

Secondaryacuteinfection
The cultured GFP-expressing P. aeruginosa were harvested and suspendedinPBStoanOD600of0.01,and13.8nLofthesuspensionwas injectedintofliesasafirstchallenge.Theflieswereputinaclimate chamberat18°Cfor24hours,thenanRFP-expressingP.aeruginosa suspensionofOD 0.1wasinjectedintotheGFP-bacteriainjection 600 recipient flies. The death of flies was subsequently recorded every 12hoursorfliesweresacrificedtomeasuretheirbacterialburdenat different time points before death. The bacterial colonies were observedunderafluorescencemicroscopetodiscriminatebetween GFP-andRFP-expressingbacteria.

Bacteriaretrievedfromhemolymphforinjection
The empty capillary needle was fixed into the Nanoject III machine (Drummond,3-000-032,USA),thenwasprickedonthethoraxofeach flytocollecthemolymphonthebasisofacapillarityextractioneffect. Theretrievedhemolymphofasingleflywasimmediatelyreinjected intoasinglenaiveflyoraflypre-injectedwithheat-killedbacteria24h earlier.

Cellwalldisruptionbacterialcellsinjection
Theovernight-grownbacterialcellswereharvestedbycentrifugation asdescribedaboveandwashedwithPBStwice.Thebacterialpelletwas suspendedindigestionbuffercontaining100µMEDTA(ornot)and 100ng/mL lysozyme for 30min, then pelleted by centrifugation at 2680g.ThebacterialcellpelletwaswashedinPBStwiceandthenits concentrationwasadjustedinPBSasrequiredpriortoinjection.

NAG/NAMinjectionassay
ForNAG/NAMco-injectionexperiments,theovernightgrownbacterial cells were harvested, washed, and adjusted at OD 0.01PBS con- 600 tainingNAG-NAM,NAG,orNAMsolution(10mg/mL)forimmediate injection.Actually,similarresultswereobtainedwhentheNAGorNAM waspre-injectedintoflies30minpriortotheinjectionofthebacterial solution.ForassessingtheroleofNAG/NAMonprimedbacteria,NAG/ NAMwasinjectedintofliesthathadbeeninjectedwithbacteria24h earlier.ToexaminetheeffectsofNAGandNAMonFimV-EGFPlocalizationinbacteriainvivo,flieswereinoculatedwithahigherbacterial dose(10^5CFU/fly)mixedwitheitherNAGorNAM(Fig.6g).

Chitinaseinjectionexperiment
The bacteria-produced chitinase powder (Macklin, Shanghai,China) wasdissolvedinwaterat25units/mLand13.8nLweretheninjected into flies. These flies were subsequently injected with P. aeruginosa suspensionaboutonehourthereafter.

Lysozymedigestionassay
Theovernightculturedbacteriawereharvestedandresuspendedin PBS,andwerethenmixedifneededwithdifferentdosesofNAG/NAM ortheirmixtureina500µg/mLlysozymesolutionin96-wellplate.The platewasputat37°C.Theturbidityofbacteriawasmeasuredbythe microplatereaderspectrophotometerat600nm.

Transmissionelectronmicroscopeassayandmorphologyassay byO-antigenstaining
Bacteria observation by transmission electron microscope was performedasdescribed9.Briefly,wecollectedhemolymphoffliescontainingbacteria;thesampleswerethen observed and images taken withatransmissionelectronmicroscope,withoutfixation,embedding orsectioning.Themorphologyofbacteriawasalsoobservedinsome experimentsbyfluorescencemicroscopyafterO-antigenstaining.In brief,thehemolymphwithcirculatingbacteriawascollectedusingthe NanojectIIIwithemptyneedlesbasedoncapillarityextraction.The bacteria associated with tissues were collected just by dissecting infectedfliestoobtaintissues.Theflytissuewithadheringbacteria werefixedwith4%paraformaldehydefor30minandthenwashedwith PBSsolutionthreetimes.Thesampleswerefirstlyincubatedwith5% BSA solution for 2h, then incubated with the O5 primary antibody (Biorbyt, orb234239) diluted 1:1000 in 5% BSA solution for 1h. The sampleswerewashedinPBSfor3times,andthenincubatedwiththe DyLight™−488labeledfluorescentsecondaryantibody(ThermoScientific,35502)diluted1:5000raisedagainstmiceIgfor1h.Thesamples were washed in PBS three times and then observed under fluorescencemicroscopy.Thelengthofbacterialcellswasquantified using Microbe J. Of note, in some cases to improve the quality of pictures,bacteriawerenotdirectlyphotographedinthetissuesbuton collectedhemolymph,asindicated.Wehave,however,firstchecked thattheshapeofbacteriawasqualitativelyandquantitativelysimilarin tissuesandincollectedhemolymph,albeitwithlowersamplinginthe tissues.Fluorescence images were acquired ona ZEISSAxioImager M2system(100×oilobjective)using488nmexcitationand525nm emission to observe the 488-conjugated antibody staining bacteria andfluorescencegreenproteinlabeledbacteria.RawfileswereprocessedinZEN.LnkandFiji.

Proteomicanalysis
WildtypePAO1,ΔhigA,ΔhigB,ΔhigBAmutants(eachinthreebiological triplicates)culturedinBHIwereharvestedbycentrifugationatexponential phase and put in dry ice for delivery to the Applied Protein Technologycompany(Shanghai,China).Atotaloftwelvesampleswere lysedusingSDTbuffer(4%SDS,100mMTris-HCl,1mMDTT,pH7.6). ProteinamountwasdeterminedbyBCAassaykit(Beyotime,P0012). Proteins(200μg/sample)weredissolvedin30μlSDTbuffer(4%SDS, 100mMDTT,150mMTris-HClpH8.0),detergentandsmallmolecules inwhichwereremovedbyultrafiltration(Microconunits,10kDacutoff). Cysteineswerealkylatedwith100mMiodoacetamidefollowingbyUA bufferwashes(3×)andNH₄HCO₃washes(2×).Trypsin(BeijingHualishi Biotech,HLSTRY001C)digestion(4μg,37°C/overnight)wasperformed in25mMNH₄HCO₃.Peptidesweredesalted(Empore™SPECartridges C18,bedI.D.7mm,volume3ml,ThermoFisher),vacuum-concentrated, andresuspendedin0.1%formicacid,whichwasdeterminedbyA₂₈₀. Peptides (100μg/sample) were labeled with TMT reagents (Thermo Scientific) following manufacturer protocols. Labeled peptides were fractionatedinto10fractionsusingaHighpHReversed-PhasePeptide FractionationKit(ThermoScientific),thendesaltedandconcentrated. Peptideswereloadedontoareversephasetrapcolumn(ThermoScientificAcclaimPepMap100,100μm*2cm,nanoViperC18)connectedto theC18-reversedphaseanalyticalcolumn(ThermoScientificEasyColumn,10cmlong,75μminnerdiameter,3μmresin)inbufferA(0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrileand0.1%Formicacid)ataflowrateof300nl/mincontrolled byIntelliFlowtechnology.MSdatawereacquiredonaQExactivemass spectrometer(ThermoScientific)indata-dependentmode:Fullscans (300–1800 m/z) at 70,000 resolution (m/z 200), followed by HCD fragmentationofthetop10precursors(17,500resolution,2m/zisolationwidth,30eVcollisionenergy).Automaticgaincontrolwassetto3e6 with 10ms maximum injection time. Raw files were analyzed using the MASCOT engine (Matrix Science, London, UK, v2.2) embedded into Proteome Discoverer 1.4 software for identification and quantitation analysis. Search parameters: Trypsin (max missed cleavages: 2); fixed modifications (TMT (Tandem Mass Tag) 16-plex (N-terminus/K)); variable modifications (TMT 16-plex(Y)); mass tolerances(±20ppm); fragment mass tolerant (0.1Da); database pattern (uniprot_Pseudomonas_aeruginosa_50503_20210715.fasta). The decoy databaseapproachcontrolledforFDR(≤1%atpeptidelevel).Protein quantificationusedmedianratiosofuniquepeptides,andtheminimum peptidelengthfordetectionis6.Proteinswithfoldchangeaboveor below1.2andp-valuelessthan0.05wereidentifiedasbeingdifferentially expressed among mutants and wild type PAO1 group. BioinformaticsanalysisincludedGO/KEGGannotation(Blast2GO),PPInetworks (STRING/Cytoscapev3.2.1),andhierarchicalclustering(Cluster3.0).

Bacterialgrowthassayinvitro
TomeasurethegrowthofthesetofhighBAmutantsascomparedto their proliferation in vivo, the overnight cultures of these bacterial strainswereinoculatedinto30mLBHBsolutioninaconicalflaskand thenplacedat18°C.Samplesweretakenfromthebacterialsolutionat differenttimepointsanddilutedtoaplateforcolonycounting.To investigatetheroleofNAGandNAMinbacterialgrowthinvitro,the overnight culture of PAO1 solution wasdiluted 1000-fold and incubatedwithdifferentdosesofNAG/NAMandculturedin96-wellplates at37°Cfor12hours.Theturbiditywasmeasuredusingamicroplate readerspectrophotometerat600nm.Todeterminetheroleofcell divisiongenesoverexpressiononbacterialgrowthinvitro,theovernight culture of some bacterial strains was inoculated in LB broth (1:100)supplementedwith50μg/mLcarbenicillinin12-wellplatesfor overnight culture at 37°C. The turbidity was measured using a microplate readerspectrophotometerat600nm.Toinvestigatethe effects of NAG and NAM on cell elongation in the FimV-EGFP- overexpressingbacterialstrain,5mg/mLofeachcompoundwassupplemented into the culture medium,and bacterial morphology was examinedatdifferenttimepointspost-inoculationofthecultures.

Twitchingassay
Thisexperimentwasperformedaccordingtothepublishedprotocol84. Inbrief,theovernightbacterialculturewasadjustedto1OD inLB 600 brothand10µLbacterialsolutionwasdepositedonthesurfaceof1% agarplates,whichwereincubated overnightat37°C.Theinterstitial colonywasvisualizedbyfloodingtheplateswiththetwitchingmotility developersolutioncontaining10%glacialaceticacidand50%methanol.

Proteinexpressionandpurification
TheopenreadingframeofthetargetgenePilJanditsligandbinding domain(LBD,residues39-303)45wereamplifiedandclonedintothe protein expression vector pET28a for sequencing. E. coli BL21 cells harbouring recombinant plasmids were cultured in LB broth and inducedbyIPTGattheoptimalconcentrationat30°C.Thebacterial cells were then harvested by centrifugation and resuspended in bindingbufferwith100μg/mllysozymepriortolysisperformedusing anultrasoniccellcrusher.TherecombinantproteinswithHis-tagwere purified according to instruction of ProteinIso® Ni-NTA Resin. The concentrationandpurificationoftheseproteinswerecheckedbySDS- PAGEandCoomassiebrilliantbluestaining.

Thermalshiftassay
Thesaltionswereremovedfromthepurifiedproteinsolutionusing Centriconfilters.Theproteinswereincubatedwithpotentialligands NAG (90μM) or NAM (30μM) in the solution buffer with diluted SYPROOrangeandthentheplatewasreadusingaBio-RadRT-qPCR machineaspreviouslydescribedaccordingtoinstructions85.

Surfacecontactactivationassay
TheovernightculturedPAO1inLBwasharvestedandresuspendedin PBSbeforebeingdepositedonthesurfaceof1.5%agaroseplateand incubatedfor5hours.Thebacteriawerenhenharvestedandchecked eitherfortheinductionofthexphAgeneusingRTqPCRandnormalizedto16SRNAortoperforminfectionexperimentusingplanktonic bacterialcellsascontrols.

Reagentsandsoftware
AllthereagentsandsoftwareusedinthisstudyarelistedinSupplementaryData4.

Statisticalanalysis
Alltheexperimentsinthisstudywereperformedatleastthreetimes unlessmentionedotherwiseandthegraphsinthefigurepanelsdisplay the pooled data, which were analyzed using the Prism Graphpad 6.0 software. The survival curves were analyzed by the Logrank (Mantel-Coxtest)andthebacterialloadsetswereanalyzedaccording totheirdistribution.Kruskal-WallistestwithDunn’spost-hoctestwere usedformultiplecomparisonsasindicatedandtheStudent’st-testwas usedforcomparisonbetweentwogroups.*,p<0.05;**,p<0.01;***, p<0.001;****,p<0.0001.n.s.,notsignificant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors proposed a new concept that “virulence is acquired through a priming process.” using a\nPseudomonas aeruginosa infection model. They claimed that priming can be initiated by exposure to extracellular N-acetyl-\nglucosamine (NAG), which is sensed through the type IV pili (TFP) chemosensor PilJ that activates cAMP signaling. While\nthis story sounds interesting, the conclusion is not very solid based their current experiment settings. I have the following\nmajor points:\nI)The authors claimed that priming first allows the bacteria to proliferate actively and second\nallows them to withstand the\naction of the IMD pathway, likely by resisting the combined antimicrobial action of the two/three AMP families. HigB toxin\nmakes the bacteria vulnerable to a combination of AMPs belonging to two/three families. It is well known that anr and pmr\noperons are induced by AMP treatment in P. aeruginosa, do these operons get induced in vivo? If so, when these operons\nget induced after infection?\nII)The pre-injection of a chitinase into flies minutes prior to a challenge with PAO1 similarly led to enhanced virulence of the\ninjected bacteria.The authors concluded that NAG likely originating from the host promotes the transition to a bacillus\nmorphology, which correlates with virulence. P. aeruginosa quorum sensing is known to regulated chitinase production\n(PMID:11717261). According to this reference “after overnight growth, the majority of the ChiC produced was found\nintracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular\npool of ChiC was largely depleted, and the protein was found in the culture medium. ” Thus it is likely a chitinase-like\nprotease is released from P. aeruginosa slowly and which is able to generate NAG from host. Do QS get inducted during this\ninfection model?\nIII)To better understand the role of the higBA operon, they authors assessed the quantitative expression of PAO1 proteins in\nΔhigA, ΔhigB, and ΔhigBA bacteria relative to those found in wild-type bacteria. However, the authors only did in vitro\nproteomics analysis as documented in their method part “Wild type PAO1, ΔhigA, ΔhigB, ΔhigBA mutants (each in\nbiological triplicates) cultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for\ndelivery to the Applied Protein Technology company (Shanghai, China).” Since the authors are\nable to isolate primed bacteria in vivo after infection, why not comparing the proteome of in vivo isolated primed bacteria with naïve bacteria of the above strains.\nWe all know in vitro and in vivo P. aeruginosa have distinct transcriptome and proteome (e.g. PMID: 35913171).\nVI) The authors suggested that PilJ may be sensing NAG but not NAM. This hypothesis is very weak. Pilus is important for\nbiofilm formation and colonization in vivo of P. aeruginosa. Type VI pili is also required for full level of QS in biofilm mode of\ngrowth. A in-depth biochemical characterization is required to test whether PilJ is able to interact with NAG such as ITC\nexperiment.\nV) The authors next examined many genes in the cAMP signaling pathway and the divisome and elongation. This part is not\nclosed linked to the higBA operon since their proteomics study does not show significant expression changes of these\ngenes. In fact, the authors written in the introduction “HigA in addition is also able to bind to DNA and functions as a\nrepressor, not only of higBA itself but also in the promoters of genes involved in virulence such as mvfR or type 3 and 6\nsecretion systems.” And T3SS and mvfR are both published important for infections in different animal models. Thus, again\nthe authors should perform the proteome of in vivo isolated primed bacteria with naïve bacteria.", "reviewer_2": "I think there are some interesting results in this study, but more experiments and analyses are needed to characterize the\nmorphological change that occurs during infection. There are also several misconceptions about virulence and biofilm\nformation in Pseudomonas aeruginosa that are stated several times throughout the manuscript. Many citations are missing\nthat could better frame results and improve the design of several experiments. Much of the observed data could likely be\ndescribed via surface-dependent cAMP activation and cellular memory.\n-lines 49-53: Both acute virulence and biofilm formation require surface contact and surface sensing via Type IV pili in\nPseudomonas aeruginosa. Surface sensing triggers cAMP activation via type IV pili interaction with the surface leading to\nactivation of the global virulence regulator, Vfr, which is critical for expression of T2SS and T3SS PMID: 26041805,\n37382531, 20494996, and many others. Type IV pili interaction with the surface also triggers c-di-GMP production leading to\nbiofilm formation PMID: 25626906, 34168081. Furthermore, type IV pili are critical virulence factors and allow for motility\nwithin epithelial cells likely triggering cAMP production PMID: 31431558. cAMP activation due to type iv pili surface sensing\nhas also been shown to persist for multiple generations within Pseudomonas aeruginosa and serve as a molecular memory\nof surface contact which explains the continued virulence of cells after being recovered from the first infection PMID:\n29559526. Also, the extent of cAMP signaling relates to the stiffness of the material to which type IV pili are attached to\nPMID: 35561220, which is a maximum when binding to surfaces with a stiffness similar to that of human tissues.\n-For all figures reporting CFU data, please change the y-axis to log10 CFU/ml.\n-Fig1. Instead of adding 1 to your Cfu data to avoid you can state \"below limit of detection\" or\"0 CFU\".\n-lines 106-107: the important control here is that higA and the higB OE strain grow at the same rate as WT. Growth curves\nare typically done via 24-hour growth curves with many time points characterizing each phase of the growth curve, not 5 time\npoints over five days. There is no description of the in vitro growth conditions in the methods.\n-lines 136-138 reference \"a lack of bacterial growth until at least 18h (Fig. S3E),\" but fig S3E is a survival curve of flies and\ndoes not show an increase in bacterial CFU over time which would be indicative of growth.\n-lines 159-166: PMID: 29559526\n-line 167: please explain how \"priming\" is different or distinct from any other typical lag phases in a new growth medium.\n-line 175-180/Fig3A-C: There needs to be some sort of quantification with some sort of image analysis software to conclude\nthat there is any significant difference in cell shape/morphology. Without any sort of quantification and statistical analysis I\nam not comfortable making any sort of conclusion. Reference 9 also lacks any sort of statistical image analysis of the\nmorphological changes of bacteria. Furthermore, if the shape of bacterial cells are to be evaluated, they should all be in the\nsame focal plane.\n-all overexpression experiments should include westerns to show stable overexpression of the protein.\n-Figure S5E: there seems to be a significant difference between Nam_higA strain w/ 0mg/ml and 10mg/ml but there are not\nstatistical tests to tell if anything is different.\n-Fig 4C: please confirm the overexpression of PilJ with a western.\n-Line 271: One of the few ligands that actually bind to the ligand binding domain of PilJ are phenol soluble modulins\nproduced by Staph aureus. To properly test if PilJ is sensing NAG, you should use PilJ with the ligand binding domain\nremoved which has been used in several studies so far: PMID 27190147, 38349934. Without this control you cannot\nconclude that PilJ is sensing NAG and not just transducing a surface signal from type IV pili.\n-line 277: the citations listed do not connect cAMP levels with cell wall remodeling\n-lines 273-287: Even though overexpression of vfr does not compensate virulence it does not mean Vfr does not have a\ndownstream role in virulence given its function. Vfr has a cAMP dependent and cAMP independent regulon PMID:\n20494996. Overexpression of Vfr without an equal activation of cAMP means it is only activating the non-cAMP regulon\nwhich are not virulence genes. Furthermore, Vfr is the only known cAMP binding protein in Pseudomonas with any\nphysiological effect. PMID 38832786. The other mutants indicate Vfr likely plays a role in a cAMP-dependent manner.\n-Fig 5E: There should be quantification of polar localization like using microbeJ or Fiji. FimV appears to be cytoplasmic\nwhen OE in liquid conditions. Surface sensing leads to an increase in the number of type iv pili as well as localization of pili\ncomponents.\n-line 313-316: The data do not support the claim made. These mutations are supposed to make the cell morphology more\nrod like and therefore virulent but in line 308-312 you state that overexpression leads to decreased virulence.\n-Type IV pili interact with surfaces and are able to bind surfaces better with EPS. Perhaps NAG can bind type iv pili and\nmimic a surface that would trigger Pil-Chp, leading to cAMP production, Vfr-cAMP dependent regulon, and increased\nvirulence. This positive feedback loop maintains high levels of cAMP over multiple generations and is a hypothesis worth\ntesting here.\n-line 352: mechanosenseing is not tested in this manuscript so this claim cannot be made.\n-line 355-357: many papers have already shown this to not be true.\n-line 383-387: The claim about bacterial shape and resistance to the IMD pathway is not supported by the last set of\nexperiments in which mutants exhibiting more rod-like morphology are still cleared by IMD.\n-FimV is necessary for cAMP activation via type iv pili and flagellar mediated surface sensing so it makes sense increased\nFimV increases virulence phenotypes. PMID: 31682637", "reviewer_3": "The present work describes the process by which the bacterial pathogen Pseudomonas aeruginosa adapts to the host\nenvironment in the initial stage of the infection. Using Drosophila melanogaster as their acute infection model, this transition\nperiod allows the bacteria to not proliferate, hiding from the host’s immune system, and switching from an ellipsoid shape to\na rod morphology. The work clearly shows a correlation between the bacillus morphology and virulence. Furthermore, the\nauthors also elucidate the probable mechanism, from the extracellular signal required to initiate the transition to the genes\nrelated to bacteria wall remodeling. Overall, the data is solid with some minor concerns over significance in terms of\nextrapolating to mammalian infection models. The authors should address this in more detail in the discussion especially in\nlight of the use of antimicrobial peptides.\nDespite this the work clearly helps expand the understanding about the molecular mechanisms involved in pathogen\nadaptation to the host during different stages of infection, here specifically during the acute stage. This knowledge is crucial\nto understand the process of colonization that results from bacterial infections and for the development of new therapies and\ntreatments targeting bacterial pathogens characterized as multi-drug resistant, such as Pseudomonas aeruginosa.\nThe design and analysis of a wide variety of mutants in P. aeruginosa and on the side of the host (flies) is an innovative\nallowing a mechanistic teasing out of the role of the NAG in remodelling cell morphology and priming of virulence. They\nclearly show the sensing of NAG is a common signal that promotes virulence through morphology changes. However, the\nauthors might consider higher level imaging such as electron microscopy to confirm the morphology experiments as it is\ndifficult to really judge the morphologies. Also, in Fig 3A-C more detail of the conditions and time frame for the images would\nbe helpful to the reader. The priming phase by NAG is mediated by the T4P chemosensing system by activating\ndownstream signaling via cAMP mediated cell wall remodeling. The authors make the case that morphology drives\nvirulence. Overall the technical quality of the manuscript is extremely high and the results consistent with the conclusions.\nAlthough the experiments as performed are of a high quality the text needs to be improved in consistency and clarity.\nSpecifically, I encourage the authors to review the consistency between the names listed in the tables and the text and\nfigures, as some of them do not match or are not shown in the tables. Then, I would recommend including a complete list of\nthe plasmids designed and/or assayed as well as for the bacteria or fly mutants. In addition to just the reference, including a\nbrief description of the constructs, such as the plasmid backbone, inducible or constitutive promoter, gene/pathway affected,\netc, would be recommended and would improve the understanding of the text.\nOverall, this is a rigorous and interesting report on P. aeruginosa priming and adaptation in the D. melanogaster model\nsystem. The results are significant in providing a foundation for the role of priming in an acute infection and open further\navenues of research in other model systems.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Rebuttal letter “N-acetyl-glucosamine primes Pseudomonas aeruginosa for virulence\nthrough a type IV pili/cAMP-mediated morphology transition”\nNature Communications NCOMMS-24-49186\nWe thank the reviewers for their insightful comments that have helped us improve our initial\nmanuscript substantially. We have tried to address their suggestions experimentally whenever\ntechnically possible. A detailed reply to each point is to be found below, with our replies written\nin dark blue.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, the authors proposed a new concept that “virulence is acquired through a\npriming process.” using a Pseudomonas aeruginosa infection model. They claimed that priming\ncan be initiated by exposure to extracellular N-acetyl-glucosamine (NAG), which is sensed\nthrough the type IV pili (TFP) chemosensor PilJ that activates cAMP signaling. While this story\nsounds interesting, the conclusion is not very solid based their current experiment settings. I have\nthe following major points:\nI) The authors claimed that priming first allows the bacteria to proliferate actively and second\nallows them to withstand the action of the IMD pathway, likely by resisting the combined\nantimicrobial action of the two/three AMP families. HigB toxin makes the bacteria vulnerable to a\ncombination of AMPs belonging to two/three families. It is well known that anr and pmr operons\nare induced by AMP treatment in P. aeruginosa, do these operons get induced in vivo? If so,\nwhen these operons get induced after infection?\nWe have measured the expression of these operons by RTqPCR and did not detect any induction\nof these operons in vivo prior to 40 hours (please, see figure below), whereas the bacteria\nbecome resistant by 24h as revealed by the transplantation experiments (Fig. 2F) as we did not\ninclude these data in our manuscript for the sake of clarity.\nII) The pre-injection of a chitinase into flies minutes prior to a challenge with PAO1 similarly led to\nenhanced virulence of the injected bacteria.The authors concluded that NAG likely originating from the host promotes the transition to a bacillus\nmorphology, which correlates with virulence. P. aeruginosa quorum sensing is known to regulated chitinase production\n(PMID:11717261). According to this reference “after overnight growth, the majority of the ChiC produced was found\nintracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular\npool of ChiC was largely depleted, and the protein was found in the culture medium. ” Thus it is likely a chitinase-like protease is released from P. aeruginosa slowly and which is able to generate NAG from host. Do QS get inducted during this infection model?\nWe have addressed this question experimentally and the data are to be found in a novel figure:\nFig. S6. We have generated a chiC mutant: it presents the same virulence as wild-type P.\naeruginosa (Fig. S6A). Furthermore, the expression of the chiC gene at 40h in wild-type or\nkenny (key) flies is actually lower than in vitro grown bacteria and the expression of the gene\nwas not detectable at earlier time points (12, 24, and 36 hours) (Fig. S6C).\nAs regards QS, we did not expect it to be involved in the early infection when priming takes\nplace since we typically inject some 100 bacteria, which do not start proliferating until primed.\nWe have also tested a lasR mutant that did not display any altered virulence as displayed below.\nAs regards mvfR mutants, they behaved also like wild-type as illustrated below.\nIII)To better understand the role of the higBA operon, they authors assessed the quantitative\nexpression of PAO1 proteins in ΔhigA, ΔhigB, and ΔhigBA bacteria relative to those found in\nwild-type bacteria. However, the authors only did in vitro proteomics analysis as documented in\ntheir method part “Wild type PAO1, ΔhigA, ΔhigB, ΔhigBA mutants (each in biological triplicates)\ncultured in BHI were harvested by centrifugation at exponential phase and put in dry ice for\ndelivery to the Applied Protein Technology company (Shanghai, China).” Since the authors are\nable to isolate primed bacteria in vivo after infection, why not comparing the proteome of in vivo\nisolated primed bacteria with naïve bacteria of the above strains. We all know in vitro and in vivo\nP. aeruginosa have distinct transcriptome and proteome (e.g. PMID: 35913171).\nWe agree with the reviewer that the in vitro and in vivo transcriptomes and proteomes may\ndiffer significantly, a statement that also applies to bacterial physiology as illustrated in this\nstudy. However, we faced a technological barrier that results from the limited number of\nbacteria we can retrieve from infected flies during the early phase of the infection. Even upon\ndeath of infected flies, that is when they succumb to bacteremia, we could only collect\n107cfu/dying whole fly (and 105-106 from hemolymph), however, we need at least 1010cfu for\neach sample. It means that we should collect the hemolymph from 10,000 to 100,000 flies: this\nwould be when the bacteria are already proliferating intensely, that is way after the switch to\nvirulence has taken place. Furthermore, we also tried to send samples of flies infected with\nPAO1 injection, however, most of the data correspond to host protein and only very few P.\naeruginosa proteins. We also note that single bacterium transcriptomics as recently reported\nstill needs a substantial number of bacteria, also about 1010cfus (Blattman et al., Nature, 2024).\nIndeed, our own attempts to perform in vivo bacterial transcriptomics analysis in collaboration\nwith the laboratory of Prof. Marvin Whiteley were a failure.\nNevertheless, the findings derived from in vitro proteomics that pinpoint an important role for\nthe T4P downstream of the HigB toxin have been extensively validated in vivo through our\ngenetic analysis with rescue of the impaired virulence of DhigA mutants by pilJ, FimV, or cyaB\noverexpression, as well as the experiments described in the second half of the manuscript.\nVI) The authors suggested that PilJ may be sensing NAG but not NAM. This hypothesis is very\nweak. Pilus is important for biofilm formation and colonization in vivo of P. aeruginosa. Type VI\npili is also required for full level of QS in biofilm mode of growth. A in-depth biochemical\ncharacterization is required to test whether PilJ is able to interact with NAG such as ITC\nexperiment.\nThis issue has also been raised by Reviewer 2 and we agree that is a highly relevant issue. We\nhave therefore performed an extended set of additional experiments to specifically address this\nissue experimentally rather than speculating about potential direct sensing of NAG by PilJ as\nwas the case in our initial manuscript (Fig. 4 K-M; Fig. S8 G-H). Briefly, we now additionally\nshow that the shape of DpilJ mutants remains unchanged in the presence of NAG, in keeping\nwith our survival data that documented that NAG was no longer promoting the virulence of\nbacteria in pilJ mutants (Fig. 4I). We have also tested a direct interaction using the technique\nof Martin-Mora et al. (mBio, 2019) in which these authors had documented a direct interaction\nbetween nitrate and the PilJ domain of the McpN chemosensor. Their data was followed by\nITC analysis to determine the binding constants of the interaction. As we failed to detect a\nthermal shift (Fig. 4L) upon the addition of NAG to a recombinant protein containing the two\nligand binding domains (LBD) of PilJ, we did not perform ITC analysis. We however\nperformed further genetic experiments of complementation using constructs that lacked the PilJ\nLBDs and found that these domains are actually dispensable for virulence. We now conclude\nthat the interaction is likely indirect and this situation is reminiscent of the interaction between\nphosphatidyl ethanolamine and PilJ for directed twitching motility (Kearns, J. Bact, 2001).\nV) The authors next examined many genes in the cAMP signaling pathway and the divisome and\nelongation. This part is not closed linked to the higBA operon since their proteomics study does not\nshow significant expression changes of these genes. In fact, the authors written in the introduction\n“HigA in addition is also able to bind to DNA and functions as a repressor, not only of higBA itself\nbut also in the promoters of genes involved in virulence such as mvfR or type 3 and 6 secretion\nsystems.” And T3SS and mvfR are both published important for infections in different animal\nmodels. Thus, again the authors should perform the proteome of in vivo isolated primed bacteria\nwith naïve bacteria.\nWe are not sure that the issues raised here by the reviewer would be solved using proteomics\nas for the divisome, the relevant information would be the assembly of the complex and its\nlocalization within the bacterial cell rather than quantitative changes. As noted above, our\nattempts at in vivo proteomics have been so far unsuccessful as currently mass-spectrometry is\nnot sensitive enough to function on a limited number of bacteria. As also noted in the Reply to\nII) above, MvfR and LasR are not involved in virulence in the fly. We had also previously\nreported that the T3SS system is not required for virulence in Drosophila (Limmer, et al.,\nPNAS, 2011). In addition, the proteomics analysis (Fig. 4A) revealed that the T3SS components\nare more strongly expressed in DhigB mutants whereas they are apparently repressed in DhigA\nmutants. That T3SS protein level were not changed in the DhigB- DhigA double mutants that\nare as virulent as DhigB mutants further rules out the T3SS as being a candidate for virulence\nas described in the Results section on proteomic analysis. Finally, we describe in detail the role\nof FimV, one of the two most repressed genes found in the proteomics analysis thus establishing\na direct link between the higBA operon and a protein identified in our proteomic analysis, which\nplays a capital role in the establishment of the shape of the bacteria as well as in the extent of\nthe elongation of the lateral cell wall.\nReviewer #2 (Remarks to the Author):\nI think there are some interesting results in this study, but more experiments and analyses are\nneeded to characterize the morphological change that occurs during infection. There are also\nseveral misconceptions about virulence and biofilm formation in Pseudomonas aeruginosa that\nare stated several times throughout the manuscript. Many citations are missing that could better\nframe results and improve the design of several experiments. Much of the observed data could\nlikely be described via surface-dependent cAMP activation and cellular memory.\nWe thank the reviewer for providing us with this alternative perspective on our data. We would\nlike to emphasize that we have no evidence for adhesion to the tissues in the systemic infection\nmodel, albeit we certainly have an association in our recently described latent infection model\n(Chen et al. PLoS Pathogens, 2024): the association with tissues is however not dependent on\nPilJ (unpublished data). We have also no evidence for the formation of biofilms (in more than\n15 years of working on P. aeruginosa we just saw once a fly that seemed to have a biofilm\nforming in the hemocoel of the fly). Thus, biofilm does not appear to be a relevant parameter\nin the current model of infections in Drosophila.\nAs much of the pathology in the systemic infection model seems to be linked to bacteremia in\nthe hemolymph rather than direct damages of tissues, we had not considered the possibility of\na transient interaction with host surfaces. We acknowledge that we cannot formally exclude\nthis hypothesis. While we had read a number of articles on surface sensing by bacteria, the\nLee et al. (PNAS, 2018) article on “cellular memory” had escaped our attention and indeed this\nhypothesis is worth considering. We therefore addressed this issue experimentally in two ways.\nFirst, we asked whether having bacteria associate with an agarose surface in vitro would prime\nthe bacteria for virulence. As shown in Fig. S8D-E, even though we did get some transcriptional\nactivation (actually increase of steady-state mRNA levels) of xphA, we did not observe any\npriming. Second, we have generated mutants that would affect T4P formation and not the\nchemosensation apparatus, namely pilA and pilC. While these mutants are expected to abrogate\nsurface sensing, they nevertheless displayed only a slightly impaired virulence that is clearly\nnot of the range that we observed in pilJ mutants (Fig. S8F). Finally, in their pioneering analysis\nof twitching motility mutants, D’Argenio et al. (J. Bact, 2001) had already made the point that\ntwitching motility and virulence were separable and had described multiple mutants, including\npilY1 and pilT, for which twitching motility was affected without influencing virulence. Taken\ntogether, these data demonstrate that the priming phenomenon we report in our manuscript is\nindependent from surface sensing and have been included in the manuscript.\nWe have now referenced in the text more articles dealing with surface sensing of bacteria.\n-lines 49-53: Both acute virulence and biofilm formation require surface contact and surface\nsensing via Type IV pili in Pseudomonas aeruginosa. Surface sensing triggers cAMP activation\nvia type IV pili interaction with the surface leading to activation of the global virulence regulator,\nVfr, which is critical for expression of T2SS and T3SS PMID: 26041805, 37382531, 20494996,\nand many others.\nVirulence is primarily defined by the impact that bacterial infection has on the survival of the\nhost in a given infection model. For instance, in the Drosophila model, we did not find any role\nfor the T3SS in virulence (Limmer et al., PNAS, 2011). In our initial manuscript, we found that\nvfR overexpression did not rescue the pilJ impaired virulence phenotype. Upon thinking about\nthis reviewer’s comments, we agree that the overexpression of VfR might not be sufficient to\ninitiate its activation, for instance if cAMP levels remain too low to activate the overproduced\nprotein, as suggested by the reviewer (see further below). We therefore decided to generate a\nDvfR mutant and analyze its virulence. As now shown in Fig. 4E-F, we found that the DvfR\nmutant displays a level of impaired virulence that is similar to that of DpilJ. Importantly, we\nalso found that these bacteria remained ellipsoid in vivo (Fig. 4G-G’). We conclude that\ncontrarily to our superficial initial conclusions, VfR does indeed play an important role for the\nvirulence of PAO1 in our systemic infection model and gratefully acknowledge the suggestion\nof the reviewer that clarified the issue.\nType IV pili interaction with the surface also triggers c-di-GMP production leading to biofilm\nformation PMID: 25626906, 34168081. Furthermore, type IV pili are critical virulence factors and\nallow for motility within epithelial cells likely triggering cAMP production PMID: 31431558. cAMP\nactivation due to type iv pili surface sensing has also been shown to persist for multiple\ngenerations within Pseudomonas aeruginosa and serve as a molecular memory of surface\ncontact which explains the continued virulence of cells after being recovered from the first\ninfection PMID: 29559526. Also, the extent of cAMP signaling relates to the stiffness of the\nmaterial to which type IV pili are attached to PMID: 35561220, which is a maximum when binding\nto surfaces with a stiffness similar to that of human tissues.\nWe have already discussed above many of the points raised by the reviewer in this paragraph.\nAs regards c-di-GMP production, it depends on PilY1 (Luo et al., mBio, 2015). As noted above,\nD’Argenio et al. (J. Bact, 2001) have already tested this mutant and found it to display a normal\nvirulence.\n-For all figures reporting CFU data, please change the y-axis to log10 CFU/ml.\nDone\n-Fig1. Instead of adding 1 to your Cfu data to avoid you can state \"below limit of detection\" or\"0\nCFU\".\n-lines 106-107: the important control here is that higA and the higB OE strain grow at the same\nrate as WT. Growth curves are typically done via 24-hour growth curves with many time points\ncharacterizing each phase of the growth curve, not 5 time points over five days. There is no\ndescription of the in vitro growth conditions in the methods.\nWe have now described our in vitro growth conditions in the Methods section. We have\nincluded in the manuscript this long-time course as it reflects the actual conditions in vivo in\nterms of temperature and length of the experiment. We include below the data for 14-hour\ngrowth curve at 37°C that did not reveal any major difference in the growth curves, except for\nthe DhigB mutant that however displays an enhanced virulence in vivo, like the DhigB-DhigA\nmutant that grows like wild-type PAO1.\n-lines 136-138 reference \"a lack of bacterial growth until at least 18h (Fig. S3E),\" but fig S3E is a\nsurvival curve of flies and does not show an increase in bacterial CFU over time which would be\nindicative of growth.\nWe thank the reviewer for pointing out this sentence that was not perfectly clear. The text\nnow reads: “The finding that ∆higA recovers its virulence in flies missing either IMD pathway\nreceptors PGRP-LC and PGRP-LE, which senses bacterial growth (Fig. S3E), further\nsupports a lack of bacterial growth until at least 18h (Fig. 1B), although we cannot formally\nexclude an active inhibition of the IMD pathway by injected PAO110” (the IMD pathway is\ntriggered upon sensing polymeric PGN or short PGN degradation fragment such as tracheal\ncytotoxin. Because polymeric PGN in INTACT bacteria is buried under the outer membrane,\nit is not accessible to the PGRP-LC transmembrane sensor. The cell wall remodeling that\naccompanies cell division leads to the release of short PGN fragments that are sensed both by\nPGRP-LC specific isoforms and PGRP-LE intracellularly. Hence, the IMD pathway gets\nactivated only upon bacterial cell division).\n-lines 159-166: PMID: 29559526\nWe understand what the reviewer had in mind, that is, that the multigenerational memory of\n“priming” to surfaces might account for the priming effect we describe in this experiment. As,\ndiscussed above, we have now rigorously tested the hypothesis of surface sensing in two\nindependent manners and have excluded this possibility.\n-line 167: please explain how \"priming\" is different or distinct from any other typical lag phases in\na new growth medium.\nPriming is distinct from a typical lag phase in that it is required only in wild-type but not\nimmunodeficient kenny flies. Also, we are not aware that there are morphologic changes of the\nbacteria when transferred to a novel growth medium. Finally, some pathogens such as Serratia\nmarcescens kill flies in less than a day upon the injection of only five bacteria (Nehme et al.,\nPLoS Pathogens, 2007).\n-line 175-180/Fig3A-C: There needs to be some sort of quantification with some sort of image\nanalysis software to conclude that there is any significant difference in cell shape/morphology.\nWithout any sort of quantification and statistical analysis I am not comfortable making any sort of\nconclusion. Reference 9 also lacks any sort of statistical image analysis of the morphological\nchanges of bacteria. Furthermore, if the shape of bacterial cells are to be evaluated, they should\nall be in the same focal plane.\nWe have now added EM data and associated quantification of length and length/width ration\n(Fig. 3A-A’’). As regards light microscopy, we have focused on measuring the length of the\nbacteria for several key experiments (Fig. 3B’, 3D’; 4G’, 4K’, 5B’, 5C’, 5F’, and 5J’).\nWe are measuring the lengths of bacteria as observed through the cuticle of the fly and few\nbacteria per fly are “measurable”, hence making quantification challenging. Please, note that\nthe figures displaying bacterial shapes are “collages” in which we have pieced together in one\npanel pictures taken from different fields that do not come necessarily from the same fly, as\nnoted in the figure legends. For each bacterium, we took the pictures at the optimal focal plane\nas much as possible.\n-all overexpression experiments should include westerns to show stable overexpression of the\nprotein.\nWe agree with the reviewer that it ideally should be done. However, we certainly do not have\naccess to antibodies for any of the constructs we have made and therefore we cannot use\nWestern blot analysis (it is administratively very difficult to import biological reagents into\nChina). Yet, in a number of cases, we do observe a biological effect that indicates that the\noverexpression construct is functional (fimV, pilJ, a number of cell division genes such as ftsK,\nftsQ, ftsW...). In addition, we note that we can rescue the pilJ mutant using the pilJ\noverexpression construct (Fig. 4M). We have also made GFP fusions for FimV, which we can\ndirectly observe in vivo and its deletion constructs as it is a key gene in our analysis (Fig. 5).\nThus, we are confident that our main conclusions are correct. We have found a number of cell\ndivision genes that do not produce a phenotype when overexpressed (ftsL, ftsI, ftsX, and ftsE).\nAs these genes are not critical for our analysis, we have simply monitored that they were indeed\noverexpressed using RTqPCR (Fig. S10G). We acknowledge in the Supplementary Figure\nLegend that the overexpressed proteins might be unstable to underscore the potential limitation\nof this experiment that yielded negative results.\n-Figure S5E: there seems to be a significant difference between Nam_higA strain w/ 0mg/ml and\n10mg/ml but there are not statistical tests to tell if anything is different.\nThere was indeed a problem with the 0 mg/mL DhigA data points, which should have given\nsimilar values for NAM and NAG (no addition). The “no NAM” sample is abnormally low due\nto water evaporation in the three wells of the 96-well plate. However, since no major differences\nhave been observed for the eleven NAM/NAG concentrations tested, we do not think this\nrepresents a significant issue. Of course, should the reviewer imperatively ask for it, we will\nrepeat the experiment.\n-Fig 4C: please confirm the overexpression of PilJ with a western.\nWhile we do not have an antibody against PilJ, we have performed genetic rescue experiments\nwith the full or domain-deleted genes and have found that the full gene (and the LBD deletions)\nare able to rescue the DpilJ mutant phenotype (Fig.4M).\n-Line 271: One of the few ligands that actually bind to the ligand binding domain of PilJ are\nphenol soluble modulins produced by Staph aureus. To properly test if PilJ is sensing NAG,\nyou should use PilJ with the ligand binding domain removed which has been used in several studies\nso far: PMID 27190147, 38349934. Without this control you cannot conclude that PilJ is sensing\nNAG and not just transducing a surface signal from type IV pili.\nWe thank the reviewer for the suggestion and have performed the suggested experiments and\nadditional ones. Namely, we have addressed the issue of direct binding of NAG to the PilJ RBD\ndomains both biochemically and genetically (see also reply to Reviewer 1 Point IV). The new\ndata are reported in Fig. 4 K-M and Fig. S8G-H. We have no altered our conclusion: the data\nshow that the RBD domain is dispensable for the action of NAG, which fails to bind to the\nRBD domains in vitro. We also compare our results with those of Kearns et al. (J. Bact, 2001)\nas regards directed twitching motility towards phosphatidylethanolamine, which also does not\ninvolve the PilJ RBD domains.\n-line 277: the citations listed do not connect cAMP levels with cell wall remodeling\nWe have modified our wording of this sentence to : “cAMP levels control bacterial cell\nmorphology and also activate the Vfr transcription factor, which regulates the expression of\nseveral virulence genes4,30,32-35.”\n-lines 273-287: Even though overexpression of vfr does not compensate virulence it does not\nmean Vfr does not have a downstream role in virulence given its function. Vfr has a cAMP\ndependent and cAMP independent regulon PMID: 20494996. Overexpression of Vfr without an\nequal activation of cAMP means it is only activating the non-cAMP regulon which are not\nvirulence genes. Furthermore, Vfr is the only known cAMP binding protein in Pseudomonas with\nany physiological effect. PMID 38832786. The other mutants indicate Vfr likely plays a role in a\ncAMP-dependent manner.\nWe agree with the reviewer, who is right, and have addressed experimentally this concern\n(please, see reply to lines 51-53 above).\n-Fig 5E: There should be quantification of polar localization like using microbeJ or Fiji. FimV\nappears to be cytoplasmic when OE in liquid conditions. Surface sensing leads to an increase in\nthe number of type iv pili as well as localization of pili components.\nWe do not feel that image analysis is required to determine the polar localization of FimV-GFP\nas the human eye is certainly faster. We do however agree that quantification is desirable and\nhave performed it. We have amended our text to mention the quantitative aspect of FimV-GFP\nlocalization in vitro and in vivo: “Strikingly, whereas tagged FimV was found distributed rather\nevenly throughout the cell internal surface in 60% of cells grown in BHB, it was found to be\nenriched at the poles of all bacteria injected into wild-type flies 24h after challenge (Fig. 5C-\nC’), which suggests that bacterial pole organization changes as a result of priming.”\n-line 313-316: The data do not support the claim made. These mutations are supposed to make\nthe cell morphology more rod like and therefore virulent but in line 308-312 you state that\noverexpression leads to decreased virulence.\nWe think there is a misunderstanding here. The statements mentioned by the reviewer actually\napply to ftsZ overexpression in a higA mutant and not in a wild-type background. The\nbacteria are rounded as shown in what is now FigS10A and reported in Table S2. The\noverexpression of ftsZ in wild-type PAO1 leads to an elongated shape in vitro as reported in the\nliterature but to an ellipsoid to rod shape in vivo, as documented also in Fig. S10 A and Table\nS2. In this case, the virulence of PAO1 is only mildly altered (Fig. S10D).\n-Type IV pili interact with surfaces and are able to bind surfaces better with EPS. Perhaps NAG\ncan bind type iv pili and mimic a surface that would trigger Pil-Chp, leading to cAMP production,\nVfr-cAMP dependent regulon, and increased virulence. This positive feedback loop maintains\nhigh levels of cAMP over multiple generations and is a hypothesis worth testing here.\nWhile we understand to some degree the line of reasoning of the reviewer (we have difficulty\nthough visualizing how NAG, which are not polymers, could form a surface), our experiments\nthat directly tested the hypothesis of surface sensing do not support this possibility, especially\nthe finding that pilA mutants have a normal virulence. However, the reviewer is right in the\nsense that NAG is not sensed directly by the PilJ tandem ligand-binding domains and do not\ncurrently understand exactly how it is perceived by bacteria through PilJ.\n-line 352: mechanosenseing is not tested in this manuscript so this claim cannot be made.\nWe now have tested in two independent manner this hypothesis and believe it is now\ndisproved.\n-line 355-357: many papers have already shown this to not be true.\nWe have now clarified the statement that applied to the study of host-pathogen interactions in\nthe Drosophila field: “In contrast to the prevailing view of immediate virulence of pathogenic\nbacteria in Drosophila systemic infection models, P. aeruginosa virulence in the host requires\nan adaptation period”.\n-line 383-387: The claim about bacterial shape and resistance to the IMD pathway is not\nsupported by the last set of experiments in which mutants exhibiting more rod-like morphology\nare still cleared by IMD.\nWe are not sure we understand the reviewer’s point here that deals with the Discussion. Is it a\nreference to the comment above on lines 313-316 that we have addressed? Or is the reviewer\nreferring to: “Strikingly, bacteria overexpressing zipA or ftsW formed filaments in vitro, yet\ndisplayed a rather rounded to ellipsoid morphology when injected into wild-type flies, yet was\nrod-shaped when injected into key flies (Fig. 5J-J’).” As shown in Fig. 5J, even though the ftsW\nor zipA overexpressing bacteria filament in vitro, they do not in vivo. They are rounded in wild-\ntype flies but present a mixture between ellipsoid to rod morphology in key mutants. While\naddressing this comment, we have noted a mistake in Table S2 that we have now corrected as\nit was incorrectly referring to a rounded to ellipsoid morphology of these bacteria in key mutants\nwhen they are rod to ellipsoid morphologies. We do observe a rod morphology upon fimV\noverexpression in wild-type bacteria, which corresponds to a slightly impaired virulence in\nwild-type flies, likely mirroring a degree of toxicity of the construct since the virulence was\nalso mildly impaired in key mutants (Table S2).\n-FimV is necessary for cAMP activation via type iv pili and flagellar mediated surface sensing so it\nmakes sense increased FimV increases virulence phenotypes. PMID: 31682637\nWe agree with the reviewer. Our point with respect to cAMP signaling however is that FimV\noverexpression has much stronger phenotype than pilJ overexpression, implying that FimV has\nother function independent of cAMP. We now propose a model in which FimV is important in\nthe determination of cell shape and virulence by providing a polarity information to the\nbacterium that favors lateral cell all extension and that cAMP signaling is equally important in\nconveying a signal that enhances lateral cell wall synthesis, as now added in the Discussion and\nour dissection of the FimV domains in relation to cell shape and virulence.\nReviewer #3 (Remarks to the Author):\nThe present work describes the process by which the bacterial pathogen Pseudomonas\naeruginosa adapts to the host environment in the initial stage of the infection. Using Drosophila\nmelanogaster as their acute infection model, this transition period allows the bacteria to not\nproliferate, hiding from the host’s immune system, and switching from an ellipsoid shape to a rod\nmorphology. The work clearly shows a correlation between the bacillus morphology and virulence.\nFurthermore, the authors also elucidate the probable mechanism, from the extracellular signal\nrequired to initiate the transition to the genes related to bacteria wall remodeling. Overall, the\ndata is solid with some minor concerns over significance in terms of extrapolating to\nmammalian infection models. The authors should address this in more detail in the discussion\nespecially in light of the use of antimicrobial peptides.\nWe have followed the reviewer’s suggestion and expended the last paragraph of the Discussion.\nIt is clear that the easiest model to address in mammals the link between bacterial shape and\nvirulence as described in our work would be to use a wounding model, likely the burn wound\nmodel. While there are over 500 publications dealing with this model retrievable in PubMed,\nwe have not read most of them, looking at the Abstracts of several studies by adding key words\nrelated to bacterial shape or morphology or T4P did not retrieve relevant references and the\nimpression is that investigators look at time points that are too late, that is 24 to 48 hours of\ninoculation. Clearly, a study of the type we have pursued in Drosophila is warranted in a\nmammalian burn wound model (mouse, rat, pig? Mice would likely be the cheapest way to go).\nWe have cited the original study as well as another one that underscores the importance of\nwounding the fascia: would this lead to the release of NAG?\nDespite this the work clearly helps expand the understanding about the molecular mechanisms\ninvolved in pathogen adaptation to the host during different stages of infection, here specifically\nduring the acute stage. This knowledge is crucial to understand the process of colonization that\nresults from bacterial infections and for the development of new therapies and treatments\ntargeting bacterial pathogens characterized as multi-drug resistant, such as Pseudomonas\naeruginosa.\nThe design and analysis of a wide variety of mutants in P. aeruginosa and on the side of the host\n(flies) is an innovative allowing a mechanistic teasing out of the role of the NAG in remodelling\ncell morphology and priming of virulence. They clearly show the sensing of NAG is a common\nsignal that promotes virulence through morphology changes. However, the authors might\nconsider higher level imaging such as electron microscopy to confirm the morphology\nexperiments as it is difficult to really judge the morphologies.\nAs described above for Reviewer 2, we have now added additional EM data in Fig. 3 and have\nquantified the length of bacteria in the key experiments of the article. Importantly, besides the\nrod shape, it seems that the absolute length of the bacteria is also an important parameter in for\nthe virulence of bacteria through their ability to withstand the action of three AMP families\nregulated by the IMD pathway.\nAlso, in Fig 3A-C more detail of the conditions and time frame for the images would be helpful to\nthe reader.\nThis remark has been addressed.\nThe priming phase by NAG is mediated by the T4P chemosensing system by activating\ndownstream signaling via cAMP mediated cell wall remodeling. The authors make the case that\nmorphology drives virulence. Overall the technical quality of the manuscript is extremely high and\nthe results consistent with the conclusions.\nAlthough the experiments as performed are of a high quality the text needs to be improved in\nconsistency and clarity.\nSpecifically, I encourage the authors to review the consistency between the names listed in the tables and the text and\nfigures, as some of them do not match or are not shown in the tables.\nWe hope this concern has been addressed.\nThen, I would recommend including a complete list of the plasmids designed and/or assayed as\nwell as for the bacteria or fly mutants. In addition to just the reference, including a brief\ndescription of the constructs, such as the plasmid backbone, inducible or constitutive promoter,\ngene/pathway affected, etc, would be recommended and would improve the understanding of the\ntext.\nDone, with new supplementary Tables.\nOverall, this is a rigorous and interesting report on P. aeruginosa priming and adaptation in the D.\nmelanogaster model system. The results are significant in providing a foundation for the role of\npriming in an acute infection and open further avenues of research in other model systems.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe do hope that the process was instructive.\nRebuttal letter (second round of revisions)\nWe thank the reviewers for their generally positive comments and have addressed\nexperimentally some of the points raised by Reviewer 1.\nReviewer #1 (Remarks to the Author):\nThe study investigates the role of N-acetyl-glucosamine (NAG) in priming Pseudomonas\naeruginosa for virulence via a T4P/cAMP-mediated morphological transition. While the revised\nmanuscript addresses some concerns and presents novel insights into host-pathogen\ninteractions, several critical issues remain unresolved. Below are detailed critiques to strengthen\nthe scientific rigor and clarity of the work:\nMajor Concerns\n1. Image Quality and Presentation\n- Several fluorescence microscopy images lack clarity (e.g., Fig. 3C, 3D, 4G, 5B–C). The use of\ncomposite panels from disparate fields creates confusion, and scale bars are inconsistently\nplaced or absent. The authors should provide single-field, high-resolution images with uniform\nscale bars (e.g., 2 µm) clearly labeled in all panels. Also, please include detailed figure legends\nexplaining image stitching (if necessary) and statistical significance (e.g., n-values, replicates).\nStandardize annotations are also required (e.g., replace “wt” with “WT,” remove underscores in\nlabels like “WT_?”).\nWe fear that this reviewer is not familiar with working on imaging bacteria in living tissues. It\nis totally different from imaging in vitro. One important limitation is that there are few bacteria\nthat we can image in tissues given their low numbers, especially at early phases of the infection.\nWhen several bacteria are present in the same field, they are not on the same focal plan when\nworking at high magnification. THUS, IT IS NOT POSSBLE TO AVOID STITCHING IF\nONE WANTS TO AVOID SHOWING JUST A SINGLE BACTERIUM PER PANEL. The\nsenior author of this study has been a Drosophila developmental biologist last millennium and\nwould never have thought that it would be possible to get such high-quality images in\nDrosophila tissues (we usually use a 63x immersion objective for imaging Drosophila embryos\nand working with a 100x objective is especially difficult). We have often used the staining of\nLPS-O-antigen to visualize bacterial shape, which is by essence somewhat fuzzy.\nWe have nevertheless attempted to improve the quality of images. One technique was to image\nbacteria retrieved from hemolymph as it makes it a bit easier to find the bacteria and to quantify\nthem using Microbe J. Of note, as noted in the Methods section, we have checked that bacteria\nretrieved from the hemolymph display shape properties similar to those observed in tissues as\nwe might have introduced a bias by using such a technique, for instance by selecting bacteria\nnot associated with tissues. We have also increased the number of bacteria analyzed by Microbe\nJ, which required an important work load since bacteria have to be imaged one by one.\nFigure legends have been improved as well as scale bars.\n2. Mechanistic Evidence for NAG Sensing by PilJ\n- The claim that PilJ indirectly senses NAG is weakly supported. While the thermal shift assay\n(Fig. 4L) shows no direct binding of NAG to the PilJ LBD, no alternative mechanism is proposed.\nThe authors should measure cAMP levels in ΔpilJ mutants exposed to NAG to test whether NAG\nactivates cAMP signaling in a PilJ-dependent manner. They should perform genetic\ncomplementation with PilJ truncation mutants (e.g., ΔLBD) to confirm if NAG sensing requires\nother domains (e.g., transmembrane or HAMP domains).\nWe have monitored cAMP signaling by measuring the steady-state transcripts of pilA. This\nanalysis revealed that cAMP signaling is switched on in vivo, independently of the presence of\nNAG (Fig. 5f, j, k). We had already performed the rescue of DpilJ by a pilJ construct lacking\nthe LBD domains, it did rescue, and also of the tandem LBDs alone. We have now additionally\nshown that co-injecting NAG with the rescued pilJ by the construct lacking LBD domains did\nenhance the virulence of the injected bacteria, further demonstrating that the LBD domains are\ndispensable for NAG action on virulence in vivo. The further delineation of the PilJ domains\nmediating the effects of NAG is outside of the scope of this study.\n3. Lack of Validation in Mammalian Models\nThe study relies solely on Drosophila infection assays. The relevance of the proposed\nNAG/PilJ/cAMP/FimV axis in mammalian models (e.g., murine acute lung infection) remains\nunaddressed. They could test virulence of Δ*higA*, Δ*pilJ*, or NAG-treated PAO1 in a mouse\nmodel (e.g., intratracheal infection). If animal experiments are infeasible, explicitly discuss the\nlimitations of extrapolating findings from flies to mammals.\nWe have discussed this issue at the end of the Discussion. A more in-depth discussion would\nunnecessarily lengthen the manuscript. This issue should be discussed more extensively in a\nCommentary article. We also note that the number of studies documenting the bacterial shape\nin animal tissues at our level of resolution is low, if not null. The onus is now on investigators\nworking with mammalian models to validate our findings, for instance in burnt-wound models.\n4. Absence of Protein-Level Validation\nThe key molecular mechanisms (e.g., FimV localization, PilJ expression in truncation mutants)\nlack validation via Western blotting or immunostaining. The authors should confirm FimV protein\nexpression and polar localization using anti-FimV antibodies (commercially available or custom-\ndesigned). They should validate expression levels of truncated PilJ constructs (Fig. 4M) via\nWestern blot to ensure genetic complementation is functional.\nThis issue had already been raised by two other reviewers and our reply was deemed\nsatisfactory (please, see Reviews to first Round of revisions). The best evidence is that the\nFimV-GFP fusion has biological activity similar to that reported for unlabeled FimV, that is an\nelongation of bacteria. We have demonstrated that the overexpression of full pilJ strongly\nrescues the DpilJ mutant phenotype, which genetically demonstrates that the construct is\nfunctional, a degree of evidence that is much stronger than a Western blot: the addition of a tag\ncan kill the biological activity of a protein, yet look very nice on a Western blot. That NAG still\npromotes the virulence of the rescue by the form lacking the LBD domains demonstrates that\nthis truncated form is also functional, a major result validating this construct.\n5. Quantification of Morphological Data\nThe central claim linking bacillus morphology to virulence relies on qualitative images (e.g., Fig.\n3A–C, 4G–K) without robust quantification. The authors could use automated tools (e.g.,\nCellProfiler, MicrobeJ) to quantify bacterial length, width, and aspect ratio across ≥100 cells per\ncondition, and include statistical comparisons (e.g., ANOVA with post-hoc tests) between groups\n(e.g., WT vs. ΔhigA in key flies).\nPlease, see Reply to Point 1. Of note, we have increased the number of quantified bacteria as\nmuch as possible. Since the differences are statistically significant, it makes no scientific sense\nto quantify more bacteria to reach this artificial threshold of 100, which may be easy to reach\nwhen working in vitro… Of note, when we perform survival experiments, we use usually\nbetween 120 to 180 flies per condition. Yet, when reviewing articles, I do not request that\ninvestigators working on mice use a similar number of animals…\nWe have changed the pictures of Fig. 3b-c, Fig. 4g, Fig. 5b, Fig. 6b, c, f, j, Fig. S9d.\nWe have increased the number of bacteria quantified: Fig. 3b’, d’, Fig. 4g’, Fig. 5b’, Fig. 6b’,\nf’, j’, Fig. S5a (quantification added), Fig. S9d’-d’’’ (quantification added, including width).\nWith respect to new data, bacteria retrieved 10h after co-injection with NAM or NAG,\nwe have\nalso monitored width to document that NAM-treated bacteria are more rounded in vivo, in\nkeeping with a loss of polarity.\n6. Fitness of Overexpression Strains\nThe partial rescue of ΔhigA virulence by pilJ, cyaB, or fimV overexpression (Figs. 4C–D, 5A) may\nreflect fitness trade-offs rather than mechanistic effects. The authors should perform growth\ncurves or competitive index assays in vitro and in vivo to rule out fitness costs.\nThis analysis has actually been performed systematically from the very first manuscript by\nmonitoring the virulence of bacteria in key immune-deficient flies in which there is no AMP\nexpression that acts on injected bacteria. We have always pointed out when the in vivo fitness\nof injected bacteria appeared to be somewhat lessened: Fig. 1e, g, Fig. 2f, Fig. 3b, d-h, Fig. 4b-\nd, g Fig. 5a, b, d, e, g, Fig. S8d (for Fig. 5j), Fig. 6a, b, e, h, i, j, Fig. S2f, Fig. S5b, c, e, f, h, i,\nFig. S6b, Fig. S6g, Fig. S7e, f, Fig. S9a-b, e, Fig. S10b-f.\n7. Unresolved Mechanism of NAM Toxicity\nThe observation that NAM abolishes virulence (Fig. 3I) is intriguing but unexplained.\nWe have now made a major step toward identifying the mechanism of NAM toxicity. It prevents\nthe association of FimV to the poles, likely by preventing the binding of FimV to the septal\npeptidoglycan, as now mentioned in the revised Discussion. What is fascinating is that NAG\ncounteracts the effect of NAG to some extent, as inferred from survival analysis. Strikingly,\nNAG does promote a faster localization of FimV-GFP to the poles (Fig. S9h-h’). Further\nstructural elucidation of a molecular mechanism by competition between NAM, but not NAG,\nand septal peptidoglycan for a peptidoglycan binding site in FimV lies outside of the scope of\nthis study.\nOf note, the rescue of the DhigA by the FimV truncated construct lacking the LysM domain (a\npeptidoglycan-binding domain) takes place in a context in which there is still some endogenous\nfull-length FimV expression that is likely enough to nucleate low quantities of endogenous\nFimV to the poles, which may allow the subsequent initiation of a weak bacterial polarity\nthrough protein-protein interactions able to compensate the absence of the LySM domain in\noverexpressed FimV. This may explain why the LysM truncated construct is still able to rescue\nrather well the DhigA mutant.\nMinor Concerns\n1. Figure Standardization\n-Color schemes: Adopt consistent color coding across all figures (e.g., use Fig. 1B’s\norange/green/blue/red scheme for WT/mutant/rescue groups).\n- Axis labels:\n- Remove redundant labels (e.g., “10” in Fig. 1A x-axis).\n- Use uniform terminology (e.g., “Bacterial load (CFU/fly)” in Fig. 1B, 2C).\n- Fonts and symbols:\n- Standardize font sizes and styles (e.g., Fig. 3B’ uses “PAO1” in a different font).\n- Replace “ns” with “n.s.” (not significant) and ensure asterisks align with statistical thresholds.\n2. Scheme Clarity\n- Simplify and standardize schematics (e.g., Fig. 3J, 4H, 5G). Move overly simplistic diagrams to\nsupplementary materials.\n3. Technical Details\n- Clarify ambiguous labels (e.g., “rpl49” in Fig. 2A should be defined as a normalization control).\n- Correct distorted fonts (e.g., Fig. 4L’) and align panel labels (e.g., “WT” vs. “key” in Fig. 3B’).\nDone.\nReviewer #2 (Remarks to the Author):\nThe authors have performed a substantial amount work and quantification to improve the\nmanuscript and have adequately addressed each of my concerns. Below are minor comments:\n-Recent studies have shown a significant difference in surface sensing, colonization, and biofilm\nformation between PAO1 and PA14, specifically with Wsp/c-di-GMP being much more important\nfor PAO1 and Pil-Chp/cAMP being more important for PA14 PMID: 34516283, 32098815. This is\none of the first studies to highlight Pil-Chp in PAO1, and I wonder how these experiments would\ndiffer if PA14 were used, although this is out of the scope of this study.\nWe have actually reproduced some of these data (DfimV and Dvfr) using the PA14 strain. We\nhave not included these data in the present study.\n-line 321-324 change PilJ gene to lowercase\nDone.\nReviewer #3 (Remarks to the Author):\nThe authors have performed a a significant number of extra experiments to address the major\nconcerns including extra qPCR experiments to ensure and and per are not induced on AMP\ntreatement, generation of a chic mutant that has a similar phenotype to WT indicating the NAG is\nnot generated by the host proteases. Moreover, they tested the cAMP activation and memory\nwith additional experiments to show priming is affected independent of surface sensing. Overall\nthe manuscript is vastly improved and contributes to our understanding of virulence priming by\nNAG.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank all reviewers for their inputs and suggestions to improve the quality of the\nmanuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The study investigates the role of N-acetyl-glucosamine (NAG) in priming Pseudomonas aeruginosa for virulence via a\nT4P/cAMP-mediated morphological transition. While the revised manuscript addresses some concerns and presents novel\ninsights into host-pathogen interactions, several critical issues remain unresolved. Below are detailed critiques to strengthen\nthe scientific rigor and clarity of the work:\nMajor Concerns\n1. Image Quality and Presentation\n- Several fluorescence microscopy images lack clarity (e.g., Fig. 3C, 3D, 4G, 5B–C). The use of composite panels from\ndisparate fields creates confusion, and scale bars are inconsistently placed or absent. The authors should provide single-\nfield, high-resolution images with uniform scale bars (e.g., 2 µm) clearly labeled in all panels. Also, please include detailed\nfigure legends explaining image stitching (if necessary) and statistical significance (e.g., n-values, replicates). Standardize\nannotations are also required (e.g., replace “wt” with “WT,” remove underscores in labels like “WT_?”).\n2. Mechanistic Evidence for NAG Sensing by PilJ\n- The claim that PilJ indirectly senses NAG is weakly supported. While the thermal shift assay (Fig. 4L) shows no direct\nbinding of NAG to the PilJ LBD, no alternative mechanism is proposed. The authors should measure cAMP levels in ΔpilJ\nmutants exposed to NAG to test whether NAG activates cAMP signaling in a PilJ-dependent manner. They should perform\ngenetic complementation with PilJ truncation mutants (e.g., ΔLBD) to confirm if NAG sensing requires other domains (e.g.,\ntransmembrane or HAMP domains).\n3. Lack of Validation in Mammalian Models\nThe study relies solely on Drosophila infection assays. The relevance of the proposed NAG/PilJ/cAMP/FimV axis in\nmammalian models (e.g., murine acute lung infection) remains unaddressed. They could test virulence of Δ*higA*,\nΔ*pilJ*, or NAG-treated PAO1 in a mouse model (e.g., intratracheal infection). If animal experiments are infeasible,\nexplicitly discuss the limitations of extrapolating findings from flies to mammals.\n4. Absence of Protein-Level Validation\nThe key molecular mechanisms (e.g., FimV localization, PilJ expression in truncation mutants) lack validation via Western\nblotting or immunostaining. The authors should confirm FimV protein expression and polar localization using anti-FimV\nantibodies (commercially available or custom-designed). They should validate expression levels of truncated PilJ constructs\n(Fig. 4M) via Western blot to ensure genetic complementation is functional.\n5. Quantification of Morphological Data\nThe central claim linking bacillus morphology to virulence relies on qualitative images (e.g., Fig. 3A–C, 4G–K) without robust\nquantification. The authors could use automated tools (e.g., CellProfiler, MicrobeJ) to quantify bacterial length, width, and\naspect ratio across ≥100 cells per condition, and include statistical comparisons (e.g., ANOVA with post-hoc tests) between\ngroups (e.g., WT vs. ΔhigA in key flies).\n6. Fitness of Overexpression Strains\nThe partial rescue of ΔhigA virulence by pilJ, cyaB, or fimV overexpression (Figs. 4C–D, 5A) may reflect fitness trade-offs\nrather than mechanistic effects. The authors should perform growth curves or competitive index assays in vitro and in vivo to\nrule out fitness costs.\n7. Unresolved Mechanism of NAM Toxicity\nThe observation that NAM abolishes virulence (Fig. 3I) is intriguing but unexplained.\nMinor Concerns\n1. Figure Standardization\n-Color schemes: Adopt consistent color coding across all figures (e.g., use Fig. 1B’s orange/green/blue/red scheme for\nWT/mutant/rescue groups).\n- Axis labels:\n- Remove redundant labels (e.g., “10” in Fig. 1A x-axis).\n- Use uniform terminology (e.g., “Bacterial load (CFU/fly)” in Fig. 1B, 2C).\n- Fonts and symbols:\n- Standardize font sizes and styles (e.g., Fig. 3B’ uses “PAO1” in a different font).\n- Replace “ns” with “n.s.” (not significant) and ensure asterisks align with statistical thresholds.\n2. Scheme Clarity\n- Simplify and standardize schematics (e.g., Fig. 3J, 4H, 5G). Move overly simplistic diagrams to supplementary materials.\n3. Technical Details\n- Clarify ambiguous labels (e.g., “rpl49” in Fig. 2A should be defined as a normalization control).\n- Correct distorted fonts (e.g., Fig. 4L’) and align panel labels (e.g., “WT” vs. “key” in Fig. 3B’).", "reviewer_2": "The authors have performed a substantial amount work and quantification to improve the manuscript and have adequately\naddressed each of my concerns. Below are minor comments:\n-Recent studies have shown a significant difference in surface sensing, colonization, and biofilm formation between PAO1\nand PA14, specifically with Wsp/c-di-GMP being much more important for PAO1 and Pil-Chp/cAMP being more important for\nPA14 PMID: 34516283, 32098815. This is one of the first studies to highlight Pil-Chp in PAO1, and I wonder how these\nexperiments would differ if PA14 were used, although this is out of the scope of this study.\n-line 321-324 change PilJ gene to lowercase", "reviewer_3": "The authors have performed a a significant number of extra experiments to address the major concerns including extra\nqPCR experiments to ensure and and per are not induced on AMP treatement, generation of a chic mutant that has a similar\nphenotype to WT indicating the NAG is not generated by the host proteases. Moreover, they tested the cAMP activation and\nmemory with additional experiments to show priming is affected independent of surface sensing. Overall the manuscript is\nvastly improved and contributes to our understanding of virulence priming by NAG.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We thank the reviewers for their generally positive comments and have addressed\nexperimentally some of the points raised by Reviewer 1.\nReviewer #1 (Remarks to the Author):\nThe study investigates the role of N-acetyl-glucosamine (NAG) in priming Pseudomonas\naeruginosa for virulence via a T4P/cAMP-mediated morphological transition. While the revised\nmanuscript addresses some concerns and presents novel insights into host-pathogen\ninteractions, several critical issues remain unresolved. Below are detailed critiques to strengthen\nthe scientific rigor and clarity of the work:\nMajor Concerns\n1. Image Quality and Presentation\n- Several fluorescence microscopy images lack clarity (e.g., Fig. 3C, 3D, 4G, 5B–C). The use of\ncomposite panels from disparate fields creates confusion, and scale bars are inconsistently\nplaced or absent. The authors should provide single-field, high-resolution images with uniform\nscale bars (e.g., 2 µm) clearly labeled in all panels. Also, please include detailed figure legends\nexplaining image stitching (if necessary) and statistical significance (e.g., n-values, replicates).\nStandardize annotations are also required (e.g., replace “wt” with “WT,” remove underscores in\nlabels like “WT_?”).\nWe fear that this reviewer is not familiar with working on imaging bacteria in living tissues. It\nis totally different from imaging in vitro. One important limitation is that there are few bacteria\nthat we can image in tissues given their low numbers, especially at early phases of the infection.\nWhen several bacteria are present in the same field, they are not on the same focal plan when\nworking at high magnification. THUS, IT IS NOT POSSBLE TO AVOID STITCHING IF\nONE WANTS TO AVOID SHOWING JUST A SINGLE BACTERIUM PER PANEL. The\nsenior author of this study has been a Drosophila developmental biologist last millennium and\nwould never have thought that it would be possible to get such high-quality images in\nDrosophila tissues (we usually use a 63x immersion objective for imaging Drosophila embryos\nand working with a 100x objective is especially difficult). We have often used the staining of\nLPS-O-antigen to visualize bacterial shape, which is by essence somewhat fuzzy.\nWe have nevertheless attempted to improve the quality of images. One technique was to image\nbacteria retrieved from hemolymph as it makes it a bit easier to find the bacteria and to quantify\nthem using Microbe J. Of note, as noted in the Methods section, we have checked that bacteria\nretrieved from the hemolymph display shape properties similar to those observed in tissues as\nwe might have introduced a bias by using such a technique, for instance by selecting bacteria\nnot associated with tissues. We have also increased the number of bacteria analyzed by Microbe\nJ, which required an important work load since bacteria have to be imaged one by one.\nFigure legends have been improved as well as scale bars.\n2. Mechanistic Evidence for NAG Sensing by PilJ\n- The claim that PilJ indirectly senses NAG is weakly supported. While the thermal shift assay\n(Fig. 4L) shows no direct binding of NAG to the PilJ LBD, no alternative mechanism is proposed.\nThe authors should measure cAMP levels in ΔpilJ mutants exposed to NAG to test whether NAG\nactivates cAMP signaling in a PilJ-dependent manner. They should perform genetic\ncomplementation with PilJ truncation mutants (e.g., ΔLBD) to confirm if NAG sensing requires\nother domains (e.g., transmembrane or HAMP domains).\nWe have monitored cAMP signaling by measuring the steady-state transcripts of pilA. This\nanalysis revealed that cAMP signaling is switched on in vivo, independently of the presence of\nNAG (Fig. 5f, j, k). We had already performed the rescue of DpilJ by a pilJ construct lacking\nthe LBD domains, it did rescue, and also of the tandem LBDs alone. We have now additionally\nshown that co-injecting NAG with the rescued pilJ by the construct lacking LBD domains did\nenhance the virulence of the injected bacteria, further demonstrating that the LBD domains are\ndispensable for NAG action on virulence in vivo. The further delineation of the PilJ domains\nmediating the effects of NAG is outside of the scope of this study.\n3. Lack of Validation in Mammalian Models\nThe study relies solely on Drosophila infection assays. The relevance of the proposed\nNAG/PilJ/cAMP/FimV axis in mammalian models (e.g., murine acute lung infection) remains\nunaddressed. They could test virulence of Δ*higA*, Δ*pilJ*, or NAG-treated PAO1 in a mouse\nmodel (e.g., intratracheal infection). If animal experiments are infeasible, explicitly discuss the\nlimitations of extrapolating findings from flies to mammals.\nWe have discussed this issue at the end of the Discussion. A more in-depth discussion would\nunnecessarily lengthen the manuscript. This issue should be discussed more extensively in a\nCommentary article. We also note that the number of studies documenting the bacterial shape\nin animal tissues at our level of resolution is low, if not null. The onus is now on investigators\nworking with mammalian models to validate our findings, for instance in burnt-wound models.\n4. Absence of Protein-Level Validation\nThe key molecular mechanisms (e.g., FimV localization, PilJ expression in truncation mutants)\nlack validation via Western blotting or immunostaining. The authors should confirm FimV protein\nexpression and polar localization using anti-FimV antibodies (commercially available or custom-\ndesigned). They should validate expression levels of truncated PilJ constructs (Fig. 4M) via\nWestern blot to ensure genetic complementation is functional.\nThis issue had already been raised by two other reviewers and our reply was deemed\nsatisfactory (please, see Reviews to first Round of revisions). The best evidence is that the\nFimV-GFP fusion has biological activity similar to that reported for unlabeled FimV, that is an\nelongation of bacteria. We have demonstrated that the overexpression of full pilJ strongly\nrescues the DpilJ mutant phenotype, which genetically demonstrates that the construct is\nfunctional, a degree of evidence that is much stronger than a Western blot: the addition of a tag\ncan kill the biological activity of a protein, yet look very nice on a Western blot. That NAG still\npromotes the virulence of the rescue by the form lacking the LBD domains demonstrates that\nthis truncated form is also functional, a major result validating this construct.\n5. Quantification of Morphological Data\nThe central claim linking bacillus morphology to virulence relies on qualitative images (e.g., Fig.\n3A–C, 4G–K) without robust quantification. The authors could use automated tools (e.g.,\nCellProfiler, MicrobeJ) to quantify bacterial length, width, and aspect ratio across ≥100 cells per\ncondition, and include statistical comparisons (e.g., ANOVA with post-hoc tests) between groups\n(e.g., WT vs. ΔhigA in key flies).\nPlease, see Reply to Point 1. Of note, we have increased the number of quantified bacteria as\nmuch as possible. Since the differences are statistically significant, it makes no scientific sense\nto quantify more bacteria to reach this artificial threshold of 100, which may be easy to reach\nwhen working in vitro… Of note, when we perform survival experiments, we use usually\nbetween 120 to 180 flies per condition. Yet, when reviewing articles, I do not request that\ninvestigators working on mice use a similar number of animals…\nWe have changed the pictures of Fig. 3b-c, Fig. 4g, Fig. 5b, Fig. 6b, c, f, j, Fig. S9d.\nWe have increased the number of bacteria quantified: Fig. 3b’, d’, Fig. 4g’, Fig. 5b’, Fig. 6b’,\nf’, j’, Fig. S5a (quantification added), Fig. S9d’-d’’’ (quantification added, including width).\nWith respect to new data, bacteria retrieved 10h after co-injection with NAM or NAG,\nwe have\nalso monitored width to document that NAM-treated bacteria are more rounded in vivo, in\nkeeping with a loss of polarity.\n6. Fitness of Overexpression Strains\nThe partial rescue of ΔhigA virulence by pilJ, cyaB, or fimV overexpression (Figs. 4C–D, 5A) may\nreflect fitness trade-offs rather than mechanistic effects. The authors should perform growth\ncurves or competitive index assays in vitro and in vivo to rule out fitness costs.\nThis analysis has actually been performed systematically from the very first manuscript by\nmonitoring the virulence of bacteria in key immune-deficient flies in which there is no AMP\nexpression that acts on injected bacteria. We have always pointed out when the in vivo fitness\nof injected bacteria appeared to be somewhat lessened: Fig. 1e, g, Fig. 2f, Fig. 3b, d-h, Fig. 4b-\nd, g Fig. 5a, b, d, e, g, Fig. S8d (for Fig. 5j), Fig. 6a, b, e, h, i, j, Fig. S2f, Fig. S5b, c, e, f, h, i,\nFig. S6b, Fig. S6g, Fig. S7e, f, Fig. S9a-b, e, Fig. S10b-f.\n7. Unresolved Mechanism of NAM Toxicity\nThe observation that NAM abolishes virulence (Fig. 3I) is intriguing but unexplained.\nWe have now made a major step toward identifying the mechanism of NAM toxicity. It prevents\nthe association of FimV to the poles, likely by preventing the binding of FimV to the septal\npeptidoglycan, as now mentioned in the revised Discussion. What is fascinating is that NAG\ncounteracts the effect of NAG to some extent, as inferred from survival analysis. Strikingly,\nNAG does promote a faster localization of FimV-GFP to the poles (Fig. S9h-h’). Further\nstructural elucidation of a molecular mechanism by competition between NAM, but not NAG,\nand septal peptidoglycan for a peptidoglycan binding site in FimV lies outside of the scope of\nthis study.\nOf note, the rescue of the DhigA by the FimV truncated construct lacking the LysM domain (a\npeptidoglycan-binding domain) takes place in a context in which there is still some endogenous\nfull-length FimV expression that is likely enough to nucleate low quantities of endogenous\nFimV to the poles, which may allow the subsequent initiation of a weak bacterial polarity\nthrough protein-protein interactions able to compensate the absence of the LySM domain in\noverexpressed FimV. This may explain why the LysM truncated construct is still able to rescue\nrather well the DhigA mutant.\nMinor Concerns\n1. Figure Standardization\n-Color schemes: Adopt consistent color coding across all figures (e.g., use Fig. 1B’s\norange/green/blue/red scheme for WT/mutant/rescue groups).\n- Axis labels:\n- Remove redundant labels (e.g., “10” in Fig. 1A x-axis).\n- Use uniform terminology (e.g., “Bacterial load (CFU/fly)” in Fig. 1B, 2C).\n- Fonts and symbols:\n- Standardize font sizes and styles (e.g., Fig. 3B’ uses “PAO1” in a different font).\n- Replace “ns” with “n.s.” (not significant) and ensure asterisks align with statistical thresholds.\n2. Scheme Clarity\n- Simplify and standardize schematics (e.g., Fig. 3J, 4H, 5G). Move overly simplistic diagrams to\nsupplementary materials.\n3. Technical Details\n- Clarify ambiguous labels (e.g., “rpl49” in Fig. 2A should be defined as a normalization control).\n- Correct distorted fonts (e.g., Fig. 4L’) and align panel labels (e.g., “WT” vs. “key” in Fig. 3B’).\nDone.\nReviewer #2 (Remarks to the Author):\nThe authors have performed a substantial amount work and quantification to improve the\nmanuscript and have adequately addressed each of my concerns. Below are minor comments:\n-Recent studies have shown a significant difference in surface sensing, colonization, and biofilm\nformation between PAO1 and PA14, specifically with Wsp/c-di-GMP being much more important\nfor PAO1 and Pil-Chp/cAMP being more important for PA14 PMID: 34516283, 32098815. This is\none of the first studies to highlight Pil-Chp in PAO1, and I wonder how these experiments would\ndiffer if PA14 were used, although this is out of the scope of this study.\nWe have actually reproduced some of these data (DfimV and Dvfr) using the PA14 strain. We\nhave not included these data in the present study.\n-line 321-324 change PilJ gene to lowercase\nDone.\nReviewer #3 (Remarks to the Author):\nThe authors have performed a a significant number of extra experiments to address the major\nconcerns including extra qPCR experiments to ensure and and per are not induced on AMP\ntreatement, generation of a chic mutant that has a similar phenotype to WT indicating the NAG is\nnot generated by the host proteases. Moreover, they tested the cAMP activation and memory\nwith additional experiments to show priming is affected independent of surface sensing. Overall\nthe manuscript is vastly improved and contributes to our understanding of virulence priming by\nNAG.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank all reviewers for their inputs and suggestions to improve the quality of the\nmanuscript."}]

Decision letter