Probing Interkingdom Signaling Molecules via Liquid Extraction Surface Analysis-Mass Spectrometry

P obing In e kingdom Signaling Molecules ia Liquid Ex ac ion Su ace Analysis − Mass Spec ome y Shaun N. Robe son, Fadi Souka ieh, Thomas M. Whi e, Miguel Cama a, Manuel Rome o,* and Rian L. G i i hs* INTRODUCTION Polymic obial bio ilms a e common h oughou heal hca e, indus ial, and en i onmen al se ings. These su ace-associ- a ed o agg ega i e mic obial communi ies a e cen al o he challenge o an imic obial esis ance due o he o e p oduc ion o ex acellula polyme ic subs ances ha can en ap and limi he di usion o ce ain an ibio ics,1 and he induc ion o physiological changes ha lead o an imic obial ole ance in cells.2 I is es ima ed ha 65% o bac e ial in ec ions a e bio ilm-associa ed 3 and while unde es ima ed, Candida albicans (C. albicans) bio ilm in ec ions a e esponsible o an es ima ed >400,000 li e- h ea ening in ec ions a yea wo ldwide wi h a mo ali y a e o 46−75%.4 In hese communi ies, mic obes con ey hei p esence by p oducing small di usible signal molecules known as au oinduce s, which can hen be de ec ed and esponded o in a popula ion-coo dina ed manne . This o m o mic obial in e cellula communica ion is called quo um sensing (QS)5 and egula es physiological p ocesses in hese communi ies and, impo an ly, has been shown o con ol bio ilm o ma ion.6 Rema kably, QS signaling mole- cules ha e p e iously been shown o ac as bioma ke s o lung in ec ion ia LC−MS o plasma om cys ic ib osis pa ien s.7 Hence, hese molecules could ep esen impo an ci cula o y indica o s o in ec ion ha could be es ed nonin asi ely. Gi en ha mos bio ilm-cen e ed in ec ions a e polymic o- bial,8,9 he possibili y o in e species mic obial communica ion can also occu and QS is hough o play a i al ole in in e species in e ac ions anging om commensalism o an agonism.8 Fo ins ance, in e kingdom QS signaling be ween he bac e ium Pseudomonas ae uginosa (P. ae uginosa)and ungi C. albicans species has been desc ibed9 as media ed ia he p oduc ion o 2-alkyl-4(1H)-quinolone (AQ) signal molecules and a nesol, espec i ely. Mo eo e , hese species a e o en p esen alongside S aphylococcus au eus (S. au eus) in heal h- ca e se ings10,11 and i is well documen ed ha P. ae uginosa displays se e e compe i ion agains S. au eus in i o. Fo example, in wound in ec ions o cys ic ib osis lung, ex acellula ac o s p oduced by P. ae uginosa ha e been shown o subjuga e S. au eus o pe sis as small colony a ian s.12 One o such ac o s is he AQ molecule 2-hep yl-4- hyd oxyquinoline N-oxide (HQNO), a cy och ome inhibi o eleased by P. ae uginosa. In u n, P. ae uginosa can sense ex acellula p oduc s sec e ed by S. au eus such as he exopolyme N-ace yl glucosamine, and in esponse, inc ease he p oduc ion o i ulence ac o s and an imic obials.5,12,13 ABSTRACT: P e iously, me aboli es di used o sec e ed om mic obial samples ha e been analyzed ia liquid ch oma og aphy−mass spec ome y (LC−MS) app oaches ollowing leng hy ex ac ion p o ocols. He e, we p esen a model sys em o g owing bio ilms on discs be o e u ilizing apid and di ec su ace sampling MS, namely, liquid ex ac ion su ace analysis, o s udy he mic obial exome abolome. One o he bene i s o his app oach is i s su ace-speci ic na u e, enabling mimicking bio ilm o ma ion in a way ha he s udy o plank onic liquid cul u es canno Candida albicans (C. albicans) ha e been s udied p e iously in isola ion, e y ew s udies conside he complexi y o he in e play be ween hese pa hogens, which a e commonly combined causa i e agen s o in ec ion. Ou model sys em p o ides a ou e o in es iga e changes in he exome abolome, such as me aboli es ha become ci cula o y in he p esence o mul iple pa hogens. Ou esul s ag ee wi h p e ious epo s showing ha 2-alkyl-4(1H)-quinolone signal molecules p oduced by P. ae uginosa a e impo an ma ke s o in ec ion and sugges ha me hods o moni o ing le els o 2-hep yl-4-hyd oxyquinoline and 2,4-dihyd oxyquinoline, as well as pyocyanin, could be bene icial in he de e mina ion o causa i e agen s in in e kingdom in ec ion including P. ae uginosa. Fu he mo e, s udying changes in exome abolome me aboli es be ween pqs quo um sensing an agonis s in ea ed and non ea ed samples sugges s supp ession o phenazine p oduc ion by P. ae uginosa. Hence, ou model p o ides a apid analy ical app oach o gaining a mechanis ic unde s anding o bac e ial signaling. A icle Simila ly, a nesol, a nesoic acid,14 and a ious amino acid- de i ed alcohol15−17 molecules sec e ed by C. albicans ha e been shown o play an impo an ole in bio ilm o ma ion18,19 and a e cu en ly unde -in es iga ed in he con ex o polymic obial bio ilms. Mass spec ome y (MS) o e s a ou e o he un a ge ed analysis o mul iple species wi hou equi ing chemical agging/modi ica ion. Su ace sampling app oaches o e he oppo uni y o di ec ly p obe solid biological samples such as issue sec ions, blood spo ca ds, and bac e ial bio ilms. Many mic obial species ha e been analyzed ia di ec su ace sampling MS app oaches. The aims o hese s udies a y om bac e ial pheno yping o d ug sc eening and unde - s anding bac e ial bio ilms. Va ious biomolecules ha e been s udied ia di e en app oaches; an imic obials, bac e ial QS signaling molecules, me aboli es, and hamnolipids ha e been s udied ia ma ix-assis ed lase deso p ion ioniza ion (MALDI) ioniza ion MS,20−22 and seconda y ioniza ion mass spec ome y (SIMS).22−28 Phospholipid analysis is widely epo ed ia apid e apo a i e ioniza ion mass spec ome y (REIMS)29 and deso p ion elec osp ay ioniza ion (DESI) app oaches, and lipids and in ac p o eins ha e been s udied by liquid ex ac ion su ace analysis−mass spec ome y (LESA- MS).30−32 Many REIMS s udies ocus on specia ion using ma hema ical algo i hms o unde s and p o iles o , e.g., bac e ial s ains including P. ae uginosa29,33 and ungi such as he Candida genus,33,34 wi hou iden i ying speci ic analy es. P e iously, p o eins in ol ed in he in e ac ion be ween P. ae uginosa and S. au eus ha e been in es iga ed ia bo om-up p o eomics/MALDI-MS imaging and co ela ed o me al ions (in ol ed in p o ein in e ac ions) by lase abla ion-induc i ely coupled plasma MS imaging.35 Di e ences in he lipid and me aboli e p o iles (including heme, po phy in, and an ibio ic compounds) o Shewanella oneidensis and Bacillus sub ilis as single species and mixed bio ilms ha e been in es iga ed ia nano-DESI.36 LESA-MS is a su ace sampling app oach ha u ilizes sol en ex ac ion in o a obo ically ope a ed pipe e ip p io o elec osp ay ioniza ion. LESA-MS o e s so ioniza ion and apid analysis ime (a ew minu es) wi h he addi ional bene i o long sp ay imes, pa icula ly use ul o s uc u al iden i ica ion (MSMS) expe imen s. P e iously, LESA-MS o mic obial colonies including P. ae uginosa, S. au eus, and ESKAPE pa hogens has been desc ibed o he analysis o in ac p o eins om a ange o mono-mic obial sys- ems.30,31,37,38 Mo e ecen ly, LESA-MS has been used o he di ec bac e ial analysis o lipids om Mycobac e- ium30−32,38 and has been sugges ed as a po en ial al e na i e o MALDI-MS in specia ion o clinical isola es. He e, we desc ibe o he i s ime exome aboli e analysis speci ically, and om mono- and poly-mic obial bio ilms. This s udy seeks o in es iga e P. ae uginosa QS signaling molecules di used and/o sec e ed om single and poly- mic obial species bio ilms using a no el sample p epa a ion me hod speci ically designed o unde s and he bac e ial exome abolome. LESA-MS is desc ibed o he i s ime o a ge ed analysis o sec e ed QS molecules; in he aga unde nea h a bio ilm g own on a disc. Di e ences in QS we e de e mined in he p esence o a mic obial compe i o (namely, S. au eus o C. albicans), and in he p esence o bo h compe i o s using he LESA-MS pla o m. Ou model allowed he de ec ion o he AQ signal 2-hep yl-4-quinolone (HHQ), p e iously iden i ied as a diagnos ic ma ke o P. ae uginosa in ec ion, and sugges ed he u ili y o ou app oach. Ou esul s also sugges ha me hods o moni o ing le els o o he AQs such as 2,4-dihyd oxyquinoline (DHQ), as well as pyocyanin, could be bene icial in he de e mina ion o causa i e agen s in in e kingdom bio ilm communi ies. Fu he mo e, he e ec o an ibio ic ea men and/o inhibi ion o he AQ-based communica ion sys em in P. ae uginosa was examined and ou s udy shows ha , in ag eemen wi h p e ious epo s, AQ inhibi o s can supp ess phenazine biosyn hesis in bio ilm communi ies including his bac e ial pa hogen. The e o e, ou model p o ides a apid analy ical app oach o gaining a mechanis ic unde s anding o QS p ocesses in he con ex o polymic obial bio ilms. EXPERIMENTAL SECTION Ma e ials and Me hods. S ain and Colony Bio ilm Cul u e Condi ions. S. au eus SH1000 and P. ae uginosa PAO1-L s ains we e ou inely g own on he lysogeny b o h (LB, Oxoid, Camb idge, UK) aga . C. albicans SC5314 was ou inely g own in he Sabou aud dex ose (SAB, Oxoid, Camb idge, UK) aga . Bac e ial and ungi pla es we e incuba ed a 37 and 30 °C, espec i ely. UVC-s e ilized polyca bona e (PC) discs (13 mm diame e ) on wells o 6-well pla es illed wi h 5 mL o media. The s ock inoculum (10 μL, added on op o one ano he o polymic obial combina ions) was pipe ed o he cen e o he PC disc. Pla es we e hen ans e ed o a s a ic incuba o and incuba ed a 37 °C o 24 h o allow colony bio ilm g ow h. Fo ea men wi h he QS inhibi o s (QSIs), SEN19 and SEN89 compounds we e supplemen ed a 10 μM in he inal s ock inoculum o PAO1- L. T ea men o 18 h PAO1-L colony bio ilms wi h cip o loxacin was pe o med by adding 20 μL o 64 μg/mL in H2O pipe ed gen ly on op o he p e o med colony bio ilm. A he endpoin , PC discs wi h a ached colony bio ilms we e asep ically emo ed and aga plugs we e used o QS signal de ec ion. T iplica e biological and echnical epea s we e conduc ed o all expe imen s p esen ed. Fu he de ails, including CFU coun ing de ails, can be ound in he Supplemen al In o ma ion. LESA Sampling. LESA was ca ied ou using he T i e sa Nanoma e (Ad ionBiosciences, I haca, NY, USA). The ex ac ion/ioniza ion sol en was 1:1 me hanol/wa e . Du ing ex ac ion, 5 μL o sol en was aspi a ed om he sol en well, be o e sampling he aga wi h 2 μL o his sol en o 5 s wice (1 mix). Finally, 2.5 μL o he sampling sol en was e- aspi a ed and in used in o he mass spec ome e a a gas p essu e o 0.3 psi and a po en ial o 2.0 kV. Mass Spec ome y. Expe imen s we e pe o med on a The mo Fishe O bi ap Q-Exac i e mass spec ome e . Mass spec a we e eco ded in ull scan mode a a esolu ion o 140,000 a m/z 400 in he m/z ange o 50−750 in posi i e ioniza ion mode. The AGC a ge was 1 × 106 cha ges wi h a maximum injec ion ime o 500 ms. Each scan consis ed o 1 mic oscan. MSMS de ails can be ound in he Supplemen al In o ma ion. Da a we e eco ded o up o 1 min and analyzed using The mo Xcalibu e sion 4.2.28.14 so wa e. MSMS spec a we e manually in e p e ed. RESULTS AND DISCUSSION Alkyl-quinolone (AQ) Quo um Sensing (QS) Signaling Molecules Di used om and Vi ulence Fac o s Sec e ed by P. ae uginosa Bio ilms De ec ed by LESA-MS. A icle Figu e 1. Exome abolome analysis o QS molecules om P. ae uginosa PAO1-L bio ilms. Pho os o (A) bio ilm g own on PC disc, (B) aga sampled a e emo al o he disc wi h loca ions indica ed (blue = cen e , o ange = edge), and (C) LESA sampling. (D) Ba cha s showing dec easing signal in ensi ies o selec ed QS molecules om he cen e and he edge o whe e he bio ilm was g own on he PC disc. (E) Schema ic o he sample p epa a ion o exome abolome analysis and LESA sampling. (F) Spec a showing LESA-MS o aga backg ound (blue) and selec ed QS molecules de ec ed in he exome abolome o P. ae uginosa bio ilms. To assess whe he LESA-MS could be used o s udy he elease o small o ganic molecules om mic obial bio ilms, we s udied he p esence o QS molecules, which a e mos likely passi ely di used, and pyocyanin sec e ed by 24 h P. ae uginosa PAO1-L monospecies bio ilms. De ails ega ding speci ic QS pa hways and molecules analyzed a e p o ided in he Supplemen al In o ma ion. To allow cell- ee me aboli e ex ac ion om aga subs a es, bio ilms we e g own on 0.2 μm po e PC discs placed on he aga medium o enable he asep ic emo al o he mic obial cells p io o LESA-MS analysis (Figu e 1). The ollowing P. ae uginosa signals we e de ec ed om he media: HQNO, C9:1-PQS, C9-PQS, C9 quinolone, C11 quinolone, and HHQ, see Figu es 1 and 2. These all in o h ee classes o AQ QS molecules: HHQ-de i ed compounds [m/z 244.1696 (HHQ), 270.1849 and 272.2007], PQS-de i ed compounds (m/z 260.1642 and 288.1955), and HQNO-de i ed compounds [m/z 260.1642 (HQNO) and 288.1955], see Table 1. No e ha some o hese compounds a e isome s o one ano he , he e o e MSMS is equi ed o elucida ion. Howe e , N-acyl homose ine lac one (AHL) signal molecules we e no de ec ed in he condi ions es ed. The i ulence ac o pyocyanin ([M + H]+ m/z 211.0863) was de ec ed and sec e ed om PAO1-L bio ilms, sugges ing ha ou no el sample p epa a ion me hodology allows de ec ion o a ious di used and/o sec e ed me aboli es ex e nal o he bio ilm. LESA-MS was p e iously desc ibed o analyzing in ac bac e ial p o eins and lipids.32−34 Howe e , o ou knowledge, QS au oinduce s ha e no p e iously been epo ed using his app oach. QS molecules ha e p e iously been analyzed using, e.g., SIMS, om bio ilms anging om 7 o 72 h o g ow h.22,27,28 AQs wi h C9 alkyl chains we e shown o be highly abundan in ea ly 7 h bio ilms o P. ae uginosa g own on silicon iles.28 Simila molecules ha e been epo ed ia MALDI imaging o 12 h bio ilms.21,39 These QS signaling molecules ha e also been epo ed in ma u e (72 h) bio ilms o P. ae uginosa g own on silicon wa e iles.40 He e, we show ha AQs de i ed om HHQ, PQS, and HQNO can be analyzed ia LESA om bio ilms g own o 24 h. The e a e nume ous bene i s o ou app oach. Fi s , p e ious epo s ha e s udied bio ilms di ec ly; hence, i is no possible o dis inguish be ween me aboli es wi hin he bio ilm and hose di using away om he bio ilm. He e, we p esen a new sample o ma speci ically designed o analyze bac e ial bio ilm exome aboli es ha also has he ad an age o unde s anding he e ec o dis ance on mic obial QS, which could be impo an in de e mining diagnos ic ma ke s o in ec ion/se e i y. Compa ison o he exome abo- li es de ec ed in he media a he si e o he colony g ow h (blue, Figu e 1B), and hen a ound he ou e edge o he PC disc (o ange, Figu e 1B, and u he away (whi e, Figu e 1C), A icle Figu e 2. Ba cha showing he mean signal in ensi y o QS molecules de ec ed ia LESA-MS in he exome abolome analysis o in e species bio ilms. Mean alues o PA−SA−CA, which we e lowe han o he bio ilms a e shown in he inse . Table 1. Me aboli es De ec ed in he Exome abolome o In e species and In e kingdom Bio ilms o P. ae uginosa (PA), S. au eus (SA), and C. albicans (CA), and in he Exome abolome o Cip o loxacin (CIP) D ug and/o (SEN019 o SEN089) Inhibi o -T ea ed PA Bio ilmsa HHQ assignmen PA SA CA CA CIP SEN019 SEN019-CIP SEN089 HQNO/C7-PQS * * - * * * - * C9:1 quinolone * * - * * * - - NHQ C9 PQS C11 quinolone * * * - - * - - pyocyanin cis-2-decenoic acid (CDA) dihyd oxyquinolone (DHQ) aKey: - = no de ec ed eliably, * = de ec ed eliably ac oss epea s. shows dec easing signal in ensi ies o he ollowing signals: m/z 260.1642 (HQNO), m/z 272.2007 (C9-quinolone/2-nonyl- 4(1H)-quinolone (NHQ)) and m/z 288.1955 C9-PQS/ NQNO, see Figu e 1A,B,D. On a p ac ical le el, ou app oach allows he analysis o biomolecules di used/sec e ed om bac e ial bio ilms wi hou needing a dedica ed ins umen in he mic obial labo a o y, subjec o he app op ia e biological sa e y measu es. Ou LESA expe imen s bene i om coupling o a high- esolu ion (O bi ap) mass analyze o e ing he ad an age o de ec ed m/z accu a e m/z ppm chemical o mula 244.1696 244.1701 2.2 C 16 H 22 NO 260.1642 260.1651 3.5 C16H22NO2 270.1849 270.1858 3.3 C18H24NO 272.2007 272.2014 2.6 C 18 H 26 NO 288.1955 288.1964 3.1 C18H26NO2 300.232 300.2327 2.3 C 20 H 30 NO 211.0863 211.0871 3.8 C13H11N2O 188.1642 188.1651 4.8 C10H10N2O2 162.0548 162.0555 4.3 C 9 H 8 NO 2 PA − PA − PA − SA − PA − PA − PA − PA − * * * * * * - - * * - * * * - - * * - - * * - - * * * - * - - - A icle - - - * - * - - - - - * - * - - accu a e mass (wi hin 5 ppm), see Table 1. P e ious su ace- sampling MS s udies in o QS di ec ly om bio ilms ha e ypically u ilized ei he SIMS o MALDI, which a e mo e commonly coupled o ime-o - ligh ins umen a ion ha does no o e he same capabili ies. Only ecen ly has O bi ap SIMS ins umen a ion become a ailable,41 epo ing he de ec ion o 33 AQs and 6 AHLs in bio ilms o P. ae uginosa cul u ed o 48 h.42 The di e ences wi h ou esul s could a ise due o di e ences in sampling ime poin s (24 s 48 h bio ilms), sampling o he exome abolome a he han di ec A icle bio ilm analysis, and/o he lowe di usion and s abili y o AHLs dispe sing o he bio ilms. Ou app oach also bene i s om MSMS capabili y, allowing s uc u al cha ac e iza ion o de ec ed molecules. Hyb id ins umen s wi h his capabili y ha e only ecen ly been desc ibed in he SIMS communi y.43 MSMS expe imen s con i med m/z 244.17 is HHQ, see he Supplemen al In o ma ion and Figu e S1. HHQ has ecen ly been shown o ha e clinical diagnos ic ele ance in he ea ly de ec ion o in ec ion om nonin asi e biological luids such as u ine and b ea h condensa e in c i ically ill pa ien s wi h in ec ions cha ac e ized by polymic obial bio ilms wi h bac e ial and ungal con ibu o s.44 HCD agmen a ion o m/z 260.16 sugges s his is a mix u e o HQNO and PQS. Upon dissocia ion o m/z 272.20, he ollowing p oduc ions we e de ec ed: m/z 159.08, 172.07; howe e , he e was no agmen a m/z 188.10 o 175.06 sugges ing ha his species is NHQ a he han C9-PQS. This was de ec ed in ela i ely high abundance and has p e iously been shown o be an impo an bioma ke o lung in ec ion ia LC−MS o plasma om cys ic ib osis pa ien s.7 This demons a es he po en ial o his app oach as a model o he s udy o sec e ed biomolecules ha en e he ci cula o y sys em and can in o m QS molecules ha could be moni o ed ia nonin asi e biological samples and a e indica i e o in ec ion. In e species and In e kingdom Exome abolome Analysis. Using ou LESA-MS app oach, we s udied he exome abolome o in e species bio ilms, including P. ae ugino- sa−S. au eus (PA−SA), and in e kingdom bio ilms o P. ae uginosa−C. albicans (PA−CA), and P. ae uginosa−S. au eus−C. albicans (PA−SA−CA) combina ions. Colony bio ilms o P. ae uginosa PAO1-L, S. au eus SH1000, and/o C. albicans SC5314 mix u es we e co-cul u ed o 24 h, and he aga subs a es we e analyzed ia LESA-MS. QS signals ha e p e iously been epo ed in in e species in e ac ions om 24 h bio ilms g own a a 5 mm dis ance.20 He e, we epo hem om bio ilm colonies g own wi h no spa ial sepa a ion. PA−SA Bio ilms. Simila AQ molecules we e obse ed in he exome abolome o PA−SA communi ies compa ed o PA bio ilms, howe e , ela i e abundances we e di e en , see Figu e 2. HHQ was pa icula ly abundan in PA−SA, wi h C7- PQS/HQNO also de ec ed in highe abundance han in PA alone. In PA bio ilms, he mos abundan AQ de ec ed was NHQ, ollowed by C9:1-quinolone/NHQ and hen HHQ and HQNO/C7-PQS. Ye , in he PA−SA, HHQ was he mos abundan , ollowed by C7-PQS/HQNO, wi h NHQ signi i- can ly dec eased. This ag ees wi h p e ious MALDI imaging s udies o simila polymic obial sys ems, which de ec ed HHQ om P. ae uginosa (DK2-P2M24-2003 s ain) in he p esence o S. au eus (JE2).20 Ou esul s also sugges ha HQNO p oduc ion om P. ae uginosa PAO1-L inc eases in he p esence o S. au eus SH1000. Bo h HHQ and C9-PQS ha e been shown o ep ess S. au eus sp eading.45 In hese assays, he e was no obse ed di e ence in he iable eco e y o bo h mic obial species when co-incuba ed a 24 h when compa ed o monospecies g ow h. Pyocyanin was de ec ed in ela i ely high abundance ia LESA-MS in PA−SA, a h ee old inc ease in compa ison o PA, see Figu e S2. Pyocyanin is ac i ely sec e ed om PA; he e o e, i is a pa icula ly use ul bioma ke o a ge , i is also oxic o SA, hence an inc ease in p oduc ion is no unexpec ed. I is p omising ha ou app oach can de e mine a signi ican change in pyocyanin. Nume ous a emp s a modi ied pyocyanin assays ha e no yielded meaning ul esul s, possibly owing o sensi i i y limi a ions. Po en ially, his highligh s a bene i o ou app oach, al hough u he alida ion is equi ed. PA−CA Bio ilms. In he p esence o C. albicans SC5314, HHQ was de ec ed howe e , mos AQ molecules we e eliably supp essed (Figu e 2), sugges ing an impo an ole o HHQ in P. ae uginosa su i al in he p esence o C. albicans o , con a y o SA compe i ion, a po en ial dis up ion in he con e sion o HHQ o PQS by CA. HHQ has p e iously been shown o a ec C. albicans bio ilm o ma ion, and i is hough o play an essen ial ole in PA−CA in e kingdom in e - ac ions.46 Mo eo e , CFU coun ing showed a educ ion in CA g ow h in he p esence o PA wi h a 1−2 log di e ence in o al cell coun s. Inhibi ion o HQNO p oduc ion sugges s po en ial dis up ion o he PpqsL enzyma ic ac i i y in he PAO1-L pa hway; howe e , his pa hway is no dis up ed in PA−SA. Pyocyanin was de ec ed, al hough a lowe abundance (∼six old dec ease compa ed o PA bio ilm yields) in he p esence o CA. Fu u e wo k will ocus on alida ing educ ions in AQs and pyocyanin as po en ial bioma ke s o in ec ion caused by PA in he p esence o CA. PA − SA − CA Bio ilms. Despi e HHQ and HQNO molecules being he mos abundan AQs p oduced by PA in polymic obial bio ilms, including SA and CA (Figu e 2), a signi ican educ ion in hese AQ yields was eco ded (wi h no AQs de ec ed in some epea s), sugges ing compe i ion om hese could a ec he PQS signaling sys em. Resul s indica e ha , unde he es ed condi ions, HHQ could be a good indica o o P. ae uginosa coloniza ion, suppo ing he po en ial o his QS signal as a p ospec i e ea ly indica o o in ec ion by his pa hogen e en in he con ex o polymic obial in ec ions. In e es ingly, he AQ DHQ, con a y o single and dual-species bio ilms including PA, was consis en ly de ec ed in he PA− SA−CA communi y exome abolome (m/z 162.0548, Δppm 4.3). In con as o HHQ and PQS, DHQ can be p oduced unde low oxygen condi ions,47 sugges ing he educ ion in HHQ and PQS yields and de ec ion o DHQ could be explained by he o ma ion o anae obic niches in PA − SA − CA communi ies. Mo eo e , DHQ has been shown o ac as a QS molecule o ac i a e he esponse egula o PqsR o ansc ip ion o he pqs ope on in P. ae uginosa in he absence o PQS and HHQ signaling and has been epo ed o alle ia e he inhibi ion o PqsR by a nesol, albei a a lowe le el compa ed wi h PQS.47 Cis-2-decenoic acid (CDA) was de ec ed a m/z 188.1642 Δppm 4.8 in his iple sys em, and no in he o he samples. Simila ly o he PA−CA sys em, a signi ican dec ease in pyocyanin was obse ed in PA−SA− CA compa ed o PA bio ilms, which is in ag eemen wi h a educ ion in he PQS sys em ac i i y as i has been epo ed ha phenazine p oduc ion in P. ae uginosa is unde he con ol o his QS egula o y sys em.48 Fu u e wo k will ocus on alida ing HHQ, HQNO, and DHQ p esence and hei po en ial use, oge he wi h pyocyanin, as bioma ke s o polymic obial in ec ions including P. ae uginosa. Summa y. O e all, a ange o alkyl-quinolones (AQs) was de ec ed in he exome abolome o PA only. Pa icula ly high le els o HHQ and pyocyanin in he exome abolome we e indica i e o PA in combina ion wi h SA causa i e agen s. Low abundances o all AQs we e de ec ed when PA was co-cul u ed wi h CA, alongside a signi ican dec ease in pyocyanin. When PA was co-cul u ed in he p esence o bo h SA and CA, again many o he AQs ha we e de ec ed in he PA-only sys em we e supp essed, whe eas DHQ and CDA we e de ec ed o A icle Figu e 3. Ba cha showing he mean signal in ensi y o QS molecules de ec ed ia LESA-MS in he exome abolome analysis o ea ed PA bio ilms. CFU measu emen s om PA, inhibi o (SEN019)- ea ed PA, and cip o loxacin d ug (CIP)- ea ed PA a e shown in he inse . he i s ime and he e o e ep esen po en ially in o ma i e ions alongside AHLs. When SA was co-cul u ed wi h CA, many ions ha we e de ec ed we e simila o hose p esen in he exome abolome o he PA−SA−CA sys em, wi h he excep ion o CDA and DHQ. Ou indings a e summa ized in Figu e S3. Pla o m o Tes ing The apeu ic S a egies: Pseudo- monas ae uginosa QS Inhibi o s, Cip o loxacin, and Adjunc i e S a egies. Changes in QS molecules and i ulence ac o p oduc ion could also po en ially be used o moni o he e ec i eness o he apeu ic in e en ions. To s udy changes in hese biomolecules upon ea men , bio ilms o P. ae uginosa PAO1-L we e cul u ed o 24 h in he p esence o he an ibio ic cip o loxacin and/o he PqsR an agonis s SEN01949 (a mode a e inhibi o ) and SEN089 (a po en inhibi o ). These QSIs in e e e wi h he binding o AQ QS signals o he PqsR esponse egula o leading o educed ac i a ion o he pqs ope on exp ession and he e o e educed biosyn hesis o AQ signals and i ulence ac o s in P. ae uginosa. Despi e CFU coun s showing a educ ion in cell iabili y in PAO1-L bio ilms ea ed wi h cip o loxacin ( ∼ 4-log educ ion), mos AQ molecules de ec ed in he exome abo- lome o un ea ed PAO1-L bio ilms we e ound in communi ies ea ed wi h he an ibio ic, e.g., HHQ (m/z 244.1696), C7-PQS/HQNO (m/z 260.1642), C9:1-quinolone (m/z 270.1849), and C9-quinolone/NHQ (m/z 272.2007). Howe e , in his expe imen , HHQ was signi ican ly educed in signal in ensi y ac oss biological and echnical epea s, and C7-PQS and C9-PQS we e sligh ly ele a ed. In con as , he P. ae uginosa i ulence ac o pyocyanin abundance was no signi ican ly al e ed by cip o loxacin ea men , see Figu e 3. In he p esence o QSI SEN089 (IC50 = 67 nM,46), no AQ molecules we e de ec ed in he PAO1-L bio ilm exome abo- lomes ac oss all expe imen al biological and echnical epea s, see Figu e 3. This sugges s ha he p esence o his inhibi o e ec i ely dis up s he AQ biosyn he ic pa hway. P e iously, QS molecules p oduc ion was shown o dec ease in plank onic cul u es in he p esence o a ious pqs inhibi o s.46,49 He e, we show simila esul s om a sampling o ma speci ically designed o he analysis o su ace-associa ed mic obial communi ies. This highligh s one o he key bene i s o ou su ace-based app oach o e al e na i e liquid-based ap- p oaches. No ably, pyocyanin sec e ion was also dec eased in PAO1-L bio ilms ea ed wi h his QSI, sugges ing he supp ession o phzAB exp ession in line wi h p e ious pheno ypic analysis done wi h hese QS an agonis s.46,49 When PAO1-L bio ilms we e ea ed wi h he less po en QSI SEN19 (IC50 = 1 μM,49), AQ molecules could s ill be de ec ed in he bio ilm exo-me abolome albei a a educed yield. Simila ly, o un ea ed bio ilms, HHQ emained he mos abundan AQ ollowed by NHQ, see Figu e 3. In addi ion, pyocyanin displayed a sec e ion dec ease a e SEN19 ea men (Figu e S3), again indica ing supp ession o phzAB exp ession. As expec ed om ea men wi h an i i ulence compounds, CFU expe imen s showed no educ ion in iable coun s in he p esence o bo h QSI inhibi o s compa ed o un ea ed PAO1-L bio ilms. CDA and DHQ we e de ec ed when PA was ea ed wi h he less po en inhibi o (SEN019), howe e , hese we e no de ec ed upon ea men wi h a mo e po en inhibi o (SEN089). In e es ingly, bio ilms cul u ed in he p esence o SEN019 inhibi o and cip o loxacin led o no de ec ion o he common AQs p esen in PAO1-L exome abolome. Mo eo e , unde combina o y ea men , no sec e ed pyocyanin could be de ec ed, sugges ing a global dis up ion o AQ-based QS ne wo ks and he po en ial o LESA-MS-based app oaches o s udying mechanisms o d ug ac ion o new an i- i ulence ea men s and adjunc i e he apies. In con as , CFU coun s showed a simila dec ease in bio ilm cell iabili y (∼4-log educ ion) compa ed o bio ilms ea ed wi h cip o loxacin only. These esul s indica e ha he combina ion o hese wo d ugs could con ibu e o a educ ion in P. ae uginosa i ulence A icle bu no o a u he educ ion in cell iabili y o ea ed bio ilms unde he condi ions es ed. CONCLUSIONS This s udy demons a es ha he LESA-MS sampling o ma desc ibed he e p o ides a sui able model o he analysis o he mic obial exome abolome; wi h a ange o clinically ele an me aboli es de ec ed in he s udied mic obial communi ies, including in e species and in e kingdom sys ems. Ou app oach has he added bene i o allowing mic obiological cell cul u e in a labo a o y wi h he app op ia e sa e y measu es o he speci ic pa hogen, wi hou he need o a dedica ed ins umen in he same lab. Fu he mo e, di used o sec e ed compounds can be analyzed using ou app oach. HHQ has p e iously been indica ed as an ea ly diagnos ic ma ke o in ec ion in P. ae uginosa; ou s udy ag ees and he e o e shows p omise as a model. Addi ionally, ou esul s sugges ha me hods o moni o ing le els o AQs such as DHQ and pyocyanin could be bene icial in he de e mina ion o causa i e agen s in in e kingdom in ec ion. Fu he in es iga ion is equi ed o alida ion; quan i a i e analysis o le els in ci cula o y bio luids ia, e.g., LC−MS would be bene icial. Finally, we show how he LESA pla o m could p o ide a ou e o assessing he e ec o d ug ea men s and adjunc i e he apies o he u u e s udy o app op ia e ea men s o bac e ial, in e bac e ial, and in e kingdom sys ems using a bac e ia- ee pla o m. We en ision ha ou model will ind use in apidly in es iga ing bac e ial exome abolome analy es such as QS molecules om bac e ial and in e kingdom sys ems ha will aid p edic ion o clinically ele an diagnos ic me aboli es o in ec ion in bac e ial, ungal, and in e species/in e kingdom sys ems ha mimic he ue complexi y o ch onic in ec ion. This has ele ance in he in es iga ion and subsequen p edic ion o clinically ele an bioma ke s ha can be moni o ed nonin asi ely om biological luids. A a ie y o in e species and in e kingdom sys ems could be s udied in he u u e using his pla o m. ASSOCIATED CONTENT * Suppo ing In o ma ion The Suppo ing In o ma ion is a ailable ee o cha ge a h ps://pubs.acs.o g/doi/10.1021/acs.analchem.2c05703. Expanded Expe imen al and Resul s sec ions, and Supplemen al Figu es 1−3 (PDF) AUTHOR INFORMATION Co esponding Au ho s Manuel Rome o − U.K. Na ional Bio ilm Inno a ion Cen e (NBIC), Biodisco e y Ins i u e, School o Li e Sciences, Facul y o Heal h and Medical Sciences, Uni e si y o No ingham, NG7 2RD No ingham, U.K.; Depa men o Mic obiology and Pa asi ology, Facul y o Biology-CIBUS, Uni e sidade de San iago de Compos ela, 15782 San iago de Compos ela, Spain; Email: [email p o ec ed] Rian L. G i i hs − Facul y o Science, School o Pha macy, Uni e si y o No ingham, NG7 2RD No ingham, U.K.; o cid.o g/0000-0002-1601-4664; Email: ian.g i i hs@ no ingham.ac.uk Au ho s Shaun N. Robe son − U.K. Na ional Bio ilm Inno a ion Cen e (NBIC), Biodisco e y Ins i u e, School o Li e Sciences, Facul y o Heal h and Medical Sciences, Uni e si y o No ingham, NG7 2RD No ingham, U.K. Fadi Souka ieh − U.K. Na ional Bio ilm Inno a ion Cen e (NBIC), Biodisco e y Ins i u e, School o Li e Sciences, Facul y o Heal h and Medical Sciences, Uni e si y o No ingham, NG7 2RD No ingham, U.K.; o cid.o g/ 0000-0002-6730-2543 Thomas M. Whi e − Facul y o Science, School o Pha macy, Uni e si y o No ingham, NG7 2RD No ingham, U.K. Miguel Cama a − U.K. Na ional Bio ilm Inno a ion Cen e (NBIC), Biodisco e y Ins i u e, School o Li e Sciences, Facul y o Heal h and Medical Sciences, Uni e si y o No ingham, NG7 2RD No ingham, U.K. No es The au ho s decla e no compe ing inancial in e es . ACKNOWLEDGMENTS R.L.G. would like o acknowledge suppo om a Uni e si y o No ingham unded Anne McLa en Fellowship. This expe - imen al wo k was unded ia a B i ish Mass Spec ome y Socie y (BMSS) Resea ch Suppo G an . T.M.W. would like o acknowledge unding om he Uni e si y o No ingham o a summe s uden ship. M.R., S.N.R., F.S., and M.C. we e suppo ed by he Na ional Bio ilms Inno a ion Cen e [BBSRC, BB/R012415/1]. MR is suppo ed by he Ma ia Zamb ano p og am om he Spanish Minis y o Uni e si ies. REFERENCES (1) Daddi Oubekka, S.; B iande , R.; Fon aine-Aupa , M. P.; S eenkes e, K. An imic ob. Agen s Chemo he . 2012, 56, 3349−3358. (2) Kusa i, P.; Kusa i, S.; Lamshö , M.; Sezgin, S.; Spi elle , M.; Kayse , O. Appl. Mic obiol. Bio echnol. 2014, 98, 7173−7183. (3) Jamal, M.; Ahmad, W.; Andleeb, S.; Jalil, F.; Im an, M.; Nawaz, M. A.; Hussain, T.; Ali, M.; Ra iq, M.; Kamil, M. A. J. Chinese Med. Assoc. 2018, 81, 7−11. (4) B own, G. D.; Denning, D. W.; Gow, N. A.; Le i z, S. M.; Ne ea, M. 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