Antimicrobial Peptides Originating from Expression Libraries of Aurelia aurita and Mnemiopsis leidyi Prevent Biofilm Formation of Opportunistic Pathogens

Ci a ion: Ladewig, L.; Gloy, L.; Lang eld , D.; Pinnow, N.; Weiland-B äue , N.; Schmi z, R.A. An imic obial Pep ides O igina ing om Exp ession Lib a ies o Au elia au i a and Mnemiopsis leidyi P e en Bio ilm Fo ma ion o Oppo unis ic Pa hogens. Mic oo ganisms 2023,11, 2184. h ps://doi.o g/10.3390/ mic oo ganisms11092184 Academic Edi o : Wol -Raine Ab aham Recei ed: 19 June 2023 Re ised: 17 Augus 2023 Accep ed: 26 Augus 2023 Published: 29 Augus 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). mic oo ganisms A icle An imic obial Pep ides O igina ing om Exp ession Lib a ies o Au elia au i a and Mnemiopsis leidyi P e en Bio ilm Fo ma ion o Oppo unis ic Pa hogens Lisa Ladewig 1,†, Leon Gloy 1,†, Daniela Lang eld 1,2, Nicole Pinnow 1, Nancy Weiland-B äue 1 and Ru h A. Schmi z 1,* 1Gene al Mic obiology, Kiel Uni e si y, Am Bo anischen Ga en 1-9, 24118 Kiel, Ge many 2Ins i u e o Clinical Molecula Biology (IKMB), Kiel Uni e si y, Am Bo anischen Ga en 11, 24118 Kiel, Ge many *Co espondence: [email p o ec ed]; Tel.: +49-431-880-4334 †These au ho s con ibu ed equally o his wo k. Abs ac : The demand o no el an imic obial compounds is apidly g owing due o he ising appea ance o an ibio ic esis ance in bac e ia; acco dingly, al e na i e app oaches a e u gen ly needed. An imic obial pep ides (AMPs) a e p omising, since hey a e a na u ally occu ing pa o he inna e immune sys em and display ema kable b oad-spec um ac i i y and high selec i i y agains a ious mic obes. Ma ine in e eb a es a e a p ima y esou ce o na u al AMPs. Consequen ly, cDNA exp ession (EST) lib a ies om he Cnida ian moon jelly ish Au elia au i a and he C enopho e comb jelly Mnemiopsis leidyi we e cons uc ed in Esche ichia coli. Cell- ee size- ac iona ed cell ex ac s (<3 kDa) o he wo lib a ies (each wi h 29,952 clones) we e consecu i ely sc eened o pep ides p e en ing he bio ilm o ma ion o oppo unis ic pa hogens using he c ys al iole assay. The 3 kDa ac ion o en indi idual clones demons a ed p omising bio ilm-p e en ing ac i i ies agains Klebsiella oxy oca and S aphylococcus epide midis. Sequencing he espec i e ac i i y-con e ing inse s allowed o he iden i ica ion o small ORFs encoding pep ides (10–22 aa), which we e subsequen ly chemically syn hesized o alida e hei inhibi o y po en ial. Al hough he pep ides a e likely a i icial p oduc s om a andom ansla ion o EST inse s, he bio ilm-p e en ing e ec s agains K. oxy oca, Pseudomonas ae uginosa,S. epide midis, and S. au eus we e e i ied o i e syn he ic pep ides in a concen a ion-dependen manne , wi h pep ide BiP_Aa_5 showing he s onges e ec s. The impac o BiP_Aa_2, BiP_Aa_5, and BiP_Aa_6 on he dynamic bio ilm o ma ion o K. oxy oca was u he alida ed in mic o luidic low cells, demons a ing a signi ican educ ion in bio ilm hickness and olume by BiP_Aa_2 and BiP_Aa_5. O e all, he s uc u al cha ac e is ics o he ma ine in e eb a e- de i ed AMPs, hei physicochemical p ope ies, and hei p omising an ibio ilm e ec s highligh hem as a ac i e candida es o disco e ing new an imic obials. Keywo ds: bio ilm; an imic obial pep ide; AMP; pa hogen 1. In oduc ion Mul icellula o ganisms e ol ed in he p esence o mic obes, which play undamen- al oles in hei heal h, de elopmen , and e olu ion [ 1 ]. Ea ly-b anching me azoans, i.e., in e eb a es, ha e equi ed complex sys ems o ecognizing and disc imina ing bene- icial and pa hogenic mic oo ganisms [ 2 – 5 ]. Howe e , unlike e eb a es, in e eb a es lack classical an ibody-based adap i e immuni y [ 2 ]. In e eb a e immuni y o igina es om de ense eac ions (i.e., inna e immune sys em), including hemocy e-media ed modules, en- capsula ion, phagocy osis, and gene a ion o an imic obial pep ides (AMPs) [ 6 ]. AMPs a e small, mainly posi i ely cha ged pep ides ha p omo e he inna e de ense mechanism by a ge ing he nega i ely cha ged memb anes o mic oo ganisms [ 7 ]. AMPs become embed- ded in he hyd ophobic egions o lipid memb anes, o en o ming po es, hus des abilizing Mic oo ganisms 2023,11, 2184. h ps://doi.o g/10.3390/mic oo ganisms11092184 h ps://www.mdpi.com/jou nal/mic oo ganisms Mic oo ganisms 2023,11, 2184 2 o 17 biological memb anes and causing cell lysis [ 8 , 9 ]. AMPs can also exhibi in e ac ions wi h in acellula a ge s, con ibu ing o hei mul i ace ed an imic obial ac i i y [ 10 ]. AMPs ha e been shown o a e se he cell memb ane and access in acellula compa men s, whe e hey can in e ac wi h a ious in acellula molecules and p ocesses. These in e ac- ions can dis up in acellula componen s, such as DNA, RNA, p o eins, and enzymes, a ec ing i al cellula unc ions [ 10 ]. Such in e ac ions may u he enhance he bac e icidal o mic obicidal e ec s o AMPs and con ibu e o hei o e all an imic obial po ency [ 11 ]. AMPs a e apidly induced in esponse o mic obes o modula e he immuno eac ions. They ha e a wide ange o inhibi o y e ec s agains bac e ia, ungi, pa asi es, and i uses [ 12 ]. Consequen ly, AMPs ha e awakened in e es as po en ial nex -gene a ion an ibio ics, since eme ging an ibio ic esis ance in pa hogenic bac e ia is a se ious challenge and has led o he need o new al e na i e bioac i e molecules less p one o bac e ial esis ance [ 13 , 14 ]. One pa icula in e es in using AMPs is o comba bio ilms o pa hogens ha ha e been demons a ed o be signi ican con ibu o s o diseases [ 15 , 16 ]. Mul iple ac o s con ibu e o he o e all esis ance o bio ilms agains an ibio ics, including educed me abolic and g ow h a es, p o ec ion by ex acellula polyme ic subs ances, and speci ic esis ance mech- anisms con e ed by he al e ed physiology o bio ilm bac e ia [ 17 , 18 ]. Focusing on ea ing he highly challenging, ad e se e ec s o ha m ul bio ilm-associa ed in ec ions, AMPs a e indica ed o ha e s ong po en ial as an imic obials and an ibio ilm agen s [15,19–21] . Ma ine in e eb a es ha e been shown o be a p ima y sou ce o na u al AMPs [ 6 ] and a e, he e o e, likely o be an excellen sou ce o no el ma ine AMPs. O e he pas decades, se e al AMPs ha e been iden i ied and isola ed om ma ine in e eb a es, including mol- lusks (de ensins, my ilins, and my icins) [ 22 – 24 ], annelids (lumb icins and a enicins) [ 25 ], a h opods (cec opins and a acins) [ 26 ], and unica es (s yelins and didemnins) [ 27 – 29 ] bu also cnida ians [ 30 ]. Cnida ians p oduce a ange o AMPs wi h di e en s uc u es and unc ions, such as ShK-1 om he sea anemone S ichodac yla helian hus [ 31 ] and Aly ese in-2a om he sea anemone Anemonia sulca e [ 32 ]. Ca helicidin-like pep ides ha e been iden i ied in se e al cnida ian species, including he sea anemones Nema os ella ec ensis and Aip asia pallida [ 33 ]. C enopho es a e so a no lis ed wi hin he exis ing an imic obial-speci ic da abases, such as In e Pep (h ps://ciencias.medellin.unal.edu.co/ g uposdein es igacion/p ospeccionydisenobiomoleculas/In e Pep/public/home_en, ac- cessed on 15 Janua y 2023). In e eb a e pep ides ha e been shown o ha e an imic obial ac i i y agains bac e ia, ungi, and some i uses, and a couple may also ha e an i umo and immunomodula o y e ec s. The di e si y and complexi y o AMPs p oduced by ma- ine in e eb a es sugges ha hese compounds may ha e b oad applica ions in medicine and bio echnology and p o ide insigh s in o he e olu ion o hos de ense mechanisms. Those pep ides possess no el and unique s uc u es by only exhibi ing a ew side e ec s [ 6 ]. Howe e , mo e esea ch is necessa y o ully unde s and he p ope ies and po en ial applica ions o AMPs [ 34 ]. Unde s anding he mechanism o ac ion o AMPs is c i ical o hei de elopmen as he apeu ic agen s [ 35 ]. The op imal condi ions o he ac i i y o AMPs, hei a ge si es wi hin mic obial cells, and how hey in e ac wi h he hos immune sys em ha e o be s udied. AMPs may ha e oxic e ec s on he hos , and he e is a need o de e mine he app op ia e dosages and po en ial side e ec s [ 36 ]. Fu he , he e icacy o AMPs may be a ec ed by ac o s such as pH, ionic s eng h, and o he en i onmen al con- di ions. The e o e, esea ch is equi ed o op imize he deli e y o AMPs o hei a ge si es. Resea ch should u he ocus on unde s anding he mechanisms o po en ial esis ance de elopmen [ 37 ]. Bu , i s and o emos , new AMPs wi h po en an imic obial ac i i y agains a b oad ange o mic oo ganisms ha e o be iden i ied, which can be acili a ed by using high- h oughpu sc eening app oaches and bioin o ma ics [38,39]. The p esen s udy aimed o iden i y no el an imic obial pep ides om lowe me a- zoans as p omising and p ospe ous sou ces o no el biologically ac i e compounds o p e en bio ilms. Consequen ly, cDNA exp ession (EST) lib a ies we e cons uc ed om he Cnida ian moon jelly ish Au elia au i a and he C enopho e comb jelly Mnemiopsis leidyi. Low-molecula -weigh ac ions (<3 kDa) de i ed om bo h EST lib a ies we e unc ionally Mic oo ganisms 2023,11, 2184 3 o 17 sc eened o iden i y (a i icial) ac i i ies p e en ing he bio ilm o ma ion o oppo unis ic pa hogens. A e iden i ying he sORFs in he espec i e inse s, he co esponding pep ides we e chemically syn hesized and es ed o hei po en ial o p e en s a ic and dynamic pa hogenic bio ilms. 2. Ma e ials and Me hods 2.1. Au elia au i a Polyp Husband y The husband y o polyps is desc ibed in de ail in p e ious s udies by Weiland-B äue e al. [ 40 , 41 ]. Polyps o he subpopula ion No h A lan ic (Rosco , F ance) we e kep in he labo a o y a 20 ◦ C in 30 PSU a i icial seawa e (T opical Sea Sal s, T opic Ma in) and ed wice a week wi h eshly ha ched A emia salina (HOBBY, G a scha - Gelsdo , Ge many). 2.2. Sampling o Au elia au i a Polyps and Mnemiopsis leidyi Medusae A. au i a polyps we e used om husband y by emo ing indi idual polyps wi h a disposable pipe e. Indi idual M. leidyi medusae (wi h a mean umb ella diame e o 4 cm) we e sampled om he Kiel Bigh , Bal ic Sea (54 ◦ 32.8 0 N, 10 ◦ 14.7 0 E) in May 2017 using a dip ne . The animals we e immedia ely anspo ed o he labo a o y and washed ho oughly wi h s e ile a i icial seawa e . 2.3. Di ec mRNA Isola ion and Cons uc ion o he cDNA Exp ession Lib a ies The mRNA o he A. au i a polyps and M. leidyi medusae we e isola ed wi h he DynaBeads ® mRNA DIRECT Mic o Ki (Ambion, Aus in, TX, USA) acco ding o he manu- ac u e ’s p o ocol “mRNA isola ion om issues”. In o al, 28 pa allel p epa a ions we e conduc ed using pools o 10 A. au i a polyps and 1 × 1 cm pa s o M. leidyi medusae (in o al 10 medusae) pe isola ion. Animal issues we e ozen in liquid ni ogen and homoge- nized wi h a mo o ized pes le (polyps) o a blende (medusae). Fo A. au i a 3µg and o M. leidyi 1.2 µ g mRNA we e used o cons uc he cDNA exp ession lib a ies wi h he Clone Mine II cDNA Lib a y Cons uc ion Ki (In i ogen, Wal ham, MA, USA) acco ding o he manu ac u e ’s p o ocol. Fi s , he mRNA was ansc ibed in o cDNA and, subsequen ly, cloned in o he pDONR222 en y ec o . Second, he en y clones we e ecombined wi h Ga eway exp ession ec o pET300/NT-DEST using he ChampionTM pET300/NT-DEST Ga ewayTM Vec o Ki (In i ogen, Wal ham, MA, USA) o c ea e exp ession clones. The esul ing plasmids e ained he o iginal alignmen and eading ame, allowing o he unc ional analysis o ull-leng h genes and en i e lib a ies. SoluBL21 elec ocompe en Esche ichia coli cells we e used o he elec opo a ion and as backg ound s ain o he exp ession (Genla is, San Diego, CA, USA). Each lib a y consis ed o 29,952 single clones and we e s o ed in 96-well mic o i e pla es a − 80 ◦ C in he p esence o 8% DMSO as c yop o ec an . 2.4. P epa a ion o Cell-F ee Size-F ac iona ed Cell Ex ac s The cDNA clones we e i s es ed as 96 clone pools o iden i y an imic obial pep- ides. Posi i e pools we e successi ely sc eened as 48 and 24 clone pools and, inally, as single clones o e icien ly sc een he wo cDNA lib a ies, each consis ing o o e 29,000 clones, in a high h oughpu p ocedu e (see e . [ 42 ]). The e o e, single cDNA clones we e g own in 200 µ L Lu ia Be ani medium (LB, Ca l Ro h, Ka ls uhe, Ge many) supplemen ed wi h 100 µg/mL ampicillin o e nigh a 37 ◦ C in 96-well mic o i e pla es. Ini ially, 96 clones we e pooled. The mix u e o bac e ial cul u es was cen i uged a 9000 × g o 5 min, and he bac e ial pelle was esuspended in 250 µ L 50 mM T is/HCl bu e (50 mM NaCl, pH 7.8 ). The suspension was ans e ed o sc ew-cap ubes (Sa s ed , Nümb ech , Ge many) con aining one glass bead o a diame e 2.7 mm and app oxima ely 50 mg o glass beads o a diame e 0.1 mm (Ca l Ro h, Ka ls uhe, Ge many). Snap- ozen (liq- uid N 2 ) bac e ial cells we e mechanically dis up ed wi h P ecellys ® (Be in ins umen s, Mon ignyle-B e onneux, F ance). The cell ex ac was s e ile- il e ed h ough a 0.2 µ m Mic oo ganisms 2023,11, 2184 4 o 17 cen i ugal il e (Amch o GmbH, Ha e sheim am Main, Ge many) ollowed by size- ac iona ion using a 3 kDa cen i ugal il e acco ding o he manu ac u e ’s ins uc ions (Me ck KGaA, Da ms ad , Ge many). The samples we e kep a 4 ◦ C o on ice h oughou he p ocess. The ac ions < 3 kDa we e collec ed, and he app oxima e p o ein concen a- ions we e measu ed using a NanoD op1000 (The mo Fishe Scien i ic, Wal ham, MA, USA) and adjus ed o he same concen a ion. The emp y pET300/NT-DEST/E. coli SoluBL21 was used as a con ol. Bio ilm-p e en ing 96-pools we e u he es ed in pools o 48 and 24 clones o apidly un a el single clone(s) esponsible o he bio ilm inhibi ion using he desc ibed p ocedu e. 2.5. Bac e ial Bio ilm P e en ion In Vi o Assay The oppo unis ic pa hogenic bac e ia K. oxy oca (DSM 7342, M5a1- ype s ain), P. ae uginosa PAO1 (DSM 1707, pa hogenic- ype s ain), S. epide midis RP62A (DSM 28319, clinical isola e om ca he e sepsis), and S. au eus (DSM 11823, clinical isola e) we e g own in 5 mL LB medium o e nigh a below-indica ed empe a u es. The cell concen- a ions o he o e nigh cul u es we e analyzed wi h Neubaue cell coun ing and se o 3×108cells/mL using GC minimal medium (wi h 1% ( / ) glyce ol and 0.3% (w/ ) casamino acids) [ 43 ] o K. oxy oca and Caso bouillon (17 g/L casein pep one, 3 g/L soybean pep one, 5 g/L NaCl, 2.5 g/L K 2 HPO 4 , and 2.5 g/L glucose) o he emaining s ains in he c ys al iole assay. Cul u es we e aliquo ed in 96-well pla es (180/195 µ L o each ca i y). Cell- ee size- ac iona ed cell ex ac s (20 µ L) and syn hesized pep ides ( a ious concen a ions, 5 µ L) we e added o he cul u es. MTPs we e closed wi h gas-pe meable memb anes (B ea he-Easy ® , Di e si ied Bio ech, Dedham, MA, USA) and incuba ed a 37 ◦C (P. ae uginosa,S. au eus, and S. epide midis) o 30 ◦ C (K. oxy oca) o 18 h wi hou shaking. Th ee biological eplica es we e pe o med, each wi h eigh echnical eplica es. Medium con ols we e pe o med o no maliza ion. Bio ilm o ma ion was moni o ed and quan i ied using he c ys al iole assay. Following an 18 h incuba ion pe iod, he cell cul u es we e emo ed, and he wells unde wen wo washes wi h H 2 O. Bio ilms ha de eloped and adhe ed o he su aces we e subjec ed o s aining using 200 µ L o a 0.1% c ys al iole solu ion (Ca l Ro h, Ka ls uhe, Ge many) o 10 min a oom empe a u e (RT). The s ained bio ilms we e le o ai d y a e elimina ing he c ys al iole solu ion h ough wo H 2 O washes. A o al o 200 µ L o 96% e hanol was in oduced in o each s ained well o solubilize he dye, and incuba ion a RT o 15 min ollowed. Bio ilm o ma ion was assessed by measu ing he abso bance o esol ed c ys al iole a 590 nm wi h he pla e eade Spec a max Plus 384 (Molecula De ices, Ismaning; Ge many) [ 44 , 45 ]. The sc eening p ocedu e is depic ed in Figu e S1. In addi ion, he po en ial g ow h-inhibi ing e ec s o syn he ic pep ides we e es ed on plank onic-g owing pa hogens. Again, 3 × 10 8 cells/mL dilu ed in LB medium we e aliquo ed in 96-well pla es (200 µ L o each ca i y). Syn he ic pep ides we e added o wo selec ed inal concen a ions o 3.5 µ g/mL and 112.5 µ g/mL a he beginning o he expe imen . MTPs we e closed wi h gas-pe meable memb anes and gen ly shaken a 80 pm and 37 ◦ C (P. ae uginosa,S. au eus, and S. epide midis) o 30 ◦ C (K. oxy oca) o 18 h . A compa a i e endpoin de e mina ion (g ow h con ol, pep ide con ol IDR-1018, and iden i ied pep ides) o he u bidi y a 600 nm was measu ed wi h pla e eade Spec a max Plus 384. An unpai ed - es was conduc ed as a s a is ical hypo hesis es o de e mine he signi icance (p- alue) o g ow h di e ences. 2.6. Plasmid P epa a ion, Inse Size De e mina ion, and Sequencing The plasmid DNA o he iden i ied bio ilm-p e en ing single clones was isola ed om 5 mL o e nigh cul u es using he P es o ™ Mini Plasmid Ki , acco ding o he manu ac u e (Geneaid, Taiwan). A es ic ion diges wi h he Bs GI enzyme was pe o med o inse size de e mina ion, ollowed by subsequen gel analysis. The plasmids we e Sange - sequenced by he Ins i u e o Clinical Molecula Biology, CAU Kiel, Ge many. Sequences we e analyzed wi h Geneious P ime so wa e ( e sion 2022.2.1) (Bioma e s, Auckland, Mic oo ganisms 2023,11, 2184 5 o 17 New Zealand). Vec o sequences and PolyA ails we e emo ed om he aw sequences. Cleaned inse sequences we e used o open eading ame iden i ica ion. The pep ide sequences wi hin he ame o he ec o coding his idine- ag we e selec ed o pep ide syn hesis. All sequence da a can be ound in Table S1. 2.7. Syn hesis o Pep ides Pep ides wi h su icien bio ilm-p e en ing ac i i y and a leng h o >5 amino acids we e syn hesized using con en ional solid-phase pep ide syn hesis wi h >94% pu i y a Gensc ip (Leiden, The Ne he lands) (Table 1). The syn he ic pep ides we e dissol ed in s e ile dis illed wa e (Ca l Ro h, Ka ls uhe, Ge many) and s o ed a − 80 ◦ C as 100 µ L aliquo s o 10 mg/mL. Table 1. Syn he ized pep ides. Pep ides we e syn hesized in 94% pu i y a Gensc ip (Leiden, The Ne he lands). The cha ac e is ics o he pep ides a e summa ized. MW CALC , calcula ed molecula weigh ; pICALC, isoelec ic poin . Pep ide Designa ion Rela ed EST Clone Amino Acid Sequence MWCALC (kDA) pICALC Agg ega ion P opensi y [46] BiP_Aa_2 A. au i a 112_6C QLFCTKLSPT 1.14 8.22 0.201 BiP_Aa_4 A. au i a 127_8E THSTMNFELHQRSIQSCSPKLL 2.55 7.95 −0.091 BiP_Aa_5 A. au i a 127_8F VRNSVLFRRRRLVC 1.78 12.18 0.150 BiP_Aa_6 A. au i a 127_8H TNFTRKFPVINSSPHQWYLKD 2.58 9.7 −0.042 BiP_Ml_3 M. leidyi 010_9A GRTTLYKKVGFFFFF 1.8 10.29 0.561 con ol IDR-1018 [47] VRLIVAVRIWRR 1.5 12.48 0.487 2.8. E ec o Bio ilm-P e en ing Pep ides on Bio ilm Fo ma ion o K. oxy oca M5aI in a Mic o luidic Flow Cell The mic o luidic low cell sys em comp ises a polyme hyl me hac yla e co pus wi h a single channel (1 × 8 × 0.1 mm). Two inle s and one ou le exis . A bo osilica e glass (24 × 60 × 0.17 mm; Ca l Ro h, Ka ls uhe, Ge many) was ixed wi h he adhesi e Black- Seal silicone (Weicon, Müns e , Ge many). Mic oli e sy inges (Inno a i e Labo Sys em GmbH, S ü zenbach, Ge many) we e connec ed ia poly e a luo oe hylene (PTFE) ubes ( 0.3 mm ×0.6 mm ) (Bola, G üns eld, Ge many) wi h he in- and ou le s and clamped in o a sy inge pump (Model: 220) (KD Scien i ic, Hollis on, MA, USA). The channel was s e ilized by insing 70% E OH o 24 h a a low a e o 20 µ L/h. A e wa ds, he emaining e hanol was washed ou wi h s e ile wa e a 20 µ L/h o 1 h. Subsequen ly, he channel was equilib a ed wi h GC medium a 20 µ L/h o 2 h. Inocula ion was conduc ed wi h a cell suspension o 1 × 10 9 cells/mL, gene a ed om an o e nigh cul u e o K. oxy oca M5aI. Ini ial cell adhesion was ensu ed by incuba ion a 30 ◦ C o 1 h wi hou any low. Nex , he channel was con inuously insed a 15 µ L/h wi h GC medium injec ed wi hin he i s inle . The syn he ic pep ides IDR-1018 (bio ilm-p e en ing con ol pep ide, [ 47 , 48 ], BiP_Aa_2, BiP_Aa_5, and BiP_Aa_6 we e dilu ed in GC medium o a concen a ion o 25 µg/mL and injec ed wi hin he second inle . Thus, he inal pep ide concen a ion was 12.5 µ g/mL, co esponding o 10 ng pep ide wi hin he 0.8 µ L channel. Bio ilm o ma ion was conduc ed a a low a e o 15 µ L/h o 24 h a 30 ◦ C. The bio ilm o ma ion o K. oxy oca M5aI was u he s udied wi hou adding pep ides. Fou biological eplica es we e conduc ed o each ea men . Bio ilms we e s ained wi h he luo escen dye Sy o9 (488 nm) (Li e Technologie, Ca lsbad, CA, USA) and analyzed using he LSM 700 con ocal lase scanning mic oscope (Zeiss, Obe kochen, Ge many). The e o e, he mic o luidic low cells we e washed wi h GC medium a 3 µ L/min o 15 min. The channel was hen illed wi h a 1:1000 dilu ion o he luo escen dye Sy o9 and incuba ed in he da k a RT o 30 min. Dye esidues we e emo ed by insing GC medium a 3 µ L/min o 15 min. Fou image s acks we e eco ded Mic oo ganisms 2023,11, 2184 6 o 17 pe low cell wi h op ical sec ions o 0.9 µ m pe z-s ep. The digi al image acquisi ion, h ee- dimensional econs uc ion, and calcula ion o he bio ilm pa ame e s we e pe o med wi h he so wa e “Zen Black” ( e sion 14.0.22.201) (Zeiss, Obe kochen, Ge many) and he mic oscopy image analysis so wa e “Ima is” ( e sion 9.9.0) (Ox o d Ins umen s, Abingdon, UK). 3. Resul s The exp ession lib a ies o he wo basal me azoans, A. au i a and M. leidyi, we e cons uc ed and sc eened o bio ilm-p e en ing pep ides agains oppo unis ic pa hogenic bac e ia (K. oxy oca,P. ae uginosa,S. epide midis, and S. au eus) using he c ys al iole assay. P omising pep ide candida es we e cha ac e ized o hei po en ial o p e en he dynamic bio ilm o ma ion o K. oxy oca in mic o luidic cells. 3.1. Cons uc ion o cDNA Exp ession Lib a ies o Two Basal Me azoans Exp ession lib a ies we e cons uc ed om mRNA de i ed om he Cnida ian moon jelly ish A. au i a and he C enopho e comb jelly M. leidyi in o he Ga eway exp ession ec o pET300/NT-DEST (In i ogen). Each lib a y consis ed o 29,952 single clones (pET300/NT- DEST in E. coli Solu BL21). Acco ding o he manu ac u e , he gene a ed plasmids o he exp ession clones e ain he o iginal alignmen and eading ame o he inse , allowing o he unc ional analysis o ull-leng h genes. Howe e , he e is also he possibili y ha andom ansla ion o sho non-na u al ORFs occu s. Plasmids o andomly selec ed clones (96 clones pe lib a y) we e cha ac e ized by es ic ion analysis, demons a ing an a e age inse size o app oxima ely 1.4 kbps and 95–98% inse ion e iciencies o bo h lib a ies. Analyzing he inse sequences o hose andomly selec ed clones e ealed ha 92% o he eads aligned o he espec i e genomes [ 46 , 49 – 51 ]. This, howe e , does no necessa ily allow o any s a emen conce ning hei ac ual unc ion in he medusae [46,49–51]. 3.2. Iden i ica ion o Bio ilm-P e en ing Clones om he cDNA Exp ession Lib a ies Bo h o he cons uc ed cDNA lib a ies we e used o iden i y bio ilm-p e en ing pep- ides de i ed om he basal me azoan hos s. Cell- ee cell ex ac s we e successi ely p e- pa ed om pools o 96, 48, 24, and single clones o he cDNA exp ession lib a y ( Figu e S1 ). No ably, cell ex ac s we e size- ac iona ed using 3 kDa cu o columns, and only he ac ions < 3 kDa we e analyzed using he c ys al iole assay. This assay was ini ially pe - o med wi h he G am-nega i e oppo unis ic pa hogen K. oxy oca and he G am-posi i e pa hogen S. epide midis. In o al, 36% o he 96 clone pools showed bio ilm-p e en ing ac i i y o a leas one o he wo es ed pa hogens. Successi ely, pools o dec easing clone numbe s we e sc eened un il single clones we e iden i ied (Figu e S1). Cell- ee size- ac iona ed cell ex ac s o single clones we e e alua ed o hei bio ilm-p e en ing po en ial, including wo addi ional oppo unis ic pa hogens, P. ae uginosa and S. au eus. Bio ilm biomasses we e measu ed using he c ys al iole assay, wi h he bio ilm o ma ion o pa hogens wi hou any added es subs ance se a 100%. O e all, en bio ilm-p e en ing single clones (six de i ed om A. au i a, and 4 om M. leidyi) we e iden i ied wi h a ying impac s on he bio ilm o ma ion o he di e en pa hogens (Table 2). Fo ins ance, clones Aa_112_4H and Aa_127_8E consis en ly showed bio ilm-p e en ing e ec s on all pa hogens es ed (Table 2). Clones Aa_127_8A and Aa_127_8F appea o ha e exclusi e e ec s on K. oxy oca and S. epide midis bio ilm o ma ion (Table 2). Clone Aa_127_8H no ably en- hances he bio ilm o ma ion o K. oxy oca, bu i educes he bio ilm o ma ion o he o he bac e ia (Table 2). Sequence analysis o he bio ilm inhibi ion-con e ing inse s iden i ied he co e- sponding small open eading ames (sORFs) (Table S1). He e, exclusi ely hose sORFs N- e minally used o he ec o -de i ed his idine- ag and longe han i e amino acids we e used o u he analysis since sho e sequences we e less likely o exhibi signi - ican bioac i i y. S uc u al models o he i e espec i e pep ides we e p edic ed wi h PEP-FOLD 3 (Figu e 1). NCBI BLASTp iden i ied hose pep ide sequences in A. au i a and Mic oo ganisms 2023,11, 2184 7 o 17 M. leidyi genomes. Howe e , nonsigni ican E- alues we e eached because o he sho inpu sequences. Table 2. Iden i ied single clones de i ed om he cDNA exp ession lib a ies o A. au i a and M. leidyi and hei impac on he bio ilm o ma ion o pa hogens. Bio ilm biomasses we e calcula ed based on he c ys al iole assay. Bio ilm o ma ion o pa hogens wi hou adding a es subs ance was se as 100%. Clone Designa ion Bio ilm Fo ma ion (%) K. oxy oca P. ae uginosa S. au eus S. epide midis Aa_112_4H 71 ±12 85 ±19 77 ±9 55 ±14 Aa_112_6C 92 ±14 79 ±24 84 ±13 49 ±9 Aa_127_8A 22 ±2 100 ±18 100 ±13 77 ±13 Aa_127_8E 80 ±3 69 ±17 78 ±13 6 ±1 Aa_127_8F 28 ±3 75 ±18 84 ±23 95 ±10 Aa_127_8H 104 ±5 77 ±18 61 ±10 40 ±12 Ml_068_11H 107 ±13 81 ±13 69 ±7 29 ±3 Ml_011_11H 27 ±2 100 ±22 67 ±3 77 ±10 Ml_010_9A 71 ±8 78 ±14 76 ±5 94 ±9 Ml_010_9G 68 ±14 75 ±18 89 ±17 91 ±7 Mic oo ganisms 2023, 11, 2184 7 o 17 Aa_112_4H and Aa_127_8E consis en ly showed bio ilm-p e en ing effec s on all pa ho- gens es ed (Table 2). Clones Aa_127_8A and Aa_127_8F appea o ha e exclusi e effec s on K. oxy oca and S. epide midis bio ilm o ma ion (Table 2). Clone Aa_127_8H no ably en- hances he bio ilm o ma ion o K. oxy oca, bu i educes he bio ilm o ma ion o he o he bac e ia (Table 2). Table 2. Iden i ied single clones de i ed om he cDNA exp ession lib a ies o A. au i a and M. leidyi and hei impac on he bio ilm o ma ion o pa hogens. Bio ilm biomasses we e calcula ed based on he c ys al iole assay. Bio ilm o ma ion o pa hogens wi hou adding a es subs ance was se as 100%. Clone Designa ion Bio ilm Fo ma ion (%) K. oxy oca P. ae uginosa S. au eus S. epide midis Aa_112_4H 71 ± 12 85 ± 19 77 ± 9 55 ± 14 Aa_112_6C 92 ± 14 79 ± 24 84 ± 13 49 ± 9 Aa_127_8A 22 ± 2 100 ± 18 100 ± 13 77 ±13 Aa_127_8E 80 ± 3 69 ± 17 78 ± 13 6 ± 1 Aa_127_8F 28 ± 3 75 ± 18 84 ± 23 95 ± 10 Aa_127_8H 104 ± 5 77 ± 18 61 ± 10 40 ± 12 Ml_068_11H 107 ± 13 81 ± 13 69 ± 7 29 ± 3 Ml_011_11H 27 ± 2 100 ± 22 67 ± 3 77 ± 10 Ml_010_9A 71 ± 8 78 ± 14 76 ± 5 94 ± 9 Ml_010_9G 68 ± 14 75 ± 18 89 ± 17 91 ± 7 Sequence analysis o he bio ilm inhibi ion-con e ing inse s iden i ied he co e- sponding small open eading ames (sORFs) (Table S1). He e, exclusi ely hose sORFs N- e minally used o he ec o -de i ed his idine- ag and longe han i e amino acids we e used o u he analysis since sho e sequences we e less likely o exhibi signi ican bi- oac i i y. S uc u al models o he i e espec i e pep ides we e p edic ed wi h PEP- FOLD 3 (Figu e 1). NCBI BLASTp iden i ied hose pep ide sequences in A. au i a and M. leidyi genomes. Howe e , nonsigni ican E- alues we e eached because o he sho inpu sequences. Figu e 1. P edic ed s uc u al models o bio ilm-p e en ing pep ides. Sequences we e analyzed us- ing he Geneious P ime so wa e ( e sion 2022.2.1) (Bioma e s, Auckland, New Zealand). Models we e c ea ed using PEP-FOLD 3. 3.3. Syn he ic Pep ides—Excluding Cy o oxic Effec s on Plank onic Bac e ia The i e iden i ied pep ides we e chemically syn hesized and designa ed as BiP_Aa_2, BiP_Aa_4, BiP_Aa_5, BiP_Aa_6, and BiP_Ml_3 (Table 1). The calcula ed molec- ula weigh s (MWs) anged be ween 1.14 (BiP_Aa_2) and 2.58 kDa (BiP_Aa_6) and he isoelec ic poin s (pIs) be ween 7.95 (BiP_Aa_4) and 12.18 (BiP_Aa_5) (Table 1), offe ing insigh s in o he pep ide sizes, beha io unde diffe en pH condi ions, solubili y, elec o- pho e ic beha io , and po en ial in e ac ions wi h o he molecules. The agg ega ion p o- pensi y was calcula ed om −0.091 (BiP_Aa_4) o 0.561 (BiP_Ml_3) (Table 1), indica ing he o ma ion o he p o ein agg ega es necessa y o assessing he biopha maceu ical and Figu e 1. P edic ed s uc u al models o bio ilm-p e en ing pep ides. Sequences we e analyzed using he Geneious P ime so wa e ( e sion 2022.2.1) (Bioma e s, Auckland, New Zealand). Models we e c ea ed using PEP-FOLD 3. 3.3. Syn he ic Pep ides—Excluding Cy o oxic E ec s on Plank onic Bac e ia The i e iden i ied pep ides we e chemically syn hesized and designa ed as BiP_Aa_2, BiP_Aa_4, BiP_Aa_5, BiP_Aa_6, and BiP_Ml_3 (Table 1). The calcula ed molecula weigh s (MWs) anged be ween 1.14 (BiP_Aa_2) and 2.58 kDa (BiP_Aa_6) and he isoelec ic poin s (pIs) be ween 7.95 (BiP_Aa_4) and 12.18 (BiP_Aa_5) (Table 1), o e ing insigh s in o he pep ide sizes, beha io unde di e en pH condi ions, solubili y, elec opho e ic beha io , and po en ial in e ac ions wi h o he molecules. The agg ega ion p opensi y was calcula ed om − 0.091 (BiP_Aa_4) o 0.561 (BiP_Ml_3) (Table 1), indica ing he o ma ion o he p o ein agg ega es necessa y o assessing he biopha maceu ical and bio echnological po en ial. As a posi i e con ol, pep ide IDR-1018 was addi ionally syn hesized. IDR-1018 is known o i s immunomodula o y and an imic obial p ope ies. I has been shown o possess an imic obial ac i i y agains a wide ange o bac e ia, including bo h plank onic- and bio ilm- o ming bac e ia. IDR-1018 can inhibi bac e ial g ow h by dis up ing bac e ial cell memb anes, in e e ing wi h essen ial cellula p ocesses, and modula ing he hos immune esponse [ 47 , 48 ]. Ini ially, he po en ial g ow h-inhibi ing e ec s o all syn he ic pep ides on plank onic-g owing pa hogens we e e alua ed o exclude he pep ides’ g ow h- inhibi o y (i.e., oxic) e ec s and ocus on an ibio ilm e ec s. Two con as ing concen a ions o 3.5 and 112.5 µ g/mL we e selec ed. None o he pep ides a ec ed he g ow h beha io o any pa hogens a a low concen a ion [ 47 , 48 ]. Acco ding o o iginal epo s [ 47 , 48 ], con ol pep ide IDR-1018 adminis e ed a high concen a ion signi ican ly educed he plank onic g ow h o all s ains (Figu e 2, see Table S2 o - es s a is ics). Hos -deduced syn he ic pep ides BiP_Aa_4 and BiP_Aa_6 did no signi ican ly inhibi plank onic g ow h, Mic oo ganisms 2023,11, 2184 8 o 17 e en a 112.5 µ g/mL; mo eo e , hey mos ly esul ed in g ow h p omo ion. The p esence o BiP_Aa_2, BiP_Aa_5, and BiP_Ml_3 led o some pa hogen-speci ic g ow h e ec s. In mo e de ail, all h ee syn he ic pep ides educed he plank onic g ow h o P. ae uginosa and S. au eus by 1.5–5%, while S. epide midis was a ec ed only by BiP_Aa_5 (3%). Howe e , all obse ed g ow h e ec s we e minimal in each case, al hough mos ly s a is ically signi ican (Figu e 2and Table S2). Mic oo ganisms 2023, 11, 2184 8 o 17 bio echnological po en ial. As a posi i e con ol, pep ide IDR-1018 was addi ionally syn- hesized. IDR-1018 is known o i s immunomodula o y and an imic obial p ope ies. I has been shown o possess an imic obial ac i i y agains a wide ange o bac e ia, includ- ing bo h plank onic- and bio ilm- o ming bac e ia. IDR-1018 can inhibi bac e ial g ow h by dis up ing bac e ial cell memb anes, in e e ing wi h essen ial cellula p ocesses, and modula ing he hos immune esponse [47,48]. Ini ially, he po en ial g ow h-inhibi ing effec s o all syn he ic pep ides on plank onic-g owing pa hogens we e e alua ed o ex- clude he pep ides’ g ow h-inhibi o y (i.e., oxic) effec s and ocus on an ibio ilm effec s. Two con as ing concen a ions o 3.5 and 112.5 µg/mL we e selec ed. None o he pep- ides affec ed he g ow h beha io o any pa hogens a a low concen a ion [47,48]. Ac- co ding o o iginal epo s [47,48], con ol pep ide IDR-1018 adminis e ed a high concen- a ion signi ican ly educed he plank onic g ow h o all s ains (Figu e 2, see Table S2 o - es s a is ics). Hos -deduced syn he ic pep ides BiP_Aa_4 and BiP_Aa_6 did no signi - ican ly inhibi plank onic g ow h, e en a 112.5 µg/mL; mo eo e , hey mos ly esul ed in g ow h p omo ion. The p esence o BiP_Aa_2, BiP_Aa_5, and BiP_Ml_3 led o some pa h- ogen-speci ic g ow h effec s. In mo e de ail, all h ee syn he ic pep ides educed he plank onic g ow h o P. ae uginosa and S. au eus by 1.5–5%, while S. epide midis was a - ec ed only by BiP_Aa_5 (3%). Howe e , all obse ed g ow h effec s we e minimal in each case, al hough mos ly s a is ically signi ican (Figu e 2 and Table S2). Figu e 2. Exclusion o po en ial g ow h-inhibi ing effec s o he syn he ic pep ides on plank onic cells. K. oxy oca, P. ae uginosa, S. epide midis, and S. au eus (3 × 10 8 cells/mL) we e g own o 18 h in 200 µL LB medium a 80 pm a e adding 112.5 µg/mL o he syn he ic pep ides. Tu bidi y was moni o ed a 600 nm o wo biological eplica es, each wi h eigh echnical eplica es, ep esen ed as he means wi h co esponding s anda d de ia ions. The g ow h effec s (p omo ing and inhibi - ing) effec s we e compa ed o a con ol wi hou he addi ion o pep ide. Signi ican p- alues a e indica ed only o g ow h-inhibi ing effec s: * p < 0.05, ** p < 0.01, and *** p < 0.001. 3.4. Syn he ic Pep ides—Ve i ica ion o he Bio ilm-P e en ing Effec s on S a ic Bio ilms The bio ilm-p e en ing po en ial o all syn he ic pep ides was es ed in a ious con- cen a ions anging om 0.4 µg/mL o 112.5 µg/mL, including he wo selec ed concen a- ions o cy o oxic effec s exclusion, agains he ou oppo unis ic pa hogens using he c ys al iole assay (Figu e 3). The p esence o he con ol pep ide IDR-1018 educed he Figu e 2. Exclusion o po en ial g ow h-inhibi ing e ec s o he syn he ic pep ides on plank onic cells. K. oxy oca,P. ae uginosa,S. epide midis, and S. au eus (3 × 10 8 cells/mL) we e g own o 18 h in 200 µ L LB medium a 80 pm a e adding 112.5 µ g/mL o he syn he ic pep ides. Tu bidi y was moni o ed a 600 nm o wo biological eplica es, each wi h eigh echnical eplica es, ep esen ed as he means wi h co esponding s anda d de ia ions. The g ow h e ec s (p omo ing and inhibi ing) e ec s we e compa ed o a con ol wi hou he addi ion o pep ide. Signi ican p- alues a e indica ed only o g ow h-inhibi ing e ec s: * p< 0.05, ** p< 0.01, and *** p< 0.001. 3.4. Syn he ic Pep ides—Ve i ica ion o he Bio ilm-P e en ing E ec s on S a ic Bio ilms The bio ilm-p e en ing po en ial o all syn he ic pep ides was es ed in a ious concen- a ions anging om 0.4 µ g/mL o 112.5 µ g/mL, including he wo selec ed concen a ions o cy o oxic e ec s exclusion, agains he ou oppo unis ic pa hogens using he c ys al iole assay (Figu e 3). The p esence o he con ol pep ide IDR-1018 educed he bio ilm o ma ion o K. oxy oca a a concen a ion o 3.5 µ g/mL on a e age by 30% and a he highes concen a ion (112.5 µ g/mL) by 70%. Howe e , no in e e ence wi h he bio ilm o ma ion o he second G am-nega i e pa hogen, P. ae uginosa, was obse ed. Fo G am-posi i e S. epide midis, he bio ilm o ma ion was educed in a ange o 10 o 30%, almos ega dless o he concen a ion, whe eas S. au eus showed a concen a ion-dependen educ ion in bio ilm o ma ion by up o 60%. Fo all o he iden i ied hos -de i ed syn he ic pep ides, he e ec s on he bio ilm o ma ion o a leas one o he pa hogenic s ains we e moni o ed (Figu e 3). Pep ide BiP_Aa_2 showed a negligible impac on P. ae uginosa and S. au eus bio ilm o ma ion a all pep ide concen a ions. K. oxy oca exhibi ed no signi ican change in bio ilm o ma ion un il 14.1 µ g/mL, bu an e ec mani es ed om 28.1 µ g/mL (up o 43% educ ion). S. epide midis displayed a 45% maximum educ ion. Mo eo e , he e ec o BiP_Aa_2 on S. epide midis bio ilms appea ed o e e se wi h inc easing concen a ions (Figu e 3). BiP_Aa_4 had Mic oo ganisms 2023,11, 2184 9 o 17 a limi ed e ec on S. au eus,P. ae uginosa, and K. oxy oca bio ilms. Mode a e inhibi ion (23–47%) appea ed in S. epide midis, showing de ia ion om concen a ion dependence (Figu e 3). BiP_Aa_5 e ec i ely inhibi ed bio ilm o ma ion o all pa hogenic s ains s udied. A he highes pep ide dose (112.5 µ g/mL), bio ilm educ ion eached 47% o P. ae uginosa, 44% o S. au eus, and 54% o K. oxy oca.S. epide midis exhibi ed a milde e- duc ion a his pep ide concen a ion han he o he s ains (22%) (Figu e 3). Fo BiP_Aa_6, S. epide midis bio ilm maximally educed by 43%. K. oxy oca showed inc easing bio ilm inhibi ion, eaching 26% a he highes adminis e ed concen a ion, while S. au eus showed a 6–23% inhibi ion. P. ae uginosa bio ilm ini ially inc eased and hen g adually dec eased, app oaching con ol le els (Figu e 3). BiP_Ml_3 had no signi ican e ec on S. au eus, P. ae uginosa, o K. oxy oca bio ilms, wi h S. epide midis showing g ow h inc ease a low pep- ide doses. Howe e , 112.5 µ g/mL pep ide p og essi ely inhibi ed S. epide midis bio ilm, culmina ing in a 38% educ ion a he highes concen a ion (Figu e 3). In gene al, he bio ilm o ma ion o P. ae uginosa was no signi ican ly impac ed, bu sligh in e e ence by BiP_Aa_5 and BiP_Ml_3 was ob ained a a high concen a ion. Depending on he pep ide, S. epide midis bio ilm o ma ion was educed by 5 up o 45%. No ably, he p e en ion occu ed almos ega dless o he pep ide concen a ion, which was also he case o he con ol pep ide. Fo S. au eus, BiP_Aa_2, BiP_Aa_4, and BiP_Ml_3 could no in e - e e wi h bio ilm o ma ion, whe eas he p esence o BiP_Aa_5 e ealed a educ ion o 40% (Figu e 3). Rela ing he g ow h inhibi ion da a o he plank onic cells (Figu e 2) when conside ing bio ilm inhibi ion (Figu e 3), ou disce nible pa e ns can be deduced ha a e speci ic o he es ed pa hogen and pep ide (only esul s o he 112.5 µ g/mL pep ide concen a ion we e ega ded). G ow h-inhibi ing e ec s on plank onic cells co espond o no ewo hy bio ilm inhibi ion (e.g., BiP_Aa_5 in P. ae uginosa and S. spp.) o , con e sely, g ow h- p omo ing e ec s align wi h augmen ed bio ilm o ma ion (e.g., BiP_Aa_4 and BiP_Aa_6 in P. ae uginosa). Fu he mo e, g ow h-p omo ing e ec s s and in con as o bio ilm inhibi o y e ec s (e.g., BiP_Aa_6 and BiP_Ml_3 in S. epide midis). Finally, BiP_Aa_2, e ealed g ow h- inhibi ing e ec s on plank onic cells bu elici ed inc eased bio ilm o ma ion. Consequen ly, we canno es ablish o e a ching pa e ns. The complexi y o co ela ing hese e ec s migh a ise om he dis inc li es yles and beha io s o plank onic and bio ilm cells, making di ec compa isons be ween hei impac s challenging. In conclusion, mos o he hos -de i ed syn he ic pep ides p e en ed bio ilms o G am- nega i e and G am-posi i e pa hogenic bac e ia in a concen a ion-dependen manne , wi h BiP_Aa_5 demons a ing he mos subs an ial e ec s on bio ilm o ma ion o all es ed pa hogens. This inding a gues ha he ini ially obse ed bio ilm-p e en ing ac i i ies o he 3 kDa cell ex ac ac ions a e mainly based on he espec i e sequence-iden i ied pep ides, al hough o he inhibi o y biomolecules p esen in he 3 kDa ac ions canno be excluded en i ely. 3.5. Syn he ic Pep ides—Bio ilm-P e en ing E ec s on Dynamic K. oxy oca Bio ilms In he i s a emp , mic o luidic low cells we e cons uc ed and es ablished o he bio ilm o ma ion o ou bio ilm model o ganism and oppo unis ic pa hogen K. oxy oca (Figu e 4A). A comp ehensi e bio ilm o ma ion o K. oxy oca was eached wi h an ini ial cell concen a ion o 8 × 10 5 cells/channel and a low a e o 15 µ L/h o 24 h a 30 ◦C . A compac bio ilm wi h a wa y su ace was o med wi h a mean bio ilm hickness o 11 ±6µm (Figu e 4B, le ba ) and olume o 112 ± 62 µ m 3 (Figu e 4B, igh ba , medium con ol). In a second s ep, he bio ilm o ma ion o K. oxy oca was analyzed in he p es- ence o he syn he ic hos -de i ed pep ides BiP_Aa_2, BiP_Aa_5, and BiP_Aa_6 and he con ol pep ide IDR-1018 (10 ng/channel) wi h ou biological eplica es, each wi h ou echnical eplica es (Figu e 4B). In con as o he medium con ol, he con ol pep ide IDR-1018 signi ican ly p e en ed bio ilm o ma ion. P edominan ly, mic ocolonies bu no 3D s uc u es we e de ec ed, esul ing in a educed maximum hickness o 6 ± 2 µ m (p= 0.0036; Figu e 4B, le ba ) and a educed olume o 32 ± 16 µ m 3 (p= 0.0046; Figu e 4B , Mic oo ganisms 2023,11, 2184 16 o 17 30. O e o-Gonzáiez, A.J.; Magalhaes, B.S.; Ga cia-Villa ino, M.; López-Aba a egui, C.; Sousa, D.A.; Dias, S.C.; F anco, O.L. An- imic obial pep ides om ma ine in e eb a es as a new on ie o mic obial in ec ion con ol. FASEB J. 2010 ,24, 1320–1334. [C ossRe ] 31. Bee on, C.; Penning on, M.W.; No on, R.S. 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An imic obial pep ides: A po en al e na i e o an ibio ics. An ibio ics 2021,10, 1095. [C ossRe ] [PubMed] Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .