Anti-biofilm molecules targeting functional amyloids

an ibio ics Re iew An i-Bio ilm Molecules Ta ge ing Func ional Amyloids Le icia Ma illa-Cuenca , Alejand o Toledo-A ana and Jaione Valle *   Ci a ion: Ma illa-Cuenca, L.; Toledo-A ana, A.; Valle, J. An i-Bio ilm Molecules Ta ge ing Func ional Amyloids. An ibio ics 2021, 10, 795. h ps://doi.o g/10.3390/ an ibio ics10070795 Academic Edi o : Thee hanka Das Recei ed: 31 May 2021 Accep ed: 25 June 2021 Published: 29 June 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 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/). Ins i u o de Ag obio ecnología (Idab), CSIC-Gobie no de Na a a, 31192 Na a a, Spain; [email p o ec ed] (L.M.-C.); [email p o ec ed] (A.T.-A.) *Co espondence: [email p o ec ed] Abs ac : The choice o an e ec i e he apeu ic s a egy in he ea men o bio ilm- ela ed in ec ions is a signi ican issue. Amyloids, which ha e been his o ically ela ed o human diseases, a e now conside ed o be p e ailing s uc u al componen s o he bio ilm ma ix in a wide ange o bac e ia. This assump ion c ea es he po en ial o an exci ing esea ch a ea, in which unc ional amyloids a e conside ed o be a ac i e a ge s o d ug de elopmen o dissemble bio ilm s uc u es. The p esen e iew desc ibes he bes -cha ac e ized bac e ial unc ional amyloids and ocuses on an i-bio ilm agen s ha a ge in insic and acul a i e amyloids. This s udy p o ides a be e unde s anding o he di e en modes o ac ions o he an i-amyloid molecules o inhibi bio ilm o ma ion. This in o ma ion can be u he exploi ed o imp o e he he apeu ic s a egies o comba bio ilm- ela ed in ec ions. Keywo ds: bio ilm; amyloids; an ibio ic esis ance; pep ides; polyphenols 1. In oduc ion 1.1. Bio ilm Rela ed In ec ions Nosocomial in ec ions ela ed o he use o medical de ices a e associa ed wi h a high isk o mo ali y and inc eased economic cos s [ 1 ]. These in ec ions a e mainly caused by oppo unis ic bac e ia such as E n e ococcus aecium, S aphylococcus au eus, K lebsiella pneumoniae, A cine obac e baumannii, P seudomonas ae uginosa, and E n e obac e species om he ESKAPE g oup, many o which a e esis an o commonly used an ibio ics [ 2 ]. The apid appea ance o an ibio ic esis ance in hese bac e ia is associa ed wi h a global inc ease in an ibio ic consump ion in he ields o heal h ca e, ag icul u e, and in he en i onmen , in addi ion o hei inapp op ia e u iliza ion ela ed o inadequa e dosing o an ibio ic selec ion [ 3 – 5 ]. The di icul y o ea ing heal hca e-associa ed in ec ions is also in ensi ied by he common ai o he oppo unis ic bac e ia o o m bio ilms. Bac e ia a e able o o m mul icellula communi ies on he su ace o bioma e ials, mucosa, issues, and sec e ions, and exp ess a sel -gene a ed ex acellula polyme ic ma ix in esponse o my iad signals. The ex acellula ma ix, composed o polysaccha ides, su ace p o eins, nucleic acids, and amyloid ibe s, p o ec s he mic obes om ex e nal insul s and ad e se en i onmen al condi ions. The bio ilm ma ix ac s as a physical and chemical ba ie ha educes he a e o an imic obial pene a ion. In addi ion, i can gene a e mic oen i onmen s ha an agonize he ac ion o an ibio ics and p omo e he abili y o bac e ia o gene a e pe sis e cells wi h inc eased d ug ole ance [ 6 – 9 ]. Thus, bac e ia in bio ilms a e less sensi i e o an ibio ics han he same s ain g owing in suspension, esul ing in a 10- o 100- old dec ease in hei suscep ibili y [ 10 ]. Because con en ional an ibio ics ail o success ully ea bio ilm-associa ed in ec ions, no el he apeu ic solu ions a e in u gen ly needed. E o s a e cu en ly unde way o de elop new he apeu ic app oaches ha ocus on p e en ing he syn hesis o he assembly o he bio ilm ma ix componen s o ende bac e ia sensi i e o an ibio ic ea men s. An ibio ics 2021,10, 795. h ps://doi.o g/10.3390/an ibio ics10070795 h ps://www.mdpi.com/jou nal/an ibio ics An ibio ics 2021,10, 795 2 o 18 1.2. Amyloids as S uc u al Sca olds o he Bio ilm Ma ix Amyloids a e adi ionally associa ed wi h se e al incu able degene a i e human diseases. Howe e , nume ous s udies ha e indica ed ha mic oo ganisms make use o amyloidogenic p o eins o a numbe o non-pa hological p ocesses [ 11 ]. In ecen decades, amyloids ha e been conside ed o be essen ial componen s o he bio ilm, p o iding con- sis ency and iscoelas ici y o he ex acellula bio ilm ma ix [ 12 – 15 ]. Amyloids a e highly o de ed ib illa p o eins wi h a β -shee seconda y s uc u e and a s ongly conse ed qua e na y c oss- β s uc u e [ 16 ]. To a lesse ex en , α -helices can eplace he β -s ands, hus o ming c oss- α ib ils [ 17 , 18 ]. The amyloid s uc u e binds a ange o speci ic dyes such as Congo ed, Thio la in T (ThT), and P o eoS a , which, combined wi h o he bio- physical echniques, such as solid-s a e NMR and Fou ie - ans o m in a ed spec oscopy (FTIR), a e no mally used o de e mine he amyloidogenic ea u es o a p o ein [ 19 – 22 ]. Due o he well-o de ed s uc u e o β -s ands ha a e aligned pe pendicula o a ib il axis, amyloids a e an e ec i e ex acellula building ma e ial. They a e mos ly esis an o ha sh dena u ing condi ions and p o eoly ic clea age. In addi ion, polyme iza ion o amyloidogenic p o eins occu s h ough a nuclea ion-dependen sel -assembly p ocess, in which s a e amyloid agg ega es p o ide a con o ma ional sca old ha acili a es he assembly o polyme ic subuni s in o he amyloid s a e in he absence o ene gy. Due o his seeding mechanism, he amyloid con o ma ion is sui able unde condi ions in which he ene gy is limi ed [12,23]. Taking in o accoun ha amyloids a e widely dis ibu ed componen s o he bio ilm ma ix in many bac e ia, hey can be conside ed excellen a ge s o an i-amyloid d ugs o educe bio ilm o ma ion. This e iew aims o summa ize he cu en knowledge o he bac e ial amyloids and he di e en mechanisms o ac ion o he an i-amyloid molecules o inhibi bio ilm o ma ion. 2. Func ional Amyloids o he Bio ilm Ma ix In ecen yea s, an inc easing numbe o amyloid componen s o he bio ilm ma ix ha e been iden i ied, pu i ied, and closely in es iga ed [ 15 ]. Based on he s uc u al and unc ional s a e o he amyloidogenic p o eins, i is becoming popula o g oup hem in o in insic and acul a i e amyloids (Table 1) [24]. 2.1. In insic Amyloids The polyme ic s uc u e o in insic amyloids is he esul o a dedica ed sys em ha con ols he sec e ion and assembly o he monome ic building blocks. Indeed, he amyloidogenic subuni s do no ha e a olded s a e p io o o ming he amyloid ibe s. The amyloid con o ma ion ep esen s he p ima y s uc u al and unc ional s a e o he p o ein. Examples o amyloids om his g oup a e desc ibed below. 2.1.1. Cu li (csgBAC-csgDEFG Genes) The cu li imb iae p esen in E. coli,Salmonella, and o he En e obac e ia is one o he mos well-cha ac e ized unc ional amyloid sys ems. The cu li-speci ic genes a e disposed in wo di e gen ope ons csgBAC and csgDEFG [ 25 ]. CsgA is he majo cu li subuni ha s uc u ally o ms he amyloid ibe s. To achie e his, he mino cu li subuni CsgB ac s as a nuclea o o CsgA assembly (Figu e 1a). To a oid he o ma ion o oxic oligome s inside he cell, he pe iplasmic p o ein CsgC ac s as a chape one ha main ains CsgA as a soluble and uns uc u ed monome , hus p e en ing he amyloid s a e [26]. The csgDEFG ope on code o p o eins ha a e in ol ed in he egula ion, s abiliza ion, and sec e ion o CsgA and CsgB subuni s [ 26 – 28 ]. CsgA and CsgB each he ex acellula milieu, due o a po e-like s uc u e o med by CsgG in he ou e memb ane [ 29 ]. The accesso y p o eins CsgF and CsgE a e associa ed wi h CsgG and con ibu e o he p ope assembly o cu li ibe s [ 30 ]. In addi ion, CsgF in e ac s wi h CsgB, ensu ing he a achmen o he subuni on he cell su ace and hus, he ibe o ma ion [ 30 ]. CsgD con ols a he ansc ip ional le el he exp ession o he csgBAC ope on [ 31 , 32 ] which is igge ed by en i onmen al and chemical An ibio ics 2021,10, 795 3 o 18 signals such as osmolali y, oxygen, and empe a u e [ 33 , 34 ]. Cu li a e implica ed in su ace coloniza ion, cell–cell con ac , bio ilm ma ix sca olding, p o ec ion agains an imic obial agen s and desicca ion. Mo eo e , cu li play an impo an ole in he in e ac ion wi h hos cell ecep o s and he immune sys em [35–39]. An ibio ics 2021, 10, 795 3 o 17 le el he exp ession o he csgBAC ope on [31,32] which is igge ed by en i onmen al and chemical signals such as osmolali y, oxygen, and empe a u e [33,34]. Cu li a e implica ed in su ace coloniza ion, cell–cell con ac , bio ilm ma ix sca olding, p o ec ion agains an- imic obial agen s and desicca ion. Mo eo e , cu li play an impo an ole in he in e ac- ion wi h hos cell ecep o s and he immune sys em [35–39]. Figu e 1. Schema ic ep esen a ion o he genes in ol ed in he o ma ion o in insic amyloids, he d ugs a ge ing such s uc u es and hei mechanism o ac ion. (a) The o ma ion o E. coli and S. Typhimu ium cu li amyloids is a ec ed by se e al d ugs, which ac by di e en mechanisms: (i) s abiliza ion o CsgA monome ic subuni s; (ii) o ma ion o amo - phous o non-amyloidogenic agg ega es; (iii) disagg ega ion o al eady- o med ibe s; (i ) p e en ion o CsgB polyme i- za ion; ( ) solubiliza ion o CsgB; ( i) ac i a ion o he cell su ace s ess esponse, which educes he exp ession o he cu li egula o CsgD. (b) Pseudomonas Fap amyloids a e inhibi ed by EGCG and PGG polyphenols, which lead FapC o o -pa hway oligome s, emodeling ibe s in o amo phous agg ega es and e aining quo um-sensing molecules. Table 1. Classi ica ion o unc ional amyloids. Amyloid Type Locus Amyloid Subuni s Bac e ia In insic amyloids Cu li csgDEFG; csgBAC CsgA, CsgB E. coli, Salmonella [40] Fap apABCDEF FapC, FapB, P. ae uginosa [41] MTP m p MTP Mycobac e ium ube culosis [42] Chaplins/Rodlins chpA, chpD, dlA, dlB; chpC, chpH; chpF, chpG; chpB, chpE ChpD-H, RdlB S ep omyces coelicolo [43,44] Facul a i e amyloids PSMs psmα1–4; psmβ1–2; hld PSMα, PSMβ, δ- oxin S. au eus, CNS 1 [17,45] TasA apA, sipW, asA TasA B. sub ilis, B. ce eus [46,47] Bap bap BapB-domain S. au eus, CNS 1 [48] Esp esp N- e minal domain E. aecalis [49] P1 p1 AgII-C123 egion S. mu ans [50] WapA wapA WapA S. mu ans [51] SMU_63C smu_63c SMU_63C S. mu ans [51] 1 CNS: coagulase nega i e s aphylococci. Figu e 1. Schema ic ep esen a ion o he genes in ol ed in he o ma ion o in insic amyloids, he d ugs a ge ing such s uc u es and hei mechanism o ac ion. ( a ) The o ma ion o E. coli and S. Typhimu ium cu li amyloids is a ec ed by se e al d ugs, which ac by di e en mechanisms: (i) s abiliza ion o CsgA monome ic subuni s; (ii) o ma ion o amo phous o non-amyloidogenic agg ega es; (iii) disagg ega ion o al eady- o med ibe s; (i ) p e en ion o CsgB polyme iza ion; ( ) solubiliza ion o CsgB; ( i) ac i a ion o he cell su ace s ess esponse, which educes he exp ession o he cu li egula o CsgD. ( b )Pseudomonas Fap amyloids a e inhibi ed by EGCG and PGG polyphenols, which lead FapC o o - pa hway oligome s, emodeling ibe s in o amo phous agg ega es and e aining quo um-sensing molecules. Table 1. Classi ica ion o unc ional amyloids. Amyloid Type Locus Amyloid Subuni s Bac e ia In insic amyloids Cu li csgDEFG; csgBAC CsgA, CsgB E. coli, Salmonella [40] Fap apABCDEF FapC, FapB, P. ae uginosa [41] MTP m p MTP Mycobac e ium ube culosis [42] Chaplins/Rodlins chpA, chpD, dlA, dlB; chpC, chpH; chpF, chpG; chpB, chpE ChpD-H, RdlB S ep omyces coelicolo [43,44] Facul a i e amyloids PSMs psmα1–4; psmβ1–2; hld PSMα, PSMβ,δ- oxin S. au eus, CNS 1[17,45] TasA apA, sipW, asA TasA B. sub ilis, B. ce eus [46,47] Bap bap BapB-domain S. au eus, CNS 1[48] Esp esp N- e minal domain E. aecalis [49] P1 p1 AgII-C123 egion S. mu ans [50] WapA wapA WapA S. mu ans [51] SMU_63C smu_63c SMU_63C S. mu ans [51] 1CNS: coagulase nega i e s aphylococci. An ibio ics 2021,10, 795 4 o 18 2.1.2. Fap ( apA-F Genes) Fap ibe s a e p oduced by Pseudomonas spp. as pa o he bio ilm ma ix [ 52 ]. The Fap ibe machine y is encoded in he ope on apA-F. The Fap ibe s a e assembled by he majo s uc u al subuni FapC, and he wo mino subuni s FapB and FapE. FapB is equi ed o ibe polyme iza ion whe eas FapE ac s as an ex acellula chape on-like p o ein ( Figu e 1b ). The h ee p o eins a e expo ed ou side he cell h ough he po e- like s uc u e o med by FapF [ 53 ]. FapA wo ks as a chape one o he ibe monome s du ing he sec e ion p ocess whe eas FapD, a cys eine p o ease loca ed in he pe iplasm, is equi ed o FapC ex e naliza ion [ 53 ]. The assembly o Fap amyloids in he bio ilm enhances cell agg ega ion and a achmen con e ing p o ec ion agains chemical and mechanical a ack [ 52 ]. In addi ion, Fap amyloids inc ease he o e all hyd ophobici y and s i ness o he bio ilm, hus p e en ing bac e ia desicca ion [54]. 2.1.3. Chaplins and Rodlins Filamen ous bac e ia, such as S ep omyces, g ow abo e humid su aces o ming b anching hyd ophobic s uc u es called ae ial hyphae ha ul ima ely esul in hyd opho- bic spo es [ 55 ]. The amyloidogenic p o eins, chaplins and odlins a e in ol ed in he o ma ion o hese [ 43 , 44 ]. The e a e eigh chaplins (ChpA–H) and wo odlins (RdlA and RdlB). The long chaplins (ChpA–C) ha e a C- e minal so ing signal o a achmen o he pep idoglycan ia a so ase-media ed co alen bound wi h he LAXTG mo i and wo chaplin domains ha a e highly hyd ophobic, whe eas he sho chaplins (ChpD-H) ha e one chaplin domain [ 56 ]. To da e, because long chaplins canno be ex ac ed om he cell walls o ae ial hyphae due o he co alen linkage, only sho chaplins ha e been shown o o m β -shee s uc u es and hus amyloid-like ibe s [ 57 ]. I is he e o e possible ha ChpA-C se e as ancho ing si es o ChpD-H chaplins o o m amyloid ibe s, and ha ChpE is esponsible o he coo dina ion o he polyme iza ion o he ibe subuni s in a high pH-dependen mechanism [ 58 ]. In addi ion o chaplins, he RodA and RodB odlins polyme ize in o he hyd ophobic s uc u e known as he odle laye , which co e s he su ace o ae ial hyphae and spo es. Amyloid chaplins a e aligned in o o de ed odle s by he ac ion RdlA and RdlB, only he la e is able o o m amyloid ib ils in i o [59]. 2.2. Facul a i e Amyloids The acul a i e amyloids include p o eins wi h a dual unc ion. They a e sec e ed in a unc ional globula olded s a e ha , unde ce ain condi ions, changes hei con o ma ion o an amyloid old [ 12 , 24 ]. Two ypes o acul a i e amyloids can be di e en ia ed. One ype comp ises sec e ed monome ic p o eins, such as S. au eus phenol-soluble modulins (PSMs) and Bacillus sub ilis TasA, which assemble in o amyloid ibe s upon con o ma ional change occu s. The o he g oup includes p o ein domains wi h amyloidogenic ea u es, which has been p e iously p ocessed om a na i e su ace p o ein (e.g., S. au eus Bap, En e ococcus aecalis Esp, and S ep ococcus mu ans P1, WapA, and SMU_63C). In hei na i e con o ma ion, he acul a i e amyloids o he o me g oup can ac as an imic obials and oxins, whe eas he la e wo k as cell su ace adhesins. Bo h u n in o ma ix sca olds when hey polyme ize in o amyloid s uc u es. 2.2.1. Phenol-Soluble Modulins (PSMs) Phenol-soluble modulins (PSMs) a e small alpha-helical amphipa hic pep ides in- ol ed in he i ulence o S. au eus [ 60 , 61 ]. S. au eus exp esses ou α -PSMs (PSM α 1– PSM α 4) encoded in he α -psm ope on, wo β -PSMs (PSM β 1 and PSM β 2) encoded in he β -psm ope on and γ -PSM ( δ - oxin) p oduced om RNAIII, a egula o y RNA o he Ag sys em [ 62 – 64 ]. PSMs lack he ypical Sec-signal and hei sec e ion is ca ied ou by an ATP- binding casse e (ABC) anspo e [ 65 ]. Once a he ex acellula media, PSMs may ei he wo k as soluble p o eins ha ing an imic obial and immunomodula ing ac i i ies [ 66 – 69 ] o change he con o ma ion and polyme ize in o unc ional amyloids (Figu e 2a). PSM α 1 and PSM α 4 o m agg ega es showing a ypical amyloid c oss- β s uc u e [ 45 ] whe eas PSM α 3 An ibio ics 2021,10, 795 5 o 18 agg ega es display an unusual c oss- α s uc u e [ 17 ]. This s uc u al polymo phism e eals di e en bac e ial unc ions. Al hough PSM α 1 and PSM α 4 pa icipa e in bio ilm o ma ion, PSM α 3 is in ol ed in cy o oxici y o human T cells, p obably due o i s pa icula amyloid a chi ec u e [17]. An ibio ics 2021, 10, 795 5 o 17 PSMα1 and PSMα4 o m agg ega es showing a ypical amyloid c oss-β s uc u e [45] whe eas PSMα3 agg ega es display an unusual c oss-α s uc u e [17]. This s uc u al pol- ymo phism e eals di e en bac e ial unc ions. Al hough PSMα1 and PSMα4 pa icipa e in bio ilm o ma ion, PSMα3 is in ol ed in cy o oxici y o human T cells, p obably due o i s pa icula amyloid a chi ec u e [17]. Figu e 2. Schema ic ep esen a ion o he genes in ol ed in he assembly o acul a i e amyloids, he d ugs a ge ing such s uc u es and hei mechanism o ac ion. (a) D ugs a ge ing S. au eus PSMs ac by se e al mechanisms ha in ol e he s abiliza ion o PSM -shee s uc u e and ibe disassembly. (b) B. sub ilis TapA polyme iza ion is inhibi ed by AA-861 and pa henolide. (c) The la onoids que ce in, my ice in, and scu ella ein inhibi polyme iza ion o he Bap amyloid ag- g ega es by s abiliza ion o he Bap p o ein. (d) Polyme iza ion o he amyloids S. mu an s WapA and AgII/C123 is inhib- i ed by AA-861, Tannic acid (TA), and EGCG and indi ec ly by he AFhPs and P1 pep ides. SMU_63c amyloids a e inhib- i ed by EGCG. Figu e 2. Schema ic ep esen a ion o he genes in ol ed in he assembly o acul a i e amyloids, he d ugs a ge ing such s uc u es and hei mechanism o ac ion. ( a ) D ugs a ge ing S. au eus PSMs ac by se e al mechanisms ha in ol e he s abiliza ion o PSM α -shee s uc u e and ibe disassembly. ( b )B. sub ilis TapA polyme iza ion is inhibi ed by AA-861 and pa henolide. ( c ) The la onoids que ce in, my ice in, and scu ella ein inhibi polyme iza ion o he Bap amyloid agg ega es by s abiliza ion o he Bap p o ein. ( d ) Polyme iza ion o he amyloids S. mu an s WapA and AgII/C123 is inhibi ed by AA-861, Tannic acid (TA), and EGCG and indi ec ly by he AFhPs and P1 pep ides. SMU_63c amyloids a e inhibi ed by EGCG. An ibio ics 2021,10, 795 6 o 18 2.2.2. TasA The B. sub ilis bio ilm con ains unc ional ibe s wi h amyloidogenic p ope ies. The main amyloid componen is TasA ( ansloca ion-dependen an imic obial spo e compo- nen ), which sel -assembles in o amyloid ibe s (Figu e 2b) [ 46 ]. The accesso y p o ein TapA (TasA ancho ing and assembly p o ein) assis s in he o ma ion o TasA ib ils on o he cell su ace [ 47 ]. SipW is a memb ane-bound pep idase ha clea es he TapA and TasA signal pep ides be o e sec e ion [ 70 ]. S uc u al analyses ha e shown ha TasA is sec e ed in a globula s a e ha ansi s o an amyloid con o ma ion unde en i onmen al condi ions such as acidic pH and hyd ophobici y [ 71 ]. In addi ion o i s s uc u al ole in he mul icellula beha io , TasA main ains he cell memb ane s abili y and p e en s excess cell dea h unde bio ilm g ow h condi ions [72]. 2.2.3. Bio ilm Associa ed P o eins (BAPs) BAPs a e high molecula weigh p o eins p esen on bac e ial cell su aces and a e cha ac e ized by a mul i-domain a chi ec u e [73,74]. The pa adigm S. au eus Bap p o ein is o ganized in se e al domains (Figu e 2c) including a pu a i e Sec-dependen signal pep ide o hei expo ollowed by a N- e minal domain, which comp ises egion A and B, he la e ca ies he EF-hand mo i s o calcium binding [ 75 , 76 ]. The eupon, a co e domain o med by andem epea s (C egion) and a egion D a he C- e minus, which con ains he LPXTG mo i o ancho ing he p o ein o he cell wall. Bap o ms amyloid ibe s unde speci ic en i onmen al ci cums ances [ 48 ]. Once he p o ein is co alen ly ancho ed o he pep idoglycan, he N- e minal egion is p ocessed and eleased o he ex acellula media. As a esul , agmen s o Bap adop an uns able mol en globule-like s a e. When acidic pH and low concen a ion o Ca 2+ a e ound in he medium, he mol en globule swi ches o an agg ega ion-p one con o ma ion ha enables sel -assembly in o amyloid ibe s ha p omo e bio ilm o ma ion (Figu e 2c) [ 48 ]. In con as , when Ca 2+ is a ailable, i binds o he EF-hand mo i s, s abilizing Bap in i s na i e olded s a e and impai ing he polyme iza ion o he amyloid ibe s and bio ilm o ma ion [ 48 , 76 ]. I is no able ha Bap ca ies ou addi ional unc ions in i s p ima y globula s a e. I ac s as an adhesin, p omo ing he p ima y a achmen o S. au eus o abio ic su aces and hus playing an impo an ole in ini ial s ages o bio ilm o ma ion [ 75 ]. In addi ion, Bap is in ol ed in he pa hogenesis o S. au eus by imp o ing he adhesion o epi helial cells o mamma y glands [ 77 ] bu exe ing a p o ec i e ole by p e en ing S. au eus en y in o epi helial cell h ough binding o he Gp96 cell hos ecep o [78]. The E n e ococcal s u ace p o ein (Esp) is a Bap-o hologous p o ein p esen in E. aecalis [ 79 , 80 ]. The mul i-domain con igu a ion o Esp is simila o ha o Bap. The Esp N- e minal domain and he egion C sha e 26% and 23% o iden i y wi h he co esponding Bap domains, espec i ely [ 49 ]. Analogously, he Esp N- e minal domain is su icien o induce bio ilm o ma ion [ 81 ]. Ou g oup has ecen ly desc ibed he capaci y o he Esp N- e minal egion o o m amyloid-like ibe s in a pH-dependen manne [ 49 ]. The simila i ies obse ed be ween Bap and Esp sugges ha he dual unc ion o amyloid/adhesin may be widesp ead among membe s o he BAP amily. 2.2.4. P1 Adhesin/WapA/SMU_63c Ano he example o a su ace-associa ed adhesin wi h amyloidogenic p ope ies is he P1 p o ein (also known as AgI/II, PAc, SpaP o an igen B) o S. mu ans. This P1 is a la ge p o ein ha is co alen ly ancho ed o he cell wall h ough i s C- e minal LPXTG mo i . P1 is able o bind ex acellula p o eins [ 82 – 84 ]. The e ia y s uc u e is a p olonged s alk wi h a globula domain ha con ains β -shee s. P1 can be p ocessed and i s C- e minal domain (AgII o C123 agmen ) can be loosely associa ed wi h he co alen ly ancho ed p o ein (Figu e 2d) [ 85 ]. The C123/AgII agmen con ains he amyloid- o ming moie y ha may be implica ed in he o ma ion o ib illa s uc u es wi h amyloid p ope ies [ 50 , 86 ]. In addi ion, he wall-associa ed p o ein A (WapA) and he sec e ed p o ein SMU_63c o S. mu ans con ibu e o he ib illa s uc u es depending on he pH [ 51 ]. As he P1 An ibio ics 2021,10, 795 7 o 18 adhesin, WapA is p ocessed and o ms amyloid ibe s a neu al pH, whe eas SMU_63c does no need o be p ocessed and o ms amyloid s uc u es a acidic pH. O e all, his e idence sugges s ha en i onmen al condi ions a e c ucial o he egula ion o S. mu ans amyloid ibe s. 3. D ugs Ta ge ing Amyloid-S uc u ed Bio ilms Amyloid ibe s appea o be common bio ilm ma ix elemen s in many pa hogenic bac e ia. Se e al app oaches ha e been ocused on sea ching o compounds ha ei he al e he exp ession o he elemen s in ol ed in he amyloid p oduc ion o in e up hei assembly. The iden i ica ion o he an i-amyloid compounds o disassemble bio ilms has been ca ied ou by means o high- h oughpu sc eening o ac i e molecule collec ions, he design o pep ides based on he s uc u al biology o he amyloidogenic segmen s o he epu posing o molecules designed agains human amyloids. The mos ep esen a i e d ugs ac i e agains amyloid- ela ed bio ilms and hei mechanisms o ac ion a e discussed below and shown in Table 2. Table 2. An i-bio ilm agen s a ge ing bac e ial amyloids. Type Amyloid Inhibi o s An i-Bio ilm E ec Bac e ial Amyloid Ta ge Euka yo ic Amyloid Ta ge Re e ence Pep ides ANK6 S. Typhimu ium CsgA Aβ[87] DB3DB3 S. Typhimu ium CsgA Aβ[87] AP90 S. au eus PSMα1 ND [88] AFhPs S. mu ans, S. sanguis, S. au eus, E. coli ND ND [89] P1 S. mu ans ND ND [89] P o eins TTR E. coli CsgA Aβ, HepF-N [89] B. sub ilis ND An ibodies 3H3 S. Typhimu ium Cu li Aβ, TTR, Tau [90] Molecula weeze s CLR01 S. au eus PSMα1 Aβ,α-syn, Tau [91] CLR05 S. au eus PSMα1 Aβ,α-syn, Tau [91] Cu licides FN075 E. coli Cu li Aβ[92] BibC6 E. coli Cu li ND [92] VA028 E. coli Cu li ND [92] Bioac i e compounds AA-861 B. sub ilis TasA New1 [93] S. mu ans P1 WapA [92] Pa henolide B. sub ilis TasA New1 [93] An ibio ics 2021,10, 795 8 o 18 Table 2. Con . Type Amyloid Inhibi o s An i-Bio ilm E ec Bac e ial Amyloid Ta ge Euka yo ic Amyloid Ta ge Re e ence Polyphenols EGCG E. coli CsgA CsgB Aβ,α-syn, Tau [94] Pseudomonas sp. FapC [95,96] S. mu ans P1 WapA SMU_63c [89] S. au eus PSMα1PSMα4 [97] PGG Pseudomonas sp. FapC Aβ[95] Tannic acid S. mu ans P1 WapA P ion P P, Aβ[51] Lu eolin E. coli CsgA Aβ,α-syn [98] Mo in E. coli CsgA Aβ,α-syn [98] My ice in E. coli,S. au eus CsgA Bap Aβ,α-syn [98,99] Que ce in E. coli CsgA Bap Aβ,α-syn [98,99] Phlo e in E. coli CsgA CsgB Aβ,αSA53T [98] ND: non de e mined; Aβ: Amyloid β;α-syn: α-synuclein; αSA53T: mu an o m o α-synuclein. 3.1. An i-Amyloid Pep ides The design o pep ides binding o he domains in ol ed in amyloid ib illa ion is one o he mos p omising s a egies used o in e up amyloid ma u a ion and consequen ly bio ilm o ma ion. Based on he s uc u al simila i ies exis ing be ween he amyloidogenic domains de i ed om CsgA and hose om human amyloids, Pe o and collabo a o s ound ha he D-enan iome ic pep ides (ANK6 and DB3DB3) inhibi ed he ib illa ion o CsgA (Table 2) [ 87 ]. ANK6 and DB3DB3 pep ides we e op imized o hei po en ial o emo e amyloid- β (A β ) oligome s associa ed wi h Alzheime ’s disease. The D-pep ides bound o CsgA, hus p omo ing he s abiliza ion o he monome subuni s o inducing he o ma ion o amo phous agg ega es, which we e unable o assemble in o amyloid ibe s (Figu e 1a). The pep ides inhibi he ib illa ion o he CsgA spine segmen s con ained a he R1 and R5 epea s. The inhibi o y ac ion o he pep ides appea s o be speci ic o he seconda y s uc u e o he p o ein because ANK6 and DB3DB3 do no ha e any e ec on c oss- α amyloid-like ib ils o med by PSM α 3 ib illa ion [ 87 ]. This dis inc ion was u he exempli ied in a pionee ing s udy ha designed pep ides ha we e complemen a y o he α -shee o PSM α 1. I is o en conside ed ha , because amyloids con ain a c oss- β - s uc u e, he in e media e agg ega i e species mus also con ain a β -s uc u e. Howe e , his is no he case o se e al amyloids, such as PSM α 1, which unde go a ansi ion om α -helix →α -shee →β -shee du ing agg ega ion [ 88 ]. The an i- α -shee pep ide AP90 a ge s he ansi ional α -shee s uc u e o PSM α 1, leading o a educ ion o amyloid ib ils in i o (Figu e 2a). Consequen ly, AP90 e ec i ely educes bio ilm o ma ion by S. au eus h ough he in e ac ion wi h PSMs [88]. Ano he in e es ing s a egy o ea ing mic obial in ec ions is based on he use o amyloidogenic pep ides de i ed om bac e ial amyloids [ 100 ]. Syn he ic agg ega ion- p one pep ides om S aphylococcus epide midis (C30, C29, Hi 1, Hi 50) pene a e bac e ia, hus causing oxic p o ein agg ega ion o polypep ides in he cy osol, which leads o bac e ial dea h [ 101 ]. In e es ingly, agg ega ing pep ides a e oxic o bac e ia, bu no o human cells. Howe e , u he in es iga ions a e needed o es whe he hese pep ides An ibio ics 2021,10, 795 9 o 18 may ha e an e ec on p o ein agg ega ion and oxici y in human cells ha exp ess amyloids such as α-synuclein o Aβ. Fu he mo e, sho pep ides de i ed om he amyloidogenic agmen C123 o he adhesin P1 o S. mu ans display s ong an i-bio ilm p ope ies wi h small o no cy o oxic e ec [ 89 ]. Simula ed amyloid o ming pep ides p edic ed om C123 ha e been syn- hesized (AFhPs). AFhPs inhibi bio ilm o ma ion in a wide ange o mic oo ganisms including G am-posi i e and G am-nega i e bac e ia and ungi. These pep ides agg ega e in o igid amyloid ibe s ha agglu ina e mic obial cells in o clus e s [ 89 ], hus leading o he o ma ion o agg ega es ha impai he es ablishmen o new bio ilms. I was p oposed ha he ca bohyd a es o he mic obial cell wall would be he binding a ge s o AFhPs a he han he su ace-exposed amyloids. Mo eo e , lipid memb anes may accele a e AFhP ib illa ion. Howe e , he ole o hese s uc u es in he an i-bio ilm ac i i y o AFhPs emains o be elucida ed. Simila ly, an an i-bio ilm ac i i y was iden i ied o he P1 pep ide, a syn he ic pep ide o 24 amino acids (di e en om he P1 p o ein o S. mu ans), which was designed om he epea ed mo i s o he Ixodes ick an i eeze glycop o ein (IAFGP) sequence [102]. The P1 pep ide is able o inhibi bio ilm o ma ion o S. mu an s and o he G am-posi i e bac e ia bu does no possess bac e icidal ac i i y [ 103 ]. The an i-bio ilm e ec is associa ed wi h P1 agg ega ion and he o ma ion o an ipa allel β -shee s. Howe e , u he expe imen s a e needed o de e mine whe he he an i-bio ilm e ec is caused by he in e molecula in e ac ions o P1 agg ega es wi h bac e ial amyloidogenic p o eins [103]. 3.2. An i-Amyloid P o eins The human amyloid p ecu so ans hy e in (TTR) is a 55 kDa homo e ame p o ein mainly p oduced by he li e . In e es ingly, TTR is s uc u ally simila o he cu li inhibi o CsgC, and bo h sha e a s able olded β -shee - ich s uc u e [ 104 , 105 ]. TTR is in ol ed in he anspo o hy oxine (T4) and e inol in plasma and ce eb ospinal luid. Mis olded p o ein monome s, de i ed om he e ame ic p o ein TTR, can be accumula ed as ib illa deposi s in di e en o gans and cause he mos common o m o he edi a y ans hy e in amyloidosis. By compa ison, he homo e ame ic o m o he p o ein has he abili y o inhibi amyloid o ma ion o amyloidogenic p o eins, such as A β , he N- e minal domain o he bac e ial hyd ogenase HypF and he human isle amyloid polypep ide [ 106 – 108 ]. Taking in o accoun he an i-amyloid p ope ies o TTR, in a ema kable wo k, Jain e al. examined he e ec o TTR on CsgA amyloid assembly [ 109 ]. The incuba ion o CsgA wi h he e ame p ecu so (W -TTR) e a ded CsgA polyme iza ion, whe eas he incuba ion wi h he monome ic o m (M-TTR) s ongly p e en ed he o ma ion o CsgA p o o ib illa and ib illa s uc u es (Figu e 1a). In e es ingly, M-TTR also inhibi ed he assembly o CsgB in o amyloid agg ega es. The e o e, bo h W -TTR and M-TTR inhibi ed w inkled pellicle bio ilms o med by E. coli; howe e , he inhibi o y e ec o M-TTR was signi ican ly mo e po en han ha p oduced by W -TTR. I is possible ha W -TTR equi es e ame dissocia ion o libe a e he monome s ha a e conside ed he ac i e o ms o ib il inhibi- ion. Howe e , he use o a highly s able TTR e ame o m (TTR T119M) inhibi ed CsgA ib illogenesis, indica ing ha he W -TTR e ame dissocia ion was no equi ed o such inhibi ion [109]. 3.3. An ibodies as Na i e-S a e S abilizing Agen s The adminis a ion o an ibodies o enginee ed a ibodies has been p oposed as one o he mos p omising he apies o educe amyloid polyme iza ion [ 110 ]. An ibody-based immuno he apy has been used o educe he amyloid amoun by s imula ing he clea ance o he mis olded agg ega es by he immune sys em cells [ 111 ]. 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