Acyl homoserine lactone-mediated quorum sensing in the oral cavity: a paradigm revisited

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Acyl homose ine lac one-media ed
quo um sensing in he o al ca i y: a
pa adigm e isi ed
And ea Mu as1, paz o e o-casal2,3, Vanessa Blanc4 & Ana o e o1 ✉
Acyl homose ine lac ones (AHLs), he quo um sensing (QS) signals p oduced by G am-nega i e
bac e ia, a e cu en ly conside ed o play a mino ole in he de elopmen o o al bio ilm since hei
p oduc ion by o al pa hogens has no been asce ained hus a . Howe e , we epo he p esence o
AHLs in di e en o al samples and hei p oduc ion by he o al pa hogen Po phy omonas gingi alis.
he impo ance o AHLs is u he suppo ed by a e y high p e alence o AHL-deg ada ion capabili y,
up o 60%, among bac e ia isola ed om den al plaque and sali a samples. Fu he mo e, he wide-
spec um AHL-lac onase Aii20J signi ican ly inhibi ed o al bio ilm o ma ion in di e en in i o bio ilm
models and caused impo an changes in bac e ial composi ion. Besides, he inhibi o y e ec o Aii20J
on a mixed bio ilm o 6 o al pa hogens was e i ied using con ocal mic oscopy. Much mo e esea ch
is needed in o de o be able o associa e speci ic AHLs wi h o al pa hologies and o indi idua e he
key ac o s in AHL-media ed QS p ocesses in den al plaque o ma ion. Howe e , hese esul s indica e
a highe ele ance o he AHLs in he o al ca i y han gene ally accep ed hus a and sugges he
po en ial use o inhibi o y s a egies agains hese signals o he p e en ion and ea men o o al
diseases.
Den al plaque is an ex emely complex bio ilm ha esul s om he accumula ion and in e ac ion o o al mic o-
o ganisms a ached o he oo h su ace, embedded in a ma ix o ex acellula polyme s. Du ing den al plaque
de elopmen , he ecological equilib ium o he mic obes in he o al ca i y is main ained h ough compe i i e and
coope a i e in e ac ions. Indeed, an imbalance o he esiden mic obio a de i ed om a change in local en i-
onmen al condi ions is esponsible o he wo majo o al bac e ial diseases: ca ies and pe iodon al diseases1–3.
The esponsibili y o he p og ession o hese o al diseases canno be a ibu ed o a single o al pa hogen, ins ead
he en i e communi y esiden in he o al ca i y, as well as i s unc ional ac i i ies, a e esponsible o hei de el-
opmen 4,5. Fu he mo e, o he impo an pa hologies, including Alzheime ’s disease and o al cance , a e also
hough o be ela ed o o al bac e ia6,7. In his con ex , he in e e ence wi h he o al bio ilm o ma ion p ocesses,
h ough he inhibi ion o bac e ial coagg ega ion and/o he educ ion o den al plaque o ma ion is supposed o
ha e bene icial e ec s on o al heal h8.
Some o he po en ial a ge s o he de elopmen o s a egies o inhibi den al plaque o ma ion a e he
bac e ial communica ion p ocesses known as Quo um sensing (QS). These cell densi y-dependen mechanisms
which con ol gene exp ession a e accep ed as essen ial o he success ul es ablishmen o bac e ial bio ilms.
Howe e , he cu en knowledge abou signalling p ocesses and mic obial in e ac ions wi hin o al bio ilms is s ill
limi ed, and hei e ec s on commensal mic obio a and he es ablishmen o dysbiosis emain unclea 9. The QS
molecules p oduced by G am-posi i e bac e ia, he Au oInduce Pep ides (AIPs), ha e been iden i ied in di e -
en o al s ep ococci. The p oduc ion o he Au oinduc o -2 (AI-2), signals an in e con e ible g oup o signalling
molecules based on a u anosyl bo a e dies e , was also de ec ed in di e en G am-posi i e and G am-nega i e
o al pa hogenic bac e ia such as S ep ococcus mu ans o Po phy omonas gingi alis10–13. Among he di e en QS
signal molecules desc ibed so a , he N-acyl homose ine lac ones (AHLs), molecules cons i u ed by a homose -
ine lac one ing (HSL) linked by an amide bond o a a y acid (be ween 4 and 20 ca bons), a e he bes s udied
and cha ac e ized. The speci ici y o hese molecules is dependen on he leng h o he molecule and p esence
1Depa amen o de Mic obioloxía e Pa asi oloxía, Facul ade de Bioloxía-CIBUS, Uni e sidade de San iago de
Compos ela, San iago de Compos ela, Spain. 2Depa amen o de Ci uxía e Especialidade Médico-Ci ú xica, Facul ade
de Medicina e Odon oloxía, Uni e sidade de San iago de Compos ela, San iago de Compos ela, Spain. 3Uni o
O al Heal h, C.S. San a Comba-Neg ei a, SERGAS, Spain. 4Depa men o Mic obiology, Den aid Resea ch Cen e ,
Den aid S.L., Ba celona, Spain. ✉e-mail: [email p o ec ed]
open
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o hyd oxy- o oxo- subs i u ions in he hi d ca bon. Howe e , a emp s o de ec he p oduc ion o he AHL
signals, ypical o G am-nega i e bac e ia, by o al pa hogens ha e emained unsuccess ul10–12. The e o e, AHLs
ha e no been assigned an impo an ole in mic obe-mic obe in e ac ions wi hin den al plaque14–16. Despi e ha ,
an inc easing numbe o di ec and indi ec e idence has been accumula ed in ecen yea s, poin ing o AHLs’
ole in den al plaque o ma ion. AHLs ha e been de ec ed in sali a and spu um samples17–20. Addi ionally, se e al
AHL-p oducing s ains o En e obac e sp., Klebsiella pneumoniae, Pseudomonas pu ida, Ci obac e amalona icus
(Le inea amalona icus) L8A and Bu kholde ia sp. ha e been isola ed om he human ongue su ace and den-
al plaque samples21–25. Fu he mo e, a homologue o he AHL-syn hase Hd S, as well as a LuxR- ype ecep o
homologue, we e iden i ied in P. gingi alis W83 and P. gingi alis ATCC33277, espec i ely26–28. In his con ex ,
p e ious s udies obse ed ha AHLs and AHL-analogues modi ied no only he p o ein exp ession bu also
slowed down he g ow h in P. gingi alis26,29. Recen ly, we ha e also demons a ed ha he exogenous addi ion
o speci ic AHLs a ec s pa hology- ela ed pheno ypes such as lac ic acid p oduc ion and p o ease ac i i y in
in i o o al bio ilm models19. In hese models, N-hexanoyl-L-homose ine lac one (C6-HSL) inc eases he ela-
i e p esence o Pep os ep ococcus and P e o ella, p oducing a shi owa ds a pe iodon al bac e ial composi ion
p o ile19. Al oge he , hese esul s poin o a possible ole o AHL-media ed QS in he o al ca i y. The con i ma-
ion o he c i ical unc ion o AHLs in o al bio ilm o ma ion would open new oppo uni ies in he p e en ion
and ea men o o al in ec ious diseases since he mos ac i e QS inhibi o s desc ibed hus a in e e e wi h
AHL-media ed QS sys ems.
Nume ous o ganisms ha e e ol ed he capaci y o inhibi QS sys ems, a p ocess gene ally known as Quo um
Sensing Inhibi ion (QSI) o Quo um Quenching (QQ), p obably because hese cell- o-cell communica ion sys-
ems play a key ole in he in e ac ions, no only be ween p oka yo es bu also wi h euka yo es. Enzyma ic QQ
is he bes -s udied QS inhibi o y s a egy30. The genes ha codi y his ype o enzymes a e classi ied in wo main
g oups: lac onases and acylases, al hough o he ypes o QQ enzymes ha e also been desc ibed31. The in e e ence
wi h QS p ocesses has become an in e es ing al e na i e o igh ing he p oblem o bac e ial an ibio ic esis ance
and has been p oposed as a p omising app oach o con olling di e en pa hogenic bac e ial ai s in o de o
p e en o ea in ec ious diseases31–35. Fu he mo e, he use o QQ compounds also inc eases bio ilm- o ming
pa hogens´ suscep ibili y o an ibio ics36. Since QQ s a egies do no in e e e di ec ly wi h bac e ial g ow h,
he p obabili ies o inducing ole ance o esis ance agains hese mechanisms a e lowe 37,38. P e ious s udies
ha e al eady epo ed he success ul app oach o using QS inhibi o s o con ol di e en ypes o bac e ial bio-
ilms35,39–42. The e o e, he con i ma ion o a possible ole o AHL- ype QS signals in den al plaque o ma ion
would open new pe spec i es in he p e en ion and ea men o o al diseases.
This s udy desc ibes he p esence o AHLs in o al samples (sali a and ex ac ed ee h) and hei p oduc ion by
P. gingi alis indica ing ha his ype o QS signal plays a po en ial ole in he es ablishmen o he o al mic obial
communi ies. Fu he mo e, in o de o e alua e he impo ance o hese QS signals in he p ocess o o al bio ilm
o ma ion, he e ec o he wide-spec um, he mos able AHL-lac onase Aii20J33, ob ained om he ma ine
bac e ium Tenacibaculum sp. 20J43, was es ed on di e en in i o o al bio ilms ob ained om sali a samples
om heal hy and unheal hy dono s. Impo an inhibi ion was obse ed using he xCELLigence moni o ing sys-
em, which allows eal- ime measu emen s o su ace-associa ed bac e ial g ow h35,44 and a modi ica ion o he
Ams e dam Ac i e A achmen bio ilm model19,45. In addi ion, he inhibi o y e ec o he QQ enzyme Aii20J was
also obse ed on in i o mul i-species bio ilms o med by six o al pa hogens. All hese da a s ongly suppo he
impo an ole AHLs play in o al bio ilm o ma ion. Howe e , much mo e esea ch is necessa y in o de o be
able o associa e AHLs wi h o al pa hologies and o indi idua e he key ac o s in AHL-media ed QS p ocesses in
den al plaque o ma ion.
Resul s
AHL- ype quo um sensing signals de ec ion in o al samples and mixed bio ilm. The p esence
o AHL- ype QS signals was e alua ed in wo di e en ypes o o al samples om he same pa ien : ex ac ed
ee h and sali a samples. The analysis o sali a ob ained om di e en pa ien s unequi ocally demons a ed he
p esence o h ee AHLs (Supplemen a y ma e ial Figs.1, 2 and 3): N-oc anoyl-L-homose ine lac one (C8-HSL),
N- e adecanoyl-L-homose ine lac one (C14-HSL) and N-oc adecanoyl-L-homose ine lac one (C18-HSL)
(Fig.1a). The QS molecule C8-HSL was no only he mos abundan AHL (4.97–200.27 ng/mL), bu i was also
p esen in all he sali a samples. Those sali a samples in which he signals C8, C14, and C18-HSL we e p esen
we e ob ained om pa ien s wi h ca ies lesions while only C8-HSL and occasionally C14-HSL we e ound in
sali a om pe iodon al pa ien s. Addi ionally, C8-HSL was also de ec ed in mos o he ex ac ed ee h (Fig.1b).
The analysis o he supe na an ob ained om a mixed biol ilmcul u e o med by he six bac e ial pa ho-
gens S. o alis, Veillonella pa ula, Ac inomyces naeslundii, Fusobac e ium nuclea um, Agg ega ibac e ac inomy-
ce emcomi ans and P. gingi alis e ealed he p esence o he QS signal N-oxo-oc anoyl-L-homose ine lac one
(OC8-HSL) (0.87 ng/mL). In o de o know i P. gingi alis was he s ain esponsible o he AHL p oduc ion,
his bac e ium was cul u ed axenically and co-cul u ed wi h he G am-posi i es S. go donii o S. o alis. The da a
showed ha a monospeci ic cul u e o P. gingi alis p oduced a small quan i y o OC8-HSL (0.30 ng/mL), bu a
highe amoun o his AHL was obse ed when his o al pa hogen was cul u ed in a dual-species bio ilm wi h S.
go donii (0.83 ng/mL) o S. o alis (1.4 ng/mL).
Quo um quenching ac i i y in he o al ca i y. As a complemen a y app oach o he analysis o AHLs
in o al samples, he p esence o QQ ac i i y was also analyzed. A o al o 567 bac e ial isola es, 295 om a heal hy
pa ien and 272 om a pe iodon al pa ien , we e ob ained om sali a and den al plaque samples (Supplemen a y
ma e ial Table1). The capaci y o his o al bac e ial collec ion o in e e e wi h he sho -chain AHLs was es ed
using a Ch omobac e ium iolaceum-based bioassay46. Among he 567 o al isola es, he abili y o quench he
sho -chain QS signal C6-HSL was obse ed as he comple e inhibi ion o iolacein p oduc ion in a su p isingly
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la ge numbe o cul i able o al bac e ia (Supplemen a y ma e ial Table1). The a e age ac i i y was highe in he
pe iodon al samples (37.42%) han in he heal hy ones (23.20%). This di e ence was e en mo ee iden i only
den al plaque isola es we e compa ed, wi h 46.71% o isola es p esen ing QQ ac i i y o he pe iodon al sample,
agains only 14% o isola es om he heal hy dono . Since ac i i y agains sho -chain AHLs is gene ally much less
equen han he ac i i y agains long-chain AHLs46, he o al QQ ac i i y in he o al cul i able bac e ia may be
e en highe . The Ag obac e ium ume aciens bioassays46 did no p oduce consis en esul s ega ding he p oduc-
ion o AHLs in hese isola es bu e ealed ha 73 s ains had an ibio ic ac i i y agains his bac e ium biosenso :
44 we e isola ed om he heal hy dono (5 om den al plaque and 39 om sali a), and 29 we e ob ained om he
pe iodon al pa ien (14 om den al plaque and 15 om sali a). This highe an imic obial ac i i y in he heal hy
pa ien (60.27%) compa ed o he alues o he pe iodon al one (39.72%) could be ela ed wi h he heal h s a us
o he dono s, al hough i should be no ed ha hese esul s a e based on isola es om a single pa ien . The deg-
ada ion o C12-HSL was ound in almos all he sali a samples analyzed, bu C6-HSL was only pa ially educed
in a ew samples (da a no shown).
E ec o he AHL-lac onase Aii20J on in i o o al bio ilm o ma ion measu ed by xCELLigence
sys em. Since he p esence o di e en AHLs was unequi ocally demons a ed in o al samples, he e ec o
he wide-spec um AHL-lac onase Aii20J on bio ilm o ma ion om sali a samples ob ained om a heal hy
pa ien was es ed using he eal- ime measu emen equipmen xCELLigence (Fig.2), as a i s “black box”
app oach, o e alua e he impo ance o hese QS signals in o al bio ilm o ma ion. The AHL-lac onase Aii20J
caused a signi ican educ ion in sali a o al bio ilms g own using ei he BHI (Fig.2a) o BHI supplemen ed wi h
suc ose 0.1% (Fig.2b) as cul u e media a e only one hou o incuba ion (S uden ’s - es , p = 0.007).
Gi en he p omising esul s ob ained in he p elimina y es s, he capabili y o Aii20 o inhibi o al bio ilms
was u he e alua ed using sali a ob ained om o he heal hy and pe iodon al pa ien s as inoculum, measu ed
wi h xCELLigence (Fig.3). Again, Aii20J caused an impo an bio ilm inhibi ion (40.47–81.59%) when di e en
sali a samples we e inocula ed in BHI. In he same way, he bio ilm o ma ion in BHI supplemen ed wi h 0.1%
suc ose (BHIs) showed a high educ ion in almos all cases (25.15–93.4%)in he p esence o he enzyme. Only
one sample (S6) was no a ec ed by Aii20J, p obably because he bio ilm o med in his case was no s ong
enough o obse e any e ec (cell index lowe han 0.08, Supplemen a y ma e ial Fig.4).
E ec o he AHL-lac onase Aii20J on in i o o al bio ilm o ma ion measu ed by Ac i e
A achmen sys em. In o de o a oid he limi a ions o he xCELLigence sys em, such as he lack o
esponse in he measu emen s a e 24 h and o allow he possibili y o e eshing he cul u e media, he e ec
o Aii20J on o al bio ilms was es ed using a modi ica ion o he Ams e dam Ac i e A achmen model19,45. This
bio ilm cul i a ion model allowed o a highe adhesion su ace (up o 1.62 cm2) and enabled obse a ion o
s uc u al changes in he bio ilm. The e ec o he e eshmen o he cul u e media on sali a bio ilm o ma ion
was e alua ed using he c ys al iole s aining, showing a clea inc ease in he bio ilm biomass when cul u e
media was exchanged e e y 12 h (Supplemen a y ma e ial Fig.5).
The addi ion o he AHL-lac onase Aii20J (20 µg/mL) caused a signi ican inhibi ion o he bio ilms o med
a 12 h (67.65%) and 24 h (58.14%) in BHI unde ae obic condi ions (S uden ’s - es , p = 0.019, p = 0.003). The
inhibi o y e ec was educed a 48 h (Fig.4a). Also, a signi ican educ ion in he bio ilm o med in BHI supple-
men ed wi h suc ose was obse ed a 24 h o incuba ion (62.72%) in he p esence o he enzyme (S uden ’s - es ,
p = 0.003). The addi ion o Aii20J in BHI-g own bio ilms unde anae obic condi ions caused a signi ican inhi-
bi ion a 24 h (52.01%) and 72 h (37.41%) (S uden ’s - es , p = 0.019, p = 0.04). The measu emen o he bio ilms
using he BHI-2 cul u e medium (BHI supplemen ed wi h 2.5 g/L mucin, 1 g/L yeas ex ac , 0.1 g/L cys eine,
Figu e 1. De ec ion o acyl homose ine lac ones (AHLs) in sali a samples (a) and ex ac ed ee h (b) ob ained
om he same pa ien s using HPLC-MS. P = pe iodon al disease; C = ca ies.
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2 g/L sodium bica bona e, 5 mg/L hemin, 1 mg/L menadione and 0.25% ( / ) glu amic acid)47 in anae obic condi-
ions could only be done a 24 h (Fig.4b) since mos o he biomass de ached om he co e slips ob ained a 48 h,
72 h and 92 h du ing he c ys al iole s aining p ocedu e (Supplemen a y ma e ial Fig.6).
Using his bio ilm cul i a ion me hod, we obse ed a high inhibi o y e ec when he AHL-lac onase Aii20J
was added o he BHI cul u e medium using samples om a heal hy pa ien . I was demons a ed ha Aii20J
caused his an i-bio ilm ac i i y since i disappea ed when he QQ enzyme was emo ed by il a ion h ough a
10 kDa memb ane and was sha ply educed when he lac onase was au ocla ed o 10 minu es (Supplemen a y
ma e ial Fig.7).
The e ec o Aii20J (20 µg/mL) on in i o bio ilms was u he e alua ed wi h se e al sali a samples ob ained
om heal hy and pe iodon al pa ien s using BHI as cul u e media. The biomass ob ained in he bio ilm o med
by he heal hy pa ien used in he p e ious expe imen s (sali a 1) was much highe han o o he pa ien s
(Supplemen a y ma e ial Fig.8) indica ing a high a iabili y in he bio ilm o ma ion be ween indi iduals. The
addi ion o he enzyme Aii20J educed bio ilm o ma ion in 50% o he samples, wi h educ ion pe cen ages in he
ange o 20.82–76.44%, al hough his educ ion was s a is ically signi ican only o sali a 1 (76.44%) and sali a 8
(26.05%). All he bio ilms ha eached an op ical densi y alue highe han 0.2 in he c ys al iole s aining assay
we e a ec ed by he addi ion o Aii20J.
E ec o he AHL-lac onase Aii20J on bac e ial di e si y o in i o o al bio ilm. Due o he p e-
iously obse ed bio ilm inhibi o y ac i i y o Aii20J, he possible in luence he AHL-lac onase on he bac e ial
di e si y o he bio ilms was u he s udied using he sali a om he same heal hy pa ien used in p e ious
expe imen s (Fig.4 and Supplemen a y ma e ial Figs.5, 6, 7 and 8) and om a pe iodon al pa ien . The inhibi o y
e ec o he AHL-lac onase on in i o o al bio ilms o med by sali a samples was con i med using di e en cul-
u e condi ions (Fig.5). A signi ican inhibi ion o he bio ilms by Aii20J was obse ed when he samples om he
Figu e 2. E ec o he AHL-lac onase Aii20J (20 µg/mL) on in i o o al bio ilm ob ained om he sali a
o a heal hy dono as measu ed using he xCELLigence sys em. The cul u e was done in BHI (a) and BHI
supplemen ed wi h 0.1% suc ose (BHIs) (b). Bio ilm o ma ion was exp essed in Cell Index uni s. Da a a e
ep esen a i e o 3 independen expe imen s.
Figu e 3. E ec o he AHL-lac onase Aii20J (20 µg/mL) on in i o o al bio ilm o med using sali a samples
ob ained om heal hy (S0 and S1), and pe iodon al pa ien s (S2-S6) in BHI and BHI supplemen ed wi h 0.1%
suc ose (BHIs) and quan i ied by he xCELLigenceon line measu emen sys em. Measu es a e shown a e 8 h
o incuba ion and exp essed as pe cen age o he con ol cul u es (n = 2).
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heal hy pa ien we e inocula ed in bo h BHI (50.08%), and BHI supplemen ed wi h 0.1% suc ose (45.60%) unde
ae obic condi ions (Fig.5a). The p esence o he lac onase also educed bio ilm p oduc ion a 24 h in anae obic
condi ions (35.40%) (Fig.5a). On he con a y, when using he c ys al iole s aining me hod in he pe iodon al
Figu e 4. Fo ma ion o in i o o al bio ilm ob ained om sali a samples om a heal hy pa ien wi h and
wi hou he AHL-lac onase Aii20J (20 µg/mL) using he Ac i e A achmen model and measu ed wi h
he c ys al iole s aining assay. (a) Bio ilms incuba ed in ae obic condi ions a 37 °C using BHI and BHI
supplemen ed wi h 0.1% suc ose (BHIs). Ae obic bio ilms we e sampled a 24 h, 48 h and 72 h; (b) Bio ilms
incuba ed in anae obic condi ions a 37 °C using BHI and BHI-2 (BHI supplemen ed wi h 2.5 g/L mucin, 1 g/L
yeas ex ac , 0.1 g/L cys eine, 2 g/L sodium bica bona e, 5 mg/L hemin, 1 mg/L menadione and 0.25% ( / )
glu amic acid). Anae obic bio ilms we e sampled a 24 h, 48 h, 72 h and 96 h a 37 °C o BHI. BHI-2 bio ilms
could only be s ained a e 24h. Signi ican ly di e en alues a e indica ed wi h an as e isk (S uden ’s - es ,
p < 0.05) (n = 3).
Figu e 5. Fo ma ion o in i o o al bio ilms ob ained om sali a samples om a heal hy (a) and a pe iodon al
(b) dono wi h and wi hou he AHL-lac onase Aii20J (20 µg/mL) using he Ac i e A achmen model and
measu ed wi h he c ys al iole s aining assay (O.D. 590 nm). The cul u es we e done in ae obic condi ions
using BHI and BHI supplemen ed wi h 0.1% suc ose (BHIs) o 24 h (n = 3) and in anae obic condi ions o
24 and 48 h using BHI (n = 1). Signi ican ly di e en alues a e indica ed wi h an as e isk (S uden ’s - es ,
p < 0,05).

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sample, he inhibi o y e ec caused by Aii20J could only be obse ed in bio ilms g own in BHI supplemen ed
wi h 0.1% suc ose (BHIs) in ae obic condi ions a e 24 h (Fig.5b). Though he c ys al iole s aining could no
de ec di e ences in some cases, he addi ion o Aii20J caused an appa en educ ion in he amoun o bio ilm
biomass ha was ha es ed o he me agenomic analysis using samples o bo h, heal hy and pe iodon al pa ien s
in he Ams e dam Ac i e A achmen sys em a e 24 h. The di e ence in biomass could be obse ed wi h he
naked eye (Supplemen a y ma e ial Fig.9).
The 16S RNA gene sequences o DNA samples ob ained om he bio ilms g own in he di e en cul u e con-
di ions we e py osequenced wi h Illumina MiSec in o de o e alua e he e ec o he enzyme Aii20J on bac e ial
di e si y. All e ie ed sequences belonged o Fi micu es, especially o genus S ep ococcus. Fu he expe imen s
in which he collec ion o samples and sequencing me hod we e op imized also e ealed ha 99.74% o he
ob ained sequences belonged o he genus S ep ococcus wi h he selec ed cul u e condi ions (BHI and BHI sup-
plemen ed wi h 0.1% suc ose). Su p isingly, o bio ilms domina ed by G am-posi i e species, bo h quan i a i e
and quali a i e changes we e obse ed in bac e ial di e si y when he AHL-lac onase Aii20J was added (Fig.6).
The analysis o he ela i e abundance in he bio ilms om he heal hy dono e ealed ha Aii20J caused a clea
and high inc ease o an OTU close o S. o alis subsp. den isani and a concomi an dec ease o a membe o he S.
es ibula is g oup in all es ed condi ions (Fig.6a). This ise in he ela i e abundance o S. o alis subsp. den isani
was highe in anae obic (34.27–37.08%) han in ae obic condi ions (18.2–25.24%). Despi e o he c ys al iole
s ain could no e eal signi ican quan i a i e changes in he enzyme- ea ed bio ilms ob ained wi h he pe io-
don al sali a (Fig.5), an inc ease o S. o alis subsp. den isani and a dec ease o S. es ibula is we e also obse ed,
al hough hese changes we e lowe han in he bio ilms ob ained om he heal hy dono (Fig.6b).
Fu he mo e, in he pe iodon al pa ien , an OTU close o S. o alis subsp. den isani inc eased sligh ly wi h he
enzyme when he bio ilm was g own in BHI (85.81% e sus 89.89%) bu dec eased i he cul u e media was sup-
plemen ed wi h suc ose (84.50% e sus 74.88%) (Fig.6b). Mo eo e , Aii20J caused an appa en educ ion o he
Lac obacillus abundance when he samples om he pe iodon al dono we e incuba ed in anae obic condi ions
(Fig.6b)
The e ec o Aii20J on he bac e ial composi ion o in i o o al bio ilms was also e alua ed using PCA anal-
ysis (Fig.7). PC1 explains he 93.49% and he 93.63% in he a iance ob ained om he heal hy (Fig.7a) and he
Figu e 6. Bac e ial di e si y o he in i o o al bio ilms ob ained om sali a samples om a heal hy (a) and
a pe iodon al (b) dono wi h and wi hou he AHL-lac onase Aii20J (20 µg/mL). The cul u es we e incuba ed
in ae obic condi ions using BHI and BHI supplemen ed wi h 0.1% suc ose (BHIs) o 24 h and in ae obic
condi ions o 24 and 48 h using BHI.
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pe iodon al (Fig.7b) bio ilms, espec i ely. Fu he mo e, in he samples ob ained om he heal hy pa ien , PC1
di e en ia es he ea ed bio ilms om hei espec i e un ea ed bio ilms and is ep esen ed mos no ably by a
di e en ial abundance o membe s close o S. o alis subsp. den isani and S. es ibula is. Howe e , in he pe iodon-
al bio ilms, PC1 allows o he di e en ia ion o he bio ilms depending on he ae obic o anae obic condi ions
due o he highe mic obial di e si y ob ained in anae obic condi ions o he pe iodon al dono .
E ec o Aii20J on mul i-species bio ilms o med by o al pa hogens. The e ec o Aii20J (20 µg/
mL) on in i o mixed-species bio ilms o med by six o al pa hogens46 was checked using con ocal mic oscopy in
o de o co obo a e he inhibi ion o he sali a bio ilms obse ed wi h he c ys al iole s aining me hod. When
he o al pa hogens A. naeslundii, A. ac inomyce emcomi ans, F. nuclea um, P. gingi alis, S. o alis and V. pa ula
we e co-cul u ed du ing 3 o 4 days, an e iden inhibi ion o bio ilm o ma ion was obse ed when Aii20J was
added o he cul u e media (Fig.8). The o al a ea, as well as he a ea o li e cells, we e s a is ically highe in con-
ol bio ilms han in he ea ed bio ilms. The highes co e ed a ea was occupied by he con ol bio ilms a day 3
and day 4 (870.70 ± 404.92 and 1,553.30 ± 1,091.14 µm2) in compa ison o he ea ed bio ilm (585.37 ± 379.57
and 546.68 ± 340.79 µm2). In he same way, he olume o li e cells was s a is ically lowe in he ea ed bio ilms
in compa ison o he con ol bio ilms. On he con a y, he hickness o he ea ed bio ilms in compa ison o
con ol bio ilms was simila on day 3 (31.97 ± 6.58 e sus 33.41 ± 9.72 µm) bu highe on day 4 (17.43 ± 5.69
e sus 26.61 ± 7.74 µm). The po osi y o hese bio ilms was also much highe han he con ol bio ilms, indica ing
impo an di e ences in he s uc u e o bo h ypes o bio ilm.
Discussion
Con i ming p e ious s udies ha ha e al eady demons a ed he p esence o AHL- ype QS signals in sali a and
spu um samples17–20, in his s udy we epo he p esence o his ype o signal molecule in di e en o al samples.
Mo eo e , he analysis o in i o mul i-species o al bio ilms o med by S. o alis, V. pa ula, A. naeslundii, F.
nuclea um, A. ac inomyce emcomi ans and P. gingi alis, and o P. gingi alis axenic cul u es e ealed he p esence
o he QS signal OC8-HSL in small amoun s. The cu en accep ed pa adigm does no assign a signi ican ole o
AHLs in den al plaque o ma ion since hese signals a e almos comple ely excluded om he li e a u e e iews
on QS mechanisms in o al bio ilms13–16. Al hough a ew s ains wi h he abili y o p oduce AHLs we e isola ed
om he human ongue su ace and den al plaque samples21–25, he ac ha AHLs could no be de ec ed in pu e
cul u es o pa hogenic o al bac e ia in p e ious s udies10–12 is he eason why a mino ole is a ibu ed o hese
signals in he o al ca i y. The low sensi i i y o he biosenso s may be pa ially esponsible o he low numbe o
AHL-p oducing s ains isola ed om he o al ca i y. A high-sensi i i y me hod such as mass spec ome y analy-
sis is needed o he unequi ocal de ec ion o AHLs p oduced by bac e ial s ains. Mo eo e , he analyses should
be pe o med using s a ic cul u es ha a ou bio ilm o ma ion and AHL p oduc ion and he cul u e medium
can also s ongly a ec AHL p oduc ion. Since su ace adhe ence o cell- o-cell adhe ence is equi ed o ac i a e
AHL syn hesis in some bac e ia34,48 and speci ic bac e ia modi y he gene exp ession in o al bio ilms4, hese
bio ilm-p omo ing cul i a ion condi ions may ha e igge ed he exp ession o QS- ela ed genes no exp essed
in axenic cul u es o unde agi a ed condi ions. Mo eo e , AHLs may be only p oduced in mixed cul u es, as
demons a ed by he inc ease in AHL p oduc ion in mixed cul u es o P. gingi alis. In his sense, some bac e ia a e
known o depend on he QS signals p oduced by o he s; o ins ance, S. go donii equi es AI-2 in o de o o m
mixed bio ilms wi h P. gingi alis49.
The mos common AHL p esen in he ex ac ed ee h and sali a samples was C8-HSL, which was ound in all
es ed samples, wi h only one excep ion. C8-HSL and OC8-HSL we e also he main AHLs ound in sali a samples
Figu e 7. P incipal componen s analysis o he species composi ion o in i o o al bio ilms ob ained om
heal hy (a) and pe iodon al (b) sali a samples wi h and wi hou he QQ enzyme Aii20J. Bio ilms we e cul u ed
in BHI o BHI supplemen ed wi h 0.1% suc ose unde ae obic and in BHI du ing 24 h and 48 h unde anae obic
condi ions. Closed symbols ep esen he con ol bio ilms while open symbols ep esen he bio ilms ea ed
wi h he QQ enzyme Aii20J. Squa es = BHI ae obic condi ions, ci cles = BHI anae obic condi ions, iangles =
BHI supplemen ed wi h 0.1% suc ose in ae obic condi ions and diamonds = 48 h BHI in anae obic condi ions
in bo h panels.
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o a heal hy dono used o gene a e di e en o al bio ilm models, al hough some a iabili y was ound o e
ime19. All pa ien s who p esen ed C8, C14 and C18-HSL in hei sali a su e om den al ca i ies while only C8
and occasionally C14-HSL could be ound in hose wi h pe iodon al disease. The highe di e si y o AHL signals
ound in den al plaque om pa ien s wi h ca ies is compa ible wi h he lowe pH ha is expec ed in hese samples
since AHLs a e mo e s able in acidic pH50. E en hough all samples we e acidi ied in o de o allow he eco e y
o he lac one ings be o e analysis, he neu al o basic pH alues in heal hy den al plaque may ha e allowed a
as e deg ada ion o he open lac one- ing AHLs by o he bac e ia. Al hough hese da a seem o indica e a co -
ela ion be ween AHL p o ile and speci ic o al pa hologies, he da a ob ained in he p esen s udy a e no enough
o es ablish an associa ion be ween he obse ed o al pa hologies and he amoun and/o ype o AHL de ec ed.
In he p esen wo k, he p oduc ion o a small amoun o OC8-HSL (0.30 ng/mL) by P. gingi alis was de ec ed
o he i s ime. The mos common AHL-dependen QS sys em is based on wo p o eins: a LuxI- ype syn hase
and a LuxR- ype ecep o 51, al hough o he AHL syn hases belonging o he amilies LuxM/AinS and Hd S ha e
been desc ibed52,53. An ORF wi h 25% iden i y and 48% amino acid simila i y wi h he AHL-syn hase Hd S
was iden i ied in P. gingi alis W8326 indica ing he p esence o a leas a pu a i e AHL-syn hase in his species.
Addi ionally, a LuxR homologue, designa ed as CdhR (Communi y De elopmen and Hemin Regula o ), ha
con ols he ansc ip ion o he hmu ope on esponsible o i on/hemin up ake was ound in his bac e ium27,28.
Fu he mo e, a ecen s udy has iden i ied a non-classical QS syn hase in an AHL-p oducing Psych obac e s ain,
indica ing he possible exis ence o ye unknown AHL-syn hases no homologous o hose desc ibed so a 54.
The e o e, he p esence o o he unknown AHL-syn hases o ecep o s in P. gingi alis canno be dis ega ded.
The in luence o di e en AHLs, as well as AHL-analogues on P. gingi alis, we e e alua ed in p e ious wo ks
showing ha when C14-HSL was ex e nally added, changes in bo h p o ein exp ession and bac e ial g ow h we e
obse ed26,29, indica ing he p esence o an AHL ecep o . Fu he mo e, in a p e ious wo k, we demons a ed
ha he exogenous addi ion o speci ic AHLs gene a ed changes, no only in he bac e ial composi ion bu also
in me abolic ac i i y o in i o o al bio ilms ob ained om sali a19. C14 and C18-HSL (bo h AHLs ound in o al
samples in he p esen wo k) educed he p oduc ion o lac ic acid in ca iogenic and commensal in i o sali a
bio ilms, espec i ely. On he con a y, he addi ion o C8-HSL, he majo AHL ound in he same samples,
caused a signi ican dec ease in he p oduc ion o lac a e in bo h ypes o bio ilm. Addi ionally, C18-HSL also
p oduced an inc ease in he p o ease ac i i y o he commensal bio ilms19. These e ec s may be pa ien -speci ic,
and he e o e u he s udies a e needed o es ablish he ole o hese signals in he de elopmen o ca iogenic
den al plaque. Fu he mo e, he addi ion o C6-HSL inc eased he ela i e abundance o Pep os ep ococcus and
P e o ella, esul ing in a shi owa ds a pe iodon al bac e ial composi ion p o ile19. All hese da a indica e ha
AHLs pe o m a c i ical ole in o al bio ilm o ma ion, e en in hose domina ed by G am-posi i es. In ano he
s udy55 he ca iogenic po en ial o he den al plaque was educed by he addi ion o a C12-HSL analogue, which
induced changes in he ela i e abundance o he bio ilm bac e ial composi ion compa ed o he con ol, dec eas-
ing S ep ococcus spp. ( om 61% o 33%) and inc easing Ac inobacillus spp. ( om 5% o 29%). Ne e heless, his
e ec could be de i ed om g ow h inhibi ion, since he oxici y o OC12-HSL and i s e amic acid deg ada ion
p oduc has been epo ed o G am-posi i e bac e ia56, and no compe i ion was obse ed be ween he analogue
and he na u al AHL.
Figu e 8. Con ocal mic oscopy isualiza ion (63×) o he e ec o he QQ enzyme Aii20J (20 µg/mL) on
mul i-species bio ilms o med by he o al pa hogens A. naeslundii, A. ac inomyce emcomi ans, F. nuclea um,
P. gingi alis S. o alis and V. pa ula using luo escence dyes Sy o9 and p opidium iodide. The bio ilms we e
cul u ed in BHI-2 du ing 3 and 4 days in anae obic condi ions a 37 °C.
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The impo ance o AHL-media ed QS sys ems in he o al ecosys em is u he suppo ed by a high p e a-
lence o QQ ac i i y agains sho -chain AHLs among cul i able bac e ia isola ed om den al plaque and sali a
ob ained om heal hy and pe iodon al dono s (14–47%). These pe cen ages may be e en highe conside ing
ha in en i onmen al samples, QQ ac i i y agains sho -chain AHLs is less equen han agains long-chain
AHLs46,57. These esul s o QQ p e alence among he cul i able o al s ains a e simila o hose ob ained in
ma ine samples43,57–61 which is he en i onmen wi h he highes QQ ac i i y desc ibed hus a . The equency o
QQ s ains was signi ican ly highe in den al plaque samples om a pe iodon al pa ien , indica ing he po en ial
impo ance o QQ ac i i y in he es ablishmen o a dysbiosis ha should be u he in es iga ed. Addi ionally,
he capabili y o sali a om se e al dono s o in e e e wi h C12-HSL was obse ed. An impo an an imic obial
ac i i y was also ound among he isola es wi h 73 o hem being able o inhibi he g ow h o he A. ume aciens
biosenso . An imic obial ac i i y was mo e abundan in he heal hy pa ien (60.27%) han in he pe iodon al one
(39.72%). This highe abundance o s ains wi h an imic obial ac i i y in he heal hy pa ien could also be ela ed
o he heal h s a us o he dono . Recen ly, he use o he p obio ic s ain S ep ococcus den isani ( eclassi ied as
S. o alis subsp. den isani) was p oposed o he con ol o o al diseases since i is p esen in a high pe cen age o
heal hy pa ien s and can inhibi he g ow h o he p ima y o al pa hogens due o bac e iocin p oduc ion and pH
bu e ing o he o al ca i y62,63.
Though se e al wo ks ha e epo ed he po en ial use o AI-2 inhibi o s in educing den al plaque35,64–68, o he
bes o ou knowledge, his is he i s s udy ha demons a es ha an AHL-lac onase inhibi s o al bio ilm o ma-
ion, s ongly suppo ing he c i ical ole his ype o QS signal plays in o al bac e ial communi ies. The addi ion
o he wide-spec um AHL-lac onase Aii20J also a ec ed he 6-species bio ilms composed o he odon opa ho-
gens A. naeslundii, A. ac inomyce emcomi ans, F. nuclea um, P. gingi alis, S. o alis and V. pa ula. The hickness, as
well as he po osi y, inc eased bu he co e ed a ea and he olume o li e cells we e lowe when he bio ilms we e
ea ed wi h he lac onase Aii20J. The bio ilm inhibi o y ac i i y o Aii20J was con i med in bio ilms gene a ed
using di e en sali a samples om heal hy and diseased pa ien s using he xCELLigence sys em (25.15–93.4%).
Fu he mo e, he e ec o Aii20J on in i o o al bio ilms was e alua ed in di e en cul u e condi ions using he
Ac i e A achmen sys em on glass co e slips and he c ys al iole s aining assay, showing a signi ican inhibi ion
o bio ilms ob ained wi h sali a samples om a heal hy pa ien when cul i a ed in bo h ae obic and anae obic
condi ions. The analysis o he ela i e abundance and PCA analysis e ealed ha he p esence o Aii20J caused
a high inc ease o S. o alis subsp. den isani and an impo an dec ease o S. es ibula is in he bio ilm o med by
sali a ob ained om a heal hy pa ien . Al hough he AHL-lac onase did no cause an inhibi ion in he quan i y
o bio ilm o med by a pe iodon al sali a sample as measu ed using he c ys al iole s aining assay, a dec ease
o he bio ilm biomass could be obse ed wi h he naked eye when he bio ilms we e ha es ed o me agenomic
analysis, indica ing a low sensi i i y o he c ys al iole s ain35,69. A shi in he bac e ial di e si y simila o ha
obse ed in he heal hy sali a sample was also obse ed. This e ec o Aii20J on he bac e ial composi ion o he
o al bio ilms could be used as a no el s a egy in heal hca e h ough he p omo ion o he an imic obial ac i i y
p esen in some commensal o al bac e ia such as S. o alis which can inhibi he g ow h o se e al o al pa hogens
such as A. ac inomyce emcomi ans, P. gingi alis and P. in e media70.
The impo an inhibi ion obse ed in mixed in i o bio ilms, which a e composed mos ly by G am-posi i e
species (99.9%), when ea ed wi h Aii20J could be explained ei he by he possible in e ac ion be ween he AHL
and G am-posi i e bac e ia o by in e ac ions wi h mino i y G am-nega i e componen s o he o al bio ilm.
Some bac e ia do no p oduce AHLs bu possess LuxR homologs. These LuxR o phans in e ac wi h he QS
molecules p esen in he en i onmen p oduced by o he mic oo ganisms71. A gene belonging o LuxR- amily o
egula o y p o eins was p edic ed in he genome o S. mu ans, sugges ing he exis ence o a LuxR o phan72. An
impo an G am-posi i e pa hogen, S aphylococcus au eus, esponds o he OC12-HSL p oduced by Pseudomonas
ae uginosa in a sa u able and speci ic manne , esul ing in he inhibi ion o he p oduc ion o exo oxins and he
enhanced exp ession o he p o ein A, an impo an su ace p o ein in ol ed in se e al i ulence mechanisms73.
Fu he mo e, since he p oduc ion o AHLs has been epo ed in se e al G am-posi i e bac e ia belonging o
Ac inobac e ia and Fi micu es phylum74–77 as well as in bac e ia ha do no possess a known AHL syn hase54
we canno exclude ha comple ely unknown AHL syn hases a e p esen in o al pa hogenic bac e ia. In addi ion,
LuxR- ype ecep o s can be ac i a ed by se e al signalling molecules besides acyl-HSLs p oduced by LuxI homo-
logues, ega ded as “dialec ” syn hases78. Hence, all hese da a indica e ha he QS ne wo k in he o al ca i y may
be much mo e complica ed han he cu en ly accep ed model in which o al bac e ial communica ion is only
media ed by AIPs and AI-2, meanwhile he AHLs’ signalling ole is conside ed o mino ele ance.
The obse a ion o he p esence o AHL- ype QS signals in o al samples as well as in in i o o al bio ilms and
he high abundance o s ains wi h QQ ac i i y s ongly indica es ha AHL-media ed QS sys ems play an impo -
an ole in he o al ca i y. This conclusion is u he suppo ed by p e ious expe imen s ha demons a ed he
impo an e ec s o he exogenous addi ion o AHLs o di e en bio ilm models19. The signi ican quan i a i e
and quali a i e e ec s o he AHL-lac onase Aii20J on sali a and mixed o al pa hogen bio ilms con i ms his
c i ical ole and opens new possibili ies in he p e en ion and ea men o o al diseases. Fu he analysis is nec-
essa y o con i m and comple e ou knowledge ega ding he ole and ele ance o AHL-media ed p ocesses in
he main enance o he ecological equilib ium o he mic obial inhabi an s o he o al ca i y h ough compe i i e
and coope a i e in e ac ions, as well as on he possibili y o applying QQ s a egies o he con ol o o al diseases.
Me hods
Bac e ial s ains and cul u e media used. The pu e cul u es o P. gingi alis ATCC33277 and co-cul-
u e o his bac e ium wi h S. o alis CECT907T o S. go donnii ATCC49818 we e pe o med in BHI-2 [BHI
supplemen ed wi h 2.5 g/L mucin, 1 g/L yeas ex ac , 0.1 g/L cys eine, 2 g/L sodium bica bona e, 5 mg/L hemin,
1 mg/L menadione and 0.25% ( / ) glu amic acid] in anae obic condi ions o 16 h a 37 °C. Mul i-species bio ilms
o med on hyd oxyapa i e discs (HA) by he six o al pa hogens S. o alis CECT907T, V. pa ula NCTC11810,