P obing In e kingdom Signaling Molecules ia Liquid Ex ac ion
Su ace Analysis
−
Mass Spec ome y
Shaun N. Robe son, Fadi Souka ieh, Thomas M. Whi e, Miguel Cama a, Manuel Rome o,*
and Rian L. G i i hs*
INTRODUCTION
Polymic obial bio ilms a e common h oughou heal hca e,
indus ial, and en i onmen al se ings. These su ace-associ-
a ed o agg ega i e mic obial communi ies a e cen al o he
challenge o an imic obial esis ance due o he o e p oduc ion
o ex acellula polyme ic subs ances ha can en ap and limi
he di usion o ce ain an ibio ics,1 and he induc ion o
physiological changes ha lead o an imic obial ole ance in
cells.2 I is es ima ed ha 65% o bac e ial in ec ions a e
bio ilm-associa ed
3
and while unde es ima ed, Candida albicans
(C.
albicans)
bio ilm
in ec ions
a e
esponsible
o
an
es ima ed
>400,000 li e- h ea ening in ec ions a yea wo ldwide wi h a
mo ali y a e o 46−75%.4 In hese communi ies, mic obes
con ey hei p esence by p oducing small di usible signal
molecules known as au oinduce s, which can hen be de ec ed
and esponded o in a popula ion-coo dina ed manne . This
o m o mic obial in e cellula communica ion is called
quo um sensing (QS)5 and egula es physiological p ocesses
in hese communi ies and, impo an ly, has been shown o
con ol bio ilm o ma ion.6 Rema kably, QS signaling mole-
cules ha e p e iously been shown o ac as bioma ke s o lung
in ec ion ia LC−MS o plasma om cys ic ib osis pa ien s.7
Hence, hese molecules could ep esen impo an ci cula o y
indica o s o in ec ion ha could be es ed nonin asi ely.
Gi en ha mos bio ilm-cen e ed in ec ions a e polymic o-
bial,8,9 he possibili y o in e species mic obial communica ion
can also occu and QS is hough o play a i al ole in
in e species in e ac ions anging om commensalism o
an agonism.8 Fo ins ance, in e kingdom QS signaling be ween
he
bac e ium
Pseudomonas
ae uginosa
(P.
ae uginosa)and
ungi
C. albicans species has been desc ibed9 as media ed ia he
p oduc ion o 2-alkyl-4(1H)-quinolone (AQ) signal molecules
and a nesol, espec i ely. Mo eo e , hese species a e o en
p esen alongside S aphylococcus au eus (S. au eus) in heal h-
ca e se ings10,11 and i is well documen ed ha P. ae uginosa
displays se e e compe i ion agains S. au eus in i o. Fo
example, in wound in ec ions o cys ic ib osis lung,
ex acellula ac o s p oduced by P. ae uginosa ha e been
shown o subjuga e S. au eus o pe sis as small colony
a ian s.12 One o such ac o s is he AQ molecule 2-hep yl-4-
hyd oxyquinoline N-oxide (HQNO), a cy och ome inhibi o
eleased by P. ae uginosa. In u n, P. ae uginosa can sense
ex acellula p oduc s sec e ed by S. au eus such as he
exopolyme N-ace yl glucosamine, and in esponse, inc ease
he p oduc ion o i ulence ac o s and an imic obials.5,12,13
ABSTRACT: P e iously, me aboli es di used o sec e ed om mic obial samples ha e been
analyzed ia liquid ch oma og aphy−mass spec ome y (LC−MS) app oaches ollowing leng hy
ex ac ion p o ocols. He e, we p esen a model sys em o g owing bio ilms on discs be o e
u ilizing apid and di ec su ace sampling MS, namely, liquid ex ac ion su ace analysis, o s udy
he mic obial exome abolome. One o he bene i s o his app oach is i s su ace-speci ic na u e,
enabling mimicking bio ilm o ma ion in a way ha he s udy o plank onic liquid cul u es canno
Candida albicans (C. albicans) ha e been s udied p e iously in isola ion, e y ew s udies conside
he complexi y o he in e play be ween hese pa hogens, which a e commonly combined causa i e agen s o in ec ion. Ou model
sys em p o ides a ou e o in es iga e changes in he exome abolome, such as me aboli es ha become ci cula o y in he p esence o
mul iple pa hogens. Ou esul s ag ee wi h p e ious epo s showing ha 2-alkyl-4(1H)-quinolone signal molecules p oduced by P.
ae uginosa a e impo an ma ke s o in ec ion and sugges ha me hods o moni o ing le els o 2-hep yl-4-hyd oxyquinoline and
2,4-dihyd oxyquinoline, as well as pyocyanin, could be bene icial in he de e mina ion o causa i e agen s in in e kingdom in ec ion
including P. ae uginosa. Fu he mo e, s udying changes in exome abolome me aboli es be ween pqs quo um sensing an agonis s in
ea ed and non ea ed samples sugges s supp ession o phenazine p oduc ion by P. ae uginosa. Hence, ou model p o ides a apid
analy ical app oach o gaining a mechanis ic unde s anding o bac e ial signaling.
A icle
Simila ly, a nesol, a nesoic acid,14 and a ious amino acid-
de i ed alcohol15−17 molecules sec e ed by C. albicans ha e
been shown o play an impo an ole in bio ilm o ma ion18,19
and a e cu en ly unde -in es iga ed in he con ex o
polymic obial bio ilms.
Mass spec ome y (MS) o e s a ou e o he un a ge ed
analysis o mul iple species wi hou equi ing chemical
agging/modi ica ion. Su ace sampling app oaches o e he
oppo uni y o di ec ly p obe solid biological samples such as
issue sec ions, blood spo ca ds, and bac e ial bio ilms. Many
mic obial species ha e been analyzed ia di ec su ace
sampling MS app oaches. The aims o hese s udies a y
om bac e ial pheno yping o d ug sc eening and unde -
s anding bac e ial bio ilms. Va ious biomolecules ha e been
s udied ia di e en app oaches; an imic obials, bac e ial QS
signaling molecules, me aboli es, and hamnolipids ha e been
s udied ia ma ix-assis ed lase deso p ion ioniza ion
(MALDI) ioniza ion MS,20−22 and seconda y ioniza ion mass
spec ome y (SIMS).22−28 Phospholipid analysis is widely
epo ed ia apid e apo a i e ioniza ion mass spec ome y
(REIMS)29 and deso p ion elec osp ay ioniza ion (DESI)
app oaches, and lipids and in ac p o eins ha e been s udied by
liquid ex ac ion su ace analysis−mass spec ome y (LESA-
MS).30−32 Many REIMS s udies ocus on specia ion using
ma hema ical algo i hms o unde s and p o iles o , e.g.,
bac e ial s ains including P. ae uginosa29,33 and ungi such as
he Candida genus,33,34 wi hou iden i ying speci ic analy es.
P e iously, p o eins in ol ed in he in e ac ion be ween P.
ae uginosa and S. au eus ha e been in es iga ed ia bo om-up
p o eomics/MALDI-MS imaging and co ela ed o me al ions
(in ol ed in p o ein in e ac ions) by lase abla ion-induc i ely
coupled plasma MS imaging.35 Di e ences in he lipid and
me aboli e p o iles (including heme, po phy in, and an ibio ic
compounds) o Shewanella oneidensis and Bacillus sub ilis as
single species and mixed bio ilms ha e been in es iga ed ia
nano-DESI.36
LESA-MS
is
a
su ace
sampling
app oach
ha
u ilizes
sol en
ex ac ion in o a obo ically ope a ed pipe e ip p io o
elec osp ay ioniza ion. LESA-MS o e s so ioniza ion and
apid analysis ime (a ew minu es) wi h he addi ional bene i
o long sp ay imes, pa icula ly use ul o s uc u al
iden i ica ion (MSMS) expe imen s. P e iously, LESA-MS o
mic obial colonies including P. ae uginosa, S. au eus, and
ESKAPE pa hogens has been desc ibed o he analysis o
in ac p o eins om a ange o mono-mic obial sys-
ems.30,31,37,38 Mo e ecen ly, LESA-MS has been used o
he di ec bac e ial analysis o lipids om Mycobac e-
ium30−32,38 and has been sugges ed as a po en ial al e na i e
o MALDI-MS in specia ion o clinical isola es. He e, we
desc ibe o he i s ime exome aboli e analysis speci ically,
and om mono- and poly-mic obial bio ilms.
This s udy seeks o in es iga e P. ae uginosa QS signaling
molecules di used and/o sec e ed om single and poly-
mic obial species bio ilms using a no el sample p epa a ion
me hod speci ically designed o unde s and he bac e ial
exome abolome. LESA-MS is desc ibed o he i s ime o
a ge ed analysis o sec e ed QS molecules; in he aga
unde nea h a bio ilm g own on a disc. Di e ences in QS we e
de e mined in he p esence o a mic obial compe i o (namely,
S. au eus o C. albicans), and in he p esence o bo h
compe i o s using he LESA-MS pla o m. Ou model allowed
he de ec ion o he AQ signal 2-hep yl-4-quinolone (HHQ),
p e iously
iden i ied
as
a
diagnos ic
ma ke
o
P.
ae uginosa
in ec ion, and sugges ed he u ili y o ou app oach. Ou esul s
also sugges ha me hods o moni o ing le els o o he AQs
such as 2,4-dihyd oxyquinoline (DHQ), as well as pyocyanin,
could be bene icial in he de e mina ion o causa i e agen s in
in e kingdom bio ilm communi ies. Fu he mo e, he e ec o
an ibio ic ea men and/o inhibi ion o he AQ-based
communica ion sys em in P. ae uginosa was examined and
ou s udy shows ha , in ag eemen wi h p e ious epo s, AQ
inhibi o s can supp ess phenazine biosyn hesis in bio ilm
communi ies including his bac e ial pa hogen. The e o e, ou
model p o ides a apid analy ical app oach o gaining a
mechanis ic unde s anding o QS p ocesses in he con ex o
polymic obial bio ilms.
EXPERIMENTAL
SECTION
Ma e ials and Me hods. S ain and Colony Bio ilm
Cul u e Condi ions. S. au eus SH1000 and P. ae uginosa
PAO1-L s ains we e ou inely g own on he lysogeny b o h
(LB, Oxoid, Camb idge, UK) aga . C. albicans SC5314 was
ou inely g own in he Sabou aud dex ose (SAB, Oxoid,
Camb idge, UK) aga . Bac e ial and ungi pla es we e
incuba ed a 37 and 30 °C, espec i ely. UVC-s e ilized
polyca bona e (PC) discs (13 mm diame e ) on wells o 6-well
pla es illed wi h 5 mL o media. The s ock inoculum (10 μL,
added on op o one ano he o polymic obial combina ions)
was pipe ed o he cen e o he PC disc. Pla es we e hen
ans e ed o a s a ic incuba o and incuba ed a 37 °C o 24 h
o allow colony bio ilm g ow h. Fo ea men wi h he QS
inhibi o s (QSIs), SEN19 and SEN89 compounds we e
supplemen ed a 10 μM in he inal s ock inoculum o PAO1-
L. T ea men o 18 h PAO1-L colony bio ilms wi h
cip o loxacin was pe o med by adding 20 μL o 64 μg/mL in
H2O pipe ed gen ly on op o he p e o med colony bio ilm.
A he endpoin , PC discs wi h a ached colony bio ilms we e
asep ically emo ed and aga plugs we e used o QS signal
de ec ion. T iplica e biological and echnical epea s we e
conduc ed o all expe imen s p esen ed. Fu he de ails,
including CFU coun ing de ails, can be ound in he
Supplemen al In o ma ion.
LESA Sampling. LESA was ca ied ou using he T i e sa
Nanoma e (Ad ionBiosciences, I haca, NY, USA). The
ex ac ion/ioniza ion sol en was 1:1 me hanol/wa e . Du ing
ex ac ion, 5 μL o sol en was aspi a ed om he sol en well,
be o e sampling he aga wi h 2 μL o his sol en o 5 s wice
(1 mix). Finally, 2.5 μL o he sampling sol en was e-
aspi a ed and in used in o he mass spec ome e a a gas
p essu e o 0.3 psi and a po en ial o 2.0 kV.
Mass Spec ome y. Expe imen s we e pe o med on a
The mo Fishe O bi ap Q-Exac i e mass spec ome e . Mass
spec a we e eco ded in ull scan mode a a esolu ion o
140,000 a m/z 400 in he m/z ange o 50−750 in posi i e
ioniza ion mode. The AGC a ge was 1 × 106 cha ges wi h a
maximum injec ion ime o 500 ms. Each scan consis ed o 1
mic oscan. MSMS de ails can be ound in he Supplemen al
In o ma ion. Da a we e eco ded o up o 1 min and analyzed
using The mo Xcalibu e sion 4.2.28.14 so wa e. MSMS
spec a we e manually in e p e ed.
RESULTS
AND
DISCUSSION
Alkyl-quinolone (AQ) Quo um Sensing (QS) Signaling
Molecules Di used om and Vi ulence Fac o s
Sec e ed
by
P.
ae uginosa
Bio ilms
De ec ed
by
LESA-MS.
A icle
Figu e 1. Exome abolome analysis o QS molecules om P. ae uginosa PAO1-L bio ilms. Pho os o (A) bio ilm g own on PC disc, (B) aga
sampled a e emo al o he disc wi h loca ions indica ed (blue = cen e , o ange = edge), and (C) LESA sampling. (D) Ba cha s showing
dec easing signal in ensi ies o selec ed QS molecules om he cen e and he edge o whe e he bio ilm was g own on he PC disc. (E) Schema ic
o he sample p epa a ion o exome abolome analysis and LESA sampling. (F) Spec a showing LESA-MS o aga backg ound (blue) and selec ed
QS molecules de ec ed in he exome abolome o P. ae uginosa bio ilms.
To assess whe he LESA-MS could be used o s udy he
elease o small o ganic molecules om mic obial bio ilms, we
s udied he p esence o QS molecules, which a e mos likely
passi ely di used, and pyocyanin sec e ed by 24 h P. ae uginosa
PAO1-L monospecies bio ilms. De ails ega ding speci ic QS
pa hways and molecules analyzed a e p o ided in he
Supplemen al In o ma ion.
To allow cell- ee me aboli e ex ac ion om aga subs a es,
bio ilms we e g own on 0.2 μm po e PC discs placed on he
aga medium o enable he asep ic emo al o he mic obial
cells p io o LESA-MS analysis (Figu e 1). The ollowing P.
ae uginosa signals we e de ec ed om he media: HQNO,
C9:1-PQS, C9-PQS, C9 quinolone, C11 quinolone, and HHQ,
see Figu es 1 and 2. These all in o h ee classes o AQ QS
molecules: HHQ-de i ed compounds [m/z 244.1696 (HHQ),
270.1849 and 272.2007], PQS-de i ed compounds (m/z
260.1642 and 288.1955), and HQNO-de i ed compounds
[m/z 260.1642 (HQNO) and 288.1955], see Table 1. No e
ha some o hese compounds a e isome s o one ano he ,
he e o e MSMS is equi ed o elucida ion. Howe e , N-acyl
homose ine lac one (AHL) signal molecules we e no de ec ed
in he condi ions es ed. The i ulence ac o pyocyanin ([M +
H]+ m/z 211.0863) was de ec ed and sec e ed om PAO1-L
bio ilms, sugges ing ha ou no el sample p epa a ion
me hodology allows de ec ion o a ious di used and/o
sec e ed me aboli es ex e nal o he bio ilm. LESA-MS was
p e iously desc ibed o analyzing in ac bac e ial p o eins and
lipids.32−34 Howe e , o ou knowledge, QS au oinduce s ha e
no p e iously been epo ed using his app oach.
QS molecules ha e p e iously been analyzed using, e.g.,
SIMS, om bio ilms anging om 7 o 72 h o g ow h.22,27,28
AQs wi h C9 alkyl chains we e shown o be highly abundan in
ea ly 7 h bio ilms o P. ae uginosa g own on silicon iles.28
Simila molecules ha e been epo ed ia MALDI imaging o
12 h bio ilms.21,39 These QS signaling molecules ha e also
been epo ed in ma u e (72 h) bio ilms o P. ae uginosa g own
on silicon wa e iles.40 He e, we show ha AQs de i ed om
HHQ, PQS, and HQNO can be analyzed ia LESA om
bio ilms g own o 24 h. The e a e nume ous bene i s o ou
app oach. Fi s , p e ious epo s ha e s udied bio ilms di ec ly;
hence, i is no possible o dis inguish be ween me aboli es
wi hin he bio ilm and hose di using away om he bio ilm.
He e, we p esen a new sample o ma speci ically designed o
analyze bac e ial bio ilm exome aboli es ha also has he
ad an age o unde s anding he e ec o dis ance on mic obial
QS, which could be impo an in de e mining diagnos ic
ma ke s o in ec ion/se e i y. Compa ison o he exome abo-
li es de ec ed in he media a he si e o he colony g ow h
(blue, Figu e 1B), and hen a ound he ou e edge o he PC
disc (o ange, Figu e 1B, and u he away (whi e, Figu e 1C),
A icle
Figu e 2. Ba cha showing he mean signal in ensi y o QS molecules de ec ed ia LESA-MS in he exome abolome analysis o in e species
bio ilms. Mean alues o PA−SA−CA, which we e lowe han o he bio ilms a e shown in he inse .
Table 1. Me aboli es De ec ed in he Exome abolome o In e species and In e kingdom Bio ilms o P. ae uginosa (PA), S.
au eus (SA), and C. albicans (CA), and in he Exome abolome o Cip o loxacin (CIP) D ug and/o (SEN019 o SEN089)
Inhibi o -T ea ed PA Bio ilmsa
HHQ
assignmen PA SA CA CA CIP
SEN019
SEN019-CIP SEN089
HQNO/C7-PQS * * - *
*
*
- *
C9:1 quinolone * * - *
*
*
- -
NHQ
C9 PQS
C11 quinolone * * *
- - *
- -
pyocyanin
cis-2-decenoic acid
(CDA)
dihyd oxyquinolone
(DHQ)
aKey: - = no de ec ed eliably, * = de ec ed eliably ac oss epea s.
shows dec easing signal in ensi ies o he ollowing signals: m/z
260.1642 (HQNO), m/z 272.2007 (C9-quinolone/2-nonyl-
4(1H)-quinolone (NHQ)) and m/z 288.1955 C9-PQS/
NQNO, see Figu e 1A,B,D. On a p ac ical le el, ou app oach
allows he analysis o biomolecules di used/sec e ed om
bac e ial bio ilms wi hou needing a dedica ed ins umen in
he mic obial labo a o y, subjec o he app op ia e biological
sa e y measu es.
Ou LESA expe imen s bene i om coupling o a high-
esolu ion (O bi ap) mass analyze o e ing he ad an age o
de ec ed
m/z
accu a e
m/z
ppm
chemical
o mula
244.1696
244.1701
2.2
C
16
H
22
NO
260.1642
260.1651
3.5
C16H22NO2
270.1849
270.1858
3.3
C18H24NO
272.2007
272.2014
2.6
C
18
H
26
NO
288.1955
288.1964
3.1
C18H26NO2
300.232
300.2327
2.3
C
20
H
30
NO
211.0863
211.0871
3.8
C13H11N2O
188.1642
188.1651
4.8
C10H10N2O2
162.0548
162.0555
4.3
C
9
H
8
NO
2
PA
−
PA
−
PA
−
SA
−
PA
−
PA
−
PA
−
PA
−
*
*
*
*
*
*
-
-
*
*
-
*
*
*
-
-
*
*
-
-
*
*
-
-
*
*
*
-
*
-
-
-
A icle
- - - * - * - -
- - - * - * - -
accu a e mass (wi hin 5 ppm), see Table 1. P e ious
su ace- sampling MS s udies in o QS di ec ly om
bio ilms ha e ypically u ilized ei he SIMS o
MALDI, which a e mo e commonly coupled o ime-o -
ligh ins umen a ion ha does no o e he same
capabili ies. Only ecen ly has O bi ap SIMS
ins umen a ion become a ailable,41 epo ing he
de ec ion o 33 AQs and 6 AHLs in bio ilms o P.
ae uginosa cul u ed o 48 h.42 The di e ences wi h ou
esul s could a ise due o di e ences in sampling ime
poin s (24 s 48 h bio ilms), sampling o he
exome abolome a he han di ec

A icle
bio ilm analysis, and/o he lowe di usion and s abili y o
AHLs dispe sing o he bio ilms.
Ou app oach also bene i s om MSMS capabili y, allowing
s uc u al cha ac e iza ion o de ec ed molecules. Hyb id
ins umen s wi h his capabili y ha e only ecen ly been
desc ibed in he SIMS communi y.43 MSMS expe imen s
con i med m/z 244.17 is HHQ, see he Supplemen al
In o ma ion and Figu e S1. HHQ has ecen ly been shown o
ha e clinical diagnos ic ele ance in he ea ly de ec ion o
in ec ion om nonin asi e biological luids such as u ine and
b ea h condensa e in c i ically ill pa ien s wi h in ec ions
cha ac e ized by polymic obial bio ilms wi h bac e ial and
ungal con ibu o s.44 HCD agmen a ion o m/z 260.16
sugges s his is a mix u e o HQNO and PQS. Upon
dissocia ion o m/z 272.20, he ollowing p oduc ions we e
de ec ed: m/z 159.08, 172.07; howe e , he e was no agmen
a m/z 188.10 o 175.06 sugges ing ha his species is NHQ
a he han C9-PQS. This was de ec ed in ela i ely high
abundance and has p e iously been shown o be an impo an
bioma ke o lung in ec ion ia LC−MS o plasma om cys ic
ib osis pa ien s.7 This demons a es he po en ial o his
app oach as a model o he s udy o sec e ed biomolecules
ha en e he ci cula o y sys em and can in o m QS molecules
ha could be moni o ed ia nonin asi e biological samples and
a e indica i e o in ec ion.
In e species
and
In e kingdom
Exome abolome
Analysis. Using ou LESA-MS app oach, we s udied he
exome abolome o in e species bio ilms, including P. ae ugino-
sa−S. au eus (PA−SA), and in e kingdom bio ilms o P.
ae uginosa−C. albicans (PA−CA), and P. ae uginosa−S.
au eus−C. albicans (PA−SA−CA) combina ions. Colony
bio ilms
o
P.
ae uginosa
PAO1-L,
S.
au eus
SH1000,
and/o
C. albicans SC5314 mix u es we e co-cul u ed o 24 h, and he
aga subs a es we e analyzed ia LESA-MS. QS signals ha e
p e iously been epo ed in in e species in e ac ions om 24 h
bio ilms g own a a 5 mm dis ance.20 He e, we epo hem
om bio ilm colonies g own wi h no spa ial sepa a ion.
PA−SA Bio ilms. Simila AQ molecules we e obse ed in
he exome abolome o PA−SA communi ies compa ed o PA
bio ilms, howe e , ela i e abundances we e di e en , see
Figu e 2. HHQ was pa icula ly abundan in PA−SA, wi h C7-
PQS/HQNO also de ec ed in highe abundance han in PA
alone. In PA bio ilms, he mos abundan AQ de ec ed was
NHQ, ollowed by C9:1-quinolone/NHQ and hen HHQ and
HQNO/C7-PQS. Ye , in he PA−SA, HHQ was he mos
abundan , ollowed by C7-PQS/HQNO, wi h NHQ signi i-
can ly dec eased. This ag ees wi h p e ious MALDI imaging
s udies o simila polymic obial sys ems, which de ec ed HHQ
om P. ae uginosa (DK2-P2M24-2003 s ain) in he p esence
o S. au eus (JE2).20 Ou esul s also sugges ha HQNO
p oduc ion om P. ae uginosa PAO1-L inc eases in he
p esence o S. au eus SH1000. Bo h HHQ and C9-PQS ha e
been shown o ep ess S. au eus sp eading.45 In hese assays,
he e was no obse ed di e ence in he iable eco e y o bo h
mic obial species when co-incuba ed a 24 h when compa ed
o monospecies g ow h. Pyocyanin was de ec ed in ela i ely
high abundance ia LESA-MS in PA−SA, a h ee old inc ease in
compa ison o PA, see Figu e S2. Pyocyanin is ac i ely
sec e ed om PA; he e o e, i is a pa icula ly use ul
bioma ke o a ge , i is also oxic o SA, hence an inc ease
in p oduc ion is no unexpec ed. I is p omising ha ou
app oach can de e mine a signi ican change in pyocyanin.
Nume ous a emp s a modi ied pyocyanin assays ha e no
yielded meaning ul esul s, possibly owing o sensi i i y
limi a ions. Po en ially, his highligh s a bene i o ou
app oach, al hough u he alida ion is equi ed.
PA−CA Bio ilms. In he p esence o C. albicans SC5314,
HHQ was de ec ed howe e , mos AQ molecules we e eliably
supp essed (Figu e 2), sugges ing an impo an ole o HHQ
in P. ae uginosa su i al in he p esence o C. albicans o ,
con a y o SA compe i ion, a po en ial dis up ion in he
con e sion o HHQ o PQS by CA. HHQ has p e iously been
shown o a ec C. albicans bio ilm o ma ion, and i is hough
o play an essen ial ole in PA−CA in e kingdom in e -
ac ions.46 Mo eo e , CFU coun ing showed a educ ion in CA
g ow h in he p esence o PA wi h a 1−2 log di e ence in o al
cell coun s. Inhibi ion o HQNO p oduc ion sugges s po en ial
dis up ion o he PpqsL enzyma ic ac i i y in he PAO1-L
pa hway; howe e , his pa hway is no dis up ed in PA−SA.
Pyocyanin was de ec ed, al hough a lowe abundance
(∼six old dec ease compa ed o PA bio ilm yields) in he
p esence o CA. Fu u e wo k will ocus on alida ing
educ ions in AQs and pyocyanin as po en ial bioma ke s o
in ec ion caused by PA in he p esence o CA.
PA
−
SA
−
CA Bio ilms.
Despi e HHQ and HQNO molecules
being he mos abundan AQs p oduced by PA in
polymic obial bio ilms, including SA and CA (Figu e 2), a
signi ican educ ion in hese AQ yields was eco ded (wi h no
AQs de ec ed in some epea s), sugges ing compe i ion om
hese could a ec he PQS signaling sys em. Resul s indica e
ha , unde he es ed condi ions, HHQ could be a good
indica o o P. ae uginosa coloniza ion, suppo ing he po en ial
o his QS signal as a p ospec i e ea ly indica o o in ec ion by
his pa hogen e en in he con ex o polymic obial in ec ions.
In e es ingly, he AQ DHQ, con a y o single and dual-species
bio ilms including PA, was consis en ly de ec ed in he PA−
SA−CA communi y exome abolome (m/z 162.0548, Δppm
4.3). In con as o HHQ and PQS, DHQ can be p oduced
unde low oxygen condi ions,47 sugges ing he educ ion in
HHQ and PQS yields and de ec ion o DHQ could be
explained by he o ma ion o anae obic niches in PA
−
SA
−
CA
communi ies. Mo eo e , DHQ has been shown o ac as a QS
molecule o ac i a e he esponse egula o PqsR o
ansc ip ion o he pqs ope on in P. ae uginosa in he absence
o PQS and HHQ signaling and has been epo ed o alle ia e
he inhibi ion o PqsR by a nesol, albei a a lowe le el
compa ed wi h PQS.47 Cis-2-decenoic acid (CDA) was
de ec ed a m/z 188.1642 Δppm 4.8 in his iple sys em, and
no in he o he samples. Simila ly o he PA−CA sys em, a
signi ican dec ease in pyocyanin was obse ed in PA−SA− CA
compa ed o PA bio ilms, which is in ag eemen wi h a
educ ion in he PQS sys em ac i i y as i has been epo ed
ha phenazine p oduc ion in P. ae uginosa is unde he con ol
o his QS egula o y sys em.48 Fu u e wo k will ocus on
alida ing HHQ, HQNO, and DHQ p esence and hei
po en ial use, oge he wi h pyocyanin, as bioma ke s o
polymic obial in ec ions including P. ae uginosa.
Summa y. O e all, a ange o alkyl-quinolones (AQs) was
de ec ed in he exome abolome o PA only. Pa icula ly high
le els o HHQ and pyocyanin in he exome abolome we e
indica i e o PA in combina ion wi h SA causa i e agen s. Low
abundances o all AQs we e de ec ed when PA was co-cul u ed
wi h CA, alongside a signi ican dec ease in pyocyanin. When
PA was co-cul u ed in he p esence o bo h SA and CA, again
many o he AQs ha we e de ec ed in he PA-only sys em
we e supp essed, whe eas DHQ and CDA we e de ec ed o
A icle
Figu e 3. Ba cha showing he mean signal in ensi y o QS molecules de ec ed ia LESA-MS in he exome abolome analysis o ea ed PA
bio ilms. CFU measu emen s om PA, inhibi o (SEN019)- ea ed PA, and cip o loxacin d ug (CIP)- ea ed PA a e shown in he inse .
he i s ime and he e o e ep esen po en ially in o ma i e
ions alongside AHLs. When SA was co-cul u ed wi h CA,
many ions ha we e de ec ed we e simila o hose p esen in
he exome abolome o he PA−SA−CA sys em, wi h he
excep ion o CDA and DHQ. Ou indings a e summa ized in
Figu e S3.
Pla o m o Tes ing The apeu ic S a egies:
Pseudo-
monas ae uginosa QS Inhibi o s, Cip o loxacin, and
Adjunc i e S a egies. Changes in QS molecules and
i ulence ac o p oduc ion could also po en ially be used o
moni o he e ec i eness o he apeu ic in e en ions. To
s udy changes in hese biomolecules upon ea men , bio ilms
o P. ae uginosa PAO1-L we e cul u ed o 24 h in he p esence
o he an ibio ic cip o loxacin and/o he PqsR an agonis s
SEN01949 (a mode a e inhibi o ) and SEN089 (a po en
inhibi o ). These QSIs in e e e wi h he binding o AQ QS
signals o he PqsR esponse egula o leading o educed
ac i a ion o he pqs ope on exp ession and he e o e educed
biosyn hesis o AQ signals and i ulence ac o s in P.
ae uginosa. Despi e CFU coun s showing a educ ion in cell
iabili y in PAO1-L bio ilms ea ed wi h cip o loxacin (
∼
4-log
educ ion), mos AQ molecules de ec ed in he exome abo-
lome o un ea ed PAO1-L bio ilms we e ound in
communi ies ea ed wi h he an ibio ic, e.g., HHQ (m/z
244.1696), C7-PQS/HQNO (m/z 260.1642), C9:1-quinolone
(m/z 270.1849), and C9-quinolone/NHQ (m/z 272.2007).
Howe e , in his expe imen , HHQ was signi ican ly educed
in signal in ensi y ac oss biological and echnical epea s, and
C7-PQS and C9-PQS we e sligh ly ele a ed. In con as , he P.
ae uginosa i ulence ac o pyocyanin abundance was no
signi ican ly al e ed by cip o loxacin ea men , see Figu e 3.
In he p esence o QSI SEN089 (IC50 = 67 nM,46), no AQ
molecules we e de ec ed in he PAO1-L bio ilm exome abo-
lomes ac oss all expe imen al biological and echnical epea s,
see Figu e 3. This sugges s ha he p esence o his inhibi o
e ec i ely dis up s he AQ biosyn he ic pa hway. P e iously,
QS molecules p oduc ion was shown o dec ease in plank onic
cul u es in he p esence o a ious pqs inhibi o s.46,49 He e, we
show simila esul s om a sampling o ma speci ically
designed o he analysis o su ace-associa ed mic obial
communi ies. This highligh s one o he key bene i s o ou
su ace-based app oach o e al e na i e liquid-based ap-
p oaches. No ably, pyocyanin sec e ion was also dec eased in
PAO1-L bio ilms ea ed wi h his QSI, sugges ing he
supp ession o phzAB exp ession in line wi h p e ious
pheno ypic analysis done wi h hese QS an agonis s.46,49
When PAO1-L bio ilms we e ea ed wi h he less po en
QSI SEN19 (IC50 = 1 μM,49), AQ molecules could s ill be
de ec ed in he bio ilm exo-me abolome albei a a educed
yield. Simila ly, o un ea ed bio ilms, HHQ emained he mos
abundan AQ ollowed by NHQ, see Figu e 3. In addi ion,
pyocyanin displayed a sec e ion dec ease a e SEN19
ea men (Figu e S3), again indica ing supp ession o phzAB
exp ession. As expec ed om ea men wi h an i i ulence
compounds, CFU expe imen s showed no educ ion in iable
coun s in he p esence o bo h QSI inhibi o s compa ed o
un ea ed PAO1-L bio ilms. CDA and DHQ we e de ec ed
when PA was ea ed wi h he less po en inhibi o (SEN019),
howe e , hese we e no de ec ed upon ea men wi h a mo e
po en inhibi o (SEN089).
In e es ingly, bio ilms cul u ed in he p esence o SEN019
inhibi o and cip o loxacin led o no de ec ion o he common
AQs p esen in PAO1-L exome abolome. Mo eo e , unde
combina o y ea men , no sec e ed pyocyanin could be
de ec ed, sugges ing a global dis up ion o AQ-based QS
ne wo ks and he po en ial o LESA-MS-based app oaches o
s udying mechanisms o d ug ac ion o new an i- i ulence
ea men s and adjunc i e he apies. In con as , CFU coun s
showed a simila dec ease in bio ilm cell iabili y (∼4-log
educ ion) compa ed o bio ilms ea ed wi h cip o loxacin
only. These esul s indica e ha he combina ion o hese wo
d ugs could con ibu e o a educ ion in P. ae uginosa i ulence
A icle
bu no o a u he educ ion in cell iabili y o ea ed
bio ilms unde he condi ions es ed.
CONCLUSIONS
This s udy demons a es ha he LESA-MS sampling o ma
desc ibed he e p o ides a sui able model o he analysis o he
mic obial exome abolome; wi h a ange o clinically ele an
me aboli es de ec ed in he s udied mic obial communi ies,
including in e species and in e kingdom sys ems. Ou
app oach has he added bene i o allowing mic obiological
cell cul u e in a labo a o y wi h he app op ia e sa e y measu es
o he speci ic pa hogen, wi hou he need o a dedica ed
ins umen in he same lab. Fu he mo e, di used o sec e ed
compounds can be analyzed using ou app oach. HHQ has
p e iously been indica ed as an ea ly diagnos ic ma ke o
in ec ion in P. ae uginosa; ou s udy ag ees and he e o e shows
p omise as a model. Addi ionally, ou esul s sugges ha
me hods o moni o ing le els o AQs such as DHQ and
pyocyanin could be bene icial in he de e mina ion o causa i e
agen s in in e kingdom in ec ion. Fu he in es iga ion is
equi ed o alida ion; quan i a i e analysis o le els in
ci cula o y bio luids ia, e.g., LC−MS would be bene icial.
Finally, we show how he LESA pla o m could p o ide a ou e
o assessing he e ec o d ug ea men s and adjunc i e
he apies o he u u e s udy o app op ia e ea men s o
bac e ial, in e bac e ial, and in e kingdom sys ems using a
bac e ia- ee pla o m.
We en ision ha ou model will ind use in apidly
in es iga ing bac e ial exome abolome analy es such as QS
molecules om bac e ial and in e kingdom sys ems ha will
aid p edic ion o clinically ele an diagnos ic me aboli es o
in ec ion in bac e ial, ungal, and in e species/in e kingdom
sys ems ha mimic he ue complexi y o ch onic in ec ion.
This has ele ance in he in es iga ion and subsequen
p edic ion o clinically ele an bioma ke s ha can be
moni o ed nonin asi ely om biological luids. A a ie y o
in e species and in e kingdom sys ems could be s udied in he
u u e using his pla o m.
ASSOCIATED
CONTENT
*
Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acs.analchem.2c05703.
Expanded Expe imen al and Resul s sec ions, and
Supplemen al Figu es 1−3 (PDF)
AUTHOR
INFORMATION
Co esponding
Au ho s
Manuel
Rome o
−
U.K.
Na ional
Bio ilm
Inno a ion
Cen e
(NBIC), Biodisco e y Ins i u e, School o Li e Sciences,
Facul y o Heal h and Medical Sciences, Uni e si y o
No ingham, NG7 2RD No ingham, U.K.; Depa men o
Mic obiology and Pa asi ology, Facul y o Biology-CIBUS,
Uni e sidade de San iago de Compos ela, 15782 San iago de
Compos ela,
Spain;
Email:
[email p o ec ed]
Rian
L.
G i i hs
−
Facul y
o
Science,
School
o
Pha macy,
Uni e si y o No ingham, NG7 2RD No ingham, U.K.;
o cid.o g/0000-0002-1601-4664; Email: ian.g i i hs@
no ingham.ac.uk
Au ho s
Shaun N. Robe son
−
U.K. Na ional Bio ilm Inno a ion
Cen e (NBIC), Biodisco e y Ins i u e, School o Li e Sciences,
Facul y o Heal h and Medical Sciences, Uni e si y o
No ingham, NG7 2RD No ingham, U.K.
Fadi
Souka ieh
−
U.K.
Na ional
Bio ilm
Inno a ion
Cen e
(NBIC), Biodisco e y Ins i u e, School o Li e Sciences,
Facul y o Heal h and Medical Sciences, Uni e si y o
No ingham, NG7 2RD No ingham, U.K.; o cid.o g/
0000-0002-6730-2543
Thomas M.
Whi e
−
Facul y o Science, School o Pha macy,
Uni e si y o No ingham, NG7 2RD No ingham, U.K.
Miguel
Cama a
−
U.K.
Na ional
Bio ilm
Inno a ion
Cen e
(NBIC), Biodisco e y Ins i u e, School o Li e Sciences,
Facul y o Heal h and Medical Sciences, Uni e si y o
No ingham, NG7 2RD No ingham, U.K.
No es
The au ho s decla e no compe ing inancial in e es .
ACKNOWLEDGMENTS
R.L.G. would like o acknowledge suppo om a Uni e si y o
No ingham unded Anne McLa en Fellowship. This expe -
imen al wo k was unded ia a B i ish Mass Spec ome y
Socie y (BMSS) Resea ch Suppo G an . T.M.W. would like
o acknowledge unding om he Uni e si y o No ingham o
a summe s uden ship. M.R., S.N.R., F.S., and M.C. we e
suppo ed by he Na ional Bio ilms Inno a ion Cen e
[BBSRC, BB/R012415/1]. MR is suppo ed by he Ma ia
Zamb ano p og am om he Spanish Minis y o Uni e si ies.
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