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Scien i ic RepoR s | 6:29677 | DOI: 10.1038/s ep29677
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The bio ilm inhibi o Ca olac on
inhibi s plank onic g ow h o
i ulen pneumococci ia a
conse ed a ge
Jannik Donne 1, Michael Reck1, Simone Be gmann2, And eas Ki schning3, Rol Mülle 4 &
I ene Wagne -Döble 1
New an ibac e ial compounds, p e e en ially exploi ing no el cellula a ge s, a e u gen ly needed o
igh he inc easing esis ance o pa hogens agains con en ional an ibio ics. He e we demons a e ha
Ca olac on, a myxobac e ial seconda y me aboli e p e iously shown o damage S ep ococcus mu ans
bio ilms, inhibi s plank onic g ow h o S ep ococcus pneumoniae TIGR4 and mul id ug- esis an clinical
isola es o se o ype 19A a nanomola concen a ions. A Ca olac on dias e eome is inac i e in bo h
s ep ococci, indica ing a highly speci ic in e ac ion wi h a conse ed cellula a ge . S. mu ans equi es
he euka yo ic-like se ine/ h eonine p o ein kinase PknB and he cys eine me abolism egula o CysR
o suscep ibili y o Ca olac on, whe eas hei homologues a e no needed in S. pneumoniae, sugges ing
a speci ic unc ion o S. mu ans bio ilms only. A bac e icidal e ec o Ca olac on was obse ed o
S. pneumoniae TIGR4, wi h a educ ion o cell numbe s by 3 log uni s. The clinical pneumonia isola e
Sp49 showed immedia e g ow h a es and cell lysis, sugges ing a bac e ioly ic e ec o Ca olac on.
Ca olac on ea men caused a educ ion in memb ane po en ial, bu no memb ane in eg i y, and
ansc ip ome analysis e ealed compensa o y eac ions o he cell. Ou da a show ha Ca olac on
migh ha e po en ial o ea ing pneumococcal in ec ions.
The esis ance o pa hogens agains an ibio ics is inc easing wo ldwide1, while he numbe o new an ibio ics in
he clinical pipeline emains o be low2. As an ibio ic ea men o en ails agains pa hogens ha ha e de eloped
esis ance, i dis u bs he human mic obiome a he same ime3, which has become a majo conce n. New an i-
bac e ial compounds a e u gen ly needed, p e e ably compounds ha ac on no el o unde exploi ed cellula
mechanisms and could be used as a s a ing poin o a ional d ug design4.
Ca olac on is a seconda y me aboli e p oduced by he myxobac e ium So angium cellulosum. I is a mac-
olide ke oca bonic acid5 ha can be syn hesised de no o6,7. Ca olac on was disco e ed o cause memb ane dam-
age o bio ilms o he ca ies pa hogen S ep ococcus mu ans a nanomola concen a ions and is non- oxic o
euka yo ic cells8. Den al composi e ma e ials inco po a ing Ca olac on main ain a s ong in i o ac i i y agains
S. mu ans bio ilms while he mechanical p ope ies o he ma e ial a e no a ec ed9, sugges ing ha such compos-
i es migh be applicable o educe seconda y ca ies o ma ion. Ca olac on has only a weak e ec on he g ow h o
S. mu ans10, bu inhibi s g ow h o S ep ococcus o alis11 and also o Esche ichia coli TolC wi h a minimal inhibi-
o y concen a ion (MIC) as low as 0.06 μ g/m l 5. Ca olac on- ea ed cul u es show inc eased sep um o ma ion,
elonga ed cells, and cell chains, sugges ing de ec s in cell di ision o cell wall syn hesis8,10–12. All chemical modi i-
ca ions o Ca olac on es ed un il now ende ed he molecule inac i e11. Based on hese s udies, we hypo hesised
ha he p ima y e ec o Ca olac on is a dis u bance o cell di ision o cell wall syn hesis h ough a s e eospeci ic
in e ac ion wi h a conse ed cellula a ge . Mo eo e , we hypo hesised ha he dea h o S. mu ans bio ilm cells,
1Depa men o Medical Mic obiology, G oup Mic obial Communica ion, Helmhol z Cen e o In ec ion Resea ch,
B aunschweig, Ge many. 2Ins i u e o Mic obiology, Technische Uni e si ä B aunschweig, B aunschweig, Ge many.
3Ins i u e o O ganic Chemis y and Cen e o Biomolecula D ug Resea ch (BMWZ), Leibniz Uni e si ä Hanno e ,
Hanno e , Ge many. 4Depa men o Mic obial Na u al P oduc s, Helmhol z Ins i u e o Pha maceu ical Resea ch
Saa land (HIPS), Helmhol z Cen e o In ec ion Resea ch and Pha maceu ical Bio echnology, Saa land Uni e si y,
Saa b ücken, Ge many. Co espondence and eques s o ma e ials should be add essed o J.D. (email: jannik.
donne @helmhol z-hzi.de)
Recei ed: 24 Ma ch 2016
accep ed: 21 June 2016
Published: 11 July 2016
OPEN
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Scien i ic RepoR s | 6:29677 | DOI: 10.1038/s ep29677
which occu s se e al hou s a e Ca olac on ea men a low pH only10, is a seconda y e ec ha migh be unique
o his species due o he s ong acidi ica ion o mu ans s ep ococci wi hin ma u e bio ilms13.
To in es iga e hese hypo heses and o unde s and he mode o ac ion o Ca olac on in mo e de ail, we s ud-
ied he esponse o S. pneumoniae o Ca olac on and compa ed i wi h ha o S. mu ans. S. pneumoniae is a
ubiqui ous colonize o he nasopha ynx14 whose ca ie s usually emain asymp oma ic. Ne e heless, in asi e
and non-in asi e pneumococcal in ec ions accoun o almos hal a million hospi aliza ions and app oxima ely
22.000 dea hs in he Uni ed S a es annually15. A con inuous inc ease in mul id ug esis ance (MDR, esis an o
≥ 3 classes o an imic obials) is being epo ed16. Pa icula ly he p e alence o S. pneumoniae s ains o se o ype
19A has inc eased d as ically om 5% in he mid-1990’s o up o 48% in 200817. Se o ype 19A isola es ha e he
highes p opo ion o MDR and ex ensi ely d ug esis an s ains (XDR, esis an o ≥ 5 classes o an imic obi-
als)18. Consequen ly, he sea ch o no el an imic obial compounds and no el cellula a ge s emains o c ucial
impo ance, especially wi h ega d o pneumococci.
In his s udy, we ocused on S. pneumoniae TIGR4 as an ex ensi ely cha ac e ized s ain19. Fo he i s ime,
we epo comple e g ow h inhibi ion o plank onically g owing cells. We in es iga ed g ow h inhibi ion by
Ca olac on o MDR and XDR clinical isola es o S. pneumoniae classi ied as se o ype 19A. To cla i y i S. pneumo-
niae sha es he molecula a ge o Ca olac on wi h S. mu ans, we es ed i s sensi i i y agains 9-epi-Ca olac on.
This Ca olac on de i a i e has he opposi e absolu e con igu a ion a C-9, he a ea o mac olac one o ma ion,
and is inac i e agains S. mu ans11. Cell dea h o S. mu ans bio ilm cells has been shown o be dependen on he
euka yo ic-like se ine/ h eonine p o ein kinase (eSTK) PknB10 and he egula o o cys eine me abolism CysR20.
The e o e, hei homologues in S. pneumoniae TIGR4 we e dele ed and hei esponse o Ca olac on de e mined.
Finally we in es iga ed he ansc ip ome, including small egula o y RNAs, o Ca olac on- ea ed cul u es o
S. pneumoniae TIGR4, cons uc ed knock-ou mu an s o s ongly di e en ially exp essed genes and es ed
hei suscep ibili y. Ou da a demons a e ha Ca olac on ac s ia he same molecula a ge in S. mu ans and
S. pneumoniae. Dea h o bio ilm cells o S. mu ans is a la e seconda y e ec . The as (wi hin 5 min) and dis inc
ansc ip ional esponse o Ca olac on ea men ep esen s a compensa o y mechanism, since dele ion o hose
genes does no ende he mu an s insensi i e o Ca olac on. Finally, he inhibi ion o g ow h o S. pneumoniae by
Ca olac on shows he cha ac e is ics o bac e icidal and in some cases e en bac e ioly ic an ibio ics.
Resul s
Ca olac on inhibi s g ow h o S. pneumoniae TIGR4 in plank onic cul u e. Ca olac on (0.25 μ g/ml
inal concen a ion) was added o cul u es o S. pneumoniae TIGR4 p io o he onse o he exponen ial phase
(OD600 = 0.15, e med “ 0”). Ca olac on- ea ed cul u es g ew a he same a e as un ea ed con ols o abou 2 h,
a e which he OD emained cons an o dec eased (Fig.1A), sugges ing a bac e icidal ac i i y21. The Ca olac on-
ea ed cul u es ypically eached a maximal OD600 o 0.55–0.60, while he con ols g ew up o an OD600 o 1.8. A
maximal inhibi ion o ~67% could be obse ed a e 9 h o g ow h. Since S. pneumoniae au olyzes a e eaching
he s a iona y phase22, inal cell densi ies a e 24 h we e e y low and almos he same in ea ed and un ea ed
cul u es. The numbe o iable cells was de e mined by coun ing colony o ming uni s (CFUs). Figu e1B shows
ha a e 3 h o Ca olac on ea men , he numbe o CFUs was educed by 1 log uni (92.4%) and dec eased u -
he by mo e han 2 log uni s (99.4%) a e 6 h. Since his dec ease in CFUs was no accompanied by a educ ion
in OD600, he da a indica e educed cell iabili y in he p esence o Ca olac on. Fo S. pneumoniae TIGR4, he
minimal inhibi o y concen a ion (MIC) o Ca olac on was 0.06 μ g/ml (Table1). Cells g own wi h Ca olac on
showed a mo e oluminous pheno ype and a ac ion o he cells showed a mode a e bu signi ican inc ease in
size (Fig.1C). On a e age, cell leng h inc eased om 1.62 ± 0.4 μ m o 2.08 ± 0.6 μ m, while cell wid h inc eased
om 0.87 ± 0.12 μ m o 0.94 ± 0.11 μ m. E en a 100- old inc ease (0.025–2.5 μ g/ml) o he Ca olac on concen a-
ion did no inc ease he inhibi ion o g ow h conside ably (Supplemen a y Fig. S1). This is in ull acco dance
wi h Kunze e al. who epo ed a concen a ion-independen e ec o Ca olac on on dea h o S. mu ans bio ilms
o e he same concen a ion ange8.
Ca olac on inhibi s plank onic g ow h o S. pneumoniae se o ype 19A clinical isola es. To
in es iga e he e ec i eness o Ca olac on ea men on S. pneumoniae s ains ound in pneumococcal disease,
h ee di e en ecen clinical isola es o S. pneumoniae, ep esen ing d ug esis an se o ype 19A s ains (Table2,
Supplemen a y Table S1), we e subjec ed o Ca olac on suscep ibili y es ing. Isola e Sp49 had been ob ained
om he blood o a sep ic pneumonia pa ien and was esis an agains wo classes o an ibio ics. Isola e Sp61 and
Sp64 we e XDR ( esis an o ≥ 5 classes o an ibio ics); hey we e isola ed om he ho ax d ainage o a pleu i is
pa ien and by swab om an ea canal o a non-symp oma ic pa ien , espec i ely. The g ow h o all h ee clinical
isola es was inhibi ed by Ca olac on, bu o a widely a ying ex en (Fig.2). The clinical pneumonia isola e (Sp49)
was mos sensi i e and showed a di e en beha iou om TIGR4: i s a ed o die immedia ely and a e 2 h i s
OD was lowe han a 0, sugges ing a bac e ioly ic e ec o Ca olac on (Fig.2A). A maximum inhibi ion o 94.9%
could be obse ed a e app oxima ely 6–7 h o g ow h, his was he same ime poin a which he con ol cul u e
o Sp49 ypically eached i s peak (OD600 ~1.2). Fo he clinical pleu i is isola e (Sp61) and he non-in asi e
mid-ea se o ype 19A isola e (Sp64), he pe iod o uninhibi ed g ow h was longe han o he TIGR4 s ain, and
hey eached highe inal cul u e densi ies han TIGR4. The pleu i is isola e Sp61 showed a maximal inhibi ion
o g ow h o abou 45% a e 6 h (Fig.2B). Fo he non-in asi e mid-ea isola e Sp64, he maximal inhibi ion was
less han ha de ec ed o he pleu i ic isola e, eaching a maximum inhibi ion o 23%. Mo eo e , he inhibi ion
peak was e a ded and obse ed a e 8 h (Fig.2C). Ca olac on had a s ong ly ic e ec on clinical isola e Sp49; he
MIC alue de e mined o his isola e was ≤ 0.03 μ g/ml and hus lowe han o TIGR4. D ug- esis an isola es
Sp61 and Sp64, in con as , we e highly ole an o Ca olac on and we e only inhibi ed a 64 μ g/ml Ca olac on
(Table1). These esul s indica e ha Ca olac on inhibi s g ow h o clinical S. pneumoniae isola es wi h a ying
e iciency. The deg ee o inhibi ion was no a ec ed by he change o se o ype om TIGR4 (se o ype 4) o he
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Scien i ic RepoR s | 6:29677 | DOI: 10.1038/s ep29677
clinical isola es (se o ype 19A). In e es ingly, g ow h inhibi ion was weake in he pleu i is isola e Sp61 and in
he non-in asi e isola e Sp64, which elici mul iple esis ances o an ibio ics compa ed o he pneumonia isola e.
The in e ac ion o Ca olac on wi h i s cellula a ge is highly s e eo-speci ic. The Ca olac on
de i a i e 9(R)-Ca olac on (epi-Ca olac on) is a dias e eome o Ca olac on exhibi ing an in e sion a C–9
[(S)→ (R)] (Fig.3A). Al hough he s uc u al changes a e minu e, epi-Ca olac on is biologically inac i e agains
S. mu ans in bio ilms11. Likewise, 0.25 μ g/ml epi-Ca olac on, a concen a ion which is gene ally applied in ou
g ow h expe imen s wi h s ep ococci, did no inhibi g ow h o S. pneumoniae TIGR4 (Fig.3B). Thus he in e -
ac ion be ween Ca olac on and i s molecula a ge mus be highly speci ic wi h espec o he s e eochemis y
Figu e 1. T ea men wi h Ca olac on inhibi s g ow h o S. pneumoniae TIGR4 and a ec s cell coun and
cell size. (A) The g ow h cu e indica es a g ow h inhibi ion o plank onically g owing cells o S. pneumoniae
TIGR4 by Ca olac on a a inal concen a ion o 0.25 μ g/ml. The s a OD600 is indica ed by a solid ho izon al
line. Depic ed is he a e age inhibi ion om h ee independen expe imen s (± s anda d de ia ion).
(B) De e mina ion o iable cell numbe s by pla ing o colony coun s. The cell numbe was de e mined
a e pla ing se ial dilu ions and coun ing he CFU. (+ ) 0.25 μ g/ml Ca olac on and (− ) con ol. Da a a e
ep esen a i e o a leas h ee biologically independen expe imen s (± s.d.). P alue: * * * = < 0.001; Mann-
Whi ney U. (C) Cell leng h dis ibu ion o cells g own wi h 0.25 μ g/ml Ca olac on o 3 h. A leng h o ≥ 3 μ m
was only obse ed o ea ed cells ( ed box). In he mic oscope image, elonga ed cells a e indica ed by a ows,
a cell showing a bulky and oluminous mo phology is ma ked by an as e isk (* ). The a e age cell leng h (L)
and wid h (W) wi h s anda d de ia ions a e shown o n cells om h ee independen expe imen s. Scale ba :
2.5 μ m. Changes in L and W we e s a is ically signi ican o P < 0.05 as de e mined by non-pa ame ic Mann-
Whi ney-U es ; P(L) < 0.001, P(W) = 0.011.
S ain Ca olac on (μg/ml)
TIGR4 0.06
Sp49 (NRZ:3198/36486) ≤ 0.03
Sp61 (NRZ:3364/39533) 64
Sp64 (NRZ:3066/35051) 64
Table 1. Minimal inhibi o y concen a ion (MIC) o Ca olac on agains plank onically g owing
S. pneumoniae s ains.
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a posi ion 9 o he molecule, hea ily in luencing he opology o he mac ocycle. Due o he s e eospeci ici y o
his in e ac ion we sugges ha he cellula a ge o Ca olac on is likely o be iden ical be ween S. mu ans and
S. pneumoniae, in spi e o he di e ences o hei physiological esponses.
The se ine/ h eonine p o ein kinase S kP and he egula o CysR a e no equi ed o g ow h
inhibi ion by Ca olac on. Gene dele ion o he signalling kinase PknB induces Ca olac on esis ance
o S. mu ans bio ilms10. The S. pneumoniae genome encodes s kP (sp_1732) as co esponding homologue o
S. mu ans pknB. The e o e, he ole o he homologue o PknB in S. pneumoniae, S kP, in Ca olac on suscep ibili y
o S. pneumoniae TIGR4 was es ed. Since he molecula a ge o Ca olac on appea s o be highly conse ed in
hose wo o ganisms, his analysis will cla i y whe he a bac e ial euka yo ic-like se ine/ h eonine p o ein kinase
(eSTK) is he di ec molecula a ge o Ca olac on. Due o he s ong indica ions o eSTKs as he p ima y a ge
o Ca olac on10, in addi ion o PCR ampli ica ion and DNA-sequencing, he absence o he S kP p o ein in he
Δ s kP isogenic mu an was con i med by immunoblo ing using polyclonal an i-S kP an ibodies23 (Fig.4A). The
S kP dele ion s ain showed o ma ion o elonga ed cells and mul iple uncons ic ed sep a, as epo ed p e i-
ously23. I s g ow h was highly impai ed; ne e heless, Ca olac on ea men led o g ow h inhibi ion (Fig.4B).
The da a show ha he Δ s kP TIGR4 s ain is suscep ible o Ca olac on, which is in sha p con as o he indings
desc ibed o S. mu ans10.
Dele ion o he cys eine me abolism egula o CysR also esul ed in a Ca olac on-insensi i e mu an
in S. mu ans20. The CysR homologue (SP_0927) was dele ed in S. pneumoniae and es ed o suscep ibili y o
Ca olac on. Simila o he Δ s kP s ain, g ow h o he Δ cysR mu an was s ongly inhibi ed by Ca olac on
(Fig.4C). Thus, he pneumococcal homologues o bo h p o eins media ing he suscep ibili y o Ca olac on in
S. mu ans, S kP and CysR, a e mos likely no di ec ly in ol ed in he Ca olac on esponse in S. pneumoniae, in
spi e o he p esumably conse ed molecula a ge o Ca olac on in hose wo species. We conclude ha PknB
and CysR a e in ol ed in he mechanisms o memb ane damage a low pH in S. mu ans, which is a la e seconda y
esponse o Ca olac on ea men . In o de o con i m his hypo hesis, he e ec o Ca olac on on memb ane
in eg i y and memb ane po en ial o S. pneumoniae TIGR4 was analysed.
E ec o Ca olac on on cy oplasmic memb ane in eg i y and memb ane po en ial in S. pneumoniae.
Fo S. mu ans i has been epo ed ha Ca olac on leads o dea h o bio ilm cells a low pH by dis u bing he
in eg i y o he plasma memb ane and he eby causing leakage o cy oplasmic p o eins and DNA10. Memb ane
damage o S. pneumoniae cells was simila ly de e mined by LIVE/DEAD s aining du ing exponen ial and s a-
iona y phase, 3 h and 6 h a e addi ion o Ca olac on, espec i ely. Hea -killed S. pneumoniae cells we e included
as a con ol and a calib a ion cu e was eco ded using low cy ome y (Supplemen a y Fig. S2). No indica-
ion o S. pneumoniae cell dea h by Ca olac on ea men was de ec ed (Supplemen a y Fig. S3). Con ols and
Ca olac on- ea ed cul u es bo h con ained a maximum o 14% cells wi h de ec i e plasma memb anes (Fig.5A).
We hen used s aining wi h he luo escen dye 3,3-Die hyloxaca bocyanine iodide (DiOC2(3)) o de e mine
changes in he MP upon Ca olac on ea men . Since he measu ed luo escence in ensi ies a e di ec ly in luenced
by he size o he s ained pa icles, and Ca olac on ea men a ec s he cell size, a size-independen a iome ic
echnique was applied24. Ca olac on ea men caused a ime-dependen depola iza ion o he memb ane po en-
ial (Fig.5B, Supplemen a y Fig. S4). App oxima ely 48% o he cells showed a memb ane depola iza ion, indi-
ca ed by a educ ion o he ed/g een luo escen a io, a e 3 h. Memb ane depola iza ion con inued du ing he
nex 3 h, hough no as as as du ing he i s ime in e al. A e 6 h, he ed/g een luo escen a io d opped o
~34% o ha measu ed o an exponen ially g owing con ol a e 3 h, and was educed o ~56% when compa ed
o he con ol a he same ime poin . These esul s indica e a Ca olac on-dependen dis u bance o he memb ane
po en ial du ing g ow h ha is no he esul o a po ous plasma memb ane.
S ain/plasmid Rele an geno ype/se o ype Desc ip ion Re e ence
S ep ococcus pneumoniae
TIGR4 (ATCC BAA-334) encapsula ed se o ype 4 wild ype 19
JD01 TIGR4 sp_1732::e mRΔ s kP s ain This wo k
JD02 TIGR4 sp_0927::e mRΔ cysR s ain This wo k
JD03 TIGR4 sp_0119::e mRΔ sp_0119 s ain This wo k
JD04 TIGR4 sp_0120::e mRΔ gidA s ain This wo k
JD05 TIGR4 sp_0119-sp_0120::e mRΔ sp_0119/Δ gidA s ain This wo k
Clinical isola es
Sp49 se o ype 19A pneumonia/sepsis isola e M. an de Linden,
NRZ Aachen
Sp61 se o ype 19A pleu i is isola e M. an de Linden,
NRZ Aachen
Sp64 se o ype 19A mid-ea isola e M. an de Linden,
NRZ Aachen
Plasmid
pALN122 e mR58
Table 2. Bac e ial s ains, clinical isola es and plasmids used in his s udy.
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Scien i ic RepoR s | 6:29677 | DOI: 10.1038/s ep29677
T ansc ip ional esponse o S. pneumoniae TIGR4 o Ca olac on. We analysed he ansc ip ome
o S. pneumoniae TIGR4 cells g owing wi h Ca olac on s a ing 5 min a e addi ion o he d ug, un il 3 h a e
Ca olac on addi ion. To al RNA was ac iona ed in o wo size classes: > 200 n and ≤ 200 n , and di e en ial
egula ion o mRNAs (30–4776 n ), small RNAs (37–486 n ) and RNAs (71–90 n ) was assessed indi idually. We
no ed a high numbe o eads o he p e iously iden i ied housekeeping sRNAs 6S RNA (194 n )25 and RNase
P (391 n )/ mRNA (348 n )26,27 in he samples con aining small ansc ip s (≤ 200 n ) o in he samples con ain-
ing la ge ansc ip s (> 200 n ), espec i ely, con i ming he applicabili y o ou sequencing app oach o eliable
sepa a ion o ansc ip s acco ding o leng h (Supplemen a y Da ase 1). Since di e en ial ansc ip ion a e
Ca olac on ea men was exclusi ely limi ed o he la e ime poin s ( 120/ 180), we chose o only sequence h ee
ime poin s ( 0, 5 and 180) o analysis o sho small RNAs/ RNAs. The RNA-seq da a o biological eplica es
showed a e y high Pea son co ela ion be ween 0.93 o small egula o y RNAs/ RNAs and 0.99 o la ge egu-
la o y RNAs/mRNAs.
None o he 58 RNAs o S. pneumoniae TIGR4 we e signi ican ly di e en ially exp essed a e Ca olac on
ea men (Supplemen a y Fig. S5, Supplemen a y Da ase 1).
Figu e 2. Ac i i y o Ca olac on agains S. pneumoniae clinical isola es o se o ype 19A. G ow h inhibi ion
o pneumococcal clinical isola es Sp49 (pneumonia/sepsis isola e, (A)), Sp61 (pleu i is isola e, (B)) and Sp64
(mid ea isola e, (C)) in THBY. Ca olac on was added a a inal concen a ion o 0.25 μ g/ml p io o he onse
o exponen ial g ow h a an OD600 o 0.15. The s a OD600 is indica ed by a solid ho izon al line. The g ow h
cu es show he a e age inhibi ion calcula ed om h ee independen expe imen s (± s.d.).

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Scien i ic RepoR s | 6:29677 | DOI: 10.1038/s ep29677
Di e en ial ansc ip ion o small egula o y RNAs. A o al numbe o 181 known small sRNAs
(≤ 200 n ) and 21 known long sRNAs (> 200 n ) we e analysed. T ansc ip ion o he as majo i y o sRNAs was
con i med by ob aining high ead coun s a e mapping (Supplemen a y Da ase 1). Se en small sRNAs we e
di e en ially ansc ibed a 180 only (Supplemen a y Fig. S5). The highes up egula ed small sRNA n0057
(log2FC~3.9) is loca ed downs eam o he sp_0119-sp_0120 ope on and i s unc ion is unknown. Ten long sRNAs
we e di e en ially egula ed a 120 and 180 (Supplemen a y Fig. S5) o which only wo (R12 and F20) ha e been
in es iga ed and we e shown o be impo an o i ulence in mice28.
Di e en ial ansc ip ion o mRNAs. RNA-seq da a was analysed o di e en ial exp ession o 1985 la ge
(> 200 n ) and 120 small (≤ 200 n ) ansc ip s o S. pneumoniae TIGR4, accoun ing o 2105 p o ein-coding genes
(Supplemen a y Da ase 2). Ca olac on caused a maximum o di e en ial gene exp ession a e 120 and 180 min.
O e all, only 1.9% o he 2105 p o ein encoding genes we e di e en ially ansc ibed (log2FC ≥ ± 2, FDR < 0.01)
(Supplemen a y Fig. S6).
Two ansc ip s smalle han 200 n we e di e en ially egula ed, a ype II DNA modi ica ion me hyl ans-
e ase (sp_0569) and he 50S ibosomal p o ein L28 (sp_0441) (Supplemen a y Fig. S5). 39 ansc ip s la ge han
200 n we e s ongly di e en ially exp essed (log2FC ≥ ± 2 and FDR < 0.01 o a leas one ime poin ) (Fig.6), 26
o which a e encoded wi hin 7 ope ons on he TIGR4 ch omosome (Table3). Di e en ial exp ession o sp_0119
and sp_0120 was de ec ed al eady 5 min a e addi ion o Ca olac on and con inued o inc ease du ing he expe -
imen o a inal log2FC o 4.4 and 4.3 a e 180 min, espec i ely. Sp_0119 codes o a Mu T/Nudix amily p o ein
and sp_0120 encodes he glucose-inhibi ed di ision p o ein A (GidA).
In luence o sp_0119 and sp_0120 on suscep ibili y o S. pneumoniae TIGR4 o Ca olac on.
Since he gene p oduc s o bo h sp_0119 and sp_0120 a e in ol ed in cen al cellula p ocesses we wonde ed
whe he an uncon olled ansc ip ional up egula ion o his ope on could cause he g ow h inhibi o y e ec o
Ca olac on. The e o e, single dele ion mu an s o sp_0119 and sp_0120 and a Δ sp_0119-sp_0120 double mu an
we e cons uc ed in S. pneumoniae TIGR4. G ow h o all h ee mu an s ains was s ill signi ican ly inhibi ed by
Ca olac on ea men (Supplemen a y Fig. S7).
Figu e 3. E ec o epi-Ca olac on on g ow h o S. pneumoniae TIGR4. (A) Chemical s uc u es o na i e
9(S)-Ca olac on ( op) and he 9(R)-epime (epi-Ca olac on, bo om) ob ained by o al syn hesis. (B) Cells we e
ea ed wi h epi-Ca olac on (black do s) a a inal concen a ion o 0.25 μ g/ml a an OD600 o 0.15. As con ol,
pneumococci we e cul i a ed wi h an equi alen olume o sol en (DMSO, whi e do s). The expe imen was
epea ed independen ly a leas h ee imes.
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Compa ison o he ansc ip ome o S. mu ans UA159 and S. pneumoniae TIGR4 in esponse
o Ca olac on. Un il now, he only o he o ganism o which he in luence o Ca olac on has been s udied
in mo e de ail is he human o al pa hogen S. mu ans. The e o e, he RNA-seq da a ob ained in his s udy we e
compa ed o mic oa ay da a p e iously ob ained o Ca olac on- ea ed S. mu ans bio ilm cells10. 22.8% o all
genes we e di e en ially ansc ibed a 180 min in S. pneumoniae a a log2FC o ≥ ± 0.8 (Supplemen a y Fig. S6),
which had p e iously been used o S. mu ans10. A his cu -o , he e ec o Ca olac on was highly pleio opic.
Supplemen a y Fig. S8 shows a di ec compa ison be ween log2FC o he mos s ongly di e en ially exp essed
genes in S. mu ans compa ed o hei homologues in S. pneumoniae. O e all, he pa e ns o ansc ip ional
changes we e simila o some, bu no all genes, and he iming and ex en o he esponses shows di e ences
(Supplemen a y Fig. S8). Fo example, up egula ion o he ComDE wo-componen sys em (TCS) o gene ic
compe ence is obse ed in bo h species, bu s onge and ea lie in S. pneumoniae, and migh ep esen a gen-
e al s ess esponse29. The mos conspicuous ansc ip ional esponse in S. mu ans was he ins an aneous s ong
up egula ion o genes o py imidine biosyn hesis. In S. pneumoniae, ansc ip ion o hese genes showed a di e -
en pa e n, indica ing ha hei egula ion in S. mu ans migh be ela ed o he memb ane damage occu ing a
la e s ages o bio ilm g ow h. In S. mu ans, down egula ion o so ase A was obse ed. In S. pneumoniae TIGR4,
nei he so ase A (s A, sp_1218) no i s h ee so ase pa alogs (s BCD, sp_0466-sp_0468)30 we e di e en ially
ansc ibed. Also in con as o S. mu ans, he VicKRX TCS was no di e en ially exp essed upon Ca olac on
Figu e 4. G ow h inhibi ion o S. pneumoniae TIGR4Δs kP and TIGR4ΔcysR by ea men wi h
Ca olac on. (A) Immunoblo analysis wi h S kP-speci ic an ibodies o S. pneumoniae TIGR4 and TIGR4Δ
s kP con i ms absence o S kP-exp ession in he TIGR4Δ s kP dele ion mu an . Pneumococcal α -enolase was
de ec ed wi h speci ic an ibodies as loading con ol. P esen ed a e wo pa s o he same blo ea ed wi h
di e en p ima y an ibodies. (B) G ow h inhibi ion o a TIGR4Δ s kP mu an (solid lines) is plo ed a e
addi ion o 0.25 μ g/ml Ca olac on (black do s) and wi hou Ca olac on as con ol (whi e do s). (C) Inhibi ion
o a plank onically g owing TIGR4Δ cysR mu an wi h (black do s) and wi hou (whi e do s) Ca olac on.
Inhibi ion o he TIGR4 wild ype s ain (do ed lines), used as con ols du ing bo h assays (Ca olac on-
ea ed = black iangles, con ol = whi e iangles), is displayed as a semi- anspa en plo in (A) and (B). All
g ow h cu es p esen he a e age o h ee independen biological eplica es.
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ea men in S. pneumoniae. Consequen ly, he VicKRX egulon also showed a di e en pa e n: only ansc ip s
o he cell shape p o eins M eC and M eD we e up egula ed, al hough ea lie and s onge han in S. mu ans. In
S. mu ans, cell wall me abolism- ela ed genes we e up egula ed, while he espec i e homologues in S. pneumo-
niae TIGR4 we e no signi ican ly di e en ially exp essed.
In e es ingly, he gidA homologue o S. mu ans (smu_2141) was among he mos s ongly up egula ed an-
sc ip s like in S. pneumoniae, al hough i s up egula ion was weake and occu ed la e han in he pneumococcus
(Supplemen a y Fig. S8).
Taken oge he , he ansc ip ional esponse o S. mu ans and S. pneumoniae o Ca olac on appea s o be a
pleio opic s ess esponse, di e en be ween species, and no a speci ic and ocused ansc ip ional esponse as
assumed a e s udies wi h S. mu ans be o e10.
Discussion
Fo he i s ime we epo on he physiological e ec o Ca olac on on he clinically ele an human pa hogen
S. pneumoniae. G ow h o plank onic cul u es o S. pneumoniae is al eady inhibi ed by Ca olac on a a concen-
a ion as low as 0.025 μ g/ml (Supplemen a y Fig. S1). G ow h inhibi ion s a s abou 2 h a e applica ion o
he d ug; a la e ime poin s, he op ical densi y emains cons an , bu he numbe o iable cells con inues o
dec ease. Such beha iou is ypically obse ed when ea ing pneumococci wi h bac e icidal an ibio ics21. G ow h
inhibi ion by Ca olac on is concen a ion-independen , which could indica e ha he p ima y cellula a ge o
Ca olac on is easily sa u a ed and hus o low abundance in cells.
He e we show ha g ow h inhibi ion by Ca olac on occu s o clinical isola es as well as o he i ulen
labo a o y s ain TIGR4 and hus appea s o be independen o he se o ype. In e es ingly, he suscep ibili y o
Ca olac on was ela ed o he d ug esis ance o he pa hogen. The sepsis isola e Sp49 (which is esis an o wo
classes o an ibio ics only) was ins an ly killed by Ca olac on, while he mul id ug- esis an isola es Sp61 and
Sp64 we e inhibi ed o a lowe deg ee han he TIGR4 s ain. Acco dingly, also he MIC alues o Ca olac on di -
e ed no ably be ween S. pneumoniae TIGR4 and among he clinical isola es. Ca olac on has a po en inhibi o y
e ec on S. pneumoniae TIGR4 and Sp49 wi h MIC alues in he ange o MICs o classical an ibio ics used o
he he apy o pneumococcal disease, such as e.g. ampicillin, cla i h omycin o i ampicin31. The exac MIC o
Sp49 emains o be de e mined and could be e en lowe han 0.03 μ g/ml. Mul id ug- esis an isola es Sp61 and
Sp64 can ole a e and g ow in he p esence o high concen a ions o Ca olac on. Mul id ug esis ance is o en
media ed by inc eased exp ession o ac i i y o mul id ug e lux pumps. Sp61 and Sp64 a e esis an o mac-
olides (cla i h omycin, eli h omycin)32 and luo oquinolones (le o loxacin)33, which is media ed by e lux in
he pneumococcus. Ca olac on is s uc u ally simila o he mac olide class o na u al compounds and migh be
expo ed in a simila way. Unspeci ic e lux o Ca olac on o e ime, as a by-p oduc o esis ance o mac olides
and luo oquinolones, could explain why he MICs o Sp61 and Sp64 a e much highe han ha o S. pneumo-
niae TIGR4. This is in acco dance wi h he obse a ion ha E. coli is insensi i e o Ca olac on, bu he g ow h o
E. coli TolC, which lacks a mul id ug e lux pump, is comple ely inhibi ed5. These indings a e impo an o u u e
Figu e 5. E ec o Ca olac on- ea men on he memb ane in eg i y and memb ane po en ial o S.
pneumoniae TIGR4. (A) Cells g own wi h 0.25 μ g/ml Ca olac on (+ ) and con ols (− ) we e s ained wi h Sy o
9/PI and analysed using low cy ome y as exempli ied in Fig. S8. E o ba s o s ained hea -killed con ols
( ed ba s) a e no isible, as con inuously mo e han 99.9% o all hea - ea ed cells we e killed and emi ed ed
luo escence. (B) Changes in memb ane po en ial we e quan i ied by low cy ome ic analysis o DiOC2(3)-
s ained plank onic cells. The ela i e memb ane po en ial was calcula ed a iome ically. Analyses we e ca ied
ou wi h 30 μ M DiOC2(3). Hea -killed cells se ed as a con ol o ully depola ized cells. The ba cha s p esen
he a e age o h ee biological eplica es (± s.d.). P alue: n.s. = > 0.05; * * = < 0.01; * * * = < 0.001; wo- ailed
S uden ’s - es .
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de elopmen o he compound and po en ial clinical applica ions. Fo example, Ca olac on could be used syne -
gis ically wi h s uc u ally un ela ed non-mac olide an ibio ics.
A Ca olac on de i a i e wi h an in e ed s e eocen e a C-9 (epi-Ca olac on) was comple ely inac i e agains
bo h S. mu ans and S. pneumoniae, as well as agains se e al o al s ep ococci11. All o he chemical modi ica ions
o he side-chain o he lac one ing es ed so a we e also inac i e11. This indica es a highly s e eospeci ic in e -
ac ion o Ca olac on wi h i s molecula a ge and led us o hypo hesise ha he a ge could be highly conse ed
and sha ed be ween S. pneumoniae and S. mu ans. I appea s unlikely ha a swi ch o he s e eochemis y a C-9
could comple ely abolish in e ac ion wi h mul iple less conse ed a ge s in bo h species. I Ca olac on would
in e ac wi h se e al weakly conse ed a ge s, we would expec a emaining ac i i y o epi-Ca olac on in one
o he es ed species, which is absen as shown he e and p e iously11. A pa e n simila o he one desc ibed
o epi-Ca olac on he e was ound o he an ibio ic os omycin, which a ge s he conse ed bac e ial cell wall
hyd olase Mu A34, bu i s s e eoisome lacks an imic obial ac i i y35. Consequen ly we in es iga ed he oles o
PknB and CysR, which bo h had been shown o be essen ial o he damage o bio ilm cells o S. mu ans a low
pH10,20, o Ca olac on suscep ibili y in S. pneumoniae. Ou da a indica e ha nei he S kP no CysR a e equi ed
o suscep ibili y o S. pneumoniae o Ca olac on and hey hus canno be i s molecula a ge i his is conse ed
in bo h s ep ococci. Ano he indica ion ha Ca olac on ac s independen ly o S kP is he po en ac i i y o
Ca olac on agains E. coli TolC, as E. coli does no possess S kP homologues36. S kP and CysR a e appa en ly
exclusi ely impo an o S. mu ans a low pH in ma u e bio ilms, which is a unique en i onmen undamen ally
di e en om plank onically g owing S. pneumoniae cul u es.
Figu e 6. Di e en ially egula ed mRNAs in S. pneumoniae TIGR4 upon ea men wi h Ca olac on. The
hea map illus a es he 39 mos di e en ially exp essed genes (mRNA > 200 n ) a e addi ion o Ca olac on
(0.25 μ g/ml). The cu -o o di e en ially egula ed genes was se a log2FC ≥ ± 2 and FDR < 0.01 o a leas
one sample du ing he ime cou se. RNA-seq da a was analysed using Rockhoppe and he edgeR package o R.
The hea map was cons uc ed using he R package gplo s. Main ope on s uc u es a e indica ed by e ical lines
on he le . In o ma ion on encoded p o eins and hei in ol emen in biological pa hways we e ob ained om
KEGG ORTHOLOGY o KEGG BRITE da abases.