Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing ...

13 Pages·2012·5.84 MB·English

RESEARCHARTICLE
Propionibacterium acnes Bacteriophages Display Limited Genetic
Diversity and Broad Killing Activity against Bacterial Skin Isolates
LauraJ.Marinelli,a,bSorelFitz-Gibbon,cClarmyraHayes,b*CharlesBowman,dMeganInkeles,cAnyaLoncaric,b*DanielA.Russell,d
DeborahJacobs-Sera,dShawnCokus,cMatteoPellegrini,cJennyKim,b,eJeffF.Miller,aGrahamF.Hatfull,dandRobertL.Modlina,b
DepartmentofMicrobiology,Immunology,andMolecularGenetics,DavidGeffenSchoolofMedicine,LosAngeles,California,USAa;DivisionofDermatology,Department
ofMedicine,DavidGeffenSchoolofMedicine,LosAngeles,California,USAb;DepartmentofMolecular,Cell,andDevelopmentalBiology,UniversityofCalifornia,Los
Angeles,California,USAc;DepartmentofBiologicalSciencesandPittsburghBacteriophageInstitute,UniversityofPittsburgh,Pittsburgh,Pennsylvania,USAd;and
DepartmentofDermatology,VAGreaterLosAngelesHealthcareSystem,LosAngeles,California,USAe
*Presentaddress:AnyaLoncaric,SoltaMedical,Inc.,Hayward,California,USA;ClarmyraHayes,CaliforniaInstituteofTechnology,Pasadena,California,USA.
ABSTRACT Investigationofthehumanmicrobiomehasrevealeddiverseandcomplexmicrobialcommunitiesatdistinctana- D
o
tomicsites.Themicrobiomeofthehumansebaceousfollicleprovidesatractablemodelinwhichtostudyitsdominantbacterial w
inhabitant,Propionibacteriumacnes,whichisthoughttocontributetothepathogenesisofthehumandiseaseacne.Toexplore n
thediversityofthebacteriophagesthatinfectP.acnes,11P.acnesphageswereisolatedfromthesebaceousfolliclesofdonors lo
a
withhealthyskinoracneandtheirgenomesweresequenced.ComparativegenomicanalysisoftheP.acnesphagepopulation, d
e
whichspansa30-yeartemporalperiodandabroadgeographicrange,revealsstrikingsimilarityintermsofgenomelength,per- d
centGCcontent,nucleotideidentity(>85%),andgenecontent.Thiswasunexpected,giventhefar-rangingdiversityobservedin f
r
o
virtuallyallotherphagepopulations.AlthoughtheP.acnesphagesdisplayabroadhostrangeagainstclinicalisolatesofP.ac- m
nes,twobacterialisolateswereresistanttomanyofthesephages.Moreover,thepatternsofphageresistancecorrelateclosely
h
withthepresenceofclusteredregularlyinterspacedshortpalindromicrepeatelementsinthebacteriathattargetaspecificsubset t
t
p
ofphages,conferringasystemofprokaryoticinnateimmunity.ThelimiteddiversityoftheP.acnesbacteriophages,whichmay :
/
/
relatetotheuniqueevolutionaryconstraintsimposedbythelipid-richanaerobicenvironmentinwhichtheirbacterialhosts m
reside,pointstothepotentialutilityofphage-basedantimicrobialtherapyforacne. b
io
IMPORTANCE Propionibacteriumacnesisadominantmemberoftheskinmicrofloraandhasalsobeenimplicatedinthepatho- .a
genesisofacne;however,littleisknownaboutthebacteriophagesthatcoexistwithandinfectthisbacterium.Herewepresent s
m
thenovelgenomesequencesof11P.acnesphages,therebysubstantiallyincreasingtheamountofavailablegenomicinformation
.
o
forthisphagepopulation.Surprisingly,wefindthat,unlikeotherwell-studiedbacteriophages,P.acnesphagesarehighlyhomo- r
g
geneousandshowastrikinglackofgeneticdiversity,whichisperhapsrelatedtotheiruniqueandrestrictedhabitat.Theyalso /

o
shareabroadabilitytokillclinicalisolatesofP.acnes;phageresistanceisnotprevalent,butwhendetected,itappearstobecon- n
ferredbychromosomallyencodedimmunityelementswithinthehostgenome.Webelievethatthesephagesdisplaynumerous A
featuresthatwouldmakethemidealcandidatesforthedevelopmentofaphage-basedtherapyforacne. p
r
il
1
0
Received16August2012 Accepted17August2012 Published25September2012 ,

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CitationMarinelliLJ,etal.2012.Propionibacteriumacnesbacteriophagesdisplaylimitedgeneticdiversityandbroadkillingactivityagainstbacterialskinisolates.mBio3(5): 0
e00279-12.doi:10.1128/mBio.00279-12. 1
9
EditorJulianDavies,UniversityofBritishColumbia
b
Copyright©2012Marinellietal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution-Noncommercial-ShareAlike3.0Unported y
License,whichpermitsunrestrictednoncommercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. g
AddresscorrespondencetoGrahamF.Hatfull,[email protected]. u
e
s
t
Theinvestigationofthemicrobiomeatspecificanatomicsitesis thedominantinhabitantofthehumanpilosebaceousunit(10),an
essential toward understanding the pathogenesis of disease invaginationwithinthehumanepidermiscontainingahairfolli-
and the development of targeted therapies. In such microbial cle, hair shaft, erector pili muscle, and associated sebaceous
communities, there is an interaction between bacterial popula- glands,whichproducesebum.Despiteitsubiquitouspresenceon
tionsandthebacteriophagesthatinfectthem.Thesevirusesinflu- theskin,P.acnesisalsothoughttoplayamajorroleinthepatho-
ence bacterial community structure and function by several genesisofacnevulgaris,inpartbyelicitingahostinflammatory
mechanisms, including their ability to kill their bacterial hosts response(11).ThereisasignificantincreaseinP.acnescoloniza-
and/ormediategeneticexchangethatcanincreasediversityand tionatpuberty,thetimeduringwhichacnecommonlydevelops,
potentiallydisseminatevirulencegenes.Virtuallyallofthebacte- and teenagers with acne can have as many as 100-fold more
riophagepopulationsthathavebeenstudieddisplayawiderange P.acnesbacteriapresentontheirskinthanhealthy,age-matched
ofgeneticdiversity(1),includingthosethatinfectMycobacterium counterparts(12).Theefficacyofantibioticsforacnetreatmentis
(2–5),Staphylococcus(6,7)andPseudomonas(8,9)species. relatedtothereductionofthenumberofP.acnesbacteriaonthe
TheGram-positiveskincommensalPropionibacteriumacnesis skin,aswellastodirectanti-inflammatoryproperties(13),which
September/October2012 Volume3 Issue5 e00279-12 ® mbio.asm.org 1
Marinellietal.
TABLE1P.acnesbacteriophagescharacterizedinthisstudy
Phage Methodofisolationorsource Donor Date Accessionno.
P1.1 Directplatingofporestripsample Acne 06/2007 JX262223
P9.1 SupernatantofporestripcultureplatedonATCC6919 Acne 01/2008 JX262215
P14.4 PorestripsampleplatedonATCC6919 Acne 01/2008 JX262216
P100A FSaporestripsampleplatedonATCC6919 Noacne 09/2010 JX262221
P100D SupernatantofFSporestripcultureplatedonATCC6919 Noacne 09/2010 JX262220
P100.1 SupernatantfromuninducedB100.1platedonB100.4b Noacne 11/2010 JX262222
P101A FSporestripsampleplatedonATCC6919 Noacne 10/2010 JX262217
P104A FSporestripsampleplatedonATCC6919 Acne 12/2010 JX262218
P105 FSporestripsampleplatedonATCC29399 Noacne 12/2010 JX262219
ATCC29399B_C ATCC Acne 1978c JX262225
ATCC29399B_T ATCC Acne 1978c JX262224
a FS,filtersterilized.
bP.acnesclinicalisolateobtainedfromsamedonorasP100A,P100D,andP100.1.
cWebsterandCummins(18). D
o
w
n
reflectsthemultifactorialetiologyofacne.Nevertheless,theemer- MorphologyofP.acnesphagevirions.P.acnesstrainATCC lo
a
genceofantibiotic-resistantstrainsofP.acnes,asmeasuredinup 6919issusceptibletoinfectionbyallofthephagesisolatedhere d
to60%ofclinicalisolates(14–16),hasresultedinclinicalchal- and was used as a host to prepare high-titer lysates for all 11 e
d
lengesandhighlightstheneedforimprovedtherapeutics(13). phages. These were analyzed by electron microscopy, and all f
r
P.acnesbacteriophagesareacommoncomponentoftheacne phageswereobservedtopossessasiphoviralmorphology,withan o
m
microbiome. In early studies, these phages were used to type isometric head, ~50nm in diameter, and a long flexible tail,

P.acnes strains, and some were found to exhibit a broad host ~150nminlength(Fig.1).Morphologically,theseresemblethe h
t
t
rangeagainstP.acnesclinicalsubtypes(17–19).However,despite P.acnesphagesreportedpreviously(18,20–23)andthuslackthe p
:
therelativeeasewithwhichP.acnesbacteriophagescanbeisolated diversity of forms observed in some other well-characterized //
m
fromhumanskin,thecompletegenomesequencesofonlythree phagepopulations.Forexample,mycobacteriophagespossessing
b
P.acnes phages have been reported (20, 21) and little is known either contractile tails (Myoviridae) or prolate heads have been io
abouteithertheirgeneticdiversityorthemolecularbasisoftheir observed (3, 5), and both the Staphylococcus and Pseudomonas .a
relationshipswiththeirbacterialhosts(20,21).Wethereforese- phage populations have members with podoviral morphologies sm
quenced 11 novel P.acnes phages from healthy individuals and (i.e.,withshort,stubbytails)(7,8). .
o
thosewithacneandpresenttheircompletegenomesequences,as Genomic characterization of P.acnes bacteriophages. To r
g
wellasadetailedcomparativegenomicanalysisandphenotypic gainamorecomprehensiveunderstandingofthegenomicdiver- /

o
characterizationofthesephages.Wefindthatthesephagespossess sity present within the P.acnes bacteriophage population, total n
astrikinglackofgeneticdiversity,whichcontrastswithwhathas genomicDNAwaspreparedfromeachofthe11phages,andtheir A
beenobservedinotherphagepopulations.Furthermore,weshow genomes were sequenced. Upon assembly and annotation, all p
r
thattheP.acnesphagesinthisstudyinfectabroadrangeofclinical werefoundtobesimilarinsizeandstructuretothethreeprevi- il
1
isolates,andphageimmunity,whenpresent,appearstobecon- ouslyreportedP.acnesphages,PA6,PAD20,andPAS50(20,21). 0
ferredbythepresenceofchromosomallyencodedelements. The14P.acnesphagesrangeinsizefrom29,017to29,739bp,with ,
2
45to47predictedprotein-codinggenes(Table2);notRNAgenes 0
RESULTS wereidentified.Ourdatafurthersuggestthatallofthesephage 1
9
IsolationofP.acnesbacteriophages.Toinvestigatethediversity genomes possess the same 11-base single-stranded 3= extension b
present within the population of bacteriophages that infect (5=-TCGTACGGCTT),whichisshorterby2basesthanthatre- y

g
P.acnes, we isolated phages and bacterial isolates from micro- portedinthegenomesofthethreeP.acnesphagespublishedpre- u
comedones(thesebaceousfolliclecontent)obtainedfromthena- viously (5=-CCTCGTACGGCTT). For each of our 11 phages, e
s
sal skin of donors either with or without acne. Nine P.acnes readsfromboththe454andtheIlluminadataeitherterminateat t
phageswereisolatedeitherbydirectcultureofthemicrocome- the3=endwiththe5=-CCorreadthroughtotheotherendofthe
donematerial,withorwithoutindicatorstrainsofhostbacteria, genomeviathe11-baseputativesingle-strandregion,suggesting
orfromthesupernatantofculturesinoculatedwithmicrocome- thatourinterpretationiscorrect.
donesandgrowntosaturation(Table1).Fiveofthesewereob- TherearetwonotablefeaturesofthepercentGCcontentofthe
tainedfromhealthydonors,andfourwereisolatedfromdonors P.acnesphagegenomes.First,thereisverylittlevariationinper-
withacne(Table1).Additionally,usingP.acnesATCC6919asa centGCcontentfromgenometogenome,astheyallfallwithina
host,werecoveredtwophagesfromaphagestockobtainedfrom narrowrangeof53.76to54.17%(Fig.2;Table2).Second,their
theAmericanTypeCultureCollection(ATCC),ATCC29399B, average percent GC content (54.1%) is substantially different
whichwenamedATCC29399B_CandATCC29399B_Ttoreflect fromthatoftheirP.acneshosts,whichis~60.0%.Thegenomesof
theirdistinctplaquemorphotypes,clearandturbid,respectively. fewotherPropionibacteriumspecieshavebeensequenced,butwe
BecauseATCC29399Bwasobtainedover30yearsagofroman notethatP.freudenreichiiisconsiderablymoreGCrich(67.3%;
acnepatientinPhiladelphia,PA(18),thetwophagespresentin Fig.2).TherestrictedpercentGCcontentoftheP.acnesphagesis
thisstockaretemporallyandgeographicallyseparatedfromthe in contrast to the considerable variation observed in phages of
otherphagesisolatedinourstudy. other hosts. For example, the percent GC content varies by as
2 ® mbio.asm.org September/October2012 Volume3 Issue5 e00279-12
DiversityandKillingActivityofP.acnesPhages
D
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FIG1 P.acnesphagevirionmorphologies.Negativelystainedelectronmicrographsofthe11P.acnesphagescharacterizedinthisstudyrevealthatallhavethe w
n
samesiphoviralvirionmorphotype.Allofthesephageshavesimilarlysizedisometricheads(~50nmindiameter)andlong,flexibletails(~150nminlength).
lo
a
d
e
muchas18to28%withineachofthelargecollectionsofphages the P.acnes phages is reflective of limited nucleotide sequence d
infectingMycobacteriumsmegmatis,Staphylococcussp.,andPseu- diversity, we performed dot plot comparisons (Fig. 3A), which f
r
domonassp.(Fig.2).Aconsequenceofthisisthatmostofthese clearlydemonstrateahighlevelofnucleotidesequencesimilarity o
m
phageshavepercentGCcontentsthataredistinctfromthoseof extendingacrossthelengthsofall14genomes.Thisisillustrated
h
theirknownhostsandcloserelativestherof,whichmayserveas bythenearlysolidlineof“dots”alongthediagonalofeachpair- t
t
potentialhosts,asisalsothecasewiththeP.acnesphages(Fig.2). wisecomparison,witheachdotrepresentinganucleotidesegment p
:
However, unlike the P.acnes phages, each of these other three thatissharedbytwophages.Thelackofdiversityobservedamong //m
groups of phages exhibits a range of percent GC contents that theP.acnesphagesisincontrasttothatseeninMycobacterium, b
overlapsthatofthehost,andtheaveragepercentGCcontentis Staphylococcus, and Pseudomonas phages (Fig. 3B), where al- io
.
similartothehostvalue,whichistobeexpectedbecauseofthe thoughsomegenomesexhibitsimilarity,mostpairwisecompari- a
s
propensityforgeneticexchangebetweenaphageanditshost. sonsdonot.Thecomparativelackofdiversityobservedamong m
There are two plausible explanations for these observations. theP.acnesphagesversusotherphagepopulationsisunlikelyto .o
First,theP.acnesphagesmayhavesufficientlybroadhostranges r
be a consequence of sample size or bias, because six randomly g
thatalthoughtheywereisolatedonP.acnes,intheirnaturalenvi- chosensetsof14mycobacteriophagesfromthetotalof221each o/
ronment, they were most recently associated with another host show a diversity considerably greater than that of the P. acnes n
organism(s).Second,thephagesmayhaveadaptedforgrowthin A
phages(seeFig.S1inthesupplementalmaterial).
the known host relatively recently, such that their percent GC p
AlignmentoftheP.acnesphagegenomemapsanddisplayof r
contenthasyettofullyamelioratetowardthatoftheirbacterial pairwise nucleotide sequence similarities, as indicated by the il 1
host(24).Collectively,thelimitedrangeofgenomesizeandper- shadingbetweenthegenomes,furtherrevealsthestrikingsimilar- 0
centGCcontentobservedintheP.acnesphagessuggeststhepos- ,
ityshownbythesephagesbutalsoidentifiesregionsofsequence 2
sibilityoflimiteddiversityinthisbacteriophagerepertoirecom- 0
variation(Fig.4).Thegreatestvariationoccursclosetotheright 1
paredtothatofotherwell-characterizedphagepopulations. 9
genomeends,withsomeadditionalvariationinthecentralpor-
LimitednucleotidesequencediversityofP.acnesphages.To b
tions.Incomparison,genomemapsdepictingasubclusterofre- y
determine whether the limited percent GC content variation of
latedmycobacteriophages(seeFig.S2Ainthesupplementalma- g
u
terial) show a lesser degree of nucleotide identity, and genome e
TABLE2P.acnesbacteriophagegenomefeatures maps from a selection of mycobacteriophages chosen to reflect st
their maximal diversity (see Fig. S2B) display almost no shared
Phage Length(bp) %GC No.ofORFs
nucleotidesequenceidentity.
PAS50 29,017 53.97 46
OrganizationoftheP.acnesphagegenomes.Thelimitedge-
PAD20 29,074 54.10 45
neticvariationamongtheP.acnesphagegenomesisalsoreflected
P105 29,202 54.17 45
P9.1 29,214 54.12 45 in their genome organizations. The DNA packaging and virion
P1.1 29,348 54.37 45 structureandassemblygenes(1to19)occupyapproximatelyhalf
P104A 29,371 53.98 45 ofthegenome(coordinates1to~15kbp)andareorganizedinto
P100A 29,505 53.83 45 anoperonthatispresumablytranscribedrightward,withfewin-
P100D 29,506 53.76 47
tergenicgaps,asiscommonamongphageswithasiphoviralmor-
ATCC29399B_T 29,516 54.01 46
photype,suchasthe(cid:1)-likecoliphagesandmanyofthemycobac-
ATCC29399B_C 29,516 54.00 46
P101A 29,574 54.14 47 teriophages (1, 3, 5) (Fig. 4 and 5; colored boxes represent
P100.1 29,612 54.07 47 predictedproteincodinggenes).Thesegenesareallhighlycon-
P14.4 29,729 54.08 47 servedatthenucleotidelevel,withthenotableexceptionofthelast
PA6 29,739 54.02 45
oftheputativeminortailproteingenes(P100Dgene19andits
September/October2012 Volume3 Issue5 e00279-12 ® mbio.asm.org 3
Marinellietal.
cobacteriophageTM4(4)—butratherit
islikelythattheirabsencereflectsacore
featureofthesegenomearchitectures.In
agreement with these observations, we
wereunabletoisolatestablelysogensfor
anyofthephagesthatwetested(datanot
shown).
Anotablegenomicfeatureisthepres-
ence of a large ((cid:1)1-kbp) noncoding re-
gion near the right end of the P.acnes
phagegenomes(Fig.4and5).Thisregion
shows the greatest variation among the
genomes(Fig.4)andisalsonotableforits
unusual base composition. The left side
ofthenoncodingregionhasalargedrop D
o
inpercentGCcontent,below25%atthe w
greatestextreme(Fig.5).Throughoutthe n
region are several surprisingly well- lo
a
conserved low-complexity runs (see d
e
Fig.S3andS4inthesupplementalmate-
d
rial). Additionally, plots of GC content f
r
skewsummedoverall14genomesshowa o
m
FIG2 PercentGCcontentsofbacteriophagesandtheirhosts.ThepercentGCcontentsofthegenomes clearexcessofG’sontheforwardstrand
h
ofindividualbacteriophagesthatinfectP.acnes,M.smegmatis,Staphylococcussp.,orPseudomonassp. overmostofthegenome,switchingtoan t
t
areplottedinorder,fromthelowestvaluetothehighest.Theanalysisincludes14phagesofP.acnes,221 excessofC’swithinthenoncodingregion p
phagesofM.smegmatis,66phagesofStaphylococcussp.,and51phagesofPseudomonassp.(seeMate- (see Fig. S3 and S4). Transcription ap- ://
rialsandMethods).ThepercentGCcontentsofhostbacteriaandtheircloserelatives,whichmayserve m
pearstohaveaminimaleffectontheGC
aspotentialhosts,werecalculatedfrompublishedgenomes;theseareshownasdottedlinesandlisted b
withpercentGCcontentsinparentheses. contentskew,withnocleardifferenceev- io
ident across the primary region where .a
s
coding switches from one strand to the m
relatives),asnotedpreviously(21).Wehavefoundthatthevari- other (position 17500 of the multiple alignment) (see Fig. S3). .
o
ationisgreatestwithinthe3=halvesofthesegenes,andtheprotein This indicates that the switch in GC content skew is related to r
g
products vary substantially in the corresponding C-terminal replication rather than transcription and suggests an origin of /

o
parts.Thehighglycinecontent(~20%)oftheseproteinsandthe replicationwithinthenoncodingregion. n
presence of variable numbers of collagen-like GXXG repeats Relationship of P.acnes phages to other phages. BLASTP A
(from 17 to 42) support their likely role as tail fibers, and the searches of predicted P.acnes phage gene products against the p
r
observedvariationisconsistentwiththepossibilitythattheseplay completeproteindatabaseshowthatalmost50%matchonlypre- il
1
rolesinhostrecognitionandthusinhostrangedetermination.A dicted proteins from other P.acnes phages or putative P.acnes 0
lysiscassettethatencodesanendolysin(gp20)andaputativeholin prophages. Many of those that do have matches outside the ,
2
(gp21)issituatedimmediatelytotherightofthetailgeneandis P.acnesphages/prophagesarerelatedtophagesofotherActino- 0
1
alsotranscribedrightward. mycetales hosts and their prophages, including mycobacterio- 9
Withtheexceptionofoneortwogenesattheextremeright phages, Streptomyces phages, and Rhodococcus phages; however, b
endsofthegenomes,theremaindersofthegenesaretranscribed noneoftheseproteinrelativesshowgreaterthan50%aminoacid y

g
leftward; these are closely spaced and are presumably cotrans- identity.Toexaminetheserelationshipsfurther,agenecontent u
cribed.Asobservedinphagesofotherhosts,onlyasmallnumber mapwasconstructedfromtheP.acnesphagesandphagesofother e
s
arerelatedtoproteinswithknownfunctions(Fig.4and5).The Actinomycetaleshosts,includingsubsetsofthosethatinfectMy- t
fewgenesforwhichputativefunctionscanbepredictedareimpli- cobacterium, Rhodococcus, Streptomyces, Gordonia, Corynebacte-
catedinDNAmetabolismandregulation,includingP100Dgenes rium,andTsukamurella(Fig.6A).Therearetwostrikingobserva-
30/31, 33, and 36 (and their relatives), which encode DNA pri- tions that emerge from this analysis, the first of which is the
mases,DnaB-likehelicases,andRecB-likeexonucleases,respec- limiteddiversityoftheP.acnesphages,especiallyincomparison
tively(Fig.4and5).P100Dgene23anditsrelativesencodelikely withthatofthemycobacteriophages.LiketheP.acnesphages,the
transcriptional regulators and contain strongly predicted helix- mycobacteriophages(withthenotableexceptionofDS6A)have
turn-helix DNA-binding motifs; HHPred also predicts a 95.9% the ability to infect a single bacterial host strain (in this case,
probability of structural similarity of P100D gp23 to the DNA- M.smegmatis),yetthediversityoftheirgenecontentsisdramat-
binding domain of the phage 186 repressor. Presumably, these icallydifferent.Thesecondistheobservationthatalthough~20%
proteinsactasthephagerepressorand/orasmodulatorsoflytic of the P.acnes phage genes show (cid:1)30% amino acid sequence
gene expression. Interestingly, the P.acnes genomes lack either identitytomycobacteriophagegeneproducts,thehomologuesare
integrationorpartitioningsystemsthatarecommontotemperate distributedbroadlyamongthemycobacteriophages,suchthatthe
bacteriophages.Itisunlikelythatthesefunctionshavebeenlost P.acnes phages are not more closely related to any particular
duringrecentevolutionaryhistory—ashasbeenreportedformy- groupofmycobacteriophagesthantotheothers.Thisisincon-
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FIG3 DotplotnucleotidesequencecomparisonsofP.acnesphages,mycobacteriophages,Staphylococcusphages,andPseudomonasphages.Thegenome
g
sequencesof14P.acnesphages,221mycobacteriophages,66Staphylococcusphages,and51Pseudomonasphageswereconcatenatedtoformfourcompiled u
sequencesof0.41,15.46,3.3,and3.88Mbp,respectively.EachwasthencomparedwithitselfusingthedotplotprogramGepard(48). e
s
t
trast to Rhodococcus phage RequPine5 and Tsukamurella phage (e.g.,P100A,P100D,andP100.1)donotnecessarilyappeartobe
TPA2, both of which are more closely related to the cluster B more related to one another than they are to the other P.acnes
mycobacteriophages (PG1, Colbert, Nigel, Cooper, Qyrzula, phages.However,werecognizethatthevariationofP.acnesphage
Rosebush,Phlyer,andPhaedrus)thantothoseofotherclusters gene content is very restricted, and therefore, these representa-
(Fig.6A). tionsaresensitivetominordifferencesingenomeannotation.
ThelimiteddiversityofP.acnesphagegenecontentisshownin P.acnesbacteriophageendolysins.Thebacteriophageendo-
finerdetailinthenetworkmapinFig.6B.Itshouldbenotedthat lysinproteinfunctionsatthecompletionofthelyticcycle.With
the diversity of the P.acnes phages on this gene content map is theassistanceoftheholinprotein,theendolysingainsaccessto
apparent only because the scale has been magnified 100 times anddegradesthebacterialcellwallpeptidoglycan,leadingtohost
comparedtothemapinFig.6A.Despitetheirlimiteddiversity, celllysisandreleaseofprogenyphage(25).Endolysinscandisplay
P.acnesphagesfromthesamegeneralsource,suchastheATCC anumberofdifferentenzymaticactivities,includingamidase,en-
phages and the two phages from Sweden, PAD20 and PAS50, dopeptidase,glucosaminidaseandlytictransglycosylaseactivities,
group together. However, phages arising from the same donor whichtargetvariouscovalentbondswithinthehostcellwall(25).
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FIG4 Whole-genomecomparisonsofP.acnesphages.Thegenomemapsofthe14completelysequencedP.acnesphagesareshownwiththepairwisenucleotide
sequencesimilaritiesdisplayedascoloredsegmentsbetweenthegenomes;thestrengthofsequencesimilarityisrepresentedaccordingtoacolorspectrumin
whichvioletisthemostsimilarandredistheleast.Thepositionsofpredictedgenesareshownasboxeseitherabove(transcribedrightward)orbelow(transcribed
leftward)eachgenome,withgenenumbersshownwithintheboxes;putativegenefunctionsarenotedatthetop.Themapsweregeneratedusingtheprogram
Phamerator(49)andadatabasenamedacnes_myco30(seetext).
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FIG5 OrganizationoftheP100DgenomeanditspercentGCcontentvariation.AgenomemapofP.acnesphageP100Disshownwithpredictedgenes m
representedascoloredboxeseitherabove(transcribedrightward)orbelow(transcribedleftward)thegenome.Thegenenumberisshownwithineachbox,and h
the“phamily”towhichthatthegenecorrespondsisdisplayedabove,withthenumberof“phamily”membersshowninparentheses.Putativegenefunctionsare t
t
notedwitharrowsindicatingtheorientationoftranscription.Alloftheothergenomesdescribedherehavethesamebasicorganization.Atthebottomisascan p
:
ofpercentGCcontentsacrosstheP100Dgenomedonewithwindowandstepsizesof100and50bp,respectively.ThesharpdeviationofpercentGCcontentat //
m
therightendofthegenomeoccursinalloftheP.acnesphages.
b
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a
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Gene20ispredictedtoencodetheP.acnesphageendolysin,and equalefficienciesofplating(Fig.8).ThreeoftheATCCstrainsand m
thisproteinishighlyconservedinallsequencedP.acnesphages 12donorisolatesderivedfromfivedifferentdonors—andthusat .
o
(Fig. 7A). Partial sequences are available for a number of other least8distinctisolatestotal—shownodiscriminationandarein- r
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P.acnesphages,andthesealsodemonstrateahighdegreeofcon- fectedequivalentlybyall11phages.Theother11strainsfromsix /

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servationinthisgenefamily(22).Databasecomparisonspredict differentdonors,plusonefromtheATCC,haveavarietyofphage n
that the P.acnes endolysin proteins contain a highly conserved sensitivities(Fig.8).Inmoststrainswherevariablephagesensitiv- A
N-terminaldomainassociatedwithN-acetylmuramoyl-l-alanine itiesareobserved,thisoccursagainstasmallnumberofphages p
r
amidaseactivity,coupledtoa110-residueC-terminalextension andthereductionsinplatingefficiencyrangefrom10-to1,000- il
1
whose function is unknown but which likely mediates cell wall fold,relativetothatofreferencestrainATCC6919.However,in 0
binding.Themostcloselyrelatedphageendolysinsarethoseen- comparison,twostrains—B66.8andB101.9—aredistinctfrom ,
2
coded by Rhodococcus phages ReqiDoc7 and REQ2 and a large the others in that they are strongly resistant to 9 and 10 of the 0
1
groupofclusterBmycobacteriophages(26).Althoughallofthese phages,respectively,withplatingefficienciesreducedbyatleast 9
haverelatedamidasedomains,theC-terminalregionsarequite 1,000,000-fold as compared to those observed on ATCC 6919 b
different, likely reflecting their proposed role in conferring cell (Fig.8). y

g
wall binding specificity. The majority of the differences among TheroleofP.acnesCRISPR(clusteredregularlyinterspaced u
P.acnesphageendolysinsarewithinthesmalllinkerregionsepa- shortpalindromicrepeat)elementsinphageresistance.Varia- e
s
rating the two domains and, to a lesser degree, within the tions in phage sensitivity could arise from a variety of mecha- t
C-terminal putative cell wall-binding region (Fig. 7B). Collec- nisms, including receptor variation, restriction-modification,
tively,thesedatashowthatP.acnesphageendolysinsarehighly abortiveinfection,lysogenicimmunity,orinnateimmunitycon-
conserved; the encoded proteins likely target essential elements ferredbyCRISPRs(28).BecauseasubsetofP.acnesisolatesfor
withintheP.acnescellwallandarepredictedtokillabroadrange which genomic sequence information is available in the NCBI
ofP.acnesisolates. databasecontainputativeCRISPRlocibelongingtothetypeI-E
P.acnesphagehostpreferences.Thelimiteddiversityofthe subfamilyandthatappeartoconferresistancetomobilegenetic
P.acnesphagesraisesthequestionofwhethertheyhavesimilar elements (29), we examined if there is any correlation between
hostpreferences.Wethustestedthephagesensitivitiesofapanel phagesensitivitiesandCRISPRelementsinthe27P.acnesstrains.
of27P.acnesstrains(Fig.8),including23isolatesrecoveredfrom TheP.acnesCRISPRlocicontaineightconservedcasgenes(29),
the donors screened for P. acnes phages (the strains and their andPCRamplificationoftwoofthese,cas3andcsc4(cas7inthe
sourcesaredescribedinTableS1inthesupplementalmaterial); nomenclaturesystemproposedbyMakarovaandcolleagues[30,
16SrRNAgenotypingconfirmsthattheseareallP.acnes(27).The 31]),identifiedCRISPRelementsinsixofourP.acnesisolates,i.e.,
11 phages tested display a broad array of host preferences, and B66.8,B69.7,B101.9,B102.1,B102.8,andB102.10.Sequencingof
only ATCC 29399B_T infects the entire set of host strains with the associated spacer/repeat loci showed that all have the same
September/October2012 Volume3 Issue5 e00279-12 ® mbio.asm.org 7
Marinellietal.
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FIG6 GenecontentrelationshipsbetweenP.acnesphagesandphagesofrelatedhosts.(A)Therelationshipsbetweenthe14P.acnesphagesandaselectionof m
phagesthatinfectotherbacteriaoftheorderActinomycetaleswereobtainedbyusingtheNeighborNetfunctioninSplitstree4(51).TheP.acnes,Mycobacterium, .
o
Rhodococcus,Corynebacterium.Streptomyces,Tsukamurella,andGordoniaphagesareshowninthered,green,orange,blue,yellow,aqua,andmauvecircles, r
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respectively.(B)DetailedviewofthedisplayinpanelAshowingtheP.acnesphages. /

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n

A
28-bp direct repeat sequence, separated by variable numbers of mismatchesinallofthephagesandthusisunlikelytocontribute p
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33-bpspacers(Fig.9;seeTableS2inthesupplementalmaterial), toresistance.Incontrast,spacer1ofB66.8matchesgene16ofthe il
1
ranging from 1 spacer in B69.7 to 10 in B101.9. Four of these phages, with only two to four mismatches (Fig. 9). Like strain 0
CRISPRs (in B69.7, B102.10, B102.1, and B102.8) contain only 101.9,B66.8issensitivetophageATCC29399_T,andallofthe ,
2
oneortwospacerseach,noneofwhichhavecorrespondingse- CRISPRspacershavethreeormoremismatches.However,B66.8 0
1
quencesinanyofthephagegenomes(seeTableS2).Accordingly, is also sensitive to phage ATCC 29399_C, suggesting that the 9
these were sensitive to the panel of phages assayed as described CRISPR spacers—including spacers 3 and 1—are ineffective at b
above.Incontrast,thetwostrainswiththemostspacers—B66.8 conferringresistance,eventhoughtheyhave0and2mismatches, y

g
andB101.9,with9and10,respectively—arealsothosethatshow respectively(Fig.8).Thisdiscrepancycannotbeexplainedbymu- u
extensive phage resistance (Fig. 8), implicating CRISPRs in this tationsintheprotospacer-adjacentmotifs(PAMs)inthisphage, e
s
phenotype. as the 10 bases downstream of the protospacers found in all 14 t
InstrainB101.9,fiveofthespacershavesequence-relatedseg- P.acnesphagegenomesfollowtheexpectedCRISPRtypeIcon-
ments (known as protospacers) in the P.acnes phage genomes, sensusmotif,TGGC/TGAAGA/CG/AT/G.Inspiteofthisunex-
with spacers 1 to 5 corresponding to genes 9, 2, 3, 7, and 29, plained observation, the strong overall correlation between the
respectively(Fig.9).However,thematchestospacers2and5are presenceofCRISPRelementsandspacermatcheswiththephage
poorandthenumbersofmismatcheslikelyprecludeanyrolein genomessuggestsstronglythattheseplayaroleinthephagere-
phageresistance.However,spacers1,3,and4haveeitheraperfect sistanceprofilesobserved.
match or a small number of mismatches in each of the phage
genomes,withthenotableexceptionofATCC29399B_T(Fig.9), DISCUSSION
theonlyoneofthesephagestowhichthisstrainissensitive. Genomicanalysisofbacteriophageshasrevealedaremarkabledi-
AsimilarpatternwasfoundinstrainB66.8.Threeofthefive versitywithinpopulationsthattargetspecifichostbacteria,pro-
spacers(spacers3,4,and5)arethesameasthoseinB101.9,butas vidinginsightsintohowtheyinteractwithandkilltheirhosts,as
noted above, spacer 5 has no close phage matches. Spacer 2 of wellastheevolutionofmicrobialcommunities.Here,theanalysis
B66.8 has a related sequence in phage gene 2 but at a location of 14 fully sequenced P.acnes bacteriophages reveals an unex-
differentfromthatofspacer2ofB101.9(Fig.9).Italsohasfive pected degree of homogeneity in terms of percent GC content,
8 ® mbio.asm.org September/October2012 Volume3 Issue5 e00279-12
DiversityandKillingActivityofP.acnesPhages
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FIG7 OrganizationofP.acnesphageendolysins.(A)OrganizationoftheP.acnesendolysinsandtheirclosesthomologuespresentinRhodococcusphages /m
ReqiDocB7andREQ2andagroupofsubclusterB1mycobacteriophagesofwhichKLucky39isrepresentative.Allhaveanamidase-2domainthatcleaves b
peptidoglycanbuthavedifferentandunrelatedC-terminaldomains,whicharepresumedtoconfercellwallbindingspecificity.TheKLucky39endolysin—like io
manyofthemycobacteriophageendolysins—containsthreedomains,includinganN-terminalM23peptidase(26).(B)AlignmentoftheP.acnesphage .
a
endolysinsshowsthattheyarecloselyrelatedanddisplaythegreatestaminoacidsequencedifferencesataroundposition200,whichlikelycorrespondstoa s
glycine-richinterdomainlinker. m
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nucleotidesequenceidentity,andgenecontent.Thisisparticu- andrecombination.Thisisincontrasttothediversephagepop- /

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larlyevidentwhenthesephagesarecomparedwithothergroups ulationsthatarefoundincomplexmicrobialcommunities,such n
ofwell-characterizedbacteriophagepopulations.Phylogeneticre- asthoseinaquaticenvironmentsorsoilandeveninotherregions A
lationshipsbasedongenecontentshowthattheP.acnesphages ofthehumanbody,includingtheoralmucosa(32)andthegut p
r
clustertogetheronasinglenode,unlikethemycobacteriophages, (33). In addition to the occupation of a specialized niche, the il
1
whichshowmultiplenodeswithextensivebranchingindicativeof P.acnesphagehomogeneity,combinedwithanaveragepercent 0
gene content diversity. Strikingly, the differences between the GCcontentthatissodistinctfromthatofthehostbacteria,might ,
2
P.acnes phages are revealed only when their node is magnified suggestthatthesephagesarosefromacommonancestorthatonly 0
1
100-foldrelativetothatoftheotherbacteriophagepopulations. recentlyacquiredtheabilitytoinfectP.acnes.Itisalsopossible 9
The lack of P.acnes phage diversity is further reflected by their thatageneticbottleneckeliminatedmuchoftheP.acnesbacte- b
broadhostrangesagainstclinicalisolates.Thissuggeststhepres- riophage diversity, leaving the one dominant genotype present y

g
enceofevolutionaryconstraintsimposeduponthispopulationof today. u
phagesandtheirbacterialhoststhatfunctiontomaintainasingle AnotherinterestingfeatureoftheP.acnesphagesisthatnone e
s
phagetypeandpreventsignificantdiversificationofP.acnesand encodeanyrecognizableproteinspredictedtobeinvolvedinthe t
thewidespreaddispersalofphageresistance. establishmentormaintenanceoflysogeny,suchasintegrasesor
It is noteworthy that the lack of genetic diversity among putativepartitioningfunctions.Wenotethatthesephagesdocon-
P.acnesphagesismaintaineddespitetheircollectionoveravaried tainapossiblecandidateforthephagerepressor,P100Dgene23
geographic and temporal range, spanning two continents and (anditsrelatives),whichencodesalikelytranscriptionalregulator
morethan30years.Oneexplanationforthisistherelativelyiso- predictedtocontainahelix-turn-helixDNA-bindingmotifand
lated niche in which they and their host bacteria are found. haspredictedstructuralsimilaritytotheDNA-bindingdomainof
P.acnesisthedominantmicrobialinhabitantofthehumanpi- theenterobacterialphage186repressor.However,intheabsence
losebaceousunit(10),inpartrelatedtotheuniquenatureofthis ofothergenesrequiredforlysogeny,andbecauseofourinability
anaerobicmicroenvironment,whichischaracterizedbyhighlipid toisolatestablelysogens,itismorelikelythatthisproteinfunc-
content.Therefore,thelackofothermicrobes—andpresumably tionstoregulatethetranscriptionoflysisgenes.Theseobserva-
their phages—in this environment diminishes the possibility of tionsdonotprecludethepossibilitythatthesephagesengageinan
recoveringmoredistantlyrelatedphagesthatmaypreferalterna- unstable “pseudolysogenic” relationship with their hosts (21).
tivehostsbutarestillabletoformplaquesonP.acnesbacterial This is supported by our observation that many of our phages
lawns.Italsoprovidesfeweropportunitiesforlateralgenetransfer formturbidplaquesonsomeP.acnesstrainsandthedetectionof
September/October2012 Volume3 Issue5 e00279-12 ® mbio.asm.org 9
Marinellietal.
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FIG8 HostpreferencesofP.acnesphages.Theefficiencyofplatingofeachofthe11P.acnesphageswascalculatedfor27bacterialisolates,including23donor p
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isolatesandfourATCCstrains;ATCC6919wasusedasareferencefornormalization.ElevenisolatesandATCC11827havevariabledegreesofphagesensitivity; //
m
mostdifferencesresultfrommodestreductionsinplatingefficiency(10-to1,000-foldrelativetoreferencestrainATCC6919),buttwoisolates,B66.8andB101.9,
b
arestronglyresistantto9and10ofthephages,respectively(platingefficienciesarereducedbyatleast1,000,000-fold).Theremaining12isolatesandthreeATCC io
strainsareinfectedequivalentlybyallofthephages. .
a
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m
.
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nonintegratedbacteriophagesequenceswithinsequencedP.acnes An alternative approach to topical phage therapy for acne r
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bacterial isolates that have been deposited in GenBank (see wouldbetodeveloptheP.acnesphageendolysinasananti-acne /

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TextS1inthesupplementalmaterial). therapeutic. The endolysin is a cell wall hydrolase predicted to n
ThelackofgeneticdiversitywithintheP.acnesphagepopula- functioninbacterialpeptidoglycanhydrolysisatthecompletion A
tion,combinedwithabroadhostrangeandaninabilitytoform ofthelyticcycle(25).TheP.acnesphageendolysin,apredicted p
r
stablelysogenswithinthehostbacteria,makesthesephagesideal amidase,isencodedinoneofthemostconservedregionsofthe il
candidatesforthedevelopmentofaphage-basedtopicalanti-acne phagegenomeandwasfoundtobe(cid:1)95%conservedattheamino 10
therapy.Thepotentialtherapeuticuseofphageshasbeenknown acidlevelinallknownP.acnesphages.Ashasbeendemonstrated ,
2
forover90years,witharecentresurgentinterestbroughtaboutby forotherGram-positivebacteria,P.acnesmaybesensitivetokill- 0
1
theemergenceofantibioticresistanceinmanypathogenicbacte- ingbyexogenousapplicationofpurifiedendolysin,withpotential 9
ria(34).Phageshavebeenadministeredtohumanswithouttox- antibacterialapplication(42).Theconservednatureofthispro- b
icity(35,36),andtheefficacyofphagetherapyhasbeendemon- teinalsostronglyimpliesthat,regardlessofwhichoneischosen, y

g
stratedinanumberofanimalmodelsforinfectionswithShigella theendolysinfromanyP.acnesphageshouldhaveactivityagainst u
dysenteriae(37),Escherichiacoli(38,39),andcutaneousStaphylo- most,ifnotall,isolatesofP.acnesbacteria.Importantly,inother e
s
coccusaureus(40,41).TheFDAhasalsoapprovedtheuseofLis- phage-hostsystems,resistancetopurifiedrecombinantendolysin t
teriaphageLMP-102tocontrolcontaminationofmeat(34). proteinhasnotbeenobserved,evenafterrepeatedroundsofex-
Onepossibleissuewithphagetherapyforacneisapotentialfor posure,perhapsbecausetheyhaveevolvedtotargetessentialcom-
theemergenceofresistantstrains.Wehavefoundthatwhenbroad ponentsofthecellwall(42).
phageresistanceisobserved,itisgenerallycorrelatedwith,and In summary, by studying the diversity of P.acnes bacterio-
thereforelikelyattributableto,thepresenceofaCRISPRelement. phages,wehavegainedinsightintothemicrobiomeofaunique
Clearly,definitiveexperimentsdemonstratingtheroleofP.acnes anatomical niche, the pilosebaceous unit. The unexpected but
CRISPRelementsinprovidingimmunitytophagelysisandelu- strikinglimiteddiversityofthesephages,whichcanlysethemajor
cidatingtherateatwhichP.acnesstrainsmayacquireCRISPR- bacterialinhabitantofthepilosebaceousunit,P.acnes,likelyre-
mediated resistance are needed. There are, however, potential flectsthecoevolutionofvirusandbacteriainadistinctandre-
strategiestocircumventCRISPR-mediatedresistance,including stricted microenvironment characterized by high lipid content
thepossibilityoftreatmentwithacocktailofphages,selectingfor andanaerobicconditions.P.acnesisalsofoundontheskinsur-
escape mutants with alterations in their protospacers and/or faceaspartofamorecomplexmicrobiome(43,44),yettheP.ac-
PAMs,orusingengineeredphagesinwhichpotentialmatchesto nesbacteriophagesisolatedfromdifferentanatomicsitesappear
theknownspacershasbeeneliminated. tobesimilarintermsoftheamidaseandmajortailproteingenes
10 ® mbio.asm.org September/October2012 Volume3 Issue5 e00279-12