Pan-genome and comparative genome analyses of Propionibacterium acnes reveal its genomic

14 Pages·2017·5.2 MB·English

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Open Access Publications
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Shuta Tomida
University of California - Los Angeles
Lin Nguyen
University of California - Los Angeles
Bor-Han Chiu
University of California - Los Angeles
Jared Liu
University of California - Los Angeles
Erica Sodergren
Washington University School of Medicine in St. Louis
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Tomida, Shuta; Nguyen, Lin; Chiu, Bor-Han; Liu, Jared; Sodergren, Erica; Weinstock, George M.; and Li,
Huiying, "Pan-genome and comparative genome analyses of Propionibacterium acnes reveal its genomic
diversity in the healthy and diseased human skin microbiome." mBio. 4, 3. e00003-13. (2013).
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Shuta Tomida, Lin Nguyen, Bor-Han Chiu, Jared Liu, Erica Sodergren, George M. Weinstock, and Huiying Li
This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/
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RESEARCHARTICLE
Pan-Genome and Comparative Genome Analyses of Propionibacterium
acnes Reveal Its Genomic Diversity in the Healthy and Diseased
Human Skin Microbiome
ShutaTomida,aLinNguyen,aBor-HanChiu,aJaredLiu,aEricaSodergren,bGeorgeM.Weinstock,bHuiyingLia,c
DepartmentofMolecularandMedicalPharmacology,CrumpInstituteforMolecularImaging,DavidGeffenSchoolofMedicine,UCLA,LosAngeles,California,USAa;The
GENOMEInstituteatWashingtonUniversityinSt.Louis,St.Louis,Missouri,USAb;UCLA-DOEInstituteforGenomicsandProteomics,UCLA,LosAngeles,California,USAc
ABSTRACT Propionibacteriumacnesconstitutesamajorpartoftheskinmicrobiomeandcontributestohumanhealth.However,
ithasalsobeenimplicatedasapathogenicfactorinseveraldiseases,includingacne,oneofthemostcommonskindiseases.Its
pathogenicrole,however,remainselusive.Tobetterunderstandthegeneticlandscapeanddiversityoftheorganismanditsrole
inhumanhealthanddisease,weperformedacomparativegenomeanalysisof82P.acnesstrains,69ofwhichweresequencedby
ourgroup.ThiscollectioncoversallknownP.acneslineages,includingtypesIA,IB,II,andIII.Ouranalysisdemonstratedthat
althoughtheP.acnespan-genomeisopen,itisrelativelysmallandexpandsslowly.Thecoreregions,sharedbyallthesequenced
genomes,accountedfor88%oftheaveragegenome.Comparativegenomeanalysisshowedthatwithineachlineage,thestrains
isolatedfromthesameindividualsweremorecloselyrelatedthantheonesisolatedfromdifferentindividuals,suggestingthat
clonalexpansionsoccurredwithineachindividualmicrobiome.Wealsoidentifiedthegeneticelementsspecifictoeachlineage.
DifferencesinharboringtheseelementsmayexplainthephenotypicandfunctionaldifferencesofP.acnesinfunctioningasa
commensalinhealthyskinandasapathogenindiseases.OurfindingsofthedifferencesamongP.acnesstrainsatthegenome
levelunderscoretheimportanceofidentifyingthehumanmicrobiomevariationsatthestrainlevelinunderstandingitsassocia-
tionwithdiseasesandprovideinsightintonovelandpersonalizedtherapeuticapproachesforP.acnes-relateddiseases.
IMPORTANCE Propionibacteriumacnesisamajorhumanskinbacterium.Itplaysanimportantroleinmaintainingskinhealth.
However,ithasalsobeenhypothesizedtobeapathogenicfactorinseveraldiseases,includingacne,acommonskindiseaseaf-
fecting85%ofteenagers.Tounderstandwhetherdifferentstrainshavedifferentvirulentpropertiesandthusplaydifferentroles
inhealthanddiseases,wecomparedthegenomesof82P.acnesstrains,mostofwhichwereisolatedfromacneorhealthyskin.
Weidentifiedlineage-specificgeneticelementsthatmayexplainthephenotypicandfunctionaldifferencesofP.acnesasacom-
mensalinhealthandasapathogenindiseases.Byanalyzingalargenumberofsequencedstrains,weprovidedanimprovedun-
derstandingofthegeneticlandscapeanddiversityoftheorganismatthestrainlevelandatthemolecularlevelthatcanbefur-
therappliedinthedevelopmentofnewandpersonalizedtherapies.
Received4January2013 Accepted26March2013 Published30April2013
CitationTomidaS,NguyenL,ChiuB,LiuJ,SodergrenE,WeinstockGM,LiH.2013.Pan-genomeandcomparativegenomeanalysesofPropionibacteriumacnesrevealits
genomicdiversityinthehealthyanddiseasedhumanskinmicrobiome.mBio4(3):e00003-13.doi:10.1128/mBio.00003-13.
EditorMartinBlaser,NewYorkUniversity
Copyright©2013Tomidaetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution-Noncommercial-ShareAlike3.0Unported
license,whichpermitsunrestrictednoncommercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
AddresscorrespondencetoHuiyingLi,[email protected].
Propionibacterium acnes is a major commensal of the human strainsofaspeciesratherthantheentirespecies,inlinewiththe
skin.Itcontributestomaintainingskinhealthbyinhibiting studiesofotherdiseases(5,6).
theinvasionofcommonpathogens,suchasStaphylococcusaureus P.acneshasbeenclassifiedintothreemajorgeneticlineages.
andStreptococcuspyogenes.Itdoessobyhydrolyzingtriglycerides StudiesbyJohnsonandCummins(7)firstrevealedtwodistinct
andreleasingfreefattyacidthatcontributestotheacidicpHofthe phenotypesofP.acnes,knownastypesIandII,thatwereabletobe
skinsurface(1).Ontheotherhand,P.acneshasbeenhistorically distinguishedbasedonserologicalagglutinationtestsandcellwall
linked to acne vulgaris, a chronic inflammatory disease of the sugar analysis. McDowell et al. (8) differentiated types I and II
pilosebaceous unit affecting more than 85% of adolescents and P.acnesbymonoclonalantibodytyping.Furthermore,theirphy-
young adults (2). Our metagenomic study previously demon- logeneticanalysisofP.acnesstrainsbasedonthenucleotidese-
stratedthatP.acneswasadominantbacteriuminthepiloseba- quences of the recA gene and a more-variable hemolysin/cyto-
ceousunitinbothhealthyindividualsandacnepatients(3,4).At toxingene(tly)demonstratedthattypesIandIIrepresentdistinct
the strain level, however, the population structures of P. acnes lineages.Theirinvestigationsalsorevealedthatstrainswithinthe
were different between the two groups. Our findings suggested typeIlineagecanbefurthersplitintotwoclades,knownastypes
thatmicrobe-relatedhumandiseasesareoftencausedbycertain IA and IB (8, 9). An additional phylogenetic group of P. acnes,
May/June2013 Volume4 Issue3 e00003-13 ® mbio.asm.org 1
Tomidaetal.
knownastypeIII,wasdescribedlater(10).Recentstudiesbased publiclyavailable(14,17–22)(Table1).Theaveragegenomesize
onmultilocussequencetyping(MLST)furthersubdividedP.ac- of these 13 P. acnes strains was 2.51 Mb (ranging from 2.48 to
nes into closely related clusters—IA1, IA2, IB, IC, II, and III or 2.56Mb),andtheGCcontentwas60%,encoding2,319ORFson
I-1a, I-1b, I-2, II, and III—some of which were associated with average(rangingfrom2,233to2,412).These13genomesinclude
variousdiseases,includingacne(11–13). sixRT1strains,twoRT2strains,fourRT3strains,andoneRT5
ThefirstcompletegenomesequenceofP.acnes,KPA171202,a strain; however, no genomes of RT4, RT6, RT8, and type III
typeIBstrain,providedinsightintothepathogenicpotentialof strains were available. Our sequencing effort significantly in-
thisGram-positivebacterium(14).Thegenomeis2.56Mbwith creasedthenumberofgenomesforeachP.acneslineageaswellas
60%GCcontent.Itencodes2,333openreadingframes(ORFs), thenumberoflineagescovered.
includingmultiplegeneproducts,suchassialidases,neuramini- P.acnespan-genome.Todeterminethegeneticlandscapeof
dases, endoglycoceramidases, lipases, and pore-forming factors, P.acnes,weestimatedthepan-genomebasedonthe82P.acnes
thatareinvolvedindegradinghostmolecules.However,these- genomes.Wefirstestimatedthenumberofnewgenesthatwould
quenceofasinglegenomedoesnotreflectthegeneticlandscapeof bediscoveredbysequencingadditionalP.acnesgenomesbyusing
theorganismandhowgeneticvariationsamongstrainsdetermine a power law regression analysis, n (cid:1) (cid:1)N(cid:2)(cid:2)(24) (Fig. 1A). Our
theirvariousphenotypesandpathogenicproperties. analysisidentified(cid:2)as0.788.Theaveragenumberofnewgenes
Tobetterunderstandthehumanmicrobiomevariationsatthe addedbyanovelgenomewasthreewhenthe82ndgenomewas
strainlevel,aspartoftheHumanMicrobiomeProject(HMP)(15, added.WethenestimatedthenumberofP.acnespan-genesthat
16),wepreviouslygeneratedthereferencegenomesequencesof wouldbeaccumulatedbysequencingadditionalP.acnesgenomes
66P.acnesstrainsselectedfromacollectionofover1,000strains byusingapowerlawregressionanalysis,n(cid:1)(cid:1)N(cid:3)(Fig.1B).The
isolatedfromacohortofhealthysubjectsandacnepatients(4). exponent(cid:3)was0.067,andP.acneshad3,136pan-genes(n(cid:1)82).
These66strainsrepresentthemajorlineagesofP.acnesfoundon Basedontheseresults,thepan-genomeofP.acnesisdefinedas
thehumanskin,includingtypesIA,IB,andII.Tocoverallthe open,astheexponent(cid:2)waslessthanoneand(cid:3)wasgreaterthan
mainP.acneslineagesintheanalysis,wenewlysequencedthree zero(24).However,since(cid:2)wasclosetooneand(cid:3)wascloseto
additional P. acnes strains, including the first available type III zero,wespeculatethatthisorganismevolvedtightlywithoutlarge
P.acnesgenome.ThirteenP.acnesgenomessequencedbyother expansions.
research groups (14, 17–22) were also available at the time of PhylogeneticrelationshipsamongtheP.acnesgenomes.Ge-
analysis.Withatotalof82genomes,weperformedacomparative nomecomparisonofthe82P.acnesstrainsrevealedthat2.20Mb
genomeanalysistocharacterizethepan-genomeofP.acnes,the (88%oftheaveragegenome)weresharedbyalltheP.acnesge-
phylogenetic relationships among different lineages, the micro- nomes,asectionwhichwerefertoasthe“coreregions.”Within
evolutionofthestrainsinthesameindividualmicrobiome,and the core regions, 123,223 unique single nucleotide polymor-
thegeneticelementsspecifictoeachlineageandtheirassociations phisms (SNPs) were detected among the strains. Twenty-
withhealthanddisease. sevenpercentoftheSNPswereuniquetotypeI,22%wereunique
to type II, and 22% were unique to type III (see Fig. S1 in the
RESULTS supplementalmaterial).Weconstructedaphylogenetictreebased
P.acnesstrainsandgeneralgenomefeatures.Tounderstandthe onthe123,223SNPsinthecoreregions(Fig.2).Thetreeshowed
genomicdiversityofthisimportantskincommensalatthestrain thattherecAtypeclassificationofthestrainswasconsistentwith
level,weanalyzedthegenomesof69P.acnesstrainsthatwese- themajorcladesbasedonthegenomes,whichwenamedclades
quenced.Amongthem,67P.acnesstrainswereisolatedfromthe IA-1,IA-2,IB-1,IB-2,IB-3,IC,II,andIII(4).TherecAtypeIA,IB,
skinofhealthyindividualsandacnepatients(4)andtwoP.acnes andIIstrains,exceptHL097PA1andPRP-38,wereallclustered
strains,HL201PA1andHL202PA1,wereisolatedfromrefractory together within each type. The only recA type III strain,
endodonticlesions(23)(Table1).These69strainscoverallthe HL201PA1, formed a separate branch from type I and type II
knownP.acneslineagesisolatedtodate.Weclassifiedthestrains strains.Thetreealsoshowedthatthe16SrRNAribotypesofthe
basedontheir16SrRNAsequences.Eachunique16SrRNAse- strains were consistent with the phylogenetic relationships in-
quencewasdefinedasaribotype(RT).AllthesequencedP.acnes ferredfromthegenomesequences.MostoftheRT1strainswere
genomes had three identical copies of 16S rRNA. Based on our clustered in clade IA-1, while all the RT4 and most of the RT5
metagenomicstudyoftheskinmicrobiomeassociatedwithacne strainswereclusteredincladeIA-2.AllsixRT8strainswereclus-
(4),amongthetoptenmajorribotypes,RT1,RT2,andRT3were teredtogetherincladeIB-1.AlltheRT3andRT16strainsexcept
the most abundant and found in both healthy individuals and SK187 were clustered together in clade IB-2. HL030PA1 and
acnepatientswithnosignificantdifferences.RT4,RT5,andRT8, KPA171202wereclusteredtogetherwith6609asadistinctIB-3
however, were enriched in acne patients, while RT6 was found cladewhichhasdifferentphenotypesfromstrainsincladesIB-1
mostly in healthy individuals. Our 69 strains included 19 RT1 and IB-2, including the lack of expression of dermatan sulfate
strains,fiveRT2strains,15RT3strains,eightRT4strains,seven binding adhesions (13). HL097PA1 and PRP-38, which are
RT5strains,fourRT6strains,sixRT8strains,fourstrainsofmi- antibiotic-resistantstrains,wereclusteredtogetherandwereclas-
norribotypes,andonetypeIIIstrain.Thesequencetypesofallthe sifiedasanoveltypeICrecentlynamedbyMcDowelletal.(22).
strainswereassignedbasedontwopublishedMLSTschemes(11– AlltheRT2strainswereclusteredincladeII,distantfromcladeI,
13)andareshowninTableS1inthesupplementalmaterial.The togetherwithRT6strains.HL202PA1,whichisanRT6strainand
averagegenomesizewas2.50Mb(rangingfrom2.46to2.58Mb), wasisolatedfromanoralsite,wasnotmuchdifferentfromthe
andtheGCcontentwas60%.Onaverage,eachgenomeencoded skinRT6isolatesandwasclusteredtogetherwiththem.Thephy-
2,626ORFs(rangingfrom2,393to2,806)(Table1). logenetic tree based on the core genome regions demonstrated
Ouranalysisincluded13additionalP.acnesgenomesthatwere that16SribotypingcanbeusedforP.acnesstrainidentification
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ComparativeGenomeAnalysisofP.acnes
TABLE1 Generalfeaturesofthe82P.acnesgenomes
Sequencing Genome No.of
Genomeno. Strainname Origin Ribotype recAtype method size(Mb) GC(%) ORFs
1 HL001PA1 Skin 2 II Illumina 2.49 60.0 2,661
2 HL002PA1 Skin 3 IB Illumina 2.48 60.1 2,549
3 HL002PA2 Skin 1 IA Illumina 2.48 60.1 2,594
4 HL002PA3 Skin 1 IA Illumina 2.48 60.1 2,565
5 HL005PA1 Skin 4 IA Illumina 2.53 60.2 2,724
6 HL005PA2 Skin 1 IA Illumina 2.48 60.0 2,645
7 HL005PA3 Skin 1 IA Illumina 2.48 60.1 2,579
8 HL005PA4 Skin 3 IB Illumina 2.47 60.0 2,607
9 HL007PA1 Skin 4 IA Illumina 2.53 60.2 2,691
10 HL013PA1 Skin 3 IB Illumina 2.48 60.0 2,618
11 HL013PA2 Skin 1 IA Illumina 2.48 60.1 2,588
12 HL020PA1 Skin 1 IA Illumina 2.48 60.1 2,554
13 HL025PA1 Skin 1 IB Illumina 2.54 60.1 2,581
14 HL025PA2 Skin 3 IB Illumina 2.48 60.0 2,616
15 HL027PA1 Skin 3 IB Illumina 2.53 60.1 2,711
16 HL027PA2 Skin 1 IA Illumina 2.48 60.1 2,629
17 HL030PA1 Skin 1 IB Illumina 2.54 60.0 2,662
18 HL030PA2 Skin 3 IB Illumina 2.51 60.1 2,647
19 HL036PA1 Skin 532 IA Illumina 2.48 60.1 2,575
20 HL036PA2 Skin 532 IA Illumina 2.48 60.1 2,565
21 HL036PA3 Skin 1 IA Illumina 2.48 60.1 2,601
22 HL037PA1 Skin 3 IB Illumina 2.48 60.1 2,617
23 HL038PA1 Skin 4 IA Illumina 2.54 60.2 2,663
24 HL042PA3 Skin 6 II Roche/454 2.53 60.1 2,610
25 HL043PA1 Skin 5 IA Illumina 2.53 60.2 2,698
26 HL043PA2 Skin 5 IA Illumina 2.53 60.2 2,688
27 HL045PA1 Skin 4 IA Illumina 2.53 60.2 2,692
28 HL046PA1 Skin 3 IB Illumina 2.48 60.0 2,599
29 HL046PA2 Skin 1 IA Illumina 2.53 60.1 2,692
30 HL050PA1 Skin 3 IB Illumina 2.48 60.0 2,652
31 HL050PA2 Skin 1 II Illumina 2.46 60.1 2,581
32 HL050PA3 Skin 3 IB Illumina 2.48 60.0 2,558
33 HL053PA1 Skin 4 IA Illumina 2.53 60.2 2,632
34 HL053PA2 Skin 8 IB Illumina 2.51 60.1 2,664
35 HL056PA1 Skin 4 IA Illumina 2.48 60.1 2,581
36 HL059PA1 Skin 16 IB Illumina 2.48 60.1 2,570
37 HL059PA2 Skin 16 IB Illumina 2.48 60.0 2,535
38 HL060PA1 Skin 2 II Illumina 2.48 60.1 2,601
39 HL063PA1 Skin 1 IA Illumina 2.48 60.1 2,520
40 HL063PA2 Skin 3 IB Illumina 2.53 60.0 2,669
41 HL067PA1 Skin 3 IB Illumina 2.53 60.1 2,633
42 HL072PA1 Skin 5 IA Illumina 2.53 60.1 2,594
43 HL072PA2 Skin 5 IA Illumina 2.53 60.1 2,672
44 HL074PA1 Skin 4 IA Illumina 2.53 60.2 2,723
45 HL078PA1 Skin 1 IA Illumina 2.58 60.1 2,785
46 HL082PA1 Skin 8 IB Illumina 2.50 60.1 2,648
47 HL082PA2 Skin 2 II Illumina 2.51 60.0 2,644
48 HL083PA1 Skin 1 IA Illumina 2.48 60.1 2,575
49 HL083PA2 Skin 3 IB Illumina 2.48 60.0 2,633
50 HL086PA1 Skin 8 IB Illumina 2.53 60.1 2,610
51 HL087PA1 Skin 3 IB Illumina 2.48 60.1 2,584
52 HL087PA2 Skin 1 IA Illumina 2.48 60.1 2,572
53 HL087PA3 Skin 3 IB Illumina 2.52 60.1 2,619
54 HL092PA1 Skin 8 IB Illumina 2.50 60.1 2,590
55 HL096PA1 Skin 5 IA Roche/454 2.49 60.0 2,393
56 HL096PA2 Skin 5 IA Illumina 2.56 60.1 2,638
57 HL096PA3 Skin 1 IA Illumina 2.56 60.0 2,651
58 HL097PA1 Skin 5 IC Illumina 2.52 60.2 2,617
59 HL099PA1 Skin 4 IA Illumina 2.58 60.1 2,735
60 HL100PA1 Skin 1 IA Illumina 2.48 60.1 2,562
61 HL103PA1 Skin 2 II Illumina 2.48 60.1 2,546
62 HL106PA1 Skin 2 II Illumina 2.48 60.1 2,533
63 HL106PA2 Skin 1 IA Illumina 2.48 60.1 2,567
64 HL110PA1 Skin 8 IB Illumina 2.51 60.1 2,667
(Continuedonfollowingpage)
May/June2013 Volume4 Issue3 e00003-13 ® mbio.asm.org 3
Tomidaetal.
TABLE1 Generalfeaturesofthe82P.acnesgenomes
Sequencing Genome No.of
Genomeno. Strainname Origin Ribotype recAtype method size(Mb) GC(%) ORFs
65 HL110PA2 Skin 8 IB Illumina 2.50 60.1 2,614
66 HL110PA3 Skin 6 II Illumina 2.54 60.1 2,806
67 HL110PA4 Skin 6 II Illumina 2.54 60.1 2,724
68 HL201PA1 Refractoryendodontic Notassigned III Illumina 2.48 60.1 2,629
lesion
69 HL202PA1 Refractoryendodontic 6 II Illumina 2.56 60.0 2,821
lesion
70 KPA171202 Plate 1 IB Sanger 2.56 60.0 2,297
71 J139 Skin 2 II Roche/454 2.48 60.0 2,364
72 J165 Skin 1 IA Roche/454 2.50 60.0 2,403
73 SK137 Skin 1 IA Roche/454 2.50 60.0 2,352
74 SK187 Skin 3 IB Roche/454 2.51 59.0 2,381
75 SK182 Skin 1 IA Roche/454 2.48 60.0 2,338
76 266 Pleuropulmonary 1 IA Roche/454 2.50 60.0 2,412
infection
77 6609 Skin 1 IB Solid 2.56 60.0 2,358
78 ATCC11828 Abscess 2 II Solid 2.49 60.0 2,260
79 P.acn17 Cornealscrape 3 IB Solid 2.52 60.0 2,266
80 P.acn31 Aqueoushumor 3 IB Solid 2.50 60.0 2,247
81 P.acn33 Aqueoushumor 3 IB Solid 2.49 60.0 2,236
82 PRP-38 Skin 5 IC Solid 2.51 60.0 2,233
andclassification.The16Sribotypesofthestrainsarealsoconsis- SNPdistributioninthecoregenomeregions.Tounderstand
tentwiththeclonalcomplexesassignedbasedonthetwoMLST whetherthereare“hotspots”formutationand/orrecombination
schemes (11, 13) with a similar resolution (see Table S1 in the intheP.acnesgenomes,wedeterminedwhethertheSNPswere
supplementalmaterial).16Sribotypingprovidesamuchhigher randomlydistributedthroughoutthegenomesorenrichedinpar-
resolutionthanrecAtyping,andinthemeantime,itismuchsim- ticularregions.WecalculatedthefrequencyofSNPsineachpro-
plerandfaster,withonlyonegenerequired,thanMLST,whichis teincodinggeneinthecoreregions.Theaveragerateofpolymor-
alaboriousprocessgenerallyrequiring7to9genes. phicsitesinthecoreregionswas5.3%,i.e.,5.3uniqueSNPsin
Thelargenumberofgenomesequencesthatwegeneratedal- every100bp.Thisrateiscomparabletotheonesfoundinmultiple
lowed us to analyze the P. acnes pan-genome at the clade level. gutbacterialgenomes(25).Amongthe1,888genesencodedinthe
Clades IA, IB, and II had 36, 33, and 12 genomes, respectively. coreregions,55geneshadhigherSNPfrequencieswithmorethan
Basedonthepowerlawregressionanalysesdescribedabove,we twostandarddeviations(SD)and47geneshadhigherfrequencies
determinedthatatthecladelevel,P.acnesalsohasanopenpan- with more than three SD (Fig. 3A). Using the Kolmogorov-
genomeforrecAtypeIAclade,typeIBclade,andtypeIIcladewith Smirnov(K-S)test,wedemonstratedthatthese102highlymu-
limitedexpansions(seeFig.S2inthesupplementalmaterial).The tated genes were not randomly distributed throughout the ge-
expansionrateswerenotsignificantlydifferentamongtheclades nome (P (cid:3) 0.01) (Fig. 3B). This suggests that P. acnes has an
andweresimilartotheoneatthespecieslevel.Thissuggeststhat evolutionaryriskmanagementstrategy.Basedontheclustersof
allthemajorlineagesofP.acnesevolvedatasimilarrate. orthologousgroups(COG)categories,thefunctionsofthese102
A B
160 3400
n) 140 n = 25.9N-0.788 n) n = 2335N0.067
es ( 120 me ( 3200
w gen 100 geno 3000
Number of ne 24680000 P. acnespan- 222468000000
0 2200
0 20 40 60 80 0 20 40 60 80
Number of genomes (N) Number of genomes (N)
FIG1 P.acnespan-genome.(A)Apowerlawregressionfornewgenes(n)discoveredwiththeadditionofnewgenomesequences(N).(B)Apowerlaw
regressionfortotalgenes(n)accumulatedwiththeadditionofnewgenomesequences(N).Circlesarethemediansofnfor200simulations.Errorbarsindicate
thestandarddeviationsforthe200simulations.
4 ® mbio.asm.org May/June2013 Volume4 Issue3 e00003-13
ComparativeGenomeAnalysisofP.acnes
FIG2 Aphylogenetictreeof82P.acnesstrainsandthedistancematrixamongthestrains.Aphylogenetictreeof82P.acnesstrainswasconstructedbasedon
the123,223SNPsinthecoreregions(2.20Mb).ThedistancesbetweenstrainswerecalculatedasnucleotidesubstitutionratesatallSNPsites,coloredaccording
tothescalebar.Thestrainsfromthesameindividuals(SSIs)belongingtothesamelineagesaremarkedwith“(cid:5).”
genesshowedasimilardistributionasthoseofall1,888genesin genomic analysis, we demonstrated that most individuals har-
thecoreregions.Therewasnoenrichmentofaparticularfunc- boredmultipleP.acnesstrainsfromdifferentlineages(4).How-
tionalcategoryinthesefrequentlymutatedgenes. ever,itisnotknownwhetherthestrainsofthesamelineageinthe
Wefurtherdeterminedwhetherthemutationsinthecorere- same individual were derived from the same ancestor. Genome
gionswereunderselectionbycalculatingtheratioofnonsynony- sequencesofthestrainsisolatedfromthesamesamplesmakeit
mous (NS) to synonymous (S) SNPs for the 1,888 genes. The possibletoexaminewhetherthestrainsfromthesameindividuals
averagerateofNSmutationswas38%.Amongthe1,888genes,54 (SSIs)evolvedfromthesameoriginviaclonalexpansion.Our69
geneshadhigherNSmutationrateswithmorethantwoSDand13 sequencedP.acnesstrainsincluded49SSIs:13duets(i.e.,13pairs
genes had higher NS mutation rates with more than three SD ofstrainsisolatedfrom13individuals),fivetrios,andtwoquar-
(Fig. 3C). These 67 genes were randomly distributed in the ge- tets. Twenty-three SSIs were clustered in the same clades, with
nomeandnotparticularlyenrichedincertainregions(P(cid:4)0.05 nineincladeIA-1,fourincladeIA-2,twoincladeIB-1,sixinclade
withtheK-Stest)(Fig.3D).Mostofthe102geneswithhigherSNP IB-2,andtwoincladeII.Wecalculatedthedistance(substitution
frequenciesdidnotoverlapwiththese67genes,suggestingthat rateatthe123,223SNPsitesinthecoreregions)betweeneachpair
independentevolutionaryeventsmightleadtothesegenealterna- ofSSIs(Fig.2).TheaveragedistanceoftheSSIsincladeIA-1was
tions.OnlytengeneshadbothhighSNPfrequenciesandhighNS 0.0014, while that of strains from different individuals in clade
mutationrates,allannotatedashypotheticalproteins. IA-1was0.0064(P(cid:3)0.001).Consistentresultswereobservedin
Evolutionaryrelationshipsofthestrainsisolatedfromthe otherclades,includingIA-2,IB-1,IB-2,andII(seeFig.S3Ainthe
sameindividuals.ThelargenumberofP.acnesstrainsisolated supplemental material). This demonstrated that the SSIs in the
fromthecohortofacnepatientsandhealthyindividualsallowed samelineageweresignificantlymoresimilartoeachotherthanthe
ustoinvestigatewhethertheP.acnesstrainsinhairfolliclesfrom strains isolated from different individuals, suggesting that they
the same individual were clonal. Based on our previous meta- wereclonalineachindividual.AmongtheRT4andRT5strains
May/June2013 Volume4 Issue3 e00003-13 ® mbio.asm.org 5
Tomidaetal.
30.0%
A c
hi 25.0%
p
r 20.0%
os
mte15.0%
ysi
ol 10.0%
p 5.0%
%
0.0%
0.3
B 0.25
c
0.2
0.15
0.1
0.05
0
-0.05
-0.1
100.0%
C
80.0%
a
t 60.0%
u
m
40.0%
S
N 20.0%
%
0.0%
0.3
D 0.25
c
0.2
0.15
0.1
0.05
0
-0.05
-0.1
0 200 400 600 800 1000 1200 1400 1600 1800 2000
1,888 ORFs encoded in the core regions
Average + 2SD >Average + 2SD >Average + 3SD
FIG3 SNPdistributioninthecoreregions.(A)SNPfrequencies(percentagesofpolymorphicsites)ofthegenesinthecoreregions.(B)K-Sstatisticsforgenes
thathadhigherSNPfrequencieswithmorethantwostandarddeviations(SD).(C)Nonsynonymousmutationfrequenciesofthegenesinthecoreregions.(D)
K-Sstatisticsforgenesthathadhighernonsynonymousmutationfrequencieswithmorethan2standarddeviations.
within clade IA-2, however, the average distance between SSIs that antibiotic-resistant propionibacteria were transmissible be-
(0.0004)wasnotsignificantlydifferentfromtheaveragedistance tweenacne-proneindividuals,includingdermatologists(26),as
betweenstrainsfromdifferentindividuals(0.0017)(P(cid:1)0.072). mostoftheantibiotic-resistantP.acnesstrainsbelongtoRT4and
Moreover, the average distance between RT4/RT5 strains from RT5(4).OuranalysisofSSIsfurthersupportsthetheorythatRT4
differentindividuals(0.0017)wassimilartotheaveragedistance andRT5strainsmaybeapathogenicfactorinacne.
betweentheSSIsincladeIA-1(0.0014)andevenshorterthanthe Todeterminewhetherthedistancesbetweenstrainpairsfrom
averagedistancesbetweentheSSIsincladesIB-1(0.0059),IB-2 thesameindividualsbutbelongingtodifferentlineagesweredif-
(0.0019),andII(0.0022)(Fig.S3A).Thissuggeststhatalthough ferentfromthosebetweenrandomstrainpairs,wecalculatedthe
isolated from different individuals, these RT4 and RT5 strains distances of any pair of SSIs from different clades. The average
seemedtobeclonalandhadevolvedfromthesamerecentances- distanceoftheSSIsbetweencladesIA-1andIA-2(i.e.,HL005PA3
tor. We observed a similar relationship between the two RT5 versusHL005PA1,HL005PA2versusHL005PA1,HL096PA3ver-
strainsincladeIC,inwhichHL097PA1andPRP-38,isolatedfrom sus HL096PA1, and HL096PA3 versus HL096PA2) was 0.039,
different individuals, were closely related to each other with a similartothatoftheisolatesfromdifferentindividuals(0.040).
distanceof0.0012.Ourmetagenomicstudyhasdemonstrateda Similarresultswereobtainedforallothercladepaircomparisons
strongassociationofstrainsofRT4andRT5withacne(4).The (seeFig.S3Binthesupplementalmaterial).Theseresultsdemon-
clonalityofthesestrainsisolatedfromdifferentindividualssug- stratedthattheSSIsfromdifferentcladesweresimilarlydifferent
geststhatRT4andRT5strainsmaybetransmittedamongindi- from each other as from the strains from different individuals.
viduals.Thisfindingisconsistentwiththepreviousclinicalreport Ouranalysissuggeststhatineachindividualmicrobiome,P.acnes
6 ® mbio.asm.org May/June2013 Volume4 Issue3 e00003-13
ComparativeGenomeAnalysisofP.acnes
Strain RT Clade
HL078PA1
HL087PA3
HL067PA1 IB-2
HL027PA1
HL072PA2
HL072PA1
HL046PA2 IA-1
J165
HL096PA3
HL096PA2
HL096PA1
HL007PA1
HL053PA1
HL043PA2
HL043PA1
HL045PA1
HHLL007348PPAA11 IA-2
HL005PA1
HL099PA1
SK137
SK182
HL106PA2
HL083PA1
HL056PA1
HL025PA1
HL005PA2
HL027PA2
HL087PA2
HL013PA2
HL036PA3
HL005PA3
HHLL100002PPAA12 IA-1
HL002PA3
HL063PA1
HL036PA2
HL020PA1
HL036PA1
266
HL037PA1
HL030PA2
HL063PA2
P.acn17
P.acn31
P.acn33
HL005PA4
HL050PA1
HL013PA1 IB-2
HL083PA2
HL002PA1
HL025PA2
HL046PA1
HL050PA3
HL059PA2
HL087PA1
HL059PA1
SK187
HL086PA1
HL053PA2
HHLL008922PPAA11 IB-1
HL110PA2
HL110PA1
HL030PA1
6609 IB-3
KPA171202
PHLR0P9378PA1 IC
HL201PA1 III
HL050PA2
HL001PA1
HL103PA1
HL106PA1
ATCC11828
HL060PA1
J139 II
HL042PA3
HL202PA1
HL082PA2
HL110PA3
HL110PA4
RT1 RT2 RT3 RT4 RT5 RT6 RT8 RT16RT532 Absent in Absent in Genomic Loci Locus Present in Genomic Locus 4
Present Absent type II type III Island 2 1 & 2 3 type II Islands 1, 3, 4
FIG4 P.acnesstrainswithineachlineageshareuniquenoncoregenomicregions.Rowsrepresent82P.acnesgenomes,andcolumnsrepresent314noncore
regionsthatarelongerthan500bp.Thegenomesandthenoncoreregionswereclusteredbasedonsimilarity.Thewidthofeachblockplottedisnotproportional
tothegenomiclengthofeachnoncoreregion.Thepresenceofanoncoreregioniscoloredinyellow,andtheabsenceiscoloredinblue.Thecolorschemesused
forRTandcladesarethesameasinFig.2.
strainsundergoclonalexpansioninthesamepopulation,while tomainlytheRT4andRT5strains(4).Theselociappeartobe
multiplestrainpopulationscanoftencoexistinthesamecommu- originatedfrommobilegeneticelements,encodeseveralvirulent
nitywithlittlerecombination. genes,andmaycontributetothevirulenceofthesestrains.Inthe
NoncoregenomeregionsintypeIstrains.Bycomparingthe meantime, the genomic island-like cluster 2 (GI2) (18) was
genomesequencesofthe82P.acnesstrains,weidentifiednoncore uniquely absent in most of the strains in this clade. Clade IB-1
genomeregionsthatwerenotsharedbyall82strains.Thetotal consistedofallRT8strains,whichwerealsohighlyassociatedwith
length of the noncore regions was approximately 0.90 Mb. The acnebasedonourmetagenomicstudy(4).Theyallhaveaunique
averageGCcontentofthenoncoreregionswasslightlylowerthan genomicisland(locus4)whichis20kblongandencodesaseries
thatofthecoreregions,58%(cid:6)6.9%,suggestingthatpartofthe ofnonribosomalpeptidesynthetases(NRPS)whichmaycontrib-
noncoreregionsmightbeoriginatedfromotherspeciesviahori- ute to increased virulence of these strains. Most RT3 and RT16
zontalgenetransfer. strainsbelongtocladeIB-2andhavefewernoncoreregionsthan
DifferentlineagesofP.acnesstrainshavedistinctnoncorere- thestrainsinotherclades.Thismaybeexplainedbythelackof
gions. Using hierarchical clustering of the noncore regions, we entirerearrangementhotspot(RHS)familyproteins,whichfunc-
showedthatthestrainsofthesameribotypeswereclusteredto- tioningenomicrearrangementsaspreviouslyimplicatedinEsch-
gether,withdistinctseparationsamongtheclades(Fig.4).Among erichiacoli(27).CladeIB-3consistedofthreestrains,including
thenoncoreregions,weidentifiedthegeneticelementsspecificto KPA171202.Threeofthefourgenomicislandsdescribedprevi-
each lineage, which may explain the phenotypic and functional ously(18),GI1,GI3,andGI4,wereuniquetothiscladeandwere
differencesofthestrainsinhealthanddisease.IncladeIA-2,we absentinallotherstrains.OuranalysissuggeststhatKPA171202,
previouslyidentifiedgenomicloci1,2,and3,whichwereunique althoughitwasthefirstsequencedcompletegenomeofP.acnes,
May/June2013 Volume4 Issue3 e00003-13 ® mbio.asm.org 7