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Tatelano ea liphatsa tsa lefutso e tsosolositse lefapha la lipatlisiso tsa mafu a tšoaetsanoang, ho senola lefu la seoa sa mafu, pathogenesis, litšebelisano-'moho le likokoana-hloko, le mokhoa oa ho iphetola ha lintho o behiloeng ho likokoana-hloko. Mycobacterium tuberculosis complex (MTBC) e nka Mycobacterium bovis e le e 'ngoe ea litho tsa eona tse ikamahanyang le liphoofolo tse bakang lefuba (TB) liphoofolong tse anyesang tsa lefatše, 'me ke mohlala o tloaelehileng oa phetoho ea baktheria. Joalo ka litho tse ling tsa MTBC, Mycobacterium bovis e nkoa e le kokoana-hloko e entsoeng ka thata, e ntseng e fetoha butle-butle, 'me ho hlakile hore ha ho na letšoao la ho kopana hape kapa ho fetisetsa liphatsa tsa lefutso tse rapameng. Mosebetsing ona, re sebelisa comparative genomics to whole genome sequence (WGS) dataset e entsoeng ka likhomo tse 70 M. tse tsoang lihlopheng tse fapaneng (Europe le Africa) ho fumana temohisiso ka mefuta-futa ea liphatsa tsa lefutso tsa likhomo M. Evolutionary power. Ho sebelisoa mekhoa e meraro e fapaneng ho hakanya matšoao a tlhophiso bocha. Lefatšeng ka bophara, palo e nyenyane ea liketsahalo tsa ho kopanya hape e 'nile ea khetholloa' me ea tiisoa ke mekhoa e 'meli e ikemetseng e nang le tšehetso e tiileng. Leha ho le joalo, ha ho bapisoa le liphetoho, ho kopanngoa hape ho na le phello e fokolang ho mefuta-futa ea M. bovis (ka kakaretso r/m = 0.037). Phapang ea r/m karolelano e fumanoeng ka har'a motsoako oa clonal oa Mycobacterium bovis sebakeng sa rona sa data e lumellana le mohopolo o akaretsang oa hore tekanyo ea ho kopanya bocha e ka fapana haholo lipakeng tsa meloko e abetsoeng mofuta o tšoanang oa taxonomic. Ho ipapisitsoe le mosebetsi ona, recombination ho Mycobacterium bovis e ke ke ea qheleloa ka thoko, ka hona e lokela ho ba taba ea boikitlaetso bo eketsehileng ba lipatlisiso tsa nakong e tlang tsa genomics, moo WGS ea li-data tse kholo tse tsoang maemong a fapaneng a lefu la seoa lefatšeng ka bophara e leng ea bohlokoa. Ka mor'a moo, tlhahlobo e eketsehileng e ile ea etsoa ho sete ea data e nyenyane ea Mycobacterium bovis (n = 42) ho tloha ho batho ba bangata ba nang le lefuba la mafu a mangata, 'me ho ile ha fumanoa li-loci tse fetang 1,800, tseo bonyane mofuta o le mong o bontšang nucleotide polymorphism (SNP) . Tse ngata (87.1%) li sebakeng sa likhoutu, 'me karo-karolelano ea lefats'e ea liphetoho tse sa tšoaneng (dN / dS) ea liphetoho tse tšoanang li feta 1.5, e leng se bontšang hore khetho e ntle ke matla a bohlokoa a ho iphetola ha lintho a sebelisoang ho M. bovis. Karolo e phahameng ea li-SNP e ile ea fumanoa ka liphatsa tsa lefutso tse nang le likarolo tse ngata tse sebetsang tsa "lipid metabolism", "cell lebota le mekhoa ea lisele", le "metabolism e bohareng le phefumoloho", e senola bokhoni ba bona ho baeloji le ho iphetola ha lintho tsa Mycobacterium bovis bohlokoa. Ho shebisisa liphatsa tsa lefutso tsa baholo-holo ba MTBC tse atisang ho fetisa liphatsa tsa lefutso tse rapameng 'me li kenyelelitsoe tsamaisong ea 3R (DNA tokiso, replication, and recombination) e senola boleng ba lefats'e ka bophara bo bobe ba teko ea ho se nke lehlakore ea Taijima, e bontšang ho hlahlojoa ha nako e fetileng.
Mycobacterium tuberculosis complex (MTBC) ke e 'ngoe ea lekhetho le atlehileng ka ho fetisisa la likokoana-hloko tsa baktheria le boemo bo tloaelehileng ba ho iphetola ha baktheria. Litho tsa eona li bonts'a boitsebiso bo phahameng ba nucleotide bo makatsang boemong ba genomic (> 99%)1,2. Mefuta e fapaneng ea li-ecotypes tsa MTBC li ka baka lefuba (TB), e leng lefu le tšoaetsanoang la granulomatous, mefuteng e mengata ea mefuta e mengata e tsoang ho liphoofolo tse anyesang tse nyenyane ho ea ho batho3,4,5. Hona joale, mokhoa ona o akarelletsa batho [M. Lefuba (Mtb), Mycobacterium africanum] le likokoana-hloko tse ikamahanyang le liphoofolo (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter , Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surika, "Bacillus chimpanzee") 5 le "6". M. canettii (eo hape a tsejoang e le "Nodobacter glabrata") Kakaretso ea boitsebiso ba nucleotide le mycobacteria e boletsoeng ka holimo ke 98%, 'me mosebetsi oa ho bapisa oa genomics o bontšitse hore M. canettii le MTBC kaofela ba sa tsoa kheloha ho tloha ho moholo-holo ea tloaelehileng.7 Ha re nahana ka khopolo ena, bangoli ba bang ba ka bitsa MTBC 8 M.
MTBC e hlalosoa ka mokhoa o hlophisehileng e le setsi se thata sa clonal, 'me sebopeho sa eona sa baahi se laoloa ka ho hlaka ke mefuta-futa e fokotsehileng, litšitiso, scanning e khethehileng, le genetic drift9,10. Ho nka khopolo ea ho iphetola ha lintho tse thata, joalo ka li-polymorphisms tse sieo, li ke ke tsa khutlisoa ka ho kopanya hape. Ho ipapisitsoe le ntlha ena, liketsahalo tse latellanang tsa ho hlakoloa ha genomic sebakeng sa phapang (RD) le TbD1 (Mtb specific deletion 1 region) li hlahisitsoe e le matšoao a molek'hule a MTBC evolution2,5,11. Papiso ea genomics le mosebetsi oohle oa tatellano ea liphatsa tsa lefutso (WGS) o tšehetsa karohano ea litho tse ikamahanyang le batho ka mela e robong (Mycobacterium tuberculosis L1 ho L4, L7, le L8; le Mycobacterium africanum L5, L6, le L9), leloko L2 ho ea ho L4 e arolelanoang hlakolwa TbD1,11,1. Ntle le moo, litho tse ikamahanyang le liphoofolo li reriloe hore li arolelane moholo-holo a le mong, e hlalosoang ke litlatsetso tse ikhethileng ho RD7, RD8, RD9 le RD102, 5, le 14.
Horizontal gene transfer (HGT) le liketsahalo tsa recombination li nkoa e le tse sa tloaelehang 'me li etsahala ho baholo-holo ba MTBC, ho e-na le histori e fapaneng ea setho sa MTBC kaofela15,16,17. Litlaleho tse peli tsa pele tsa Hughes le basebetsi-'moho (2002) le Gutacker le basebetsi-'moho (2006) ba ile ba fana ka maikutlo a hore liketsahalo tsa recombination li ka thusa ho theha li-polymorphisms tse tšoaeang loci e itseng ho M. lefuba strains18,19. Mabaka a ho haella ho totobetseng ha recombination ho MTBC ke: (1) ts'ebetso ea mechine le tahlehelo ea bokhoni ba HGT; (2) ho se fumanehe ha liketsahalo tsa HGT; (3) ha ho na monyetla oa liketsahalo tsa recombination ho MTBC niche14,17. Haufinyane tjena, liphuputso tse ling tsa whole-genome sequencing (WGS) tse sebelisitsoeng ho MTBC strain 20 le Mycobacterium bovis 21 li fane ka bopaki ba ho kopanya hape, ea pele e bonts'ang hore mefuta ea MTBC hangata e fapanyetsana likhechana tse nyane tsa DNA, empa ka lebaka la phapano e fokolang ea tatellano ea nucleotide, liketsahalo tsena li ntse li sa hlokomeloe.
Mycobacterium bovis ke setho sa MTBC se atisang ho fumanoa ho tsoa mehlapeng (haholo-holo likhomo), leha e ka boela ea aroloa ho liphoofolo tse hlaha tse sa tsitsang le tse etselitsoeng terata4,22,23,24. M. bovis e ile ea fetoha li-clonal complexes tse hlano tse kholo [European 1 (Eu1), European 2 (Eu2), European 3 (Eu3), African 1 (Af1) le Africa 2 (Af2)], ho ea ka spoligotyping profile, ho hlakoloa ka ho khetheha And single nucleotide polymorphisms (SNPs) 25, 7, 292 in specific genes. Mehaho ena ea clonal e bonts'a sebopeho se fapaneng sa palo ea batho ba Mycobacterium bovis le kamano ea eona le libaka tsa tikoloho. Ntle le moo, mosebetsi oa morao-rao oa WGS o entsoeng ke Zimpel le bathusi (2020) o thehile phylogeny e ipapisitseng le SNP ea Mycobacterium bovis, e nang le liphatsa tsa lefutso tse fetang 1,900, e bonts'ang hore bonyane ho na le mela e mene e fapaneng (e bitsoang Lb1 ho Lb1 ho Lb4), ha e hlalosoe ka botlalo le geographic30 e rarahane. Bangoli bana ba ile ba etsa tlhahlobo e fapaneng ea ho ratana ha phylogeny le molek'hule, empa ha baa ka ba ithuta recombination30.
Mosebetsi o fetileng o sebelisa mekhoa e fapaneng ea limolek'hule, tse kang spoligotyping, MIRU-VNTR (mycobacterial interspersed repeat unit-variable tandem repeat number), le ho thaepa ha morao tjena SNP ho ile ha senola boemo bo itseng ba mefuta-futa ea liphatsa tsa lefutso har'a mefuta ea M. bovis 31,32,33, 34,35. Phapang ea phapang ea liphatsa tsa lefutso e fetohile sesebelisoa sa bohlokoa thutong ea lefu la seoa sa mafu, e leng thuso bakeng sa kutloisiso e tebileng ea pathogenesis, virulence le phetiso ea mafu. Ho hlaha ha mokhoa oa WGS ho fana ka monyetla oa ho senola lintlha tsa ho khanna tsa ho iphetola ha lintho tse behiloeng ke Mycobacterium bovis genome nakong ea ho ikamahanya le maemo le ho phehella ho mabotho a fapaneng le maemo a lefu la seoa.
Mosebetsing ona, re sebelisa tlhahlobo e bapisang ea genomic ho li-datasets tse fapaneng tsa Mycoplasma bovis (n=70), ho kenyelletsa le li-isolate tse tsoang ho li-clonal complexes tse fapaneng, ho fumana leseli mabapi le ts'ebetso ea ho iphetola ha lintho tsa Mycoplasma bovis, haholo-holo ho rarolla likamano tsa phylogenetic Le liketsahalo tsa ho kopanya hape. E le tlatsetso tlhahlobisong ena, sete e nyane ea M. bovis e itšehla thajana (n = 42) e fumanoeng sebakeng se tsebahalang sa lefuba se nang le batho ba bangata naheng ea Portugal 31,36 e ile ea hlahlojoa ka ho eketsehileng ho fana ka boitsebiso bo sa tsejoeng Tekano pakeng tsa karo-karolelano ea kutloisiso (dN) ho synonymous (dS) nucleotide substitutions, hammoho le liphatlalatso tsa li-nucleotide tse boletsoeng. 37,38 e fumanoeng ke baholo-holo ba MTBC ka HGT, le encode 3R (DNA repair, replication, and recombination) likarolo tsa liphatsa tsa lefutso tsa tsamaiso 39. Khetha liphatsa tsa lefutso tse fumanoang ka HGT hobane li ka 'na tsa emela li-polymorphisms tsa khale, kahoo ho lebeletsoe hore li ka ba le karolo e phahameng ea liphetoho tse tšoanang. Liphatsa tsa lefutso tse kenyelletsoeng tsamaisong ea 3R li khethiloe hobane mosebetsi o fetileng oa mathata a M. lefuba o bontšitse likhetho tse akaretsang tse mpe / tlhoekiso tse sebetsang liphatseng tsena tsa lefutso, 'me li ka' na tsa phetha karolo ea bohlokoa ho iphetola ha lintho 39. Sepheo se seng sa mosebetsi ona ke ho hlahisa boteng ba liketsahalo tsa ho hlophisa bocha. Ka lebaka lena, ha re nahana hore dataset ea rona e tsoang Portugal e na le liphatsa tsa lefutso tsa European clone complex 2 le mefuta e sa kang ea abela clone complex, re nkile qeto ea ho kenyelletsa lintlha tsa genome tse fumanehang phatlalatsa hore qetellong re fumane moemeli oa li-clone complexes tsohle, le ho Ntlafatsa matla le bophara ba liphetho.
42 Mycoplasma bovis genomes tse sa tsoa tatellano tse tsoang sebakeng sa Sepotoketsi sa lefuba le nang le batho ba bangata (lintlha tse ka tlase), tse neng li tsejoa ho latela pono ea lefu la seoa36, ke setsi sa mosebetsi ona. Ha ho nahanoa hore datha tse tsoang Portugal li na le baemeli ba li-clone tsa European 2 le mefuta e se nang li-complexes tse khethiloeng, lintlha tse fumanehang phatlalatsa tsa tatellano ea liphatsa tsa lefutso li kentsoe ho atolosa sete ea data e kenyeletsang baemeli bohle ba M. bovis clone complexes. Ka hona, mehloli e meraro ea tatellano ea liphatsa tsa lefutso e sebelisitsoe mosebetsing ona: kopano e feletseng / e hlophisitsoeng ea genome, ho fihlela ho li-scaffolds tsa 10 tse bolokiloeng ho NCBI (Setsi sa Sechaba sa Boitsebiso ba Biotechnology) (n = 15 isolates); bolokoa ka SRA ( The Illumina fastq faele ea tatellano bala archive) emela mefuta e fapaneng e rarahaneng ea M. bovis clones (n = 12 isolates)30; le liphatsa tsa lefutso tse 42 tse sa tsoa tatellano tse tsoang Portugal. Mycobacterium bovis BCG (Bacille Calmette-Guerin) e ne e qheletsoe ka thoko ho patlisiso ea NCBI. M. bovis AF2122/97 hangata e sebelisoa joalo ka genome ea litšupiso e tla kenyeletsoa pokellong ea data. Ka lebaka la ho se fumanehe ha sechaba ha tatellano eohle ea liphatsa tsa lefutso e emeloang ke 1 ea African cloning complex, le palo e nyenyane ea liphatsa tsa lefutso tse tsoang mefuteng e emelang ea Af2 le Eu1, data ea pele ea tatelano e fanoeng ke SRA e ile ea sebelisoa maemong ana. Mosebetsi oa Zimpel le basebetsi-'moho le eena (2020) o thusitse ho khetholla genome ho tsoa ho motsoako oa cloning o boletsoeng ka holimo 'me o thusitse ho khetha Mycobacterium bovis bakeng sa ho kenyelletsoa sete ea data. Bakeng sa Eu3, ho hlalosoa mofuta o le mong feela oa genome (Branger et al., 2020), kahoo genome eo re e kenyelletsang ke moemeli ea arohaneng oa Eu3 complex.
Lefats'eng ka bophara, sehlopha sena sa lintlha se kenyelletsa likhomo tse 70 tsa M. bovis tse arohaneng le mefuta e 8 ea liphoofolo, e ajoang linaheng tse 12 ho tloha ka 1985 ho ea ho 2016. Mefuta e 36 e khetholloa e le Eu2, mefuta e 7 ke Eu1, mefuta e 1 ke Eu3, mefuta e 3 ke Af1, mefuta e 4 ke Af2 le 19 ha e rarahane. Lintlha tse felletseng (ho kenyeletsoa le nomoro ea ho kena) ea Mycobacterium bovis e sebelisitsoeng thutong ena e bontšoa ho Lethathamo la 1 le Lethathamo la Tlatsetso la 1.
Liphatsa tsa lefutso tse 42 tse sa tsoa hlophisoa tsa Mycobacterium bovis tse tsoang libakeng tse nang le lefuba la liphoofolo tsa Portugal 'me li ajoa ka lilemo tse fetang 12 ke setsi sa thuto ena, kaha mekhoa e ka bang teng ea mafu a liphoofolo tse hlaha e 'nile ea behoa leihlo khafetsa 31,36 ( Tlatsetso ea Fig. 1). Ho ea ka mekhoa e latelang, mefuta ena e ne e arotsoe ho likhomo (n = 14), likhama tse khubelu (n = 16) le likolobe-moru (n = 12) ho tloha 2003 ho ea ho 2015: bokella le ho sebetsana le liphoofolo ho latela tataiso e khothalletsoang ea protocol. mahareng. Litloaelo li kenngoa ka 37 ° C 'me kholo e hlahlojoa hang ka beke bonyane libeke tse 12. Likolone li bolokoa ka kotloloho ka har'a motsoako oa glycerol ho -80ºC. Sebakeng se khethang sa Mycobacterium (Middlebrook 7H9, BD Diagnostics), lisampole tsa mantlha tse bolokiloeng li ile tsa fetisoa ka phasejeng e le 'ngoe ea in vitro ho fumana DNA ea lenaneo la WGS. Bakeng sa sena, tharollo ea stock stock e leqhoa e ile ea ntlafatsoa ka 5% sodium pyruvate le 10% ADS (50 g albumin, 20 g glucose, 8.5 g sodium chloride metsing a 1 L) ho Middlebrook 7H9 ka 37 ° C Retrain. Ka mor'a libeke tse 4 tsa kgolo, mokhoa o mocha o ile oa nchafatsoa 'me setso se ne se lula se behiloe leihlo ho fihlela khōlo e hlokomeloa. Lisele li ile tsa kotuloa ka centrifugation, pellet e ile ea tsosolosoa ka 500 µL phosphate buffered saline (PBS), e futhumatsoa ka 99 ° C ka metsotso e 30, centrifuged, 'me supernatant e bolokiloe ho -20 ° C ho fihlela WGS. Mekhoa eohle e etsoa maemong a 3 a biosafety.
Laeborari ea liphatsa tsa lefutso ea WGS e kopantsoeng ka bobeli e lokiselitsoe ho sebelisoa index e ikhethang ea sampole ka 'ngoe ea DNA, 'me e sebelisa Illumina MiSeq (2 × 250 pb) (sampole tse 40) le HiSeq (2 × 150 pb) (tse peli tse isolates) (Eurofins Genomics, Jeremane) bakeng sa ho latellana. Ho latela litaelo tsa moetsi, sebelisa Illumina Genome Analyzer e nang le sehokelo sa mojule se nang le likarolo tse peli ho latela DNA ea genomic, 'me u sebelise Nextera XT DNA Library Prep Kit ho tloha Illumina ho haha laeborari.
Ha ho nahanoa ka lintlha tse fumanoeng ho SRA (n = 12), boitsebiso ba motsoako oa clone bo ka sebelisoa e le metadata ea khatiso e lumellanang le 30, 41, 43. Ha ho nahanoa ka genome e feletseng, ntle le Mycobacterium bovis AF2122/97 le Mycobacterium bovis 3601, e leng litho tsa Eu3 2 tsa Eunal1, tse tsejoang e le litho tsa Eunal1. 29, ka ho latellana, e tšoana le genome e feletseng ea Mycobacterium tuberculosis H37Rv (NCBI accession number NC_000962.3). Ho lumellana ha liphatsa tsa lefutso ho etsoa ho sebelisoa MAFFT (lenaneo la ho tsamaisana le mefuta e mengata ea amino acid kapa nucleotide sequence, version 7.458) le parameter -addfragments48. Ebe, batla ho ba sieo ha li-clonal complexes tse fapaneng le/kapa boteng ba likarolo tsa SNP.
Mycobacterium bovis e sa tsoa tatellano (n = 42) le lipalo tsa mantlha tsa moralo o kopaneng oa genome (n = 3) li hokahana le motsoako oa genome Mycobacterium tuberculosis H37Rv ka phaephe ea vSNP le boteng ba ho hlakolwa le/kapa likarolo tsa SNP tsa clones tse fapaneng Ho ile ha etsoa lipatlisiso.
Bokella tlhahisoleseling ho tsoa ho khaello ea likarolo le / kapa boteng / ho ba sieo ha SNP le li-profiles tsa spoligotyping ho abela data ea genomic ho komporo e lumellanang ea clonal. Bakeng sa likopano tse 'nè tsa moralo, profil ea spoligotyping e ke ke ea hlalosoa, ka hona e kenyelelitsoe sehlopheng sa "no complexity".
Ts'ebetso ea bioinformatics e lateloang ke mosebetsi ona e qala ho tloha kopanong ea de novo le ho etsa 'mapa ho ea ho leano la litšupiso, ka sepheo sa ho hlahloba liketsahalo tsa ho kopanya bocha le li-polymorphisms tse khethehileng tsa genome. Setšoantšo sa 1 se fana ka lethathamo la mehato e lateloang. Bakeng sa tlhahlobo ea recombination, liphatsa tsa lefutso tsohle li sebelisetsoa ho eketsa matla a litšupiso le matšoao a amanang.
E le ho fokotsa liphoso tlhahisong ea tatellano ea tumellano ea liphatsa tsa lefutso, re ile ra qala ka ho fumana kopano ea de novo, ebe re fumana litlhophiso tsa mantlha tse ngata. Unicycler pipeline e fumaneha hona joale ho https://github.com/rrwick/Unicycler49 'me e sebelisetsoa ho etsa kopano ea de novo ea li-genome tse 54 tse latellanang (42 tse sa tsoa tatellano le lifaele tse 12 tsa fastq tse fumanoeng ho SRA). Ka bokhutšoane, pele e bokana ho tloha qalong, tlhahlobo ea boleng ba ho bala e ne e etsoa ka mofuta oa FastQC 0.11.7 (https://github.com/s-andrews/FastQC), le mofuta oa Trimmomatic 0.36 (khetho ea "ho khaola li-adapter le tatellano e khethehileng ea mabone ho tsoa ho tse baloang" le "Khaola metheo ea boleng bo tlase" haeba e le ea boleng bo tlase ho 0. sebelisoa) (http://www.usadellab.org/cms/?page= trimmomatic) 50. Joale, SPAdes optimiser49 e ile ea sebelisoa bakeng sa kopano ea genome, 'me Pilon version 1.1851 e ne e sebelisetsoa ho ntlafatsa ka mor'a kopano. Ho ile ha khethoa mokhoa o tsitsitseng oa borokho ho qoba kopano e fosahetseng, 'me boholo ba k-mer bo ile ba phenyekolloa le ho khethoa pakeng tsa 20% le 95% ea bolelele bo baloang. Latela litataiso tsa SPAdes 'me u nahane ka boholo ba ho bala, hlakola li-contigs tse nyane ho feta 300 bp,' me u thehe 20 bala botebo ba khaso cutoff ea 52. Leanong la kopano ea de novo, libaka tsa genomic tse kang proline-glutamate (PE) le proline-proline glutamate (PPE) paralogs ha lia ka tsa tlosoa.
Boleng ba kopano ea de novo bo lekoa ka phaephe ea QUAST (http://quast.sourceforge.net/quast.html), e thusang nchafatso ea contig le M. bovis AF2122/97 referense genome (NCBI accession number LT708304.1) Mapping (sheba1 Supplementary Table parameter).
Ka thuso ea phaephe ea vSNP (https://github.com/USDA-VS/vSNP), faele ea FASTQ ea M. bovis e sa tsoa hlophisoa e tsoang ho Illumina sequencing e bapisoa le M. bovis AF2122/97 reference genome (LT708304.1) . Ho ea ka likhothaletso tse ntle ka ho fetisisa tsa Genome Analysis Toolkit (GATK) 53, 54, 55 sebelisa li-parameter tse tloaelehileng tsa filthara kapa lintlha tse fapaneng tsa boima bakeng sa ho nchafatsa. Liphetho li tlhotliloeng ho sebelisoa palo e tlase ka ho fetisisa ea SAMtools ea boima ba 150 le AC = 2. Hape sebelisa Kraken (http://ccb.jhu.edu/software/kraken/) ho hlahloba lipalo ho thibela tšilafalo. Pipe ea vSNP e sebelisoang ho etsa 'mapa ho hlophisa maano mosebetsing oa rona e hlahloba letoto la li-SNP tse hlalositsoeng le lipheo, hape ha e kenyelle maemo a ts'oaetso a tsoakiloeng. Ts'ebetso ea genome e baloang e betere ho feta 99% (Letlapa la 1 la Tlatsetso).
E le ho qoba liphoso tsa 'mapa le li-SNP tse fosahetseng, sefa phapang maemong a latelang: (1) e tšehetsoa ke ho baloa ka tlase ho 20, (2) e fumanoa ka makhetlo a ka tlaase ho 0.9, (3) e bonyane Mathata a le mong, empa bonyane ho na le likheo tsa mofuta o mong. Mofuta o kopaneng oa genomics viewer (IGV) 2.4.19 (http://software.broadinstitute.org/software/igv/)56 o ile oa sebelisoa ho netefatsa li-SNP le maemo a nang le mathata a 'mapa kapa a ho tsamaisana. Kaha proline-glutamate (PE) le proline-proline glutamate (PPE) liphatsa tsa lefutso li kopitsoa haholo 'me ke karolo ea lelapa la mefuta e mengata ea liphatsa tsa lefutso, li utloisisoa habonolo ka ho latellana ha Illumina le ho senya, kahoo li khethoa The mycobacterial bioinformatics workflow e tlositse litho tsa lefuba le rarahaneng ha li sebelisa leano la ho etsa limmapa ho netefatsa ho SNP. Ka hona, re sefa liphatsa tsa lefutso tsa PE/PPE le li-indels ho tsoa tlhahlobong.
Ho ea ka Bovilist (http://genolist.pasteur.fr/BoviList/), li-SNP tsohle li arotsoe ka lihlopha tse sebetsang. Pipeline ea SnpEff (https://pcingola.github.io/SnpEff/) e sebelisetsoa ho fana ka liphello tsa SNP (liphetoho tse tšoanang kapa tse sa tsitsang). E thehile database e ncha ea Mycobacterium bovis AF2122/97 genome (LT708304.1).
The core genome multiple alignment e entsoe ka Parsnp v1.2, hajoale e fumaneha ho https://github.com/marbl/parsnp57, ho sebelisoa likopano tse 69 tse felletseng tsa genomes/draft (ka khetho -c) le M. bovis AF2122/97 (LT708304.1) e sebelisoa joalo ka sesupo. Ho ile ha etsoa litlhophiso tse 'nè tsa mantlha tse ngata: ke litho feela tsa Eu2 cloning complex (n = 37), ho kenyelletsa le litho tsohle tsa European cloning complex (n = 44), ho kenyeletsoa sebaka sa mateano sa European le African cloning complex (n = 51), 'me e kenyelletsa bovis bohle ba Mycobacterium thutong ena (n = 70).
Khokahano ea mantlha e hlahisoang ke Parsnp e sebelisetsoa ho infer monyetla o moholo oa sefate sa phylogenetic (ML) ho sebelisa CIPRES Science Gateway v3.3 (http://www.phylo.org/)58 ho sebelisa RAxML, le ho etsa liphetolelo tse tataisoang tse 1000.
Ho sebelisoa li-algorithms tse tharo tse fapaneng le lisebelisoa tsa bioinformatics ho lekola boteng ba liketsahalo tsa ho kopanya bocha ka ho ts'oana: software ea SplitsTree4, Gubbins (leloko le sa tsitsang ka ho kopanya hape ka tatellano ea li-nucleotide), le RDP4 (lenaneo la ho lemoha bocha, mofuta oa beta 4.101) software.
Mokhoa oa ho bola oa ho arohana o kentsoeng ho SplitsTree4 v4.15.1 (http://www.splitstree.org/)59 o sebelisetsoa ho bala marang-rang a se nang metso a phylogenetic, ho sebelisoa tlhahlobo ea Phi bakeng sa netefatso ea lipalo-palo, 'me moeli oa bohlokoa ke p = 0.05. Tlhahlobo ea mantlha ea li-multi-alignment ea Parsnp e sebelisoa e le ho kenya letsoho, 'me ho senyeha ha ho arohana e le mokhoa oa marang-rang ho phethahala.
Gubbins pipeline v2.3.1 (https://github.com/sanger-pathogens/gubbins60 e tsamaisana le li-parameter tsa kamehla e le mokhoa o mong oa ho lekola phello ea recombination ho Mycobacterium bovis. Algorithm e kentsoeng tšebetsong e theha bocha leloko la clone le loketseng LT708304.1) li kopane; 'me u hlahlobe boemo ba SNP lekaleng le leng le le leng la sefate ho lemoha sehlopha sa SNP se emelang ketsahalo ea ho kopanya hape.
Qetellong, molemong oa ho netefatsa ketsahalo ea tlhophiso e ncha e hlahisitsoeng ke phaephe ea Gubbins, li-algorithms tse tšeletseng tse kentsoeng ts'ebetsong ho RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 le SiScan66) li sebelisoa ho tsamaellana ea mantlha ea Parsnp tlasa maemo a kamehla. Re entse qeto ea hore bonyane litharollo tse tharo tse kentsoeng tšebetsong ho RDP4 li tlameha ho lula li bonts'a lets'oao la bohlokoa ho netefatsa ketsahalo e 'ngoe le e 'ngoe ea ho kopanya bocha.
Ha ho nahanoa hore li-Gubbins le RDP software li batla matšoao a ho kopanya hape ka ho hlahloba li-alignments tse ngata ka fensetereng ho fihlela ho 500 bp, le ho netefatsa hore ho kenyelletsoa ha liphatsa tsa lefutso tsa PE / PPE nakong ea kopano ea de novo ho ke ke ha kena-kenana le lipontšo tsa ho kopanya hape tse fumanoeng, tlhahlobo e eketsehileng e etsoa ka homolinearity Hlahloba sebaka se haufi le ketsahalo ea ho kopanya bocha. 'Mapa oa synlinear o sebelisang genome e felletseng o hahiloe ho sebelisoa MAUVE-multi-genome alignment (http://darlinglab.org/mauve/mauve.html) ho qhelela ka thoko phetisetso ea lehae ea genome kapa inversions. Ho feta moo, genome eohle e ne e sebelisetsoa ho etsa tlhahlobo ea homolinearity ka tatellano ea amino acid ka seva sa marang-rang sa SyntTax (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
Tlhahlobo e tebileng ea boitsebiso ba liphatsa tsa lefutso tse fumanoang tsamaisong ea lefuba ea Sepotoketsi e nang le batho ba bangata ke ho hlahloba polymorphism ea liphatsa tsa lefutso tse boletsoeng libukeng. Liphatsa tsa lefutso tsena ke 37,38 le gene encoding 3R e fumanoeng ke baholo-holo ba MTBC ka HGT (DNA tokiso, replication and recombination) likarolo tsa tsamaiso 39. Sebelisa ClustalX v2.1 (http://www.clustal.org/clustal2/) 'me u sebelise DnaSP v6.12.03 (http://u/dub divers divers and recombination). mefuta-futa ea nucleotide (π) le tlhahlobo ea paramethara ea teko ea D ea Tajima.
Ho na le monyetla o moholo (ML) oa sefate sa phylogenetic se thehiloeng ho 69 Mycoplasma bovis isolates le liphatsa tsa lefutso tsa referense li ile tsa fumanoa (Setšoantšo sa 2A). Ha ho bapisoa le lifate tse nang le liphatsa tsa lefutso kapa lifate tse nang le mefuta e mengata, leano lena le lumella ho hlahisa lifate tse matla haholo tse sa nkeng phapang ea genome eohle 'me kahoo li bonts'a bokhoni bo fokolang ba ho khetholla pakeng tsa mefuta ea 68,69. Sebopeho sa topological sa sefate sa ML hangata se lumellana le lihlopha tse rarahaneng tsa clones. Genome ea Eu2 e kopane ka lekala, 'me genome ea Af1 e boetse e kopantsoe hammoho (Setšoantšo sa 2A). Sephetho se boetse se lumellana le kamano e tsebahalang ea ho iphetola ha lintho ea Mycobacterium bovis, ke hore, ho na le phapang e kholo lipakeng tsa setho sa Eu1 le sehlopha se nang le li-clonal complexes le liphatsa tsa lefutso tse ling kaofela, empa motsoako oa clonal 30 ha oa boleloa. Ho se lumellane ho honyenyane pakeng tsa motsoako oa clonal le kamano e hlokometsoeng sefateng sa phylogenetic ho ka hlalosoa ke taba ea hore motsoako oa clonal o hlalosoa ho itšetlehile ka libaka tse itseng tsa genomic, ha sefate sa phylogenetic se itšetlehile ka litlhophiso tse ngata tsa liphatsa tsa lefutso tse emelang genome eohle.
Boholo ba monyetla oa sefate sa phylogenetic (GTR) se hahiloe ho latela tekano ea mantlha ea genome ea Mycobacterium bovis genome pele (A) le ka mor'a (B) ho tlosoa ha sebaka sa ho kopanya hape. Mebala ea makala e emela Mycobacterium bovis clone complex: Europe 1 e pherese, Europe 2 e khubelu, Europe 3 e putsoa, Afrika 1 ke lamunu, le Afrika 2 e tala. Sefate se metse ka metso 'me se huleloa sekaleng,' me bolelele ba lekala bo lekanngoa e le sebaka sa sebaka ka seng.
Mycobacterium tuberculosis complex e hlalosoa e le clonally evolution, 'me boholo ba bopaki bo bokelletsoeng ho theosa le lilemo bo tšehetsa khopolo ea hore liketsahalo tse tsoelang pele tsa HGT le ho kopana hape li ke ke tsa etsahala boemong bo ka bonoang ba MTBC15,17,18.
Mosebetsi o fetileng o bontšitse hore ho ka 'na ha e-ba le kopano e fokolang pakeng tsa MTBC strain20,21, ha ba bang ba hlōlehile ho tseba liketsahalo tse ka lekanyetsoang tsa recombination70,71. Buisana hape ka taba ena ka ho tsepamisa maikutlo ho Mycobacterium bovis, e fapaneng le mosebetsi o fetileng o neng o nka feela lefuba la Mycobacterium 70,71; kapa nahana ka MTBC ka kakaretso, hoo e batlang e se M. bovis e emelang 20; kapa u nahane feela ka likaroloana tse thibelang likhomo. Dataset ea mycobacteria, mosebetsing ona, ho na le mefuta eohle ea 70, e emelang li-clonal complexes tsohle, tse sebelisetsoang ho shebella bakeng sa ho kopanngoa hape. Seteishene sa data se lekantsoe ho ea ka mekhahlelo e mene ea koketso: (1) Litho tsa Eu2, (2) litho tsohle tsa European clone complex (ke hore Europe), (3) Europe le African clone complex (Eu + Af) le (4) Lipokello tsa data kaofela (ho kenyeletsoa liphatsa tsa lefutso tse sa kenyelletsoeng ho li-clone complexes tse seng li hlalositsoe).
Ho tsoela pele ho ithuta khopolo-taba ena, marang-rang a arohaneng a ne a etsoa ho hlahloba ho ba sieo ha liketsahalo tsa ho kopanya hape pakeng tsa liphatsa tsa lefutso, hobane mokhoa ona o ka bona kamano ea baholo-holo pakeng tsa batho ka bomong le ho bontša lipontšo tse hanyetsanang tsa phylogenetic. Lintlha tsohle tse 'nè tsa tlhahlobo li netefalitse ho ba teng ha li-loops ka marang-rang (ke hore, libaka tse sa fetoheng sefateng se le seng), empa tlhahlobo ea Phi ha e na tšehetso ea lipalo-palo (Eu2, p = 0.0956; Europe, p = 0.1637; Eu + Af p = 0.2774; data eohle e behiloeng pganiza1), e leng se fanang ka bopaki bo fokolang ba 0.2 (Fiegu 20.2) 3A-D).
Europe 2 liphatsa tsa lefutso (n = 37) (A), liphatsa tsa lefutso tsa Europe (n = 44) (B), liphatsa tsa lefutso tsa Europe le tsa Afrika (n = 51) (C) le data eohle e behiloeng (n = 70) (D).
Kamora tlhahlobo ena, le ho ela hloko litebello tsa cyclic ho marang-rang ohle, algorithm ea kaho bocha e kentsoeng phaepeng ea Gubbins e ile ea sebelisoa ho aha bocha leloko la clonal le ho tlatselletsa khakanyo ea phello ea ho kopanya bocha ho genome ea M. bovis. Nahana ka palo e akaretsang ea liketsahalo tsa ho kopanya bocha, tseo bongata ba tsona li etsahetseng makaleng a ho qetela (ke hore, ka genome e le 'ngoe) (Letlapa la 2). Lipontšo tsena li bontša ho lumellana ha data eohle e behiloeng 'me li bontša hore khafetsa liketsahalo tsa ho kopanya hape ke makhetlo a 200 ho isa ho a 300 a liphetoho. Hang ha parameter ea rho / theta e emelang litekanyetso tse lekanyelitsoeng tsa recombination le liphetoho tsa ntlha lekaleng li bonahala li le pakeng tsa 0.0037 le 0.0056 (Letlapa la 3). Haufinyane tjena, mosebetsi o hatisitsoeng oa mofuta oa 38 M. bovis o bonts'itse boleng bo phahameng ba rho/theta (rho/theta = 0.1) ho feta bo fumanoeng lethathamong lena la data, empa mosebetsi oa Patané le basebetsi mmoho le bona ba sebelisitse kopano e thehiloeng ho litšupiso ho fana ka methati ea ho kopanya hape
Ka mor'a moo, parameter ea r / m e emela karolelano ea mefuta-futa ea ho kopanya bocha le ho hlahisa phetoho, 'me boleng ba eona bo pakeng tsa 0.025 le 0.037, ho bontša hore ha ho bapisoa le liphetoho, ho kopanya ho na le phello e fokolang ka kakaretso ho mefuta-futa ea liphatsa tsa lefutso tsa M. bovis (Letlapa la 3) . Bakeng sa papiso e batsi, ho ile ha sebelisoa mokhoa o ts'oanang ho hakanya r/m paramethara bakeng sa dataset ea MTBC e entsoeng ka liphatsa tsa lefutso tse 23, e bonts'ang boleng ba karolelano ea 0.48620, ha ho Patané le basebetsi-'moho le dataset ea 38 M. bovis, e pakile boleng ba kakaretso Ke 0.98. Phuputsong ea pele, ke tse peli feela tsa liphatsa tsa lefutso tsa 23 tse kenyelletsoeng mosebetsing oa M. bovis (M. bovis BCG le khatello ea maikutlo), kahoo boleng bo fumanoeng bo ka 'na ba e-ba leeme ka lebaka la ho feteletsa ha lefutso la M. lefuba. Tlalehong ea bobeli, palo e hlahlobiloeng ea Mycobacterium bovis e ile ea fumanoa haholo United States le liphoofolo tse ruiloeng. Ka lehlakoreng le leng, setsing sa rona sa boitsebiso, libaka tse ngata tsa libaka le mefuta e mengata ea libaka li emeloa, 'me liphatsa tsa lefutso tse hlophisitsoeng ka lihlopha tse fapaneng tsa clonal tse nang le litšobotsi tse fapaneng tsa liphatsa tsa lefutso tsa baahi li boetse li sebelisoa, kahoo ho finyella tsebo e tebileng le e pharaletseng ea baahi. Phapang r / m karolelano ea boleng bo fumanoeng ka sete ea rona ea data e lumellana le khopolo ea hore tekanyo ea ho kopanya bocha e fapana haholo pakeng tsa meloko e abetsoeng mefuta e tšoanang ea taxonomic, kahoo liphetho tsena li bontša hore M. bovis clone complex e ka 'na ea bontša phapang ea recombination Tšusumetso e boetse e hlahisoa ke Didelot & Maiden72. Leha ho le joalo, ho atolosa haholo data ena e behiloeng ka ho kenyelletsa palo e kholoanyane ea liphatsa tsa lefutso tsa M. bovis ho tla lumella tlhaloso e eketsehileng ea ntlha ena. Ka bobeli li-parameter tsa r/m le rho/theta li bontša phapang pakeng tsa makala, 'me sephetho sena se lumellana le litlaleho tsa mefuta e meng ea baktheria72,73.
Qetellong, e le ho netefatsa liketsahalo tsa tlhophiso e ncha tse khethiloeng ke phaephe ea Gubbins, li-algorithms tse tšeletseng tse fapaneng li ile tsa sebelisoa ho software ea RDP4 ho leka ka boikemelo lipapiso tse fapaneng tsa mantlha tse fapaneng. Lefatšeng ka bophara, ka tlase ho halofo ea liketsahalo tse hloailoeng ke Gubbins li netefalitsoe ke RDP4 (Lethathamo la 4 le la 5). Ha ho nahanoa ka boitsebiso bohle ba boitsebiso, liketsahalo tse tharo tsa ho kopanya li ile tsa tiisoa, tse peli tse amanang le li-node tsa ka hare le tse ling tse amanang le genome e le 'ngoe lekaleng la terminal, tseo li-clonal complexes li neng li ke ke tsa abeloa ho tsona (Letlapa 4 le 5). Ho tsebahatsa diketsahalo makaleng a ho qetela ho ka bontsha hore ho kopanngwa botjha ho ntse ho tswelapele ka mefuta ya sejwalejwale ya M. bovis kapa sephetho se behilwe hampe70. Sebakeng sena sa khopolo-taba ea ho kopanya hape, hoo e ka bang 20% ea maemo a na le li-nucleotide tse sa hlalosoang (N), kahoo li ama pontšo ea ho kopanya hape (Setšoantšo sa Tlatsetso 2). Ho feta moo, sebaka sena se ama mofuta oa rrs, o kenyelletsang 16S ribosomal RNA e lebelletsoeng ho bolokoa haholo, kahoo lets'oao lena la ho kopanya bocha e kanna ea ba litholoana tsa tatellano ea liphoso kapa ho se tsamaisane hantle. Joale ho ile ha etsoa tumellano eohle ea genome pakeng tsa Mb0003 le Mycobacterium bovis AF2122/97, 'me ho na le li-nucleotide tse sa hlalosoang le li-SNP li tiisitsoe, kahoo mathata a ka bang teng a amanang le ho lumellana ka tsela e fosahetseng ha aa ka a bakoa ke boitsebiso ba likokoana-hloko bo kentsoeng tšebetsong ena E hlahile ka mor'a ho ithuta lenaneo.
Ha ho likheo kapa li-nucleotide tse sa hlalosoang tse fumanoeng libakeng tsa ho kopanya li-node tsa ka hare (Figures 4 le 5). Mabapi le liketsahalo tsena, e 'ngoe e na le genome ea Eu2 feela mme e ama mofuta oa pks12, o kenyelletsang polyketide synthase e ka khonehang; ha e 'ngoe e ngolisitsoe ho genome ea Eu1 'me e ama mofuta oa narX o kenyelletsang ho khoneha ho fokotsa nitrate (Letlapa la 4). Ka kakaretso, tlhahlobo ea ho kopanya bocha e bontša hore ho na le palo e lekanyelitsoeng ea likhechana tsa ho kopanya hape tse nang le tšehetso ea lipalo-palo, 'me matšoao a boletsoeng a bontša hore ho kopanngoa ho na le tšusumetso e tlaase ho leloko la M. bovis. Letšoao la ho kopanya bocha le lebelletsoe ho ba tlase, empa ho bohlokoa ho khetholla lets'oao la 'nete la ho iphetola ha lintho ho tsoa lerata le ka morao, e leng mosebetsi o boima. E le ho fokotsa lets'oao la lerata le hlahisitsoeng ke kopano e thehiloeng ho litšupiso le mathata a fapaneng a 70, 71, tse ling kaofela ntle le genome e felletseng li ile tsa bokelloa ho tloha qalong, 'me boleng ba kopano bo ile ba hlahlojoa le ho netefatsoa ke tlhahlobo ea lipeipi tsa QUAST (Letlapa la Tlatsetso 1). Ho feta moo, ho ile ha etsoa letoto la litlhahlobo tsa tlatsetso ho fana ka matla le ho nepahala ha phuputso e akaretsang. Ka hona, boleng ba tatellano ea liphatsa tsa lefutso tsa narX le pks12 bo ile ba hlahlojoa ka ho bala 'mapa khahlano le Mycobacterium bovis AF2122/97. Boemo ba SNP bo khothalletsoang sebakeng sa ho kopanya hape bo tiisitsoe ka ho sebelisa mekhoa e boletsoeng karolong ea mokhoa (bonyane ho baloa ha 20 le 0.9 ho fetola maqhubu). Polymorphism ea mofuta oa narX e tiisitsoe ka botlalo ho liphatsa tsa lefutso tse peli (Mb1792361 le Mb7240415; 2.3%) le liphatsa tsa lefutso tsa pks12 genome: Mb0891, Mb1711, Mb1789, Mb1870, Mbnes, M1706 le M1704. Leha ho le joalo, bakeng sa genome Mb2043, maemo a tšeletseng ho tse robeli ha a finyelle tekanyo ea botebo ba ho bala hobane SNP e tšehetsoa ke palo e kholo ea 17 e baloang, e ka tlase ho boleng bo thehiloeng ba cutoff ea 20. Ka hona, ho tsosolosoa ha liphatsa tsa lefutso tse tšeletseng (8,6%) sebakeng sena sa genome ho ka tiisoa (Figure 4 le 5).
Ponahalo e qaqileng ea khokahano ea sebaka sa recombination ea dataset ea Mycobacterium bovis e ama mofuta oa narX o kenyelletsang phokotso ea nitrate e ka bang teng. Ha ho likheo kapa li-nucleotide tse sa hlalosoang tse ileng tsa fumanoa sebakeng sa ho kopanya li-node tse ka hare. Ketsahalo ena e ngolisitsoe ho genome ea Eu1. Boleng ba tatellano ea mofuta oa narX bo ile ba hlahlojoa ka ho rala lipalo tsa Mycobacterium bovis AF2122/97. Netefatsa sebaka sa SNP se khothalelitsoeng sebakeng sa ho kopanya hape ka ho sebelisa mekhoa e boletsoeng karolong ea mokhoa (bonyane ho baloa ha 20 le makhetlo a 0.9 a fetohang). Polymorphism ea mofuta oa narX e tiisitsoe ka botlalo liphatseng tsa lefutso tsa Mb1792361 le Mb7240415 (2.3%).
Ponahalo e qaqileng ea khokahano ea sebaka sa recombination ea dataset ea Mycoplasma bovis e amang mofuta oa pks12. Ha ho likheo kapa li-nucleotide tse sa hlalosoang tse ileng tsa fumanoa sebakeng sa ho kopanya li-node tse ka hare. Mabapi le ketsahalo e amang pks12 gene encoding polyketide synthase, e na le genome ea Eu2 feela. Boleng ba tatellano ea pks12 bo hlahlobiloe ke 'mapa o baloang oa Mycobacterium bovis AF2122/97. Netefatsa sebaka sa SNP se khothalelitsoeng sebakeng sa ho kopanya hape ka ho sebelisa mekhoa e boletsoeng karolong ea mokhoa (bonyane ho baloa ha 20 le makhetlo a 0.9 a fetohang). Li-polymorphisms tsa genomes Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, le Mb1960 li netefalitsoe ka botlalo.
Liphatsa tsa lefutso tsa PE le PPE li na le libaka tse ipheta-phetang tse baloang habonolo ke Illumina ho latela tatellano le ho fosahala, kahoo hangata li hlakoloa ho bioinformatics workflow ea litho tsa M. lefuba feela ha ho sebelisoa leano la 'mapa-to-sequence. Khokahano ea liketsahalo tsa recombination e sebelisitsoeng mosebetsing ona e ipapisitse le kopano ea de novo ntle le ho sefa PE/PPE. Re lumela hore ka ho kenya ts'ebetsong mekhoa e meraro e fapaneng ea tlatsetso le li-algorithms ka SplitsTree, Gubbins pipeline le RDP4 software, maano a sebelisitsoeng a tiile ho sebetsana le ho sefa libaka tse hlophisitsoeng bocha tse bakoang ke matšoao a phoso. Leha ho le joalo, e le hore u se ke ua kenyelletsa ho kena-kenana ha liphatsa tsa lefutso tsa PE / PPE ho Gubbins le software ea RDP4 ho khetholla lihlopha tsa SNP, ka hona ho khetholloa ha libaka tsa recombination tse reretsoeng ho ama liphatsa tsa lefutso tsa narX le pks12, sebaka sa boahelani sa liphatsa tsa lefutso tsena se ile sa hlahlojoa (Supplementary Fig. 3-5). Ho M. bovis AF2122/97, mofuta oa narX o arotsoe ke narK2 le Mb1764c, ha pks12 e pota-potiloe ke Mb2075c e Mb2073c (Setšoantšo sa Tlatsetso 3-5). 'Mapa o hlahisitsoeng ho sebelisoa' mapa oa synline oa MAUVE oa genome e felletseng e fana ka tlhaiso-leseling mabapi le paballo ea tatellano ea liphatsa tsa lefutso le tlhophiso e ncha, e bonts'ang li-collinear blocks, 'me ha ho na matšoao a phetoho ea genome kapa ho fetoha. Ntle le moo, tlhahlobo ea tlatsetso ka tatellano ea amino acid e pakile thuto ea homology ho liphatsa tsa lefutso tse felletseng, mme ha ho PE/PPE e fumanoeng libakeng tse haufi tsa narX kapa pks12. Bakeng sa narX, genome e le 'ngoe (Mb0030) e na le lintlha tse tlase tsa synonyme hobane mofuta oa narX o ile oa khetholloa e le likhechana tse peli (likaroloana tsa 1891 le 1890). Bakeng sa pks12, ka lebaka la ho tšoana, Mb0030 le Mb003 li bontšitse lintlha tse tlaase tsa synlinearity, ha pks12 e ne e khetholloa ka likhechana tse peli le tse tharo, ka ho latellana, tse emelang libaka tse fapaneng tsa protheine (Supplementary Figure 3-5). Ha re ela hloko tlhahisoleseding ena, 'me Gubbins le RDP4 software ka bobeli ba etsa tlhahlobo, hlahloba ho lumellana ha li-multiple tsa mantlha tsa boholo ba 500 bp ka fensetere, re tiisitse hore mofuta oa PE / PPE o ke ke oa kena-kenana le pontšo ea ho kopanya hape e amang narX le pks12.
Le hoja matšoao a recombination a fumanoeng sethalong sena sa data a ka 'na a nkoa e le masala, ke' nete hore recombination ho M. bovis e ke ke ea hlakoloa, kahoo e lokela ho tsoela pele ho ba taba ea tlhahlobo e tsoelang pele, moo liphatsa tsa lefutso tse tsoang maemong a fapaneng a epidemiological a latellanang ho ea Bohlokoa.
Ho bapisa lifate tsa ML phylogenetic tse fumanoeng pele le ka mor'a ho lokisoa hape (Setšoantšo sa 2A, B) ha sea ka sa lebisa liphetohong tse kholo kamanong ea phylogenetic, 'me mefuta ea M. bovis e ne e kopantsoe sehlopheng se le seng.
Ka mor'a ho etsa 'mapa 42 e sa tsoa tatellano ea M. bovis e baloa le genome ea referense ea M. bovis AF2122/97, ho ile ha fumanoa tumellano ea SNP e nang le 1816 polymorphic positions. Boholo ba li-SNP (87.1%) li sebakeng sa coding, 'me liphatsa tsa lefutso tse amehileng li khetholloa ho ea ka lihlopha tse sebetsang tse bontšitsoeng ho Bovilist (Setšoantšo sa 6A, B). Ha ho nahanoa ka palo e felletseng ea liphatsa tsa lefutso sehlopheng ka seng se sebetsang, liphatsa tsa lefutso tsa "lipid metabolism" li bonts'itse li-SNP tse ngata, tse lateloang ke "metabolism e bohareng le phefumoloho", e bonts'a hore li ho M. bovis ho iphetola ha lintho.
Tlhahlobo ea hierarchical ea dataset ea M. bovis e tsoang Portugal (n = 42). Palo eohle ea li-SNP tse ngolisitsoeng le liphatsa tsa lefutso tse amehileng bakeng sa sehlopha ka seng sa tšebetso (A). Palo eohle ea liphetoho tse tšoanang le tse sa ts'oaneng tse ngolisitsoeng ka sehlopha sa tšebetso (B).
Lefatšeng ka bophara, karolelano ea dN / dS e molemo ho feta 1.5, e bontšang hore khatello ea lefats'e ea ho iphetola ha lintho ke ho tlosa boemo ba baholo-holo 'me e emela boemo bo botle (bo fapa-fapaneng kapa bo laoloang) le / kapa bo phutholohileng ba khetho ea tlhoekiso. Likarolong tsa "virulence, detoxification, adaptation", "ho kenya tatellano le phages", le "liprotheine tsa taolo", tse fetang tse peli ho tse tharo tsa SNP ha li na mantsoe a tšoanang (Setšoantšo sa 6B).
Likarolong tsohle, ho na le liphatsa tsa lefutso tse nang le li-SNP tse ngata, e leng se etsang hore ho be le tekanyo e tloaelehileng ea phetoho (ke hore, SNP e tloaelehileng ka liphatsa tsa lefutso) e kholo ho feta 1 (Setšoantšo sa 6A). Pks12 (Mb2074c) e nang le 15 SNPs le fas (Mb2553c) e nang le 8 SNP e na le litekanyetso tse phahameng tsa phetoho. Ka bobeli liphatsa tsa lefutso tsena li ameha ho metabolism ea mafura a acid. Mofuta oa pks o kenyelletsa polyketide synthase (PKS), e leng enzyme e sebetsang ka bongata e amehang ho mycobacterial cell wall lipid biosynthesis74,75. Mofuta ona o kenyelletsa polypeptide e sebetsang ka bongata e kenyang letsoho kholisong ea mycoketides74,76. Fas gene e ameha ho qaleng ha mycolic acid. Ka bobeli liphatsa tsa lefutso tsena li phetha karolo ea bohlokoa ho biosynthesis ea lerako la lisele ho kopana le moamoheli.
E le ho tsoela pele ho ithuta ka ho iphetola ha Mycobacterium bovis, lihlopha tse peli tsa liphatsa tsa lefutso li ile tsa hlahlojoa. Mesebetsi e hatisitsoeng pele e sebelisa mokhoa oa ho latellana le mekhoa ea phylogenetic e khethollang liphatsa tsa lefutso tse ileng tsa fumanoa ke baholo-holo ba MTBC ka HGT pele ho mefuta-futa37,38. Liphatsa tsa lefutso tsena li thathamisitsoe ho Supplementary Table 2. Kabo ea SNP ea kakaretso ea liphatsa tsa lefutso tsa 77 tse ka 'nang tsa amana le HGT li ile tsa hlahlojoa,' me libaka tsa 26 tsa polymorphic li ile tsa khetholloa, tseo hangata (78%) li ileng tsa fella ka liphetoho tse sa tsejoeng (NS) (Supplementary Table 2). Mosebetsi o fetileng ho MTBC genome o bontšitse hore sebaka sa HGT se behang se bonts'a karo-karolelano e phahameng ea NS SNP ha e bapisoa le genome eohle. Haeba motho a nahana hore libaka tsena tsa recombination li ile tsa fumanoa ke baholo-holo ba MTBC, ka hona, li emela li-polymorphisms tsa boholo-holo, joale karolo ea liphetoho tse tšoanang e lebeletsoe ho ba holimo, hobane li-substitutes tsa NS li lebeletsoe hore li felisoe ka khetho e mpe ka lebaka la liphetoho tsa amino acid E ka fetola mosebetsi oa protheine. Ka hona, liphetho tsa rona li bonts'a hore litlamorao tsa ts'ebetso li ka bakoa ke ho nkeloa sebaka ha liphatsa tsa lefutso tse kang HGT, tse bonts'ang bohlokoa ba tsona bakeng sa mefuta e fapaneng ea liphatsa tsa lefutso tse feto-fetohang.
Ka mokhoa o ts'oanang le tlhahlobo ena, liphatsa tsa lefutso tse kenyang likarolo tsa 3R (DNA tokiso, replication, and recombination) tsamaiso li ile tsa hlahlojoa ka botlalo ho latela lenane le hatisitsoeng pele ke dos Vultos le basebetsi-'moho (2008)39. Phapanyetsano ea likaroloana tse tšoanang tsa DNA e ke ke ea hlokomeloa ka ho toba, le hoja e ka 'na ea e-ba ts'ebetso ea khafetsa ha libaktheria tse amanang haufi-ufi li ameha, joalo ka tabeng ea sete sena sa data; ho feta moo, ts'ebetso ena e kanna ea ba senotlolo sa mekhoa ea ho lokisa DNA72, ka hona E phetha karolo ea ho kopanya homologous. Kakaretso ea maemo a 26 a polymorphic a ajoang ke liphatsa tsa lefutso tse 54 li ile tsa khetholloa (Tlaleho ea Tlatsetso 3). Sehlopheng sena sa liphatsa tsa lefutso, liphetoho tsa NS li ile tsa etsa hoo e ka bang 65% ea liphello, tse lumellanang le litlaleho tse fetileng tsa mafu a lefuba a Mycobacterium.
Nako ea poso: Oct-21-2021