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Beskikbaarheid van 'n databasis wat die proteïene van Vibrio cholerae en hul ooreenstemmende geenvolgorde bevat

Beskikbaarheid van 'n databasis wat die proteïene van Vibrio cholerae en hul ooreenstemmende geenvolgorde bevat


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Ek wou 'n lys hê van al die proteïene (aminosuurvolgordes) en hul ooreenstemmende geenvolgorde van Vibrio cholerae. Ek het probeer om in patriek te soek, maar die aantal aminosuurvolgordes en geenvolgordes stem nie ooreen nie, aangesien hulle die hele genoom verskaf het. Ek moet weet of daar 'n databasis is waar al die proteïene van Vibrio cholerae en hul ooreenstemmende geenvolgorde in 'n enkele lêer of in 'n paar lêers verkry kan word. Copy pasting 4000-5000 genoom- en proteïenvolgordes individueel vanaf die internet lyk vir my omslagtig.


Jy wil na PATRIC > Data > Download Tool gaan.

Van die Taksonomie-boom, vind Vibrio cholerae, kies Tipe annotasie as óf PATRIC/RefSeq óf albei, kies die tipe lêer wat jy wil hê (gbk of faa) en laai daarvandaan af.


Karakterisering en genetiese variasie van Vibrio cholerae Geïsoleer van kliniese en omgewingsbronne in Thailand

Affiliasie Nasionale Voedselinstituut, Tegniese Universiteit van Denemarke, Navorsingsgroep vir Genomiese Epidemiologie, WGO-samewerkingsentrum vir antimikrobiese weerstand in voedselgedraagde patogene en genomika en Europese Unie-verwysingslaboratorium vir antimikrobiese weerstand, Kgs. Lyngby, Denemarke

Affiliasie Nasionale Voedselinstituut, Tegniese Universiteit van Denemarke, Navorsingsgroep vir Genomiese Epidemiologie, WGO-samewerkingsentrum vir antimikrobiese weerstand in voedselgedraagde patogene en genomika en Europese Unie-verwysingslaboratorium vir antimikrobiese weerstand, Kgs. Lyngby, Denemarke

Affiliasie Departement Mikrobiologie, Fakulteit Openbare Gesondheid, Mahidol Universiteit, Bangkok, Thailand

Affiliasie Nasionale Voedselinstituut, Tegniese Universiteit van Denemarke, Navorsingsgroep vir Genomiese Epidemiologie, WGO-samewerkingsentrum vir antimikrobiese weerstand in voedselgedraagde patogene en genomika en die Europese Unie-verwysingslaboratorium vir antimikrobiese weerstand, Kgs. Lyngby, Denemarke

Affiliasie Fakulteit Openbare Gesondheid, Thammasat Universiteit, Rangsit Sentrum, Pathumthani, Thailand

Affiliasie Nasionale Voedselinstituut, Tegniese Universiteit van Denemarke, Navorsingsgroep vir Genomiese Epidemiologie, WGO-samewerkingsentrum vir antimikrobiese weerstand in voedselgedraagde patogene en genomika en Europese Unie-verwysingslaboratorium vir antimikrobiese weerstand, Kgs. Lyngby, Denemarke


2 DIE PARADIGM: DIE CHEMOSENSORIESE STELSEL IN DIE MODEL ORGANISME Escherichia Coli

Chemosensories word die beste verstaan ​​as die chemotaxis-weg in die modelbakterie E coli (Parkinson et al., 2015), Figuur1. Hierdie organisme het 'n enkele chemosensoriese sisteem wat die bindingstoestand van lokmiddels en afweermiddels aan vier transmembraanreseptore waarneem, genaamd metiel-aanvaardende chemotaxis-proteïene (MCP's). Hierdie reseptore bespeur voedingstowwe, seinmolekules en gifstowwe wat direk of indirek aan hul periplasmiese domeine bind (Milburn et al., 1991 Tam en Saier, 1993 Englert et al., 2010). 'n Addisionele vyfde reseptor is 'n metilering-onafhanklike redokssensor wat funksioneer deur die regulering van oksidasie en reduksie van 'n flavien-adenien-dinukleotied-eenheid wat aan 'n periplasmiese periode sirkadiese proteïen, arielkoolwaterstofreseptor kernvertalerproteïen, enkelgedrewe proteïen (PAS)-domein bind. (Bibikov et al., 2004 ).

Die bindingstoestand van die reseptore word gekommunikeer deur 'n histidienkinases, adenilaatsiklases, metielaannemende proteïene en fosfatases (HAMP) domein na hul sitoplasmiese punte. Hier reguleer hulle die outofosforilering van die histidienkinase CheA. Hierdie kinase is 'n vyf-domein ensiem wat homodimere vorm. Elke domein speel 'n duidelike rol in die funksie van CheA: P1 dra die substraat histidien wat die fosforielgroep ontvang tydens outofosforilering, P2 is die bindingsplek vir die responsreguleerder CheY. Beide die P1- en P2-domeine is aan die res van die proteïen geheg via buigsame skakelaars. P3 is die dimerisasie domein, P4 is die kinase domein met die ATP bindende sak, en P5 is die reseptor-bindende domein (Muok et al., 2020 ).

By aktivering dra die kinase die fosforielgroep oor na CheY. Gefosforileerde CheY bind op sy beurt aan die flagellêre motor waar dit 'n skakelaar in die rigting van flagellêre rotasie veroorsaak (Stock en Da Re, 2000). Wanneer die vlakke van lokmiddels toeneem, word die kinase afgeskakel en CheY word nie gefosforileer nie. In die afwesigheid van CheY-P, draai die motors antikloksgewys (ccw). Dit veroorsaak 'n bundeling van die veelvuldige flagella wat saam die sel vorentoe dryf, wat lei tot 'n gladde swempatroon ('n sogenaamde "hardloop"). Met die afname van lokmiddels of toename van afweermiddels, word die kinase deur die reseptore geaktiveer. CheY word gefosforileer, wat lei tot 'n omkering van motoriese draairigting by CheY-P-binding. Dit veroorsaak dat die flagellêre bundel skei en die selle hulself lukraak heroriënteer (hulle "tuimel"). Om die algehele CheY-P-vlak sinchroniseer met CheA-aktiwiteit te hou, word CheY-P vinnig gedefosforileer deur die fosfatase CheZ (Parkinson) et al., 2015 ).

Die chemotaxisisteem kan aanpas by huidige toestande via die regulering van reseptormetilering by spesifieke glutamielresidue in die sitoplasmiese gedeelte van die MCP's (Sourjik en Wingreen, 2012). Die metielesterase CheB word ook geaktiveer deur CheA-fosforilering. Saam met die konstitutief aktiewe metieltransferase CheR beheer hierdie ensieme die metilering van die reseptore wat presiese aanpassing moontlik maak deur die aktiveringstoestand van die reseptore op 'n tydsvertraagde wyse teen te werk: 'n Volledig gemetileerde reseptor lei tot hoë kinase-aktiwiteit, terwyl 'n volledig gedemetileerde reseptor afreguleer die kinase (Parkinson et al., 2015). Die chemotaxisisteem laat die selle toe om die duur en frekwensie van hardloop- en tuimelfases te beheer. Uiteindelik, deur die balans tussen hierdie twee toestande te verander, beweeg die selle in 'n "bevooroordeelde ewekansige stap" op aantreklike gradiënte en afwerende gradiënte.

Die reseptore vorm saam groot trosse by die selpole. In E coli, vorm reseptore goed geordende seskantige skikkings met CheA en CheW CheW en die CheW-homologe P5-domein van CheA vorm afwisselende ringe wat aan die reseptorpunte gebind is. Die P3 dimerisasie domeine van CheA vorm 'n brug tussen naburige seshoeke. Die stoïgiometrie van CheA tot CheW wissel van 1:1 tot 1:2. Hierdie veranderlikheid van CheW is afkomstig van die argitektuur van die skikking, daar is ses seshoeke wat elk drie CheA monomere bevat, rondom een ​​wat die histidienkinase ontbreek (Briegel) et al., 2012 Liu et al., 2012 Cassidy et al., 2015). Hierdie CheA-vrye seshoek kan bykomende CheW bevat.

Die rangskikking van reseptore in hoogs geordende skikkings is nie uniek aan E coli, maar is gevind in alle afgebeelde chemotaktiese bakterieë en archaea tot dusver (Briegel et al., 2009 2015 ).

Terwyl ons 'n deeglike begrip van die chemosensoriese stelsel in E coli, ons begin net struktuur en funksie van sulke stelsels in ander organismes ontrafel. In hierdie oorsig sal ons die huidige kennis oor die komplekse chemosensoriese stelsels in V. cholerae, 'n sleutelmodelstelsel as 'n menslike patogeen met 'n komplekse lewensiklus. Ons sal spesifiek ooreenkomste en verskille met die modelstelsel in E coli, en skets sommige van die oorblywende oop vrae.


Resultate

Ontwikkeling van verslaggewerstamme om die frekwensie van verlies van GI's in V. cholerae.

Vorige werke wat die eksisieverskynsel van GI's bestudeer het in V. cholerae het die PCR-toets aangeneem (19, 20). Hierdie toets is egter nie vaardig in die monitering van die verlies van GI's nie en is nie in staat om bakteriële selle te isoleer van 'n heterogene bevolking wat die GI van hul genoom verloor het nie. In hierdie studie het ons die genoom van verskillende gemanipuleer V. cholerae stamme om die frekwensie van verlies van verskillende GI's onder in vitro en in vivo groeitoestande te monitor. Elke GI (VPI-1, VPI-2, VSP-1 en VSP-2) en die profeë is gemerk met veelvuldige kiesbare (kat), teen-kiesbaar (sakB), en chromogenies (lacZ) allele in die kliniese V. cholerae stam N16961 en sy afgeleides (Fig. 2). Aangesien N16961-genoom sonder SXT is, het ons hierdie element gemerk met soortgelyke kiesbare en teen-kiesbare merker in 'n V. cholerae O1 El Tor kliniese isolaat 41081 (Tabel 1). Die alleel sacB-kat of sacB-cat-lacZ of sacB-aadA is in die GI's of SXT ingebring deur opeenvolgende alleliese uitruilmetode deur gebruik te maak van afgeleides van selfmoordvektor pKAS32 wat 'n selekteerbare merker bevat bla en teen-kiesbare merker rpsL in die plasmied ruggraat (SI Aanhangsel, Tabel S1). Eerste oorkruis intermediêre stam dra vektor ruggraat (pKAS32) en verslaggewer alleel (sacB-kat of sacB-cat-lacZ of sacB-aadA) is bevestig deur antibiotiese weerstand (Cam R of Spc R en Amp R ) en vatbaarheid vir streptomisien en sukrose. Die verlangde verslaggewer stamme SB30 (VPI-1::sacB-kat), SB26 (VPI-2::sacB-kat), SB23 (VSP-1::sacB-kat), DD1(VSP-2::sacB-kat), BB2 (CTXΦ::sacB-kat), en JV3 (SXT::sacB-aadA) is bevestig deur antibiotika weerstand, sukrose vatbaarheid, kolonie kleur, PCR toets en DNA volgordebepaling (Fig. 3). Aangesien integrasie van CTXΦ en RS1Φ onomkeerbaar is, is die TLC-CTX-RS1-profaag-skikking in die genoom van N16961 vir die eerste keer deur spektinomisienweerstandskasset vervang deur alleliese uitruilmetode. Vervolgens, CTXΦ::sacB-kat element is ingestel in die lacZ-dif1 alleel deur plekspesifieke rekombinasie om die rekombinante bakteriële stam BB2 te ontwikkel (Tabel 1). Antibiotiese weerstandfenotipe, PCR-toets en blou-wit sifting het die verlangde stam BB2 bevestig.

Skematiese voorstelling van die genetiese organisasie van die genomiese eilande (GI's), integrerende konjugatiewe element (ICE), en profeë en ligging van verslaggewergene in die gemerkte genoom. Kiesbaar (kat), teen-kiesbaar (sakB), en chromogenies (lacZ) allele is ingevoer in die V. cholerae genoom, wat 'n alleliese uitruilmetode aanneem. Asterisk dui dit aan Vibrio patogenisiteit eiland-1 (VPI-1) is gemerk met sacB-kat sowel as sacB-cat-lacZ allele.

Relevante genotipe en fenotipe van wild-tipe en geneties gemodifiseerde Vibrio cholerae stamme wat in die studie gebruik is

Genetiese organisasie van chromosomale integrasieplekke (attB) voor en na uitsny van al vier genomiese eilande uit die genoom van N16961. (A) attL en attR terreine is gevorm as gevolg van plekspesifieke rekombinasie tussen genomiese eilandhegtingsplek (attP) en chromosomale aanhegtingsplek (attB). Baan nommers 17 en 18 het onderskeidelik 1-kb en 100-bp DNS-lere bevat. (B) Polimerase kettingreaksie (PCR) analise van N16961 en sy afgeleides in die teenwoordigheid en afwesigheid van genomiese eilande (GI's). Geamplifiseerde PCR-produkte is in 'n agarosegel opgelos. Baannommer en ooreenstemmende amplikon word in genoem A. Blou pyle dui primers aan wat aan die stroomop en stroomaf van GI bind, en groen pyle dui primers aan wat aan die GI bind.

Bepaling van in vitro en in vivo frekwensie van verlies van GI's, geïntegreerde konjugatiewe element en CTX profeet.

Om die stabiliteit van al die vier GI's, CTX-profaag en SXT-element onder in vitro groeitoestande te monitor, is die verslaggewerstamme wat in hierdie studie gemanipuleer is (SB30, SB26, SB23, DD1, BB2 en JV3) in voedingsryke medium gekweek. (Luria–Bertani-bouillon LB) vir 12 uur by 37 °C. Kulture van die verslaggewerstamme is uitgeplaat op Luria agar (LA) plate wat aangevul is met sukrose (15%) en streptomisien (100 μg/ml). Kolonies wat op die seleksieplate gegroei is, is gemonitor vir hul sensitiwiteit vir chlooramfenikol of spektinomisien afhangende van die weerstandskasset wat gebruik is om die GI's, CTX-profaag en SXT-element te merk. Die uitgesnyde kolonies is verder aan PCR-bevestiging onderwerp deur 'n chromosomale aanhegtingsgebied van elk van die GI's te versterk (Fig. 3). Aanhegselreekse (attB) vir elke GI en SXT is bevestig deur DNA-volgordebepaling. Totale aantal bakteriële selle in 'n oornag-gegroeide bakteriese kultuur is bepaal deur dieselfde kultuur op 'n LA-plaat wat met streptomisien aangevul is, te plaat. Ons het verskillende frekwensie van verlies waargeneem vir verskillende GI's (Tabel 2). Die hoogste frekwensie van verlies onder in vitro groeitoestande is waargeneem vir die VPI-1, terwyl geen verlies vir CTX-profaag opgespoor is nie (Tabel 2).

In vitro en in vivo verlies frekwensie van verskillende genomiese eilande

Om die frekwensies van verlies van verskillende GI's onder in vivo groeitoestande te monitor, het ons die geligate konyn ileale lus eksperimentele model aangeneem. Verslaggewerstamme wat gemerkte GI's in hul genoom bevat, is in 'n geligate ileale lus geënt, en die frekwensies van verlies van elke GI is na 12 uur gemeet deur selle op seleksieplate te plaas, aangevul met sukrose (15%) en streptomisien (100 μg/ml) . Soos die in vitro eksperiment, is die totale aantal bakteriële selle in die geligeerde ileale lusvloeistof gekwantifiseer deur die vloeistof op die LA-plaat wat met streptomisien aangevul is, te plateer. Die hoogste frekwensie van verlies is waargeneem vir die VPI-2 in vergelyking met enige ander GI's (Tabel 2). Geen verlies is waargeneem vir die CTX-profaag nie (Tabel 2). Verslaggewerstamme wat delesie dra van GI's wat uit geligate ileale lusse geïsoleer is, is bevestig deur die kolonies weer op die Vibrio-spesifieke selektiewe medium TCBS en LA aangevul met streptomisien (weerstandig) of chlooramfenikol (sensitief). Interessant genoeg het ons opgemerk dat die frekwensie van verlies van al die GI's van die V. cholerae genoom was baie hoog onder in vitro toestand in vergelyking met in vivo-gegroeide selle (Tabel 2).

V. cholerae Stamme sonder genomiese eilande en profeë is lewensvatbaar en kan beide in ryk en minimale media vermenigvuldig.

Ons het uitgebreide genoom-ingenieurswese van N16961 gedoen om al sy GI's en profege uit sy genoom te verwyder. Eerstens het ons die GI's gemerk met sacB-kat of sacB-cat-lacZ en dan geïsoleer V. cholerae selle wat die gemerkte GI verloor het deur die selle op seleksiemedium aangevul met sukrose te plateer (Tabel 1). Om stamme te isoleer wat nie verskillende kombinasies van MGE het nie, soos ΔVPI-1 (SB31), ΔVPI-2 (SB27), ΔVSP-1 (SB25), ΔVSP-2 (DD2), ΔTLCΦ-CTXΦ-RS1Φ (BS1), ΔVPI-1ΔVPI- 2 (SB52), ΔVSP-1ΔVSP-2 (SB44), ΔVSP-1ΔVSP-2ΔVPI-1 (SB47), ΔVSP-1ΔVSP-2ΔVPI-2 (SB48), ΔVSP-1ΔVSP-2ΔVPI-1Δ5VPI-2, en ΔVSP-1ΔVSP-2ΔVPI-1ΔVPI-2ΔTLCΦ-CTXΦ-RS1Φ::lacZ-dif1 (BB1) stam (Tabel 1), het ons gebruik gemaak van voorwaardelike toksisiteit van sakB aan die gasheerbakterieë.

Ons het die groeitempo van N16961 en sy afgeleides met of sonder GI's en profage gemeet in 'n voedingsryke (LB) of minimale (M9) medium (voedingsarm). V. cholerae selle sonder enige GI's en profeë kon in beide media groei (Fig. 4 A en B). Maar groei van SB50 (Δvsp-1Δvsp-2vpi-1vpi-2) was aansienlik verminder (P < 0,01) gedurende 5, 6 en 7 uur se groei in vergelyking met sy ouerstam N16961 (Fig. 4)A). Ons het ook aansienlike vermindering waargeneem (P < 0.05) in groei van die gemanipuleerde stam SB47 (Δvsp-1Δvsp-2vpi-1) gedurende 5, 6 en 7 uur se inkubasie. Interessant genoeg, BB1, die afgeleide van SB50 dra verskil 1 in die Chr1, maar sonder al die vier GI's en profeë in die chromosoom, het 'n soortgelyke groeiprofiel as die WT-stam N16961 getoon (Fig. 4) A en B). Ons het geen groot veranderinge in groeiprofiel in M9 of LB tussen N16961 en BB1 stamme waargeneem nie (Fig. 4) A en B). Daar is voorheen berig dat die V. cholerae O1 El Tor-stam N16961 het 'n beduidende groeivoordeel bo die klassieke biotipe-stam O395 in stilstaande-fase-kultuur wanneer beide stamme saamgekweek is in LB onder optimale groeitoestande (27). Die groot verskil tussen El Tor en klassieke biotipes met betrekking tot GI is die afwesigheid van beide VSP-1 en VSP-2 in laasgenoemde. Ons het ondersoek of O1 klassieke stam O395 kan meeding met O1 El Tor stam SB60 sonder VSP-1 en VSP-2 eilande. Om die SB60- en O395-stamme in die kokultuur te onderskei, het ons twee verskillende weerstandsgeenkassette bekendgestel, kat en sh ble, in die verskil 1 terrein van El Tor en klassieke stamme, onderskeidelik. Die gemengde kultuur is onder optimale groeitoestande in LB of M9 medium gekweek, en die aantal bakteriese selle is op verskillende tydpunte gemeet (Fig. 4) C en D). Ons bevinding dui daarop dat O395 klassieke biotipe kan meeding met die VSP-1 en VSP-2-geskrap El Tor biotipe wanneer hulle saam gekweek is in M9 medium. Die VSP-1- en VSP-2-geskrap El Tor-stamme kan egter O395 in die LB-medium uitkom. Hierdie bevinding dui aan dat die verkryging van VSP-1 en VSP-2 in die genoom van V. cholerae sekere metaboliese eienskappe verskaf wat moontlik die sewende pandemie El Tor-biotipe gehelp het om die sesde pandemie-veroorsakende klassieke biotipe te oortref. Egter addisionele rol van oriC funksies kan ook die El-biotipe help om die klassieke biotipe in voedingryke omgewing uit te kompeteer, selfs in die afwesigheid van sulke metaboliese eienskappe (28). Daar is voorheen berig dat die oriC van die klassieke biotipe dra 'n enkelbasismutasie (T→G) in sy gekonserveerde AT-ryke 13-meer R-herhalingsgebied in vergelyking met die oriC van El Tor-biotipe (28). Dit is gedemonstreer dat die minichromosoomkopiegetal en sy transformasiedoeltreffendheid aansienlik verminder word as gevolg van substitusiemutasie in die oriC van die klassieke biotipe (28). Dit kan een van die redes wees vir minder bevoegdheid van klassieke biotipe terwyl dit saam met die El Tor-biotipe in die voedingsryke medium gekweek word.

Groeikromme van wilde tipe en geneties gemanipuleer V. cholerae stamme in ryk (LB) en voedingsgedefinieerde kultuurmedium (M9). Die El Tor-stam N16961 en sy afgeleides is individueel in monokulture in LB (A) en M9 (B), en die groei van die bakteriese selle is gemonitor deur die optiese digtheid van die kultuur by 600 nm (OD) te meet.600) in 'n spektrofotometer op verskillende tydpunte. Groeikompetisie tussen ΔVSP-1ΔVSP-2 N16961 en O395 is ook gedoen in ryk (C) en minimaal medium (D) in 'n saamgekweekte medium deur 'n soortgelyke aantal selle saam te ent (1:1). CFU-telling van elke stam is bepaal deur die kultuur uit te plaat in ryk medium (LA) aangevul met spesifieke antibiotika. TCR = TLCΦ-CTXΦ-RS1Φ.

Integrasiedoeltreffendheid van verskillende GI's, SXT en CTXΦ in die teenwoordigheid en afwesigheid van verworwe genoom.

Studies oor integrasiedoeltreffendheid van GI's is ingewikkeld as gevolg van hul groot grootte en nie-repliserende aard van hul uitgesnyde sirkelvorm. Daarom, om hierdie kompleksiteit te vermy, het ons die integrerende module van GI's, SXT en CTXΦ uit sy uitgesnyde sirkelvorm versterk, wat aanhegtingsplek insluit (attP) met of sonder rekombinases (Fig. 5). Die integrasiemodules is gekloon in 'n voorwaardelik replikatiewe konjugatiewe vektor (SI Aanhangsel, Tabel S1). Rekombinante vektor wat integrasiemodule van GI's, SXT of CTXΦ bevat, is ingestel deur vervoeging in V. cholerae stamme wat inheemse chromosomale aanhegtingsplek dra (attB) met of sonder ander GI's. Integrasie van integrerende module in die chromosoom van V. cholerae is bepaal op grond van die antibiotika (chlooramfenikol of zeosien) weerstandsprofiel van ontvangerselle. Integrasie van 'n GI by spesifieke chromosomale lokus is verder bevestig deur PCR-toets en blou-wit kolorimetriese toets. Konjugasie van verskillende rekombinante vektore wat nie-replikatiewe integrasiemodule van verskillende GI's, SXT en CTXΦ dra, het aanleiding gegee tot verskillende getalle transkonjugante (Tabel 3). Onder niereplikatiewe GI's en SXT is die hoogste aantal transkonjugante verkry met die rekombinante vektor wat die integrasiemodule van SXT dra (Tabel 3). Afwesigheid van GI's en profeë het die integrasiedoeltreffendheid van SXT verhoog. 'n Minimum aantal transkonjugante is verkry met rekombinante vektor wat die integrasiemodule van VSP-1 dra (Tabel 3). In vergelyking met die integrasiedoeltreffendheid van GI's en SXT met die replikatiewe en integrerende module van CTXΦ, is die hoogste aantal transkonjugante egter met die faaggenoom verkry (Tabel 3). Om te onderskei tussen V. cholerae selle wat die integrerende of replikatiewe vorm van die RS2-module van CTXΦ dra, het ons die chromosomale aanhegtingsplek saamgesmelt verskil 1 in die koderingsgebied van die lacZ geen geamplifiseer vanaf E. coli. Die lacZ-dif1 alleel het funksionele β-galaktosidase geproduseer en blou kolonies gegenereer in die teenwoordigheid van chromogene stof X-gal in die seleksieplaat. Ons het toe endogene verwyder lacZ geen van V. cholerae selle, en die TLC-CTX-RS1-profaag-skikking is vervang met lacZ-dif1 alleel (SI Aanhangsel, Fig. S3). Die verslaggewerstam wat die replikatiewe RS2-module van CTXΦ dra, het blou geword in die teenwoordigheid van X-gal op die seleksieplaat, terwyl die stam wat integrerende RS2 in die verskil 1 in die chromosomaal lacZ-dif1 alleel het wit kolonies geproduseer as gevolg van inaktivering van lacZ geen en die afskaffing van β-galaktosidase produksie (SI Aanhangsel, Fig. S3). Die aantal transkonjugante met dieselfde rekombinante vektor in die teenwoordigheid en afwesigheid van ander GI's in die gasheerselle het verskillende uitkomste getoon (Tabel 3). Die integrasie doeltreffendheid van CTXΦ en sy afgeleides is gemeet in die afwesigheid van TLCΦ en ander profeë. Die integrasiemodule van SXT en VPI-1 kan byna met dieselfde doeltreffendheid integreer in die teenwoordigheid en afwesigheid van ander GI's in die V. cholerae genoom. Integrasie doeltreffendheid van VPI-2 is verminder in die afwesigheid van ander GI's. Interessant genoeg is integrasiedoeltreffendheid van VSP-1 en VSP-2 verhoog in die afwesigheid van GI's (Tabel 3).

Genetiese gereedskap wat ontwerp is om die integrasiedoeltreffendheid van verskillende GI's, ICE (SXT) en CTXΦ te meet. Die integrasiemodule van al die elemente is versterk vanaf hul uitgesnyde sirkelvorm, wat aanhegtingsplek (attP) met of sonder tyrosienrekombinases. Die oorspronklike vektor (pSW23T) kon nie in repliseer nie V. cholerae as gevolg van sy voorwaardelike ori (R6K) funksie.

Integrasie frekwensies van verskillende genomiese eilande, CTXΦ en SXT element in die teenwoordigheid en afwesigheid van verworwe genetiese elemente

Ons het ook die integrasiedoeltreffendheid van die niereplikatiewe RS2-module van CTXΦ in die verskil 1 webwerf van Chr1 in V. cholerae verslaggewerstam BS1 en BB1. Ons het soortgelyke integrasiedoeltreffendheid van niereplikatiewe RS2-module waargeneem in die teenwoordigheid en afwesigheid van verskillende GI's. Die integrasiegebeurtenis van niereplikatiewe RS2-module was plekspesifiek (≥99.3%) en onomkeerbaar, beide in die teenwoordigheid en afwesigheid van ander GI's (Tabel 3).

Kruisgesprek tussen horisontaal verkry genetiese elemente en kerngenoom.

Kruisgesprek tussen GI's en profage in V. cholerae is 'n bekende verskynsel (29, 30). Die invloed van verworwe funksies in die funksionaliteit van gene teenwoordig in die kerngenoom is egter nie bekend nie. Begrip van die molekulêre basis van noodsaaklikheid van LexA in V. cholerae is van spesiale belang as gevolg van sy bydrae in die biologie van horisontaal verworwe MGEs, veral in die replikasie, integrasie en produksie van ekstrachromosomale CTXΦ van profeë en eksisie en verspreiding van SXT. Ons het die raaisel van LexA-essensaliteit opgelos in V. cholerae deur die te skrap lexA geen van die chromosoom in die gemanipuleerde V. cholerae stam BB1 geskrap met GI's en profage (Tabel 1). Ons het 'n rekombinante vektor pAP17 wat kanamisien weerstand kasset bevat (aff3) flankeer deur die stroomop en stroomaf streke van lexA om die uit te vee lexA geen deur alleliese uitruilmetode. Om die sleutel genetiese element verantwoordelik vir noodsaaklikheid van die te identifiseer lexA geen in V. cholerae genoom, het ons enkele of meervoudige GI of profeet-geskrap stamme geselekteer (Tabel 1) gevolg deur 'n poging om te skrap lexA in elk van hierdie gemanipuleerde stamme. Verbasend genoeg het sulke keuring ons toegelaat om te ontdek dat die lexA geen is nie noodsaaklik in TLC-CTX-RS1-profaag-geskrap nie V. cholerae selle, maar dit is steeds noodsaaklik in enkel- of meervoudige GI-negatiewe stam (Tabel 4). Om te bepaal of TLC-CTX-RS1-skikking of enige spesifieke een van hierdie fage verantwoordelik is vir die noodsaaklikheid van lexA, elkeen van hulle is opeenvolgend uitgevee, gevolg deur 'n poging om te skrap lexA in daardie gemanipuleerde stamme. Interessant genoeg is dit geïdentifiseer dat lexA is nie net noodsaaklik in CTX-profaag-geskrap selle nie (Tabel 4). CTX-profaag is saamgestel uit RS2 en kernstreek. Terwyl RS2 dra rstR-rstA-rstB gene, kernsegment dra sewe verskillende gene, insluitend CT-kodering ctxAB gene. Aan die ander kant is die RS1Φ identies aan die RS2 behalwe dat dit 'n ekstra geen dra, genaamd rstC. Die rstA, rstB, en rstR geenprodukte is onderskeidelik noodsaaklik vir faagreplikasie, -integrasie en transkripsieregulering. Sedert die uitdrukking van rstR, rstA, en rstB is onder beheer van LexA van PrstA promotor (31), het ons dus die funksie van elk van hierdie proteïene ontleed om hul bydrae, indien enige, in die essensie van lexA geen in V. cholerae. RstB is 'n enkelstrengige DNA-bindende proteïen, wat help met die integrasie van CTXΦ en die RS1Φ by die chromosomale verskil werf. RstR is 'n transkripsiereguleerder, wat die transkripsie van rstA, rstB, en rstC van die PrstA promotor (SI Aanhangsel, Fig. S3). RstA is 'n Rep-proteïen, wat DNA-kerwe in die orictx volgorde en help CTXΦ om ekstrachromosomale-faaggenoom vanaf die profaag te genereer. Ons het veronderstel dat, in die afwesigheid van LexA-gemedieerde onderdrukking, daar oortollige produksie van RstA vanaf die CTX- en RS1-profage kan wees, wat op sy beurt gereelde inkepings in die CTX- en RS1-profaag-DNS genereer, wat lei tot dodelike chromosomale breuke. Om hierdie hipotese te toets, het ons probeer om CTXΦ of CTXΦ-RS1Φ in tandem te integreer in V. cholerae ΔlexA ΔCTXΔRS1 mutant, maar kon nie veelvuldige kopieë van CTXΦs of CTXΦ-RS1Φ in tandem integreer nie. In skerp kontras, integrasie van veelvuldige kopieë van CTXΦs of CTXΦ-RS1Φ in tandem in die verskil 1 terrein van die V. cholerae ΔCTXΔRS1 maar lexA-positiewe stam is maklik verkry (SI Aanhangsel, Fig. S1). Aangesien RecA ingewikkeld geassosieer word met LexA-stabiliteit en dus profagegeen-uitdrukking, het ons 'n ΔlexAΔrecA dubbel-mutante stam JV5 en het die CTX-profaagnommer in die verskil 1 plek na die invoering van die faag deur vervoeging. Soos ΔlexA stam, was die enkelkopie CTX-profaag ook dominant in ΔlexAΔrecA dubbel mutant (SI Aanhangsel, Fig. S1). Om verdere bewyse te verskaf dat die veelvuldige kopie van funksionele rstA word geassosieer met die noodsaaklikheid van LexA in V. cholerae, het ons plekgerigte mutagenese uitgevoer om die katalitiese tirosienresidu van die RIYNK-motief van RstA-proteïen met fenielalanienresidu te vervang. As gevolg van hierdie puntmutasie kon die gemanipuleerde CTXΦ wat katalities onaktiewe RstA Y237F mutante proteïen kodeer nie rollende sirkel replikasie inisieer in V. cholerae. 'n Enkele kopie van CTXΦ wat nie-funksionele RstA dra, is egter stabiel gelysogeniseer, beide in die teenwoordigheid en afwesigheid van LexA. Hierdie resultate dui dus sterk daarop dat, wanneer dit funksioneel is rstA geen is teenwoordig in V. cholerae genoom, skrapping van lexA is dodelik vir die bakterie. Monitering van fenotipes (antibiotiese weerstand) sowel as genotipes (PCR-toets en volgordebepaling) het die egtheid van hierdie V. cholerae mutante.

Noodsaaklikheid van lexA vir die lewensvatbaarheid van Vibrio cholerae in die teenwoordigheid en afwesigheid van genomiese eilande en profeë

Core Genome-Encoded RecA help CTXΦ om gasheerimmuniteit te oorkom en weer te besmet V. cholerae.

Anders as faag-gekodeerde RstA, speel chromosomaal gekodeerde UvrD-helikase en LexA ook 'n belangrike rol in CTXΦ replikasie (30). Daar is gerapporteer dat RstR, die CTXΦ-gekodeerde transkripsiefaktor, aan die rstA promotor (PrstA) en onderdruk die uitdrukking daarvan vanaf die profaggenoom (31). Volledige onderdrukking vind egter plaas deur samewerkende interaksie met LexA, die meesterreguleerder van bakteriële SOS-reaksie. Die belangrikheid van RecA, die tweede deurslaggewende proteïen vir die bakteriële SOS-reaksie, in die replikasie van CTXΦ in V. cholerae is nie ondersoek nie. Ons het die replikasie-doeltreffendheid van El Tor, klassieke en omgewingstipes CTXΦs gemeet in die teenwoordigheid en afwesigheid van RecA in verskillende V. cholerae stamme (Fig. 6). Die genoom van N16961 huisves 'n CTXΦ ET-profaag in sy groot chromosoom. Ons het die replikasie- en integrasiemodule van CTXΦ ET (RS2 ET ) en CTXΦ Cl (RS2 Cl ) in RecA-positiewe N16961 ingebring en transkonjugante op 'n seleksieplaat aangevul met chlooramfenikol geïsoleer. Ons het 2.44 × 10 4 en 3.32 × 10 5 transkonjugante verkry wat onderskeidelik RS2 ET (pBS66) en RS2 Cl (pBS22) dra (Fig. 6). Ons het ook volledige CTXΦ ET, RS2 ET, RS2 Cl, RS2 Env en RS1Φ in 'n verslaggewer bekendgestel V. cholerae stam BS1 wat nie die CTXΦ-profaag in sy genoom het nie, wat 'n funksionele RecA het. Dit is opmerklik om te noem dat soortgelyke getalle transkonjugante verkry is vir elk van die fage wat ingebring is (Fig. 6). Toe ons elkeen van hierdie fage bekendgestel het in die BS1-afgeleide wat CTXΦ ET-profaag by die verskil 1 loci, hier het ons ook 'n soortgelyke aantal transkonjugante verkry wat replikatiewe en integrerende faaggenome of RS-modules dra. Die resultate dui daarop dat herinfeksie deur CTXΦ moontlik is selfs in die teenwoordigheid van biotipe-spesifiek rstR en funksioneel recA gene in die gasheergenoom (Fig. 6).

Analise van die replikasie-doeltreffendheid van verskillende CTXΦ in die teenwoordigheid en afwesigheid van RecA-funksie. Die stawe verteenwoordig getalle CFU's wat replikatiewe module van CTXΦ dra. Replikatiewe modules is oorgedra na die gasheer bakterieë deur 3 uur konjugasie. Transkonjugante is geselekteer op grond van die antibiotika-weerstand fenotipes. Tweerigting RM-ANOVA is gebruik om statistiese betekenisvolheid van die verskille van CFU-getalle af te lei. Foutbalkies dui SD's aan. Wt, wilde tipe ET, El Tor (A) Cl, klassiek (B) Env, omgewing (C) TCR, TLC-CTX-RS1-profaag. * = CTXΦ ET Cam.

Aangesien die funksie van RecA ingewikkeld geassosieer word met die SOS-reaksie en LexA-outoproteolitiese aktiwiteit moduleer, het ons ons studie uitgebrei om die rol van RecA in CTXΦ replikasie te ontsyfer. Om dit te doen, is al die genetiese elemente, naamlik CTXΦ ET , RS2 ET , RS2 Cl , en RS2 Env , in die recA-geskrapte BS1-stam, en die getalle van transkonjugante op die seleksieplate is vergelyk. Soortgelyke getalle transkonjugante wat replikatiewe en integrerende faaggenome of RS-modules dra, is verkry in die teenwoordigheid van funksionele RecA (SI Aanhangsel, Tabel S2). Ons het toe die recA geen in N16961 en het CTXΦ ET, RS2 ET, RS2 Cl en RS2 Env bekendgestel om replikasiedoeltreffendheid van verskillende faag-afgeleide elemente te monitor. Tot ons verbasing, terwyl geen transkonjugant vir die CTXΦ ET- of RS2 ET-element verkry is in die afwesigheid van recA, transkonjugante vir RS2 Cl (4.6 × 10 5 ) of RS2 Env (5 × 10 4 ) element was verkrygbaar deur dieselfde V. cholerae mutante stam (Fig. 6). Ons het hierdie fenotipe verder bevestig deur gebruik te maak van die N16961-afgeleide BS11-stam, wat nie recA en al die profeë. Sodra CTXΦ ET bekendgestel is, het dit ook immuniteit getoon teen herinfeksie met die soortgelyke tipe CTXΦ (Fig. 6). Ons het ook soortgelyke fenotipes in klassieke (O395) en omgewings (VCE232) waargeneem. V. cholerae strains when we introduced CTXΦ Cl or CTXΦ Env replication and integration module to the strains lacking recA gene but containing CTXΦ Cl or CTXΦ Env in the chromosome, respectively (Fig. 6 and SI Appendix, Table S2). In both cases, CTXΦ can replicate in the absence of recA but presence of different biotypes of CTX prophages in the chromosome. We also ensured that the transmission process is not reduced in the absence of recA gene function by using a replicative conjugative plasmid pFX497 (SI Appendix, Table S1) as a control, which gave a similar number of transconjugants in the presence or absence of recA. However, RecA function was not linked with the integration efficiency of CTXΦ, since it is able to integrate at the dif site with almost equal efficiency in presence or absence of RecA function (SI Appendix, Table S2). The findings of the present study demonstrate that RecA helps CTXΦ replication in the presence of preexisting CTX prophage in the host chromosome.

Contribution of GIs in Modulating Cellular Proteome.

For identification, quantification, and comparison of whole-cell proteomes of wild type V. cholerae strain N16961 and its derivative BB1 devoid of GIs and prophages, we performed experiments using a high-throughput liquid chromatography (LC) system (Eksigent 2D) coupled with Triple TOF 5600 mass spectrometer (AB Sciex). We have identified a total of 1,864 proteins in both strains (Dataset S1). Based on the proteome profile of N16961 and BB1, we categorized the protein pool into three classes: (i) proteins with lower abundance (>1.5 log fold changes) or absent in BB1, (ii) proteins with higher abundance (>1.5 log fold changes) in BB1, and (iii) proteins with similar abundance in both strains. Proteins that are not detectable in BB1 but found in the N16961 strain were analyzed to find their coding sequences. We have identified similar numbers of proteins in N16961 (n = 1,765) and BB1 (n = 1,724) strains. A total of 141 proteins were detected only in N16961, of which 41 proteins are encoded by the genes present in Chr2. More than 10 proteins are encoded by the 4 GIs, VPI-1 (n = 1), VPI-2 (n = 4), VSP-1 (n = 3), VSP-2 (n = 1), and CTXΦ (n = 2), which were detected in N16961 but not in BB1. We observed that 131 proteins were differentially expressed (log fold change 1.5 and P value <0.05) between N16961 and BB1 strains. Out of 131 differentially expressed proteins in BB1, 59 and 72 proteins were up- and down-regulated, respectively. Proteins down-regulated in BB1 belong to uncharacterized proteins (n = 8), outer membrane proteins (n = 2), flagellar biosynthetic proteins (n = 2), aspartate modifying enzymes (n = 2), and several others. Enzymes involved in succinate metabolic pathways (n = 3), RNA degradation (n = 2), and ABC transporters (n = 2) are up-regulated in the absence of MGEs. Both small- and large-subunit ribosomal proteins are up- and down-regulated in the absence of MGEs. We observed that 61 proteins encoded by the Chr2 were detectable either in N16961 (n = 25) or BB1 (n = 36).


Bespreking

Most genomic studies on V. cholerae have focused on serogroup O1, but the global genomic landscape for the non-O1/non-O139 serogroups, the genetic diversity of the O-PS gene clusters, and the evolutionary associations with O1 and O139 serogroups have not been adequately studied. Here, we sequenced three strains from two O159 and O170 serogroups, together with 25 strains from 16 other serogroups to reveal their O-PS gene cluster organizations, genomic features and the evolutionary associations among the different serogroups.

The ANI scores between the different strains reflect the level of global similarity among them. Based on our results, serogroups O159 and O39 are most similar in terms of their ANI score, as are serogroups O170 and O89. Among all the representative strains, most share high similarity scores with each other and only serogroup O115 has an ANI score lower than 95% when compared with all the other strains.

O-PS contains genetic signatures that allow the genetic clones of strains within a species to be distinguished. The genes related to O-PS biosynthesis are generally organized together to form a cluster. All the O-PS biosynthesis genes from the 18 serogroups from this study clustered together and were chromosomally located between the gmhD en rjg genes, suggesting that site-specific transfer has occurred among the different serogroups and strains. Within the O-PS gene clusters, several genes are grouped together and appear in the O-PS gene clusters in the different serogroups, possibly showing that frequent recombination has occurred in these gene clusters. Thus, genetic variability in the O-PS biosynthesis genes, coupled with frequent genetic transfers and recombination may have generated the many serogroups of V. cholerae. Serogroups O1 and O139 cause cholera epidemics, but only one gene in the O-PS genetic region of serogroup O159 was found to share sequence homology with a serogroup O1 gene. It is interesting that serogroup O170 shares the most sequence homology in the O-PS genes with O139, suggesting that O139 and O170 O-PS genetic regions have similar sub-structures.

By visualizing their vertices and edges, networks are a useful way of displaying complicated relationships among vertices in an intuitive way, and analysis of a specified network may provide new insights into biological systems. Here, we constructed a relationship network containing different V. cholerae serogroups and their associated O-PS genes. Previous preliminary research has shown there is variety in the O-PS genetic region in the different serogroups [19], and our network analysis on the homologous genes in the present study supports the idea that there is great diversity in the O-antigen among different serogroups, a viewpoint supported by our 2-dimensional hierarchical clustering analysis. This analysis made further efforts to decipher the detailed relationship between the different serogroups and the contributions of the constituent O-PS genes in each serogroup. The O-PS gene cluster size and gene count showed great divergence among the serogroups. Except for the left and right junction genes in the wb* regions encoding O-PS synthesis, no genes were shared by all the serogroups, further indicating the genetic diversity of the O-PS in V. cholerae. Based on the network analysis, some genes shared by more than two serogroups were identified, showing that exchange and recombination has occurred in the genes and even in the gene clusters among the O-PS genetic regions from the different serogroups. This is further supported by the finding that the O-PS genetic regions comprise the combinations of several smaller gene sets with different origins [20]. Our data have revealed that the O-PS biosynthesis gene pool contains 405 homologous gene clusters, and the network analysis results suggest the hot-spots for serogroups or homologous genes that link other serogroups or homologous genes to a high degree. We suggest that the genes in the gene pool facilitate O-antigen shifts in the different V. cholerae serogroups. Serogroup conversion of the O1 recipient by the O139 donor has been validated in the laboratory, and it is proposed that the exchange of serogroup-specific gene clusters could potentially occur between the different O serogroups of V. cholerae [21].

Our phylogenetic analysis revealed that most toxigenic O1 V. cholerae strains and two classical strains form two separate clusters in the tree. One O139 strain clustered together with most toxigenic O1 V. cholerae strains. Two non-O1, non-O139 strains, V52 (O37) and 981–75 (O65), appear in the same cluster with toxigenic O1 V. cholerae strains, indicating the possibility of O serogroup conversions from O1 to non-O1/non-O139 [20, 22]. Most of the non-O1/non-O139 strains were distributed in the other part of the tree, a finding consistent with that of a previous study [20]. Furthermore, we investigated whether the O-PS biosynthesis gene clusters had undergone co-evolution with the whole genomes in the different V. cholerae serogroups. The genomes and O-PS genes had different phylogenic structures in these strains, revealing that asynchronous genetic variation had occurred during their evolution. We also found that several serogroup pairs had consistent relationships between core-genes or pan-genes and the O-PS genetic region. The consistency between the core gene-based and O-antigen region-based genetic distance indicates that these serogroups had the same common ancestors before gaining similar O-PS genetic regions. In addition, the consistency between the pan gene-based and O-PS genetic region-based distance indicates that these serogroups have undergone some horizontal gene transfer events and acquired similar foreign genes before gaining similar O-PS genetic regions.

The virulence-related genes in the V. cholerae strains displayed the highest levels of sequence similarity with those from P. aeruginosa, V. vulnificus, H. somnus en H. influenzae. As V. cholerae has an aboriginal habitat in the environment, these data suggest that V. cholerae can exchange virulence-related genes with the above-named species under certain environmental conditions.

Changes that occur in mobile genetic elements may relate to the emergence, drug resistance, fitness and virulence of a strain. Here, we analyzed the profiles of the mobile genetic elements (i.e., VPI-1, VPI-2, VSP- I, VSP-II, SXT and CT) in the different representative strains. We found O159 had a nearly complete VSP-II and partial VPI-1 and VPI-2 sequences, whereas the O170 strain carried partial VPI- 1 and VPI- 2 sequences. VSP-II is considered to be a genetic marker of epidemic V. cholerae therefore, gaining VSP- II may allow a strain to obtain a survival advantage during epidemics [23]. VPI is one of two essential virulence gene clusters in epidemic-causing toxigenic V. cholerae. Several non-O1/non-O139 strains contain full or partial VPI-1 and VPI-2 islands, indicating their potential to evolve into epidemic strains. Because alteration of the O-antigen in pathogenic strains will boost the emergence of new epidemic strains, a combined description of the O-PS genetic region in the non-O1/non-O139 serogroups and their virulence elements, such as CT and TCP, will provide further information on the genesis mechanism of new emerging strains.

Positive selection is known to be an important aspect of the evolution of many pathogens [24, 25]. Indeed, one study showed that the diversification of clinical and environmental V. cholerae isolates from Haiti was driven by positive selection [26]. Selection pressure analysis of strains within the different V. cholerae serogroups should provide detailed information on their evolutionary trends. Here, we found that several of the strains share genes that appear to have evolved under positive selection and are involved in environmental adaptation (e.g., chemotaxis, Na + resistance and cell wall synthesis). These functions may represent the common mechanisms used by strains within the different serogroups to adapt to environmental changes. Serogroup-specific selected genes were also found, and some positively-selected genes may exist in more than one serogroup. Thus, the genetic complexity of the different serogroups and strains may reflect their adaptability to different niches.


Availability of a database containing the proteins of Vibrio cholerae and their corresponding gene sequences - Biology

We showed previously that chitin catabolism by the marine bacterium Vibrio furnissii involves at least three signal transduction systems and many genes, several of which were molecularly cloned, and the corresponding proteins were characterized. The predicted amino acid sequences of these proteins showed a high degree of identity to the corresponding proteins from Vibrio cholerae, whose complete genomic sequence has recently been determined. We have therefore initiated studies with V. cholerae. We report here a novel ATP-dependent glucosamine kinase of V. cholerae encoded by a gene designated gspK. The protein, GspK (31.6 kDa), was purified to apparent homogeneity from recombinant Escherichia coli. The product of the reaction was shown to be GlcN-6-P by matrix-assisted laser desorption/ionization-time of flight (MALDI mass spectrometry) and NMR. The Km values for GlcN, ATP, and MgCl2 were 0.45, 2.4, and 2.2 m m , respectively, and the Vmax values were in the range 180–200 nmol/μg/min (∼6 nmol/pmol/min). Kinase activity was not observed with any other sugar, including: galactosamine, mannosamine, Glc, GlcNAc, GalNAc, mannose, 2-deoxyglucose, and oligosaccharides of chitosan. The enzyme is also ATP-specific. The kinase can be used to specifically determine micro quantities of GlcN in acid hydrolysates of glycoconjugates. The physiological function of this enzyme remains to be determined.

Published, JBC Papers in Press, February 15, 2002, DOI 10.1074/jbc.M107953200

This work was supported by Grant GM51215 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: ISM Biopolymer, 220 Denison E. Granby, Quebec J2H 2R6, Canada.


Bespreking

In this study, we adopted reverse vaccinology based reductive screening and fished out five immunogenic proteins harboring 10 peptide epitopes as potential vaccine candidates in the V. cholerae proteome. Reverse vaccinology is a genome/proteome based approach for vaccine development that has been proved effective (Giuliani et al., 2006). Reductive screening is performed based on parameters i.e., protein essentiality, subcellular localization, host homology and effective immunogenicity for predicting an effective vaccine candidate. A computer-aided screening process is more convenient, accurate and fast in comparison with the contemporary vaccine development which depends on a hit and trial approach. This strategy studies key aspects of the pathogen i.e., genome, essential metabolism, virulence and protein-protein interactions and incorporates this information for determining the prospective vaccine candidates prior to any wet lab experimentations (Naz et al., 2015). One of the key limitation is that this strategy is primarily focused on prediction of peptide epitopes based on amino acid sequences of the proteins. Hence, the long known immunogenic potential of nonprotein antigens (i.e., Lipopolysaccharides) couldn’t be accounted in this strategy (Lüderitz et al., 1982 McGhee et al., 1980 Del Barrio et al., 2015). But the addition of such known epitopes as adjuvants is a good approach for overcoming this limitation (Caucheteux et al., 2017 Noguchi et al., 2017). Another prominent limitation could be the high mutation rate of the viral surface proteins (Steinhauer & Holland, 1987 Echave, Spielman & Wilke, 2016). The prospects of reverse vaccinology approach have been discussed in detail in our previous study (Rashid et al., 2017).

Peptide vaccines theoretically have several advantages over conventional and recently developed DNA vaccines (Ingolotti et al., 2010). Lesser cost and convenient synthesis with improved safety and stability are the key features which have been demonstrated in various studies (Firbas et al., 2006 Jagannath et al., 2009). Conventional vaccines are overburdened with unnecessary antigens which divert immune response resources thus might result in a chaos which lacks the required dedicated for eliminating the threat thus impedes the vaccine efficacy (Czerkinsky & Holmgren, 2015). As in case of cholera, whole cell vaccines were only able to impart varying protective efficiency (39–60%) in studies conducted in Bangladesh and Vietnam (Clemens et al., 1990 Thiem et al., 2006). While live attenuated vaccine was unsuccessful in generating long term protective response (Fournier, 1998). One interesting inconsistency is the comparative efficacy of cholera vaccines in developed and developing countries (Czerkinsky & Holmgren, 2009), while a notable recent exception was observed in South Sudan (Bekolo et al., 2016). Considering these factors, the need for novel strategy is vital to achieve protection against this pathogen.

Reported prioritized targets included lipoprotein NlpD, outer membrane protein OmpU, accessory colonization factor AcfA, putative porin, and outer membrane protein OmpW. These predicted proteins are involved in important virulence mechanisms of V. cholerae. Role of lipoprotein NlpD, has been studied in reference to cell division and intestinal colonization by the pathogen. Septal peptidoglycan (PG) amidase, AmiB is involved in separation of daughter cells at the end of cell division process (Yakhnina, McManus & Bernhardt, 2015). AmiB is regulated by NlpD in V. cholerae (Möll et al., 2014). Both of these processes are important for pathogen’s survival in host intestine. Another predicted potential target accessory colonization factor AcfA is of peculiar interest as it has been subjected to edible vaccine (Sharma et al., 2008). Targeting NlpD en AcfA could provide passive therapeutic potential as immune inactivation would impede the pathogen’s ability to colonize and multiply in the small intestine.

Among these vaccine candidates, we obtained two outer membrane proteins (OMPs), OmpU and OmpW that also serve as antibiotic resistance determinants. In vibrio species OMPs are studied to play vital roles as porins in iron, phosphate and sugar acquisition as well as in bacterial attachment to solid surfaces (Aeckersberg et al., 2001). While OmpU has been reported to be involved in conferring polymyxin B sulfate resistance (Mathur & Waldor, 2004). We consider OmpU as an important vaccine candidate selected via our computational framework as it is not only involved in host cell invasion but also confers antibiotic resistance (Duperthuy et al., 2011). Moreover, it has also been used as an effective vaccine candidate in other vibrio species such as V. alginolyticus en V. harveyi in Lutjanus erythropterus en Scophthalmus maximus, respectively (Cai et al., 2013 Wang et al., 2011). Such studies provide good examples of how a reverse vaccinology strategy can be used for systematic vaccine design against drug resistant microbial pathogens.

Another important predicted potential vaccine candidate is OmpW. It’s a characteristic outer membrane protein expressed by V. cholerae and has been used to identify infectious agent via different PCR based detection techniques. Studies have reported this protein to be conserved and harbors immunogenic properties (Nandi et al., 2005 Jalajakumari & Manning, 1990). Considering its abilities, OmpW could be a good candidate for developing a broad spectrum and effective vaccine.

Interestingly, when we analyzed our screened results with a recent antibody profiling study of the V. cholerae O1 protein immunome, nine overlapping antigens were observed (Charles et al., 2017). These antigens were: Organic solvent tolerance protein (VC0446), outer membrane protein OmpU (VC0633), toxin co-regulated pilin (VC0828), outer membrane protein OmpV (VC1318), neuraminidase (VC1784), hemolysin-related protein (VC1888), and flagellar proteins/components (VC2142, VC2143, VC2187). Among these nine, outer membrane protein OmpU (VC0633) was common in the most effective antigens reported in the final selection of the both studies. While toxin co-regulated pilin (VC0828) was among our initial screening list but it is reported as one of the most effective by Charles et al. (2017). One possible reason of the screening results could be the difference in the adopted screening strategies. Our strategy was purely computational, with the calculations all derived using only the peptide sequences of the proteins. A shortcoming to this is that it could only be applied for peptide antigens, while on the other hand antigens other than proteins do have their immuno-protective potential i.e., the O polysaccharide, LPS, etc. These overlapping proteins in the two investigations provide confidence to our prediction.


Present address: The 306th Hospital of PLA, Beijing, 100101, China

Present address: CAS key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

Affiliations

The 306th Hospital of PLA, Beijing, 100101, China

Yong Yi, Liping Jia, Hua Jing, Hu Xia & Yan Cui

CAS key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

Na Lu, Fei Liu, Jing Li, Ruifen Zhang, Baoli Zhu & Yongfei Hu

Beijing Key Laboratory of Microbial Drug Resistance and Resistome, Beijing, 100101, China

Na Lu, Fei Liu, Jing Li, Ruifen Zhang, Baoli Zhu & Yongfei Hu

Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, 100730, China


Background

Identification of conserved protein domains that span a wide range of biological functions provide deep insights regarding the origin and evolution of complex biological systems, These versatile conserved domains often have catalytic or structural roles that can be utilized, with small variations, in different contexts. The P-loop-containing nucleotide phosphatase fold represents one such catalytic domain that is utilized in almost every conceivable biological system in all the three superkingdoms of life [1,2]. Folds such as the SH3-like barrels, the PAS-like fold, the OB fold, the double-stranded β-helix, the β-propeller and rubredoxin-like zinc ribbons are predominantly non-catalytic domains that are widely represented in multiple functional contexts, with roles such as small-molecule binding, nucleic-acid binding and interaction with other proteins [3,4,5] (see also the SCOP [6] and CATH [7] databases). Versatile globular domains appear to have emerged fairly early in evolution in various fold classes, such as the α/β or α+β mixed folds, or the all-α and all-β folds [5,8]. Comparative genomics and evolutionary studies indicate that many of these versatile folds probably emerged in contexts related to RNA binding in the ancient translation system and were subsequently re-utilized in other biological systems [9,10].

Of particular interest in this context are the small all-β folds that assume conformations such as barrels or β-helices [3,4]. These structures have considerable potential for functional versatility, because they are able either to accommodate small molecules within cavities formed by the curved β-sheets or to interact with various larger molecules, especially nucleic acids or proteins, via the external surfaces of the sheets. A few ancient and widespread β-rich folds such as the SH3-like barrel and the OB fold appear to have colonized multiple functional niches early in evolution, although their earliest versions may have had roles related to RNA metabolism [9,11,12,13]. We were interested in identifying other such functionally versatile β-rich folds that could be traced back to the early stages of life's evolution. The availability of extensive genome sequence data and advances in structure determination over recent years allow the successful application of comparative genomics, sequence and structure comparisons to identify any such folds that may be somewhat less widely represented than the OB or SH3-like folds.

Here, we identify one such β-barrel fold typified by the globular domain of the H subunit of the photosynthetic reaction center (PRC-H) from purple proteobacteria such as Rhodopseudomonas viridis [14,15]. The purple bacterial photosynthetic reaction center consists of three primary subunits, of which PRC-L and PRC-M primarily bind the pigments involved in photochemistry, whereas PRC-H appears to be a key regulator of electron transfer between the quinones in photosynthetic reaction centers [16]. So far, homologs of the H subunit have only been found in photosynthetic proteobacteria [17,18,19] and the carboxy-terminal globular domain of PRC-H shows a distinct β-barrel fold that is structurally unrelated to other characterized β-barrels. This raises the important question of the evolutionary provenance of this unique domain. Here we use sequence-profile analysis and comparative genomics to show that the β-barrel domain of PRC-H defines a novel, widespread superfamily of β-barrel domains that is represented in several bacterial, plant and archaeal genomes. We also show that this β-barrel domain is found in the conserved protein RimM, which is involved in RNA processing and ribosomal assembly in the course of translation. Thus we provide evidence for an unexpected evolutionary connection between RNA metabolism, translation and the redox reactions in photosynthesis in the form of a shared functionally versatile β-barrel domain.


Bespreking

The genomic island of V. parahaemolyticus TS 8-11-4 was deemed a PAI due to the presence of virulence genes on this island, despite its environmental origin (Schmidt and Hensel 2004 Hasan et al. 2010 Dobrindt et al. 2004). The thermostable direct hemolysin gene (tdh) was found on this island, as well as genes involved in the type three secretion system II (T3SS2). Both the tdh gene and T3SS2 complex are the two major virulence factors implicated in V. parahaemolyticus pathogenesis (Makino et al. 2003 Park et al. 2004 Yanagihara et al. 2010). A collagenase gene was found on the island collagenase is thought to be involved in V. parahaemolyticus virulence (Gode-Portratz et al. 2011). The genomic island of V. parahaemolyticus strain TS-8-11-4 is a PAI, and more specifically, because it contains tdh and T3SS2 genes, we designate this island as a VPAI-7 (VPaIα or tdhVPA) (Makino et al. 2003 Sugiyama et al. 2008 Xu et al. 2017).

Four genes involved in capsule production, as well as one integrase gene, and a Na + /H + antiporter (nhaA) were also found on this PAI. Capsules aid pathogens in evasion of host immune defenses, establishing infections, and survival in harsh environments, such as the stomach. V. parahaemolyticus virulence is correlated with capsule production (Broberg et al. 2011 Letchumanan et al. 2014). One capsule gene had high homology with Gram positive capsule production genes. This is interesting because vibrios are Gram negative organisms, so this gene may have been acquired laterally. An integrase gene was found near the center of the island. Integrase genes are associated with PAIs and function to integrate foreign DNA into the genome (Hacker and Kaper 2000). Usually VPAI-7 does not contain an integrase gene, but a few transposon genes instead (Ceccarelli et al. 2013). Finally, we determined that a nhaA gene is located on this genomic island. nhaA genes encode Na + /H + antiporters, which transport ions to balance pH. Na + /H + antiporters aid V. cholerae in environmental persistence (Vimont and Berche 2000) and are essential for Yersinia pestis virulence (Minato et al. 2013).

Similar to V. parahaemolyticus, the two islands found for the V. vulnificus WR-2-BW strain are characterized as PAIs due to the presence of virulence genes and virulence-related genes. Two of these genes had virulence-related functions, a putative LPS biosynthesis protein gene and an O-antigen flippase wzx gene. These genes are virulence-associated factors, as they do not directly cause host cell damage, but they do contribute to pathogenesis, aiding in the establishment of infections. Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram negative bacteria, and is a known pyrogen (fever-producing agent) (McPherson et al. 1991 Jones and Oliver 2009). Phylogenies show that the LPS biosynthesis protein gene from V. vulnificus WR-2-BW was closely related to an LPS biosynthesis protein gene from a Vibrio coralliilyticus species. The O-antigen flippase wzx gene is part of the major class of O-antigen gene clusters, and it encodes a hydrophobic protein with 12 potential transmembrane segments (Liu et al. 1996).

A cytolysin secretion gene, vvhB, was found also found on the 143 kbp V. vulnificus island. Cytolysins lyse erythrocytes by forming small pores in the cytoplasmic membrane or binding to cholesterol to interrupt potassium and sodium ion channels (Choi et al. 2002). In V. vulnificus, the expression and mechanism of cytolysins vvhA en vvhB are not fully understood, however, they are both believed to play a role in pathogenicity (Choi et al. 2002). They are homologous to a known V. cholerae El Tor hemolysin (Choi et al. 2002 Yamamoto et al. 1990). Phylogenies show that the vvhB gene in the V. vulnificus WR-2-BW strain was 99% identical to other V. vulnificus vvhB genes from other strains.

Other genes of interest on the 143 kbp PAI include a chitinase gene, tldD/tldE proteolytic complex genes, and type IV secretory pathway components. In Escherichia coli, it was shown that the TldD and TldE proteins could be involved in regulating gyrase function as well as aiding in proteolytic activity (Allali et al. 2002). The chitinase gene had a 99% blast identity score to the chitinase gene found in the V. vulnificus YJ016 strain however, the chitinase gene in YJ016 is located on the first chromosome and WR-2-BW’s chitinase gene is located in the second chromosome. Chitinous exoskeletal materials of invertebrates can be a source of carbon and nitrogen for bacteria vibrios in particular have a well-known association with marine copepods (Kaneko and Colwell 1975 Lovell 2017). V. cholerae has a well-studied association with copepods, which commonly serve as a vector of cholera infections in Bangladesh water systems (Tamplin et al. 1990). Chitinase has been identified as part of the mechanism for adsorption and attachment to copepods, which relates to its ability to colonize its host and degrade the host exoskeleton, increasing the overall ecological fitness of the vibrios (Huq et al. 1983 Nalin et al. 1979 Bhowmick et al. 2006).

Vibrio diabolicus had a large genomic island that did not contain any virulence factors or virulence associated genes, which we defined as a fitness island, as it contained genes that would aid the organism in persistence in the environment. Toxin–antitoxin (TA) systems are found either on plasmids, genomic islands, or within the chromosome and are made up of closely linked toxin and antitoxin genes. The encoded labile antitoxin protects the host from the stable toxin, while competitor cells that do not have the TA system (and respective antitoxin) are eliminated (Hayes 2003 Van Melderen and Saavedra 2009). Sometimes TA systems are referred to as “addiction modules” because the host cell is dependent on the antitoxin (Van Melderen and Saavedra 2009). The toxin and respective antitoxin loci are usually found neighboring each other, often overlapping (Hayes 2003). Seven type II TA toxins were found on JBS-8-11-1’s fitness island, along with their neighboring respective antitoxins. Type I TAs include RNA antitoxins, while type II TAs have protein antitoxins (Hayes 2003). The relE, yafQ, and yoeB toxin genes encode mRNA interferase endoribonucleases all three of these toxin genes were detected on this fitness island. The doc toxin gene (death on curing) inhibits translation by blocking translation elongation at the 30S ribosomal subunit (Lui et al. 2008) three copies of the doc toxin gene and three copies of its antitoxin partner gene, phd (prevent host death) were found on JBS-8-11-1’s fitness island. doc toxin genes and phd antitoxin genes are widespread in vibrios and were also found on V. parahaemolyticus strain TS-8-11-4’s PAI as well as V. vulnificus strain WR-2-BW’s PAI (Fig. 2).

Maximum-likelihood phylogeny (Kimura 2-parameter model) of doc toxin genes and phd antitoxin genes. Bold indicates sequences obtained from this study. The bootstrap values represent 1000 replications, and values of less than 50 are not shown. The reference sequences were acquired from NCBI GenBank

Lateral gene transfer in environmental strains

PAIs are present in environmental Vibrio strains and are most likely acquired via lateral gene transfer. All four of the islands described here have significant lower GC content than the rest of the genome, providing evidence that these islands originated from a foreign source and were transferred into these genomes relatively recently. Additional evidence includes mobile genetic elements, such as phage and plasmid genes, integrases, and transposons. Virulence loci on VPAI-7 have been detected in environmental species that do not cause human infections: Vibrio mimicus, Vibrio harveyi, and Vibrio natriegens (Gennari et al. 2011 Klein et al. 2014). Clearly, lateral transfer of individual virulence loci and/or entire PAIs is occurring between and among environmental vibrios. It is well documented that V. cholerae enters a natural competency state in the presence of chitin or under low-nutrient conditions (Hazen et al. 2010 Metzger and Blokesch 2016) however, less is known about uptake of exogenous DNA by other Vibrio species. Further studies examining the rates of lateral transfer among vibrios in the environment are needed. Vibrios survive, persist and can undergo rapid population expansions (bloom) in coastal ecosystems. Consequently, the pathogenicity loci (and potential of said loci to be transferred laterally) of naturally occurring environmental strains are clearly important.


Kyk die video: Vibrio Cholerae Cholera - Pathophysiology - Symptoms - Diagnosis - Treatment (September 2022).


Kommentaar:

  1. Neshakar

    kan nie wees nie

  2. Wazir

    in die oond

  3. Rafael

    Ek is jammer, dit het inmeng ... ek het onlangs hier.Maar hierdie tema is baie na aan my. Is gereed om te help.

  4. Vudoshakar

    hartseer

  5. Kajilkree

    I personally did not like it !!!!!

  6. Stamford

    Wacker, by the way, that phrase just came up



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