Name: high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection
Uploaded: 2017-12-18T12:30:00
Description: Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

Detta arbete stöds av en veteraner frågor Merit Award (till D.A.H.) och en National Institute of Health bevilja R01 AI 034431 (D.A.H.).

FÖRSIKTIGHET: Patogena stammar av Leptospira spp. måste hanteras under biosäkerhetsnivå 2 (BSL-2) inneslutning förfaranden. Lämplig personlig skyddsutrustning (PPE) måste bäras. En klass II biosäkerhet skåp måste användas för alla manipulationer av patogena Leptospira spp.
1. skapande av Transposon Mutant bibliotek15
<ol><li>
Överföring av transposon till Leptospira spp. av konjugering (<strong class=...

<ol>
	<li>Murray, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+transposon+mutagenesis+in+pathogenic+Leptospira+species.">Genome-wide transposon mutagenesis in pathogenic Leptospira species.</a> Infect Immun. 77 (2), 810-816 (2009).</li><li>Adler, B., Lo, M., Seemann, T., Murray, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+leptospirosis:+the+influence+of+genomics.">Pathogenesis of leptospirosis: the influence of genomics.</a> Vet Microbiol. 153 (1-2), 73-81 (2011).</li><li>Marcsisin, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+high-throughput+screen+to+identify+Leptospira+mutants+unable+to+colonize+the+carrier+host+or+cause+disease+in+the+acute+model+of+infection.">Use of a high-throughput screen to identify Leptospira mutants unable to colonize the carrier host or cause disease in the acute model of infection.</a> J Med Microbiol. 62, 1601-1608 (2013).</li><li>van Opijnen, T., Bodi, K. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn-seq:+high-throughput+parallel+sequencing+for+fitness+and+genetic+interaction+studies+in+microorganisms.">Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.</a> Nat Methods. 6 (10), 767-772 (2009).</li><li>van Opijnen, T., Lazinski, D. W., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+fitness+and+genetic+interactions+determined+by+Tn-seq,+a+high-throughput+massively+parallel+sequencing+method+for+microorganisms.">Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms.</a> Curr Protoc Microbiol. 36, 1E.3.1-1E.3.24 (2015).</li><li>Troy, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+barriers+to+Borrelia+burgdorferi+dissemination+during+infection+using+massively+parallel+sequencing.">Understanding barriers to Borrelia burgdorferi dissemination during infection using massively parallel sequencing.</a> Infect Immun. 81 (7), 2347-2357 (2013).</li><li>Wunder, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+reveals+rapid+dissemination+of+Leptospira+interrogans+after+intraperitoneal+and+conjunctival+inoculation+of+hamsters.">Real-time PCR reveals rapid dissemination of Leptospira interrogans after intraperitoneal and conjunctival inoculation of hamsters.</a> Infect Immun. 84 (7), 2105-2115 (2016).</li><li>Lourdault, K., Aviat, F., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+quantitative+real-time+PCR+for+studying+the+dissemination+of+Leptospira+interrogans+in+the+guinea+pig+infection+model+of+leptospirosis.">Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis.</a> J Med Microbiol. 58, 648-655 (2009).</li><li>Coutinho, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+Leptospira+interrogans+infection+in+hamsters+after+intradermal+and+subcutaneous+challenge.">Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge.</a> PLoS Negl Trop Dis. 8 (11), e3307 (2014).</li><li>Athanazio, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rattus+norvegicus+as+a+model+for+persistent+renal+colonization+by+pathogenic+Leptospira+interrogans.">Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans.</a> Acta Trop. 105 (2), 176-180 (2008).</li><li>Ratet, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+bioluminescent+Leptospira+interrogans+in+mice+reveals+renal+colonization+as+a+stealth+escape+from+the+blood+defenses+and+antibiotics.">Live imaging of bioluminescent Leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics.</a> PLoS Negl Trop Dis. 8 (12), e3359 (2014).</li><li>Gutierrez, M. G., Yoder-Himes, D. R., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+identification+of+virulence+factors+required+for+respiratory+melioidosis+using+Tn-seq+mutagenesis.">Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis.</a> Front Cell Infect Microbiol. 5 (78), (2015).</li><li>Gawronski, J. D., Wong, S. M., Giannoukos, G., Ward, D. V., Akerley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+insertion+mutants+within+libraries+by+deep+sequencing+and+a+genome-wide+screen+for+Haemophilus+genes+required+in+the+lung.">Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.</a> Proc Natl Acad Sci U S A. 106 (38), 16422-16427 (2009).</li><li>Gallagher, L. A., Shendure, J., Manoil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+identification+of+resistance+functions+in+Pseudomonas+aeruginosa+using+Tn-seq.">Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq.</a> MBio. 2 (1), (2011).</li><li>Slamti, L., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+library+of+random+mutants+in+the+spirochete+Leptospira+biflexa+using+a+mariner+transposon.">Construction of a library of random mutants in the spirochete Leptospira biflexa using a mariner transposon.</a> Methods Mol Biol. 859, (2012).</li><li>Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugative+transfer+between+Escherichia+coli+and+Leptospira+spp.+as+a+new+genetic+tool.">Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool.</a> Appl Environ Microbiol. 74 (1), 319-322 (2008).</li><li>Lourdault, K., Matsunaga, J., Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+parallel+sequencing+to+measure+fitness+of+Leptospira+interrogans+transposon+insertion+mutants+during+acute+infection.">High-throughput parallel sequencing to measure fitness of Leptospira interrogans transposon insertion mutants during acute infection.</a> PLoS Negl Trop Dis. 10 (11), e0005117 (2016).</li><li>Ellinghausen, H. C., McCullough, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrition+of+Leptospirapomona+and+growth+of+13+other+serotypes:+Fractionation+of+oleic+albumin+complex+and+a+medium+of+bovine+albumin+and+polysorbate+80.">Nutrition of Leptospirapomona and growth of 13 other serotypes: Fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80.</a> Am J Vet Res. 26, 45-51 (1965).</li><li>Johnson, R. C., Harris, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+pathogenic+and+saprophytic+letospires.+I.+Growth+at+low+temperatures.">Differentiation of pathogenic and saprophytic letospires. I. Growth at low temperatures.</a> J Bacteriol. 94 (1), 27-31 (1967).</li><li>Demarre, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+mobilizable+suicide+plasmids+based+on+broad+host+range+R388+plasmid+(IncW)+and+RP4+plasmid+(IncPa)+conjugative+machineries+and+their+cognate+Escherichia+coli+host+strains.">A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPa) conjugative machineries and their cognate Escherichia coli host strains.</a> Res Microbiol. 156 (2), 245-255 (2005).</li><li>Vallenet, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroScope:+a+platform+for+microbial+genome+annotation+and+comparative+genomics.">MicroScope: a platform for microbial genome annotation and comparative genomics.</a> Database (Oxford). , bap021 (2009).</li><li>Goodman, A. L., Wu, M., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+microbial+fitness+determinants+by+insertion+sequencing+using+genome-wide+transposon+mutant+libraries.">Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries.</a> Nat. Protocols. 6 (12), 1969-1980 (2011).</li><li>Miller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spirochetes in body fluids and tissues manual of investigative methods. , 22-23 (1971).</li><li>Ristow, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OmpA-like+protein+Loa22+is+essential+for+leptospiral+virulence.">The OmpA-like protein Loa22 is essential for leptospiral virulence.</a> PLoS Pathog. 3 (7), (2007).</li><li>Croda, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+in+pathogenic+Leptospira+species:+disruption+of+the+LigB+gene+does+not+affect+virulence+in+animal+models+of+leptospirosis.">Targeted mutagenesis in pathogenic Leptospira species: disruption of the LigB gene does not affect virulence in animal models of leptospirosis.</a> Infect Immun. 76 (12), 5826-5833 (2008).</li><li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hamster+model+of+leptospirosis.">Hamster model of leptospirosis.</a> Curr Protoc Microbiol. 12, 2 (2006).</li><li>Giardine, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy:+a+platform+for+interactive+large-scale+genome+analysis.">Galaxy: a platform for interactive large-scale genome analysis.</a> Genome Res. 15 (10), 1451-1455 (2005).</li><li>Goecks, J., Nekrutenko, A., Taylor, J., Galaxy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy:+a+comprehensive+approach+for+supporting+accessible,+reproducible,+and+transparent+computational+research+in+the+life+sciences.">Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences.</a> Genome Biol. 11 (8), R86 (2010).</li><li>Blankenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galaxy:+a+web-based+genome+analysis+tool+for+experimentalists.">Galaxy: a web-based genome analysis tool for experimentalists.</a> Curr Protoc Mol Biol. (Chapter 19), 11-21 (2010).</li><li>Afgan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Galaxy+platform+for+accessible,+reproducible+and+collaborative+biomedical+analyses:+2016+update.">The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update.</a> Nucleic Acids Res. 8 (44), (2016).</li><li>McCoy, K. M., Antonio, M. L., van Opijnen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAGenTA;+a+Galaxy+implemented+tool+for+complete+Tn-Seq+analysis+and+data+visualization.">MAGenTA; a Galaxy implemented tool for complete Tn-Seq analysis and data visualization.</a> Bioinformatics. , 1367 (2017).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biol. 10 (3), R25 (2009).</li><li>Kamp, H. D., Patimalla-Dipali, B., Lazinski, D. W., Wallace-Gadsden, F., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+fitness+landscapes+of+Vibrio+cholerae+at+important+stages+of+its+life+cycle.">Gene fitness landscapes of Vibrio cholerae at important stages of its life cycle.</a> PLoS Pathog. 9 (12), e1003800 (2013).</li><li>Poggi, D., Oliveira de Giuseppe, P., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+markers+for+genetic+manipulations+of+Leptospira+spp.">Antibiotic resistance markers for genetic manipulations of Leptospira spp.</a> Appl Environ Microbiol. 76 (14), 4882-4885 (2010).</li><li>van Opijnen, T., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+insertion+sequencing:+a+new+tool+for+systems-level+analysis+of+microorganisms.">Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.</a> Nat Rev Microbiol. 11 (7), 435-442 (2013).</li><li>Pappas, C. J., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+gene+expression+in+Leptospira+spp.+by+Transcription+activator-like+effectors+demonstrates+a+potential+role+for+LigA+and+LigB+in+Leptospira+interrogans+virulence.">Control of gene expression in Leptospira spp. by Transcription activator-like effectors demonstrates a potential role for LigA and LigB in Leptospira interrogans virulence.</a> Appl Environ Microbiol. 81 (22), 7888-7892 (2015).</li><li>Koskiniemi, S., Sun, S., Berg, O. G., Andersson, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection-driven+gene+loss+in+bacteria.">Selection-driven gene loss in bacteria.</a> PLoS Genet. 8 (6), e1002787 (2012).</li><li>Troy, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Tn-seq+analysis+of+carbohydrate+utilization+and+vertebrate+infectivity+of+Borrelia+burgdorferi.">Global Tn-seq analysis of carbohydrate utilization and vertebrate infectivity of Borrelia burgdorferi.</a> Mol Microbiol. 101 (6), 1003-1023 (2016).</li><li>Ramsey, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+genetic+screen+identifies+previously+uncharacterized+Borrelia+burgdorferi+genes+important+for+resistance+against+reactive+oxygen+and+nitrogen+species.">A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species.</a> PLoS Negl Trop Dis. 13 (2), e1006225 (2017).</li></ol>

Även om resultaten från vår pilot experiment för hamster utmanas intraperitonealt med 42 L. interrogans mutanter presenteras17, förväntar vi oss att större pooler av mutanter kan kontrolleras av Tn-följande punkter Eftersom frekvensen av transconjugants är låg (100-200 transconjugants/parning), flera parningar är nödvändiga för att generera ett tillräckligt antal mutanter för stora Tn-Seq experiment. Att upprätthålla ett stort antal mutanter i flytande kulturer presenter...

Identifiering av virulensgener för vissa bakterier, såsom Leptospira spp., är svårt på grund av det begränsade antalet genetiska verktyg tillgängliga. En vanligt förekommande metod är skapandet av en samling av mutanter av slumpmässiga transposon mutagenes följt av identifiering av insticksstället i varje mutant och virulens provning av enskilda transposon mutanter i en djurmodell. Denna metod är tidskrävande, dyra och kräver ett stort antal djur.
När slumpmässig mutagenes utvecklades först för patogen Leptospira interrogans, identifierades genernas virulens genom att testa enskilda mutanter i en djurmodell1. Mutanter valdes utifrån kriterier som deras potentiella roller i signalering eller motilitet eller deras förväntade yttre membran eller markrättigheter. Eftersom flesta leptospiral gener koda hypotetiska proteiner av okänd funktion2, välja mutanter baserat på dessa kriterier begränsar förmågan att upptäcka roman leptospiral virulensgener.
Mer nyligen, pooler av L. interrogans transposon mutanter som screenades för smittsamhet i hamster och mus modeller3. Varje djur ifrågasattes med en pool av upp till 10 mutanter. Smittsamhet av en mutant gjorde var så positiv om det upptäcktes av PCR av kulturer som erhållits från blod och njure. PCR-test var arbetskrävande eftersom det krävs en individuell PCR-reaktion för varje mutant i poolen. Eftersom frekvensen av varje mutant i kulturer inte kvantifierades, var metoden partisk mot identifiering av mycket försvagat mutanter.
Vi beskriver en transposon sekvensering (Tn-Seq) teknik, som en strategi för att mer effektivt skärmen för virulensgener. TN-seq består av skapandet av ett bibliotek av mutanter av transposon mutagenes följt av massiva parallella sekvensering4,5,6. Transposon mutanter är kort, poolade, inokuleras i djur och senare återhämtat sig från olika organ (utdata pooler). DNA från utdata poolerna är extraherade och smält med restriktionsenzym eller klippt av ultraljudsbehandling. Två omgångar av PCR inriktning korsningar av transposon införande platserna utförs. Detta steg kan tillägg av adaptrarna som behövs för sekvenseringen. De resulterande PCR-produkterna analyseras av hög genomströmning sekvensering att identifiera insättningsstället transposon på varje mutant av poolen tillsammans med deras relativa överflöd, som jämfört med den ursprungliga sammansättningen av av mutant.
Den främsta fördelen med denna metod är möjligheten att samtidigt visa ett stort antal mutanter med ett litet antal djur. TN-Seq kräver inte den tidigare kunskap av transposon införande platser vilket ökar chanserna att upptäcka nya Leptospira-specifika gener inblandade i virulens med mindre tid och större effektivitet. Eftersom leptospiral börda i vävnader är relativt hög i gnagare modeller mottagliga för dödliga infektion (vanligtvis 104 till 108 bakterier/g vävnad)7,8,9 samt för liksom i reservoaren värdar 10,11, vävnader kan analyseras direkt utan att behöva kultur, minska fördomar på grund av in vitro- tillväxt.
I Tn-Seq studier med de flesta bakteriella patogener beskrivs hittills, den höga frekvensen av insertionsmutationer tillåtna infektion med stora pooler som innehåller mutanter kollektivt med flera tätt radavstånd transposon infogningar inom varje gen4 ,12,13,14. TN-Seq har också utvecklats för en bakterie som mutagenes frekvensen är mycket lägre6. Med Leptospira, kan ett bibliotek av transposon mutanter genereras genom att införa transposon på en mobilizable plasmid av konjugering som beskrivs av Slamti et al15. Frekvensen av transposon mutagenes av L. interrogans är dock låg. När den Himar1 transposon introducerades på en konjugering plasmid, rapporterades transconjugant frekvensen vara bara 8,5 x 10-8 per mottagare cell med Lai stam L. interrogans16 och sannolikt är Likaså fattiga med de flesta andra stammar av L. interrogans. Protokollet beskrivs här är delvis baserat på som utvecklats för Borrelia burgdorferi, där frekvensen av transposon insertionsmutationer är också låg6.
För vår pilot experiment med de protokoll17genomförde vi transposon mutagenes med L. interrogans serovar Manilae stam L495 på grund av framgången för andra grupper i isolera transposon införande mutanter i stam tillsammans med dess låga LD50 (dödlig dos) för virulens1. Vi visades 42 mutanter av Tn-Seq och identifierat flera mutant kandidater defekt i virulens, varav två med infogningar i en kandidat adenylatcyklas gen. Individuell testning av två mutanter i hamstrar bekräftade att de var bristfällig i virulens17.

<table>
	<tbody>
		<tr>
			<td>Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td>Sigma-Aldrich</td>
			<td>K4000</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-diaminopimelic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S4014</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Lab A1 microscope with a darkfieldcondenser</td>
			<td>Zeiss</td>
			<td>490950-001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy blood and tissue kit</td>
			<td>Qiagen</td>
			<td>69504/69506</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification</td>
			<td>Qiagen</td>
			<td>28004/28006</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104/28106</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 505 Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>FB-505</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5&#34; Cup horn</td>
			<td>Fisher Scientific</td>
			<td>FB-4625</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Ruptor 24</td>
			<td>Omni International</td>
			<td>19-010</td>
			<td>Step 3.1.2.4</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase</td>
			<td>Promega</td>
			<td>M828C</td>
			<td></td>
		</tr>
		<tr>
			<td>Master mix Phusion</td>
			<td>Thermo Scientific</td>
			<td>F531</td>
			<td>Preparation of genomic libraries, step 3.4.</td>
		</tr>
		<tr>
			<td>DreamTaq Master Mix</td>
			<td>Thermo Scientific</td>
			<td>K9011/K9012</td>
			<td>Identification of the transposon insertion site, step 1.2.</td>
		</tr>
		<tr>
			<td>dCTP</td>
			<td>Thermo Scientific</td>
			<td>R0151</td>
			<td></td>
		</tr>
		<tr>
			<td>ddCTP</td>
			<td>Affymetrix/ USBProducts</td>
			<td>77112</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Invitrogen</td>
			<td>Q32851/Q32854</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit assay tubes</td>
			<td>life technologies</td>
			<td>Q32856</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>PBS pH 7.2 (1X)</td>
			<td>Gibco</td>
			<td>20012-027 20012-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel No10</td>
			<td>Feather</td>
			<td>2975#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic K2 EDTA 2 ml tubes</td>
			<td>BD vacutainer</td>
			<td>367841</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe U-100 with 26G x &#189;&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>329652</td>
			<td>IP challenge, step 2.2.1.</td>
		</tr>
		<tr>
			<td>3 mL Luer-Lok tip syringe</td>
			<td>BD vacutainer</td>
			<td>309657</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25G X 5/8&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>305901</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25 mm fritted glass base with stopper</td>
			<td>EMD Millipore</td>
			<td>XX1002502</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>25 mm aluminum spring clamp</td>
			<td>EMD Millipore</td>
			<td>XX1002503</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>15 ml borosilcate glass funnel</td>
			<td>EMD Millipore</td>
			<td>XX1002514</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>125 ml side-arm Erlenmeyer flask</td>
			<td>EMD Millipore</td>
			<td>XX1002505</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm)</td>
			<td>EMD Millipore</td>
			<td>VVLP02500</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection

I detta manuskript beskriver vi en transposon sekvensering (Tn-Seq) teknik för att identifiera och kvantifiera Leptospira interrogans mutanter ändras i fitness under infektion i gyllene syriska hamstrar. TN-Seq kombinerar slumpmässiga transposon mutagenes med kraften av hög genomströmning sekvenseringsteknologi. Djur utmanas med en pool av transposon mutanter (ingång pool), följt av upptagning av blod och vävnader några dagar senare för att identifiera och kvantifiera antalet mutanter i varje organ (utdata pooler). Utdata poolerna jämförs ingående poolen att utvärdera i vivo lämplighet varje mutant. Denna metod gör det möjligt för screening av en stor pool av mutanter i ett begränsat antal djur. Med smärre ändringar, kan detta protokoll utföras med någon djurmodell av leptospiros, reservoar värd modeller såsom råttor och akut infektion såsom hamstrar som in vitro- studier. TN-Seq erbjuder ett kraftfullt verktyg till skärmen för mutanter med i vivo och in vitro- fitness defekter.

Skapandet av ett bibliotek av transposon mutanter i L. interrogans av konjugering kräver en filtrering enhet, som visas i figur 1. Vi återhämtade sig 100-200 transconjugants från varje parning.
Transposon insticksstället identifieras i varje mutant av sekvensering av PCR-produkten genereras av semi-slumpmässigt PCR som mål i slutet av transposon och intilliggande värd sekvenser<sup ...

Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

Här beskriver vi en teknik som kombinerar transposon mutagenes med hög genomströmning sekvensering att identifiera och kvantifiera transposon leptospiral mutanter i vävnader efter en utmaning av hamstrar. Detta protokoll kan användas till skärmen mutanter för överlevnad och spridning i djur och kan också tillämpas på in vitro- studier.

Hög genomströmning parallella sekvensering att mäta konditionen av Leptospira interrogans Transposon införande mutanter under gyllene syriska Hamster infektion

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

The overall goal of this high-throughput transposon sequencing technique is to identify and quantify transposon mutants with in vivo and in vitro fitness defects, and thereby, identify Leptospira genes involved in virulence. This method can help answer key questions in the Leptospira field, such as identifying genes involved in colonization, dissemination, and survival of the bacteria in the host. The main advantage of this technique is that a large number of mutants can be screened with a limited number of animals.Though this method was designed to examine the fitness of Leptospira interrogans mutants in the hamster model with acute leptospirosis, it can also be applied through a chronic infection models, in vitro studies, and other strains of Leptospira. Demonstrating the procedure will be Kritel Lourdault and Karen Evangelista, both post-docs from my laboratory. First, assemble a filtration unit.Place the base on a 125 milliliter side arm Erlenmeyer flask. Position an acetate cellulose filter at the base and clamp a funnel onto the base. Connect the filtration unit to a vacuum system.Next, add five milliliters of Leptospira culture and 0.5 milliliters of E.coli culture into the funnel. Turn on the vacuum to filter the solution. Then place the filter with bacteria-side up onto an EMJH plate, supplemented with DAP.Incubate the plate at 30 degrees Celsius over night. The next day, transfer the filter into a 15 milliliter tube containing one milliliter of EMJH, and vortex the tube for 10 seconds to release the bacteria in the suspension. Then spread the suspension onto five EMJH plates, supplemented with kanamycin using glass beads or a spreader.Wrap the plates in parafilm and incubate them upside down at 30 degrees Celsius until colonies are visible. When colonies are visible, collect and transfer them individually into three milliliters of EMJH containing kanamycin. Then incubate the tubes at 30 degrees Celsius with 150 rpm agitation for seven to 10 days, or to a density of 100 million cells per milliliter.To identify the transposon insertion site, perform nested PCRs. First, prepare the mix for the first PCR. Then, add the samples from culture to the PCR mixes.After running the first reaction, prepare the mix for the second PCR. Add two microliters of PCR product from the first PCR to the complete the reaction mixes. After running the PCR, examine the products on an agarose gel.Proceed by sequencing the products to determine the transposon insertion site. To begin the animal experiment, first grow individual mutants in EMJH, supplemented with kanamycin. Then determine the culture densities using a dark field microscope.Dilute each cultured mutant strain in EMJH to the same density. Then, pool an equal volume of all the mutants to make the input pool. Now challenge the animals as described in the text protocol.The number of animals required depends on the size of the input pool. Be sure to consult a biostatistician to determine the number of animals to inoculate. Allow the infection to develop for four days, then harvest blood and tissues as described in the text protocol.To extract the DNA from the blood sample, use a commercial extraction kit. For tissue, dice 50 to 80 milligrams of tissue into one millimeter cubes. Then, transfer the diced tissues to a screw cap tube and weigh them on a precision balance.Next, add 500 microliters of sterile PBS into the tube. Homogenize it using a disrupter for one minute. Then use a volume corresponding to 25 milligrams of tissue to extract the DNA.Use a commercial kit according to the manufacturer's instructions. Dilute the DNA in 100 microliters of elution buffer. Next, transfer 50 microliters of extracted DNA into a 1.5 milliliter microcentrifuge tube.Then shear the DNA. Place the tube into a sonicator cup horn filled with water at four degrees Celsius. Run the sonicator for three minutes while wearing ear protection.Next, load 2.5 microliters of the sheared DNA into a 2%agarose gel to confirm that most of the DNA fragments are under 600 base pairs. Next, prepare the reaction mix to add cytosine tails to the DNA fragments. Incubate the reaction for an hour at 37 degrees Celsius.Then, inactivate the enzyme by increasing the temperature to 75 degrees Celsius for 20 minutes. When the reaction is done, clean it up using a PCR purification kit and elute the DNA with 12 microliters of elution buffer. Run two PCRs to create the genomic libraries.For the first PCR, prepare mixes with TnkN3 and olj376 primers. Primer TnkN3 is specific to the transposon and primer olj376 is specific to the C-tail. For each sample, combine 22 microliters of mix with three microliters of C-tailed purified DNA.Then, run the PCR as described in the text protocol. For the second PCR, use one microliter of the first PCR product as a template. For primers, use pMargent2 to target the transposon and use the IP primers.The IP primers each have a six base pair barcode and specific sequences recognizable by the next generation sequencing platform. Next run out three microliters of the second PCR products on a 2%agarose gel. The library should show a smear with the majority of the signal between 200 to 600 base pairs, and no amplification should be observable in the control reaction.Next, clean the genomic libraries with a PCR purification kit and elute the DNA with 30 microliters of elution buffer. Then measure the DNA concentration with a fluorometer. Now, calculate the volume of each library needed for 15 to 20 nanograms of DNA.Mix the libraries and determine the DNA molar concentration in the pool, using a publicly available calculator. Now send the pooled genomic libraries out for high-throughput sequencing. Later, process the FASTQ files obtained from sequencing using open source software and analyze the results.Eight hamsters were challenged with one milliliter of pooled transposon mutants and the infection was allowed to develop for four days. After extracting the DNA collected from these hamsters'livers, it was sheared and characterized on an agarose gel. Most of the fragments were between 200 and 600 base pairs.After adding C-tail to the sheared DNA, PCRs were used to prepare genomic libraries. Electrophoresis was performed to confirm the size of the PCR products. The size of most of the PCR products was between 200 and 600 base pairs.After processing the sequencing reads, the output/input ratios were determined using the transposon sequencing approach. As expected, mutants known to be virulent, such as flaA1 and IlgB do not show any change in fitness. In contrast, mutants known to be attenuated, such as loa22 show a decrease in fitness.Mutants with a reduced in vivo fitness level were identified, such as lic12327. Follow up experimentation showed the virulence of these mutants to be attenuated in hamsters. After watching this video, you should have a good understanding of how to generate genomic DNA libraries in order to quantify and identify mutants with altered in vivo fitness.After mutants with in vivo fitness defects are identified, they can be tested individually in hamsters to determine if their virulence is attenuated. Don't forget that Leptospira interrogans can be lethal and must be handled using BSL2 containment. Personal protective equipment including gloves and a lab coat must be worn while handling the bacteria.

Research

JoVE Journal

Immunology and Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

High-throughput Detection Method for Influenza Virus

High-throughput Detektion Metod för Influensa Virus-

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Fastställande Jord-sända helmintinfektion status och fysiska lämplighet skolbarn

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation och multi fenotypisk Hög halt Screening av<em&gt; Coxiella burnetii</em&gt; Transposon Mutanter

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

High-throughput Gene Tagging in Trypanosoma brucei

High-throughput Gene Tagging in Trypanosoma brucei

Hög genomströmning Gene taggning i<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Skapandet av en tät Transposon införande bibliotek med bakteriell konjugation i Enterobacterial stammar som Escherichia Coli eller Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Replikering av beställt, Nonredundant bibliotek av Pseudomonas aeruginosa stam PA14 Transposon införande mutanter

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Hög genomströmning mätning av plasmamembranet återförslutning effektivitet i däggdjursceller

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

En hög genomströmning analysen att bedöma och kvantifiera neutrofila extracellulära fälla bildandet

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

En hög genomströmning Shigella-specifika bakteriedödande analysmetod

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Konstruera mutanter i Serotyp 1 Streptococcus pneumoniae stam 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...