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

Dette arbejde blev støttet af en veteraner anliggender Merit Award (til D.A.H.) og en National Institute of Health give R01 AI 034431 (D.A.H.).

Forsigtig: Patogene stammer af Leptospira spp. skal behandles efter biosikkerhedsniveau 2 (BSL-2) indeslutning procedurer. Passende personlige værnemidler (PPE) skal bæres. En klasse II biosikkerhed kabinet skal bruges til alle manipulationer af patogene Leptospira spp.
1. oprettelsen af Transposon Mutant bibliotek15
<ol><li>
Overførsel af transposon til Leptospira spp. af konjugation (figur ...

<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>

Selv om resultaterne fra vores pilotprojekt for hamster udfordret intraperitoneal med 42 L. interrogans mutanter præsenteres17, forventer vi, at større puljer af mutanter kan blive screenet af Tn-FF. Da hyppigheden af transconjugants er lav (100-200 transconjugants/parring), flere parringer er nødvendige for at generere et tilstrækkeligt antal mutanter for store Tn-Seq eksperimenter. Opretholde et stort antal mutanter i flydende kulturer præsenterer logistiske udfordringer, der skal ...

Identifikation af virulens gener for nogle bakterier, som Leptospira spp., er vanskelig på grund af det begrænsede antal genetiske værktøjer til rådighed. En almindeligt anvendt metode er oprettelsen af en samling af mutanter af tilfældige transposon mutagenese efterfulgt af identifikation af indsættelsesstedet i hver mutant og virulens afprøvning af individuelle transposon mutanter i en dyremodel. Denne tilgang er tidskrævende, dyrt og kræver et stort antal dyr.
Når tilfældig mutagenese blev først udviklet for patogenet Leptospira interrogans, blev gener involveret i virulens identificeret ved at teste individuelle mutanter i en dyremodel1. Mutanter blev udvalgt på grundlag af kriterier såsom deres potentielle roller i signalering eller motilitet forudsagte ydre membran eller overflade placering. Som fleste af leptospiral gener koder for hypotetiske proteiner af ukendt funktion2, vælge mutanter baseret på disse kriterier grænser evne til at opdage roman leptospiral virulens gener.
Mere nylig, puljer af L. interrogans transposon mutanter blev screenet for infektivitetsniveauet i hamster og mus modeller3. Hvert dyr blev udfordret med en pulje af op til 10 mutanter. Smitteevne af en mutant blev scoret som positiv, hvis det blev opdaget af PCR kulturer fremstillet af blod og nyrerne. PCR test var besværlige, fordi det krævede en individuel PCR reaktion til hver mutant i poolen. Da hyppigheden af hver mutant i kulturerne ikke var kvantificeres, var tilgangen forudindtaget mod identifikation af stærkt svækkede mutanter.
Vi beskriver en transposon sekventering (Tn-Seq) teknik, som en strategi til mere effektivt skærmen for virulens gener. TN-seq består af oprettelsen af et bibliotek af mutanter af transposon mutagenese efterfulgt af massive parallelle sekventering4,5,6. Kort, transposon mutanter er samlet, podes i dyr, og senere inddrives fra forskellige organer (output pools). DNA fra output puljer er udvundet og fordøjes med restriktionsenzymer eller forskydes ved hjælp af sonikering. To runder af PCR målretning knudepunkter af transposon indsættelse websteder er udført. Dette trin giver mulighed for tilsætning af de adaptere nødvendige for Sekventeringen. De resulterende PCR produkter analyseres af høj overførselshastighed sekvensering til at identificere webstedet transposon indsættelse af hver mutant pool sammen med deres relative forekomst, som er i forhold til den oprindelige sammensætning af puljen af mutant.
Den primære fordel ved denne tilgang er evnen til at samtidig skærm et stort antal mutanter med et lille antal dyr. TN-Seq kræver ikke forudgående kendskab til transposon indsættelse websteder som øger chancerne for at opdage nye Leptospira-specifikke gener involveret i virulens med mindre tid og større effektivitet. Fordi leptospiral byrde i væv er forholdsvis høj i gnavere modeller modtagelige for dødelig infektion (typisk 104 til 108 bakterier pr. g væv)7,8,9 så godt som i reservoiret værter 10,11, væv kan analyseres direkte uden at skulle kultur, reducere bias som følge af in vitro- vækst.
I Tn-FF. undersøgelser med de fleste bakterielle patogener beskrevet til dato, den høje frekvens af pattedyrsceller mutagenese tilladt infektion med store swimmingpools der indeholder mutanter kollektivt har flere nært afstand transposon indrykninger inden for hvert gen4 ,12,13,14. TN-Seq er også blevet udviklet til en bakterie som mutagenese frekvens er meget lavere6. Med Leptospira, kan et bibliotek af transposon mutanter genereres ved at indføre transposon på en kan mobiliseres plasmid af konjugation som beskrevet af Slamti al.15. Hyppigheden af transposon mutagenese af L. interrogans er dog lav. Når Himar1 transposon blev indført på et konjugering plasmid, blev transconjugant hyppighed rapporteret at være kun 8,5 x 10-8 pr. modtager celle med Lai stamme af L. interrogans16 og forventes at være tilsvarende fattige med de fleste andre stammer af L. interrogans. Protokollen beskrevet her er i en del baseret på udviklet til Borrelia burgdorferi, hvor hyppigheden af transposon pattedyrsceller mutagenese er også lav6.
For vores pilot eksperiment med protokol17gennemførte vi transposon mutagenese med L. interrogans serovar Manilae stamme L495 på grund af succesen med andre grupper i isolere transposon indsættelse mutanter i stammen, der sammen med sin lave LD50 (dødelig dosis) for virulens1. Vi screenet 42 mutanter af Tn-FF. og identificeret flere mutant kandidater defekt i virulens, herunder to med indsættelser i en kandidat adenylate cyclase genet. Individuelle test af de to mutanter i hamstere bekræftet at de var mangelfuld 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 dette manuskript beskriver vi en transposon sekventering (Tn-Seq) teknik for at identificere og kvantificere Leptospira interrogans mutanter ændret i fitness under infektion af Golden syriske hamstere. TN-Seq kombinerer tilfældige transposon mutagenese med magt af høj overførselshastighed sekventering teknologi. Dyr bliver udfordret med en pulje af transposon mutanter (input pool), efterfulgt af høst af blod og væv et par dage senere for at identificere og kvantificere antallet af mutanter i hvert organ (output pools). Output pools er i forhold til de input pool at vurdere hver enkelt mutant i vivo egnethed. Denne tilgang gør det muligt for screening af en stor pulje af mutanter i et begrænset antal dyr. Med mindre ændringer, kan denne protokol udføres med en dyremodel for leptospirose, reservoir vært modeller som rotter og akut infektion modeller såsom hamsters, såvel som in vitro- undersøgelser. TN-Seq giver en kraftfuld værktøj til skærmen for mutanter i vivo og in vitro- fitness defekter.

Oprettelse af et bibliotek af transposon mutanter i L. interrogans af konjugation kræver en filtrering enhed, som vist i figur 1. Vi genvundet 100-200 transconjugants fra hver parring.
Transposon indsættelsesstedet er identificeret i hver mutant ved sekventering af PCR produkt genereret af semi-tilfældigt PCR, som er rettet mod slutningen af transposon og tilstødende vært 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

Vi beskriver her en teknik, der kombinerer transposon mutagenese med høj overførselshastighed sekvensering til at identificere og kvantificere transposon leptospiral mutanter i væv efter en udfordring af hamstere. Denne protokol kan bruges til skærm mutanter for overlevelse og udbredelse i dyr og kan også anvendes til in vitro- undersøgelser.

Høj overførselshastighed parallelle sekvensering til foranstaltning Fitness af Leptospira interrogans Transposon indsættelse mutanter under Golden syriske Hamster infektion

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

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 Detection Metode til Influenza 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

Bestemmelse Jord-transmitteret Helminth Infektion status og fysiske egnethed skolebørn

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 og Multi-fænotypisk High-indhold Screening af<em&gt; Coxiella burnetii</em&gt; Transposon Mutants

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

High-throughput Gene Tagging 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

Oprettelsen af en tætte Transposon indsættelse bibliotek ved hjælp af bakteriel konjugation i Enterobacterial stammer 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 af bestilt, Nonredundant bibliotek af Pseudomonas aeruginosa stamme PA14 Transposon indsættelse 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

Høj overførselshastighed måling af Plasma membran genforsegling effektivitet i pattedyrceller

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 høj overførselshastighed analyse til at vurdere og kvantificere neutrofile ekstracellulære fælde dannelse

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 høj overførselshastighed Shigella-specifikke bakteriedræbende Assay

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

Konstruktion af mutanter i Serotype 1 Streptococcus pneumoniae stamme 519/43

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