This work was supported by a Veterans Affairs Merit Award (to D.A.H.) and a National Institute of Health grant R01 AI 034431 (to D.A.H.).

CAUTION: Pathogenic strains of Leptospira spp. must be handled under Biosafety Level 2 (BSL-2) containment procedures. Appropriate personal protective equipment (PPE) must be worn. A Class II biosafety cabinet must be used for all manipulations of pathogenic Leptospira spp.
1. Creation of the Transposon Mutant Library15
<ol>
	<li>Transfer of the transposon into Leptospira spp. by conjugation (Fi...

<ol>
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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. 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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. 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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. 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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. 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The authors have nothing to disclose.

Although results from our pilot experiment for hamster challenged intraperitoneally with 42 L. interrogans mutants are presented17, we expect that larger pools of mutants can be screened by Tn-Seq. Because the frequency of transconjugants is low (100-200 transconjugants/mating), several matings are necessary to generate a sufficient number of mutants for large Tn-Seq experiments. Maintaining a large number of mutants in liquid cultures presents logistical challenges that must be addressed...

Identification of virulence genes for some bacteria, such as Leptospira spp., is difficult because of the limited number of genetic tools available. One commonly used approach is the creation of a collection of mutants by random transposon mutagenesis followed by the identification of the insertion site in each mutant and virulence testing of individual transposon mutants in an animal model. This approach is time-consuming, expensive, and requires a large number of animals.
When random mutagenesis was first developed for the pathogen Leptospira interrogans, genes involved in virulence were identified by testing individual mutants in an animal model1. Mutants were selected based on criteria such as their potential roles in signaling or motility or their predicted outer membrane or surface location. As the majority of leptospiral genes encode hypothetical proteins of unknown function2, selecting mutants based on these criteria limits the ability to discover novel leptospiral virulence genes.
More recently, pools of L. interrogans transposon mutants were screened for infectivity in the hamster and mouse models3. Each animal was challenged with a pool of up to 10 mutants. Infectivity of a mutant was scored as positive if it was detected by PCR of cultures obtained from blood and kidney. PCR testing was laborious because it required an individual PCR reaction for each mutant in the pool. Because the frequency of each mutant in the cultures was not quantified, the approach was biased towards identification of highly attenuated mutants.
We describe a transposon sequencing (Tn-Seq) technique, as a strategy to more efficiently screen for virulence genes. Tn-seq consists of the creation of a library of mutants by transposon mutagenesis followed by massive parallel sequencing4,5,6. Briefly, transposon mutants are pooled, inoculated into animals, and later recovered from different organs (output pools). The DNA from the output pools is extracted and digested with restriction enzymes or sheared by sonication. Two rounds of PCR targeting the junctions of the transposon insertion sites are performed. This step enables the addition of the adaptors necessary for the sequencing. The resulting PCR products are analyzed by high-throughput sequencing to identify the transposon insertion site of each mutant of the pool along with their relative abundance, which is compared to the initial composition of the pool of mutant.
The primary advantage of this approach is the ability to simultaneously screen a large number of mutants with a small number of animals. Tn-Seq does not require the prior knowledge of the transposon insertion sites which increases the chances of discovering new Leptospira-specific genes involved in virulence with less time and greater efficiency. Because leptospiral burden in tissues is relatively high in rodent models susceptible to lethal infection (typically 104 to 108 bacteria/g of tissue)7,8,9 as well as in reservoir hosts10,11, tissues can be directly analyzed without the need to culture, reducing biases due to in vitro growth.
In Tn-Seq studies with most bacterial pathogens described to date, the high frequency of insertional mutagenesis allowed infection with large pools containing mutants collectively having multiple closely-spaced transposon insertions within every gene4,12,13,14. Tn-Seq has also been developed for a bacterium for which the mutagenesis frequency is much lower6. With Leptospira, a library of transposon mutants can be generated by introducing the transposon on a mobilizable plasmid by conjugation as described by Slamti et al15. However, the frequency of transposon mutagenesis of L. interrogans is low. When the Himar1 transposon was introduced on a conjugative plasmid, the transconjugant frequency was reported to be only 8.5 x 10-8 per recipient cell with the Lai strain of L. interrogans16 and is likely to be similarly poor with most other strains of L. interrogans. The protocol described here is in part based on that developed for Borrelia burgdorferi, in which the frequency of transposon insertional mutagenesis is also low6.
For our pilot experiment with the protocol17, we conducted transposon mutagenesis with L. interrogans serovar Manilae strain L495 because of the success of other groups in isolating transposon insertion mutants in the strain along with its low LD50 (lethal dose) for virulence1. We screened 42 mutants by Tn-Seq and identified several mutant candidates defective in virulence, including two with insertions in a candidate adenylate cyclase gene. Individual testing of the two mutants in hamsters confirmed that they were deficient in virulence17.

<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

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

Creation of a library of transposon mutants in L. interrogans by conjugation requires a filtration unit, as shown in Figure 1. We recovered 100-200 transconjugants from each mating.
The transposon insertion site is identified in each mutant by sequencing the PCR product generated by semi-random PCR that targets the end of the transposon and adjacent host sequences15 ...

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

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

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

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.

high-throughput-parallel-sequencing-to-measure-fitness-leptospira

Research

JoVE Journal

Immunology and Infection

8.8K Görüntüleme.  Veterans Affairs Greater Los Angeles Healthcare System. 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 li...

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