Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

In vivo screen of Francisella tularensis subsp. novicida mutants in sequence defined transposon library

ABSTRACT: We used an inhalation mouse model of infection to query a collection of 2149 mutants in a Francisella tularensis subsp. novicida background for genes required for growth, survival and systemic dissemination. A microarray-based genome-wide negative selection screen (Microarray tracking of transposon mutants = MATT) allowed us to monitor the behavior of transposon insertions in 1371 unique genes. Interestingly most of these genes persisted in lung and colonized liver and spleen. We found 44 (35%) genes negatively selected in lung and 81 (65%) genes negatively selected in liver and/or spleen. If negative selection in lung occurred, the attenuated mutants in general persisted at 24h after infection, disseminated to liver and/or spleen and appeared to be lost in lung after 48 to 72h of infection. These genes with a strong phenotype in lung but also potential for dissemination might be attractive vaccine or drug candidates. Keywords: Genome-Wide Negative Selection Screen 185 arrays, no duplicates/replicates Filtered data (per organ/timepoint) provided as a supplementary file

ORGANISM(S): Francisella tularensis subsp. novicida

SUBMITTER: Allison Mitchell

PROVIDER: E-GEOD-11297 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice.

Kraemer Petra S PS Mitchell Allison A Pelletier Mark R MR Gallagher Larry A LA Wasnick Mike M Rohmer Laurence L Brittnacher Mitchell J MJ Manoil Colin C Skerett Shawn J SJ Salama Nina R NR

Infection and immunity 20081027 1

Francisella tularensis is a gram-negative, highly infectious, aerosolizable facultative intracellular pathogen that causes the potentially life-threatening disease tularemia. To date there is no approved vaccine available, and little is known about the molecular mechanisms important for infection, survival, and dissemination at different times of infection. We report the first whole-genome screen using an inhalation mouse model to monitor infection in the lung and dissemination to the liver and ...[more]

PMID: 18955478

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Project description:The transposon site hybridization (TraSH) technique (Sassetti, CM et al. 2001. PNAS 98:12712-7) was utilized to identify genes important for the survival of Y. pestis within murine macrophages. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool) in order to identify Y. pestis genes important for survival in macrophages. Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs can be used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe. A transposon library was created with ~31,500 Y. pestis KIM6+ insertion mutants. A portion of the Y. pestis transposon insertion mutant library was used to infect BMMs and the surviving bacteria (output pool) were recovered. TraSH was used to compare the output pool to a portion of the library that was not subjected to selection (input pool). Each end of the transposon used for mutagenesis contains an outward-reading T7 RNA polymerase promoter. RNAs transcribed from the T7 promoters are complementary to the chromosomal DNA flanking each transposon in the library, so the RNAs was used as “targets” to identify the approximate position of each transposon insertion in the mutant pool. Differentially labeled targets generated from the output and input pools are competitively hybridized to the 70-mer oligonucleotide microarrays obtained from Pathogen Functional Genomics Resource Center/J. Craig Venter Institute. Genes important for survival of Y. pestis in macrophages are identified by determining the ratio of the signal intensities for the output and input targets hybridizing to a given probe.

Dataset Information

In vivo screen of Francisella tularensis subsp. novicida mutants in sequence defined transposon library

Publications

Genome-wide screen in Francisella novicida for genes required for pulmonary and systemic infection in mice.

Similar Datasets

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