Faculty Collaboration Database

Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages. Infect Immun 2007 Nov;75(11):5376-89

Date

08/08/2007

Pubmed ID

17682043

Pubmed Central ID

PMC2168294

DOI

10.1128/IAI.00238-07

Scopus ID

2-s2.0-35648989527 (requires institutional sign-in at Scopus site)   75 Citations

Abstract

Francisella tularensis, the etiologic agent of tularemia in humans, is a potential biological threat due to its low infectious dose and multiple routes of entry. F. tularensis replicates within several cell types, eventually causing cell death by inducing apoptosis. In this study, a modified Himar1 transposon (HimarFT) was used to mutagenize F. tularensis LVS. Approximately 7,000 Km(r) clones were screened using J774A.1 macrophages for reduction in cytopathogenicity based on retention of the cell monolayer. A total of 441 candidates with significant host cell retention compared to the parent were identified following screening in a high-throughput format. Retesting at a defined multiplicity of infection followed by in vitro growth analyses resulted in identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in seven hypothetical genes were screened in a mouse model of infection, and all strains tested appeared to be attenuated, which validated the initial in vitro results obtained with cultured macrophages. Complementation and reverse transcription-PCR experiments suggested that the expression of genes adjacent to the HimarFT insertion may be affected depending on the orientation of the constitutive groEL promoter region used to ensure transcription of the selective marker in the transposon. A hypothetical gene, FTL_0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed to be a gene involved in O-antigen expression in F. tularensis LVS and Schu S4. These and other studies demonstrate that therapeutic targets, vaccine candidates, or virulence-related genes may be discovered utilizing classical genetic approaches in Francisella.

Author List

Maier TM, Casey MS, Becker RH, Dorsey CW, Glass EM, Maltsev N, Zahrt TC, Frank DW

Author

Dara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Cell Line
Colony Count, Microbial
DNA Transposable Elements
Female
Francisella tularensis
Genes, Bacterial
Genetic Complementation Test
Macrophages
Mice
Mice, Inbred BALB C
Mutagenesis, Insertional
Survival Analysis
Tularemia
Virulence
Virulence Factors