Identification of Francisella tularensis Himar1-based Transposon Mutants Defective for Replication in Macrophages

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, and the Mathematics and Computer Science Division of the Computational Biology Group, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439

^* To whom correspondence should be addressed. Email: frankd{at}mcw.edu.

	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 7000 Km^r clones were screened in J774A.1 macrophages for a reduction in cytopathogenicity based on the retention of the cell monolayer. Candidates (441) with significant host cell retention compared to the parent were identified following screening in a high throughput format. Retesting at a defined MOI followed by in vitro growth analyses resulted in the identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in 7 hypothetical genes were screened in a mouse model of infection and all strains tested appear attenuated, which validated the initial in vitro results in cultured macrophages. Complementation and RT-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, FTL0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed as being 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.