IAI Accepts, published online ahead of print on 3 November 2008

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Infect. Immun. doi:10.1128/IAI.01113-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification of an essential Francisella tularensis subsp. tularensis virulence factor

AIPING QIN, DAVID W. SCOTT, JENNIFER A. THOMPSON, and BARBARA J. MANN^*

Department of Medicine, and Department of Microbiology, University of Virginia, Charlottesville, VA, U.S.A. 22908

^* To whom correspondence should be addressed. Email: bjm2r{at}virginia.edu.

Francisella tularensis, the highly virulent etiologic agent of tularemia, is a low dose intracellular pathogen that is able to escape from the phagosome and replicate in the cytosol. Although there has been progress in identifying loci involved in pathogenicity, the genome sequence has revealed few obvious virulence factors. We previously reported the isolation of a F. tularensis subsp. tularensis strain Schu S4 transposon-insertion mutant in a predicted hypothetical lipoprotein, FTT1103, that was deficient in intracellular replication in HepG2 cells. In this report, a mutant with a defined nonpolar deletion in FTT1103 was created and characterized with respect to phenotype, virulence, and vaccine potential. A phagosomal integrity assay, and LAMP-1 colocalization revealed that FTT1103 mutant bacteria were defective in phagosomal escape. FTT1103 mutant bacteria were maximally attenuated in the mouse model; mice survived, without visible signs of illness, challenges greater than 10¹⁰ CFU by an intranasal route, and 10⁶ CFU by intraperitoneal, subcutaneous, or intravenous routes. The FTT1103 mutant bacteria exhibited dissemination defects. Mice that were infected by an intranasal route had low levels of bacteria in the livers and spleens, and these bacteria cleared by 3 days post-infection. Mutant bacteria given by a subcutaneous route failed to disseminate to the lungs. BALB/c or C57BL/6 mice, intranasally vaccinated with 10⁸ CFU of FTT1103 mutant bacteria, were protected against the subsequent challenge with wild-type Schu S4. These experiments identified the FTT1103 protein as an essential virulence factor, and also demonstrated the feasibility of creating defined attenuated vaccines based on type A strain.