Abstract
The highly virulent intracellular pathogen Francisella tularensis is a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis of Francisella virulence in the Fischer 344 rat, we have constructed an F. tularensis Schu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growth in vitro Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. This in vivo selection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-type F. tularensis Schu S4 strain.IMPORTANCE The intracellular bacterial pathogen Francisella tularensis causes a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches. F. tularensis is one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important for F. tularensis under in vitro and in vivo conditions, providing candidates that can be evaluated for vaccine or antibacterial development.
Highlights
The highly virulent intracellular pathogen Francisella tularensis is a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia
Three subspecies have been identified: Francisella tularensis subsp. tularensis occurs in North America, the less virulent Francisella tularensis subsp. holarctica is found in North America and Europe, and Francisella tularensis subsp. mediasiatica is found in central Asia
Sequencing of transposon-genome junctions that had been selectively enriched by PCR generated approximately 15 million reads for each replicate, of which 92% to 96% mapped to the F. tularensis Schu S4 genome (Table 1)
Summary
The highly virulent intracellular pathogen Francisella tularensis is a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis of Francisella virulence in the Fischer 344 rat, we have constructed an F. tularensis Schu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen This in vivo selection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-type F. tularensis Schu S4 strain. Generating live vaccine strains with multiple lesions continues to pose significant challenge
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