Global Analysis of Genes Essential for Francisella tularensis Schu S4 Growth In Vitro and for Fitness during Competitive Infection of Fischer 344 Rats

Abstract

The highly virulent intracellular pathogenFrancisella tularensisis 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 ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu 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 growthin 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. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis 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 forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

Tn-Seq reveals hidden complexity in the utilization of host-derived glutathione in Francisella tularensis

“…tularensis subsp. tularensis SCHU S4 [ 22 ]. In principle, this screen should define genes important for multiple aspects of infection, including bacterial uptake, immune evasion, and intracellular proliferation.…”

“…Nevertheless, comparative analysis between the results obtained in the present study with those generated by Ireland et al demonstrate a high degree of overlap, suggesting that many of the genes important for infection in the host are involved in intramacrophage growth. In relation to genes important for the utilization of host-derived GSH, ggt , dptA and chaC were found to play critical roles during infection of the rat with SCHU S4 as assessed by Tn-Seq [ 22 ]. Thus, the proteins that play central roles in the metabolism of GSH, which are highly conserved amongst different subspecies of F .…”

“…The comprehensive nature of our screen is highlighted by our identification of all thirteen genes encoding purine biosynthesis enzymes as either critical for survival in vitro or critical for growth or survival within macrophages (S2 Fig). Purine biosynthesis is critical for F. tularensis intracellular survival, yet prior screens had individually identified only up to eight genes in this pathway as important for the process [19][20][21][22][23][24][25][26][27][28][29][30]. In addition, the gene encoding PigR, a transcription regulator critical for intramacrophage growth and virulence [34], was a strong hit in our screen despite its small Each line corresponds to one mutant with a single insertion; the line height corresponds to the relative abundance of sequencing reads identified for each individual insertion.…”

“…Recently a Tn-Seq study was performed to define genes essential for rat colonization of the highly virulent type A strain F. tularensis subsp. tularensis SCHU S4 [22]. In principle, this screen should define genes important for multiple aspects of infection, including bacterial uptake, immune evasion, and intracellular proliferation.…”

“…While several screens have been employed to identify genes important for the intracellular replication of multiple F. tularensis subspecies [19][20][21][22][23][24][25][26][27][28][29][30], a comprehensive description of genes that are specifically required for the intramacrophage growth of this organism has been lacking. Here, we used transposon insertion sequencing (Tn-Seq) to identify the genes that are required for the intracellular growth of the live vaccine strain of F. tularensis (LVS).…”