Francisella Tularensis Ferric-siderophore and Ferrous Iron Transport Systems are Necessary for Iron Acquisition and Viability

Francisella tularensis is a Gram-negative intracellular bacterium and the causative agent of the disease tularemia. F. tularensis is designated by the CDC as a Tier One Select Agent, due to its low infection dose, aerosol transmissibility, lack of a licensed vaccine, and for its potential use as an agent of biological warfare. Virulent Schu S4 and the attenuated live vaccine strain (LVS) are model strains used to study F. tularensis pathogenesis in tissue culture and in the mouse model of disease. Despite the genetic similarities between LVS and Schu S4, these strains have differences in iron uptake capability and virulence. Within the host, iron is tightly controlled and sequestered by iron binding proteins. Pathogenic bacteria, include F. tularensis, have devised methods for acquiring available ferrous and ferric iron from their hosts and they are frequently associated with virulence.
A common method for ferric acquisition is the production of siderophores, which are low molecular weight iron binding chelators that are secreted into the environment and have high affinities for ferric iron. F. tularensis strains LVS and Schu S4 secrete an identical siderophore and encode genes required for siderophore biosynthesis and transport (fslABCDEF also referred to as figABCDEF). An in-frame deletion of fslC was generated in the LVS background (LVS ΔfslC), and similar to the previously published fslA mutant (LVS ΔfslA), this mutant was defective for siderophore production as determined by the liquid Chrome-Azurol S (CAS) assay. A fslABCDEF locus deletion mutant, LVS Δfsl, was complemented with plasmids containing wild-type copies of the fsl genes and was used to determine that F. tularensis siderophore biosynthesis requires FslA and FslC, and siderophore export requires FslB. The LVS fsl mutants were not defective for growth in tissue culture cells thus suggesting that another iron acquisition system is sufficient at acquiring necessary iron for Francisella growth and survival. My dissertation studies also investigated the ferrous iron uptake system (Feo) in the Francisella strains Schu S4 and LVS as an alternative iron acquisition mechanism for growth and survival. The Feo system has been demonstrated in other bacterial systems to regulate the entry of available ferrous iron. Partial deletion mutants of feoB (ΔfeoB’) were generated in LVS and Schu S4. 55Fe uptake assays demonstrated that the Feo systems in both LVS and Schu S4 are dependent on FeoB for ferrous iron uptake at both high (0.1 μM) and low (3.0 μM) affinity iron concentrations. These ΔfeoB’ mutants did utilize the ferric-siderophore iron acquisition system which was observed through CAS and 55Fe-siderophore uptake assays. My dissertation studies demonstrated that the Feo system is specific for ferrous iron transport and is the sole ferrous iron acquisition system in this bacterium. Studies with ΔfslA, ΔfeoB’, and ΔfslA ΔfeoB’ were used to delineate iron acquisition preferences and the potential presence of an unidentified iron acquisition system in Francisella. The double deletion mutants in both strains were only able to grow and survive in the presence of F. tularensis siderophore. Through intracellular replication assays and mouse infection studies it was observed that both iron acquisition systems contribute to optimal iron acquisition, growth, and virulence of F. tularensis and each iron acquisition system can compensate for the loss of the other. Both LVS and Schu S4 double deletion mutants were attenuated for virulence in C57BL/6 mouse infection studies and provided protection upon lethal challenge with wild-type LVS and Schu S4, respectively. The work presented here clarifies our understanding of the iron acquisition mechanisms utilized by this pathogenic bacterium and their role in growth and virulence in the mouse model of infection. Additionally, the LVS and Schu S4 double deletion mutants are potential candidates for vaccine development.