Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS

Abstract

Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. We have shown that F. tularensis subspecies holarctica strain LVS prevents NADPH oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. Herein, we used random transposon mutagenesis to identify LVS genes that affect neutrophil activation. Our initial screen identified carA, carB, and pyrB, which encode the small and large subunits of carbamoylphosphate synthase and aspartate carbamoyl transferase, respectively. These strains are uracil auxotrophs, and their growth was attenuated on cysteine heart agar augmented with sheep blood (CHAB) or in modified Mueller-Hinton broth. Phagocytosis of the uracil auxotrophic mutants triggered a respiratory burst in neutrophils, and ingested bacteria were killed and fragmented in phagosomes that contained superoxide. Conversely, phagocytosis did not trigger a respiratory burst in blood monocytes or monocyte-derived macrophages (MDM), and phagosomes containing wild-type or mutant bacteria lacked NADPH oxidase subunits. Nevertheless, the viability of mutant bacteria declined in MDM, and ultrastructural analysis revealed that phagosome egress was significantly inhibited despite synthesis of the virulence factor IglC. Other aspects of infection, such as interleukin-1␤ (IL-1␤) and IL-8 secretion, were unaffected. The cultivation of carA, carB, or pyrB on uracil-supplemented CHAB was sufficient to prevent neutrophil activation and intramacrophage killing and supported escape from MDM phagosomes, but intracellular growth was not restored unless uracil was added to the tissue culture medium. Finally, all mutants tested grew normally in both HepG2 and J774A.1 cells. Collectively, our data demonstrate that uracil auxotrophy has cell type-specific effects on the fate of Francisella bacteria.

Subversion of Host Recognition and Defense Systems by Francisella spp

“…Although Francisella uses the mechanisms described above to significantly suppress activation of the NADPH oxidase, low levels of ROS are produced in the phagosome during infection (203). Like many pathogens, Francisella can directly detoxify ROS using proteins, including catalase (134,151,214) and superoxide dismutases (15,16,151), whose specific mechanisms of action are reviewed extensively elsewhere (135).…”

“…Upon phagocytosis of a microbe, the cytosolic subunits traffic to the phagosome and assemble with the membrane subunits to create the active NADPH oxidase that then produces ROS. Similar to the case for numerous extracellular and intracellular bacterial species, including Helicobacter pylori and Salmonella spp., multiple Francisella species block NADPH oxidase assembly in neutrophils and macrophages (4,89,145,156,203).…”

Working toward the Future: Insights intoFrancisella tularensisPathogenesis and Vaccine Development

“…This increase in prostaglandin E 2 has also been confirmed in the lung in vivo (219). Finally, Francisella has been shown to infect and replicate within neutrophils and inhibit the respiratory burst, thus evading neutrophil killing mechanisms (127,179). Though the precise contributions of these findings to infection remain unclear, it is likely that immune evasion and/or suppression is essential to the highly virulent nature of Francisella and differences between subspecies.…”

“…This system expands upon the utility of current transposon tools by allowing unmarking of the antibiotic resistance determinant following transposition into the genome and inclusion of promoterless lux and lacZ alleles for analyzing gene expression using reporter gene technology (23). Mutagenesis using this transposon system has allowed identification of genes from F. tularensis LVS that are required for inhibition of the respiratory burst in neutrophils and for intracellular growth or survival within this cell type (179). Additionally, this system was used to identify FevR, a novel regulator of iglB (22).…”

Putting the Jigsaw Together - A Brief Insight Into the Tularemia

“…More than 300 genes considered to be virulence factors have been identified so far. Among these are genes involved in adhesion to host cells [43]; genes associated with capsule biosynthesis contributing to serum resistance; and genes, including those from the Francisella pathogenicity island coding probably of the type VI secretion system, enabling the "neutralization" of intraphagosomal milieu [44][45][46][47], escape into the cytosol, and proliferation inside the host cell [48][49][50]. F. tularensis is also able to delay cell death to increase its survival and replication through activation of Ras by the SOS2/GrB2/PKCα/PKCβI quaternary complex, which stimulates cell survival through the downregulation of caspase-3 activation [51].…”