Identification, Recombinant Expression, Immunolocalization in Macrophages, and T-Cell Responsiveness of the Major Extracellular Proteins of Francisella tularensis

Abstract

A safer and more effective vaccine than the previously developed live attenuated vaccine is needed for combating Francisella tularensis, a highly infectious bacterial pathogen. To search for potential candidates for inclusion in a new vaccine, we characterized the proteins present in the culture filtrates of a virulent recent clinical isolate and the attenuated live vaccine strain of F. tularensis using a proteomic approach. We identified a total of 12 proteins; among these, catalase-peroxidase was much more abundant in the culture filtrate of the virulent clinical isolate, whereas bacterioferritin was more abundant in the culture filtrate of the live vaccine strain. Streptolysin O treatment of infected human macrophages indicated that catalase-peroxidase and the heat shock protein GroEL are released intracellularly by actively growing F. tularensis. Mice immunized with F. tularensis developed significant cellmediated immune responses to catalase-peroxidase, the heat shock protein GroEL, and bacterioferritin as measured by splenic lymphocyte proliferation and gamma interferon production. Finally, we expressed the major culture filtrate proteins that are promising vaccine candidates in Escherichia coli at high levels in soluble form to facilitate study of their immunobiology and potential role in vaccines.

Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines

“…However, an oxidative stress regulator OxyR is present in F. tularensis that regulates the expression of KatG and AhpC. 4 F. tularensis KatG, SodC, and AhpC are induced in response to oxidative stress (52) and are secreted in abundance in the extracellular milieu and into the cytosol of F. tularensis-infected macrophages (27). Expression of these primary antioxidant enzyme genes starts immediately upon phagocytosis of F. tularensis and remains significantly up-regulated during phagosomal and cytosolic phases (52), suggesting that F. tularensis experiences oxidative stress at both of these intracellular locations.…”

“…The antioxidant enzymes of F. tularensis are strategically localized to protect it from macrophage-derived oxidative insult. The iron-containing superoxide dismutase (SodB) is constitutively expressed and secreted (26,27); and the copper-zinc-containing SodC is located in the periplasmic space. Previous studies from our laboratory with mutants of F. tularensis LVS in genes encoding for the antioxidant enzymes sodB, sodC, sodBC, and those of Lindgren et al (28)., with catalase (katG), have shown that these antioxidant enzymes are required for intramacrophage survival and virulence in mice (26,29,30).…”

O-Antigen-Deficient Francisella tularensis Live Vaccine Strain Mutants Are Ingested via an Aberrant Form of Looping Phagocytosis and Show Altered Kinetics of Intracellular Trafficking in Human Macrophages

“…Signals were developed by reacting with chemiluminescence substrate (Pierce) and detected by exposure to X-ray film. The nitrocellulose membrane was treated with Restore Plus Western Blot Stripping Buffer (Pierce) and reprobed with rabbit polyclonal antibodies specific for F. tularensis catalase/peroxidase (KatG) or bacterioferritin (Bfr) (18) and subsequently with goat anti-rabbit IgG (Bio-Rad, Hercules, CA), and signals were developed by reacting with chemiluminescence substrate as described above.…”

Interactions of Francisella Tularensis With Components of the Host Fibrinolytic System

“…The susceptibility of mice to FT LVS and the ability to elicit protective immunity following a sublethal infection with this organism have allowed researchers in the field an experimental model to tease apart the interaction between FT and the host adaptive immune responses outside of BSL-3 conditions. Recent studies have begun to shed light on the extracellular proteins and potential secretion mechanisms of various strains of F. tularensis (133)(134)147) . Our studies have indicated that immunization of mice with precipitated FT LVS culture filtrate (CF) components can protect Balb/c mice against homologous bacterial challenge.…”

“…Until recently, little was known about the extracellular proteins and potential secretion systems of Francisella (133)(134)147). Additionally, there have been no studies on the immunological response to protein antigens found in the media during culture of FT.…”