Uncovering the components of the Francisella tularensis virulence stealth strategy

Abstract

Over the last decade, studies on the virulence of the highly pathogenic intracellular bacterial pathogen Francisella tularensis have increased dramatically. The organism produces an inert LPS, a capsule, escapes the phagosome to grow in the cytosol (FPI genes mediate phagosomal escape) of a variety of host cell types that include epithelial, endothelial, dendritic, macrophage, and neutrophil. This review focuses on the work that has identified and characterized individual virulence factors of this organism and we hope to highlight how these factors collectively function to produce the pathogenic strategy of this pathogen. In addition, several recent studies have been published characterizing F. tularensis mutants that induce host immune responses not observed in wild type F. tularensis strains that can induce protection against challenge with virulent F. tularensis. As more detailed studies with attenuated strains are performed, it will be possible to see how host models develop acquired immunity to Francisella. Collectively, detailed insights into the mechanisms of virulence of this pathogen are emerging that will allow the design of anti-infective strategies.

Keywords: FPI virulence genes; Francisella tularensis; capsule mutant; phagosome escape; stealth strategy; unique LPS; virulent Schu S4.

Figure 1

F. tularensis strains produce O-antigen glycosylated proteins. One ml of F. tularensis broth cultures was centrifuged at 8000 × g for 2 min before resuspending the pellet in Buffer Part A, (6 mM Tris, 10 mM EDTA, and 2% [wt/vol] sodium dodecyl sulfate [pH 6.8]) and heated to 65°C to sterilize cultures. Bacterial lysates were incubated with, or without, proteinase K (New England Biolabs, Ipswitch, MA) at 37°C for 24 h before lyophilizing. Approximately 14 mg of bacterial material from each sample was mixed with NuPage (Life Technologies, Carlsbad, CA) sample reducing agent and buffer, boiled, and loaded into a 4–12% Bis-Tris NuPage gel and electrophoresed using NuPage MES SDS running buffer (Life Technologies, Carlsbad, CA). For immunoblots, samples were transferred to nitrocellulose and probed with FB11 primary antibody to detect O-antigen attached to LPS or to protein (QED Bioscience, San Diego, CA). Bands were visualized using goat anti-mouse IgG (H+L) conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) and SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). (A) Western blot analysis of LPS preparations from F. tularensis LVS, virulent type B F. tularensis subsp.holarctica strains (1547 and 1623) and virulent type A F. tularensis subsp. tularensis strains (Schu S4, MA00, and WY96) using the O-antigen FB11 antibody for detection of bands containing O antigen, without or with proteinase K (pk) treatment. (B) Western blot of whole cell lysates of F. tularensis Schu S4, F. tularensis Schu S4 waaY::TrgTn and F. tularensis Schu S4 waaL::TrgTn, F. holarctica LVS, F. holarctica LVS waaY::TrgTn and F. holarctica LVS waaL::TrgTn probed with the anti-O antigen monoclonal antibody FB11 to detect the presence of LPS O antigen laddering in these strains. (C) Western blot analysis of F. tularensis Schu S4, F. tularensis Schu S4 waaY::TrgTn and F. tularensis Schu S4 waaL::TrgTn whole cell lysates without or with (pk) proteinase K treatmenst, using O-antigen FB11 antibody.

Figure 2

Comparison of the intracellular growth strategies of wild type F. tularensis and a F. tularensis capsule/LPS mutant. Both wild type and mutant organisms are internalized into a host cell, although the absence of capsule and LPS allows increased internalization. Both strains can escape from the phagosome. The wild type strain grows to high numbers in the host cell cytosol before inducing death of the cell. At late stages, organisms can be found in Francisella-containing vacuoles (FCVs). In contrast, after mutant organisms escape the phagosome, they grow for a short time in the cytosol before the cell dies preventing additional growth of the organisms.