Uncovering the components of the Francisella tularensis virulence stealth strategy

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

Francisella tularensis Schu S4 lipopolysaccharide core sugar and o-antigen mutants are attenuated in a mouse model of tularemia

The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD(50)) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections

Scoring of growth patterns observed in confocal imaging.

<p>Infected cells were classified into three separate categories based on the amount of bacteria each cell contained at 24 hpi. Cells infected with Schu S4 had significantly more bacteria per cell compared to cells infected with LVS 24 hpi (*P <. 001). Percentages were calculated from greater than 300 cells containing organisms from three independent experiments.</p

TEM of <i>F</i>. <i>tularensis</i> in dying AT-II cells <i>in vivo</i>.

<p>TEM images of infected mouse lungs at both 32 and 48 hpi infected with Schu S4. (A-C) AT-II cells containing <i>Francisella</i> going through cell death characterized by lack of electron density of the cytosol and swollen mitochondria. (D-F) A separate AT-II cell undergoing cell death infected with <i>Francisella</i>. The open arrows identify the bacteria in each field and the solid black arrows indicate lamellar bodies in the cells.</p

Growth of <i>F</i>. <i>tularensis</i> Schu S4, <i>F</i>. <i>tularensis waaY</i>::TrgTn and <i>F</i>. <i>tularensis</i> LVS in cultured human alveolar type II cells.

<p>Immortalized primary AT-II tissue culture cells were used to perform gentamicin protection assays. The ability of the three <i>Francisella</i> strains to enter and replicate with these epithelial lung cells was measured after infecting at an MOI 100:1. Fold growth was calculated as the difference between the number of bacteria surviving gentamicin treatment at 4 hours and 24 hours post-infection. (A) Data are a single experiment representative of three separate experiments. (B) Data are an average of four independent experiments.</p

Schu S4 <i>waaY</i>::TrgTn increases cellular debris within the alveoli.

<p>(A-C). TEM imaging of 24 hours demonstrating clean airspace with the alveoli in lung tissue that is infected with either PBS (A), Schu S4 (B), or LVS (C) and airspace that is representative of lung tissue infected with Schu S4 <i>waaY</i>::TrgTn (D-F). B. Graphical representation of scoring of the airspace for cellular debris. There was an increase in cellular debris in lungs infected with Schu S4 <i>waaY</i>:TrgTn compared to PBS control (<i>P</i> < 0.001). Averages were calculated from analysis of greater than 50 scored airspaces from 2 separate lung sections per strain and time point.</p

Model of early <i>Francisella</i> infection within the lung.

<p>(1) <i>Francisella</i> enters into the alveoli from an aerosolized infection where it either gets phagocytosed by alveolar macrophages (2a) or interacts with AT-II cells (2b). Upon uptake into alveolar macrophages there are at least two possible outcomes, either alveolar macrophages allow bacterial growth and release into the airspace (2a) or alveolar macrophages detach and are removed from the alveoli by the mucociliary escalator (2c). Growth and release from the alveolar macrophages allows reinfection with surrounding tissue including AT-II cells (3). Internalization with AT-II cells acts as a mechanism to get past the epithelial barrier and allows for interaction with endothelial cells and eventually dissemination to the liver and spleen.</p

Electron microscopy images of <i>Francisella tularensis</i> strains within murine alveolar type II epithelial cells.

<p>TEM images of infected mouse lungs at 24 hours post-infection. Mice were infected intranasally with either <i>F</i>. <i>tularensis</i> Schu S4 (A-F), <i>F</i>. <i>tularensis</i> Schu S4 <i>waaY</i>::TrgTn (G-K), and <i>F</i>. <i>tularensis</i> LVS (I,L). Each image shows an AT-II cell containing organisms. The AT-II cell can be identified by the presence of microvilli at the cell-air interface and by the presence of lamellar granules. It is also worth noting that each infected AT-II cell was immediately adjacent to a pulmonary capillary. The area containing the internalized bacteria is within the white rectangle which is shown at higher magnification immediately beneath the corresponding image. The arrows identify the bacteria in each field.</p