Abstract
The lipopolysaccharide (LPS) and O-antigen polysaccharide capsule structures of Francisella tularensis play significant roles in helping these highly virulent bacteria avoid detection within a host. We previously created pools of F. tularensis mutants that we screened to identify strains that were not reactive to a monoclonal antibody to the O-antigen capsule. To follow up previously published work, we characterize further seven of the F. tularensis Schu S4 mutant strains identified by our screen. These F. tularensis strains carry the following transposon mutations: FTT0846::Tn5, hemH::Tn5, wbtA::Tn5, wzy::Tn5, FTT0673p/prsA::Tn5, manB::Tn5, or dnaJ::Tn5. Each of these strains displayed sensitivity to human serum, to varying degrees, when compared to wild-type F. tularensis Schu S4. By Western blot, only FTT0846::Tn5, wbtA::Tn5, wzy::Tn5, and manB::Tn5 strains did not react to the capsule and LPS O-antigen antibody 11B7, although the wzy::Tn5 strain did have a single O-antigen reactive band that was detected by the FB11 monoclonal antibody. Of these strains, manB::Tn5 and FTT0846 appear to have LPS core truncations, whereas wbtA::Tn5 and wzy::Tn5 had LPS core structures that are similar to the parent F. tularensis Schu S4. These strains were also shown to have poor growth within human monocyte derived macrophages (MDMs) and bone marrow derived macrophages (BMDMs). We examined the virulence of these strains in mice, following intranasal challenge, and found that each was attenuated compared to wild type Schu S4. Our results provide additional strong evidence that LPS and/or capsule are F. tularensis virulence factors that most likely function by providing a stealth shield that prevents the host immune system from detecting this potent pathogen.
Highlights
Francisella tularensis is the causative agent of the human disease tularemia
Identification of F. tularensis Genes Involved in Capsule and O-antigen Synthesis A total of nine strains were identified by screening a Tn5 transposon library of F. tularensis Schu S4 with monoclonal antibodies produced against F. tularensis capsule preparations
Determining In Vitro Growth Phenotypes of Mutants in Human monocyte derived macrophages (MDMs) and Murine bone marrow derived macrophages (BMDMs) Since our previous work has shown that F. tularensis strains defective in LPS and/or capsule O-antigen biosynthesis have growth defects in human MDMs (Lindemann et al, 2011), we examined whether the strains that are the subject of this work display a similar phenotype in either MDMs or BMDMs
Summary
Francisella tularensis is the causative agent of the human disease tularemia. This small Gram negative organism is able to cause disease with as few as 10 colony forming units (CFU) when inhaled, and without treatment, upwards of 60% of those infected may die (Saslaw et al, 1961; Tarnvik and Berglund, 2003; Oyston, 2008). There are relatively few naturally acquired Francisella infections per year in the United States (Carvalho et al, 2014), a primary concern is that the organism will be used in a bioterrorist attack This is not an unfounded concern as Francisella species were weaponized by Japan, United States, and the former U.S.S.R., during and after World War II (Oyston et al, 2004; Sjostedt, 2007). These concerns have highlighted the need to develop a vaccine to prevent disease and associated deaths from the potential use of Francisella as a bioterrorist weapon
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