Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

Abstract

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.

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

“…An effective vaccine for tularemia will likely require multiple antigens, but, as an initial step in determining whether Ft-glycOMVs might be a candidate vaccine component for a multivalent vaccine, we evaluated their protective efficacy in mice infected with the highly virulent F. tularensis type A strain Schu S4, which has an LD 50 of <10 colony-forming units (cfus) in mice (48). BALB/c mice were immunized by an i.p.…”

Metabolic Reprogramming of Host Cells by Virulent Francisella tularensis for Optimal Replication and Modulation of Inflammation

“…tularensis SchuS4 (Ftt) (Dr. Jeannine Peterson, CDC, Fort Collins, CO), SchuS4Δ1238, SchuS4Δ1236, SchuS4Δ1464c (Dr. Bradley Jones, University of Iowa, Iowa City IA) were generated and utilized as previously described (8). SchuS4Δ1238 (Δ1238) and SchuS4Δ1236 (Δ1236) were previously characterized as capsule mutants due to deletions in waa locus encoding enzymes responsible for adding O-antigen subunits to the LPS core or adding sugars to LPS core, respectively (13). SchuS4Δ1464c (Δ1464c) was also previously described and is devoid of capsule due to the mutation in the dTDP-glucose 4,6-dehydratase that enables formation of 6-deoxy sugars that make up the capsular structure (17).…”

“…Together, these features suggest capsule contributes to the virulence of the bacterium. Indeed, defined mutations in capsule synthesis genes results in modest attenuation of Francisella in vivo (13). Despite these advances, the contribution of capsule in the evasion suppression of pro-inflammatory responses is largely unexplored.…”

Identification and characterization of capsule and/or O-antigen mutants of Francisella tularensis Schu S4

“…Work to identify unique biosynthetic genes for the O-antigen capsule has only identified mutant strains that are disrupted for both capsule and LPS biosynthesis or LPS alone (8,173). This indicates that the majority of genes involved in the biosynthesis, assembly and secretion of polysaccharides in Francisella are also required for LPS biosynthesis.…”

“…The idea that the capsular O-antigen is most likely assembled on a lipid besides the core-lipid A LPS structure, suggests that there is a gene (or genes) specifically devoted to capsule biosynthesis independent of LPS assembly machinery. Efforts to find genes responsible for unique biosynthetic steps are ongoing (14,118,173).…”