Francisella tularensisSchu S4 O-Antigen and Capsule Biosynthesis Gene Mutants Induce Early Cell Death in Human Macrophages

Abstract

Francisella tularensis is capable of rampant intracellular growth and causes a potentially fatal disease in humans. Whereas many mutational studies have been performed with avirulent strains of Francisella, relatively little has been done with strains that cause human disease. We generated a near-saturating transposon library in the virulent strain Schu S4, which was subjected to high-throughput screening by transposon site hybridization through primary human macrophages, negatively selecting 202 genes. Of special note were genes in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238. Mutants with mutations in these genes demonstrated significant sensitivity to complement-mediated lysis compared with wild-type Schu S4 and exhibited marked defects in O-antigen and capsular polysaccharide biosynthesis. In the absence of complement, these mutants were phagocytosed more efficiently by macrophages than wild-type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic and quantitative analyses of macrophages infected with mutant bacteria revealed that these macrophages exhibited signs of cell death much earlier than those infected with Schu S4. These data suggest that FTT1236, FTT1237, and FTT1238 are important for polysaccharide biosynthesis and that the Francisella O antigen, capsule, or both are important for avoiding the early induction of macrophage death and the destruction of the replicative niche.

Subversion of Host Recognition and Defense Systems by Francisella spp

“…Francisella strains with mutations in the wbt locus are attenuated during infection of both macrophages (130,135,221) and mice (129,153,204,221,232). Furthermore, mutants lacking genes necessary for O-antigen production that are outside the wbt locus are also attenuated for intracellular replication and in vivo virulence compared to the wild type (130).…”

“…This is important to note, since recent evidence has suggested that the O antigen and capsule of Francisella are structurally similar, with the capsule consisting of a polymer of O-antigen tetrasaccharide subunits (9) as well as a glycoprotein component termed a "capsulelike complex" (18). As such, some mutants deficient in LPS O-antigen production are also impaired in the production of capsule (9,130), including specific wbt operon mutants (wbtA, wbtC, wbtM, and wbtI). wbtK and lpxL mutants lose the ability to synthesize LPS O antigen but maintain the polysaccharide capsule (9).…”

“…infection of host cells in culture, while "in vivo" refers to infection of mammalian hosts such as mice) (110,130,153,232). This is important to note, since recent evidence has suggested that the O antigen and capsule of Francisella are structurally similar, with the capsule consisting of a polymer of O-antigen tetrasaccharide subunits (9) as well as a glycoprotein component termed a "capsulelike complex" (18).…”

“…Francisella, like many other bacteria, including Neisseria (95), Pseudomonas (179), and Salmonella (23), encodes at least one known multidrug efflux pump that allows resistance to a number of different antimicrobials and detergents, the AcrAB/TolC efflux pump. In addition, other predicted efflux pump proteins have been identified in screens for Francisella virulence genes, including FTN_1066, FTN_1217, FTN_1277, FTN_1654, and FTN_1657 (130,140,232). While the exact functions of these predicted efflux proteins are unknown, they may play a role complementary to the known functions of the AcrAB/TolC efflux pump.…”

“…Many Francisella genes annotated as being involved in metabolism, for both energy production and biosynthesis of macromolecules, have been identified in screens for genes important in intracellular replication and in vivo virulence (130,135,140,214,232). These screens identified genes required for growth during infection but not in rich media, and they therefore suggest that Francisella must utilize distinct metabolic pathways in order to successfully survive and replicate within the host, compared to those required for growth in broth.…”