| 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.Much of the recent interest in Francisella tularensis, the etiological agent of tularemia, is due to concern about its potential use as an agent of bioterrorism coupled with an incomplete understanding of the molecular basis of its pathogenicity. F. tularensis is highly pathogenic by the pneumonic route, causing disease in humans with an inoculum as small as 10 organisms, and infection by this route carries a mortality rate of 30 to 60% if untreated (43, 67). Due to its extreme virulence and ease of aerosol dissemination, several nations have weaponized F. tularensis and the U.S. Centers for Disease Control and Prevention have classified this organism as a category A select agent (20). F. tularensis has a remarkably broad host range: it is capable of infecting over 250 known species from across the entire phylogenetic tree, including amoebae, insects, small mammals (such as rodents and lagomorphs), and primates (51). Tularemia is primarily a zoonosis, and humans are thought to be accidental hosts (23). The majority of human infections, the pneumonic infections reported on Martha''s Vineyard in 2000 (21) being an notable exception, are cutaneous, lead to ulceroglandular disease, and ensue following exposure to infected animals or animal products (47). The ability of F. tularensis to infect such a wide range of eukaryotes suggests that this organism either co-opts cellular mechanisms common to all hosts, has the requisite virulence genes to adapt to many different intraorganismal environments, or both.Despite the infectivity of Francisella for disparate hosts, relatively little is known about its virulence genetics. F. tularensis invades and replicates within many cell types: phagocytes, such as primary macrophages (human monocyte-derived macrophages [MDMs] and mouse bone marrow-derived macrophages) and macrophage-like cell lines (J774A.1 and THP-1), as well as in nonphagocytic cells such as bronchial airway epithelial cells, hepatocytes, human umbilical vein endothelial cells, and epithelium-derived tissue culture cell lines (i.e., HEp-2, A549, HBE, and HepG2) (17, 24, 31, 40, 61). Macrophages are known to bind and phagocytose F. tularensis using at least three receptors: complement receptor 3 (CR3), which binds to complement 3b (C3b) protein deposited upon the bacterium when exposed to fresh serum (14) (60); mannose receptor (MR) (60); and scavenger receptor A (53). Once internalized, F. tularensis organisms are able to alter intracellular trafficking of their phagosomes and prevent fusion with the lysosome, acquiring late endosomal markers such as lamp-1 transiently and altogether avoiding endosomes containing cathepsin D and S (15). Shortly thereafter, F. tularensis escapes from the phagosome and grows in the macrophage cytosol. The majority of genes known to be important for phagosomal escape and intracellular growth lie within the duplicated Francisella pathogenicity island (FPI), an ∼30-kb region of the chromosome carrying the igl and pdp operons (46). Mutants with mutations in many of the genes in the FPI, such as iglABCD, vgrG, and iglI, as well as pdpA and pdpD, are defective for intracellular growth in both primary and tissue culture cells in vitro, and these genes may encode a novel type VI secretion system (4). Mutational analysis implicates genes such as mglA, sspA, fevR, fslA, pmrA, and migR in regulation of FPI genes (5-8, 10, 38). However, knowledge of other virulence genes residing outside the FPI that play important roles in intracellular growth is limited.We undertook a high-throughput approach to identify genes important for the growth of virulent F. tularensis Schu S4 in primary human macrophages. F. tularensis has two major biovars: type B (F. tularensis subsp. holarctica), which is found throughout the Northern hemisphere, and type A (F. tularensis subsp. tularensis), which is found exclusively within North America and typically causes a more severe disease in humans than type B isolates. These biovars are further subdivided into clades that exhibit differences in virulence phenotypes (45). A significant research effort to date has focused on the virulence properties of the non-human pathogens Francisella novicida and the F. tularensis live vaccine strain (LVS), an attenuated F. tularensis subsp. holarctica variant generated in the Soviet Union in the 1950s. Although both of these strains are avirulent in healthy humans, they can grow in some human cells and cell lines in vitro and are virulent in the mouse model of infection, properties that make them attractive models for studying the pathogenesis of tularemia. Several random transposon mutant libraries generated in F. novicida and F. tularensis LVS (41, 50, 55, 66, 68, 70) have been screened using both in vitro tissue culture models and mice. In addition, Qin and Mann generated an ∼700-member Tn5 (EZ-TN) mutant library in the type A strain F. tularensis Schu S4 and screened each clone through the hepatocyte-like cell line HepG2 to detect mutants defective in intracellular growth. Among the genes identified in this screen is FTT1236 (55).Lipopolysaccharide (LPS) is a major component in the outer membranes of Gram-negative organisms, and O antigens (O Ags) are known virulence factors of many pathogenic bacteria that function in part to protect against damage by serum complement and antimicrobial peptides as well as mask bacterial surface antigens (16). LPS is composed of a lipid A moiety that secures it to the Gram-negative outer membrane on which a core polysaccharide and O Ag are assembled. Whereas the LPS of most bacterial species is recognized as a pathogen-associated molecular pattern (PAMP) through MD-2/TLR4 and is a potent activating signal for the innate immune system, the LPS of F. tularensis is nearly inert (27). Of note, the lipid A of F. tularensis has an atypical structure, does not bind to LPS-binding protein (LBP), and is therefore not recognized by TLR2 or TLR4 (3, 28, 52). In addition, the F. tularensis O Ag is structurally distinct from that of F. novicida (43). Although our understanding of the genetics of O-Ag biosynthesis in F. tularensis is incomplete, it is known that wbt operon mutants of F. tularensis LVS do not express an O Ag. These strains bind C3b more avidly and are more sensitive to bile salts and to complement-mediated lysis than wild-type strains (13, 39). These mutants are also defective for intracellular growth in J774A.1 cells and exhibit reduced virulence and dissemination in mice (41, 56, 62, 69). Of note, an LVS FTL0706 (designated FTT1238 in F. tularensis Schu S4) mutant lacks an O Ag and exhibits decreased replication within, and is cytotoxic to, J774A.1 cells (41). A corresponding Schu S4 FTT1238 mutant also lacks an O Ag, but its virulence properties have not yet been described.In many pathogenic bacteria, capsular polysaccharides also contribute to serum resistance yet, unlike O Ags, diminish phagocytosis (16). Francisella has long been thought to have an extracellular structure that resembles that of a capsular polysaccharide. Observed by Hood in 1976 (30), the nature of this capsule has remained elusive. Acridine orange treatment was used to produce an undefined LVS mutant that appears to lack an extracellular polysaccharide structure (58). This strain was designated Cap− (alternately termed “rough” in more recent work) and is serum sensitive (13, 62). Furthermore, expression of the capsular polysaccharide was observed to be increased by repeated passage of LVS on Chamberlain''s defined medium, which in turn increased virulence in mice (12). Recently, our group has identified a capsular polysaccharide using a new monoclonal antibody that recognizes this structure as distinct from LPS O Ag. This capsule has immunological properties distinct from those of purified LPS and is also a potential vaccine (2).Advances in transposon delivery systems in our lab and others allowed us to generate a near-saturating random transposon mutant library in strain Schu S4 that is capable of high-throughput screening using the transposon site hybridization (TraSH) system previously used by Weiss et al. for F. novicida (70). Here we describe the utilization of this genetic system to identify and characterize new virulence genes important for F. tularensis Schu S4 entry and growth in human MDMs. Among the genes we identified is a locus required for LPS O-Ag and capsular polysaccharide biogenesis and for serum resistance and intracellular growth within MDMs. We further show that defects in intracellular growth are due to their induction of premature macrophage death, which deprives mutant organisms of their replicative niche. |
