Tularemia is a zoonosis of humans caused by infection with the facultative intracellular bacterium Francisella tularensis. Interest in F. tularensis has increased markedly in the past few years because of its potential use as an agent of bioterrorism. Five subspecies of this organism are found in the Northern hemisphere, but only F. tularensis subsp. tularensis and subsp. holarctica cause disease in humans. This review summarizes what is known about the pathogenesis of tularemia with a focus on bacterial surface components such as lipopolysaccharide and capsule as well as information obtained from the F. tularensis subsp. tularensis SCHU S4 genome. In particular, the mechanisms of action of recently identified virulence factors are discussed in the context of bacterial replication in macrophages and manipulation of the host inflammatory response. Throughout this report shared and unique features of F. tularensis subsp. tularensis, subsp. holarctica, and subsp. novicida are discussed.

We have investigated the lipid A of Francisella tularensis subsp. holarctica strain 1547-57, a type B strain, by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, nanoelectrospray quadrupole ion-trap mass spectrometry, and chemical methods. In accordance with the previously published structures of the lipid A from F. tularensis live vaccine strain (LVS) (ATCC 29684) (E. Vinogradov et al., Eur. J. Biochem. 269:6112-6118, 2002), all of the major lipid A forms from strain 1547-57 were tetraacylated. As in the LVS strain, the major fatty acids detected in the F. tularensis 1547-57 lipid A sample included 3-hydroxyoctadecanoic acid, 3-hydroxyhexadecanoic acid, hexadecanoic acid, and tetradecanoic acid. However, several of the lipid A components present in strain 1547-57 were of higher molecular weight than the previously published structures. A major component with an M r of 1,666 was found to contain three C 18:0 (3-OH) fatty acids, one C 16:0 fatty acid, one phosphate group, and one 161-Da moiety. This 161-Da moiety could be removed from the lipid A by treatment with aqueous hydrofluoric acid and was identified as galactosamine following peracetylation and analysis by gas chromatography-mass spectrometry. Detailed investigations of the M r -1,666 species by ion-trap mass spectrometry with multiple stages of fragmentation suggested that the galactosamine-1-phosphate was linked to the reducing terminus of the lipid A. Similar to the modification of lipid A with arabinosamine, lipopolysaccharide species from F. tularensis containing a phosphate-linked galactosamine could potentially influence its intracellular survival by conferring resistance to antimicrobial peptides.Francisella tularensis is an encapsulated gram-negative bacterium that causes tularemia, a severe disease of humans and other mammals (12,24). Severity of disease depends on the host immune response and the route of infection, including intradermal inoculation via zoonotic transmission, ingestion of contaminated meat or water, and inhalation (7). F. tularensis is a facultative intracellular bacterium, and several studies have shown that a cell-mediated immune response may be required for controlling the infection (22,24). Recently, interest in F. tularensis has increased because of its suitability for use as an agent of biological warfare (6).F. tularensis possesses a lipopolysaccharide (LPS) that, when compared to Escherichia coli LPS, is not biologically active (1,8,23). The only biological activity attributed to F. tularensis LPS in vitro is the ability to activate complement (8). The relatively nonendotoxic nature of the LPS is putatively attributed to the unusual structure of the lipid A molecule. A recent study showed that the lipid A molecule of F. tularensis live vaccine strain (LVS) is not only tetraacylated but also lacks phosphate substituents (27). These structural features may contribute to its low toxicity. It has been shown that mutations affecting the degree of acylation or the acylation pattern of lipid...

Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-α-D-GalNAcAN-(1->4)-α-D-GalNAcAN-(1->3)-β-D-QuiNAc-(1->2)-β-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 µg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 µg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.

Background Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized.Methods and FindingsA capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32°C in 7% CO2. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSΔ1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSΔ1423/1422 and subsequent passage in broth restored CLC expression. LVSΔ1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD50, respectively. Following immunization, mice challenged IN with over 700 times the LD50 of LVS remained healthy and asymptomatic.ConclusionsOur results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain.

In the beginning of the 21st century, the frequency of antimicrobial resistance (AMR) has reached an apex, where even 4th and 5th generation antibiotics are becoming useless in clinical settings. In turn, patients are suffering from once-curable infections, with increases in morbidity and mortality. The root cause of many of these infections are the ESKAPEE pathogens (Enterococcus species, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli), which thrive in the nosocomial environment and are the bacterial species that have seen the largest rise in the acquisition of antibiotic resistance genes. While traditional small-molecule development still dominates the antibacterial landscape for solutions to AMR, some researchers are now turning to biological approaches as potential game changers. Monoclonal antibodies (mAbs)—more specifically, human monoclonal antibodies (Hu-mAbs)—have been highly pursued in the anti-cancer, autoimmune, and antiviral fields with many success stories, but antibody development for bacterial infection is still just scratching the surface. The untapped potential for Hu-mAbs to be used as a prophylactic or therapeutic treatment for bacterial infection is exciting, as these biologics do not have the same toxicity hurdles of small molecules, could have less resistance as they often target virulence proteins rather than proteins required for survival, and are narrow spectrum (targeting just one pathogenic species), therefore avoiding the disruption of the microbiome. This mini-review will highlight the current antibacterial mAbs approved for patient use, the success stories for mAb development, and new Hu-mAb products in the antibacterial pipeline.