Attenuated virulence of a Francisella mutant lacking the lipid A 4'-phosphatase

Abstract

Francisella tularensis causes tularemia, a highly contagious disease of animals and humans, but the virulence features of F. tularensis are poorly defined. F. tularensis and the related mouse pathogen Francisella novicida synthesize unusual lipid A molecules lacking the 4'-monophosphate group typically found in the lipid A of Gram-negative bacteria. LpxF, a selective phosphatase located on the periplasmic surface of the inner membrane, removes the 4'-phosphate moiety in the late stages of F. novicida lipid A assembly. To evaluate the relevance of the 4'-phosphatase to pathogenesis, we constructed a deletion mutant of lpxF and compared its virulence with wild-type F. novicida. Intradermal injection of 10(6) wild-type but not 10(8) mutant F. novicida cells is lethal to mice. The rapid clearance of the lpxF mutant is associated with a stronger local cytokine response and a greater influx of neutrophils compared with wild-type. The F. novicida mutant was highly susceptible to the cationic antimicrobial peptide polymyxin. LpxF therefore represents a kind of virulence factor that confers a distinct lipid A phenotype, preventing Francisella from activating the host innate immune response and preventing the bactericidal actions of cationic peptides. Francisella lpxF mutants may be useful for immunization against tularemia.

Fig. 1.

Structures of lipid A molecules synthesized by E. coli and F. novicida. (A) E. coli lipid A is synthesized by a system of nine constitutive enzymes (2), orthologs of which are present in Francisella (15). Numbers indicate the lengths of the predominant fatty acyl chains. (B) Structure of the predominant form of lipid A (compound A1) extracted from F. novicida wild-type U112 grown to late log phase at 37°C on 3% trypticase soy broth, supplemented with 0.1% cysteine (12, 48). Compound A2, which represents ≈10% of the total, is modified with a glucose residue at position 6′. (C) Structure of the predominant lipid A species (compound A4) present in XWK4, the lpxF mutant of F. novicida. When the 4′-phosphate moiety is not removed, the 3′-O-acyl chain stays in place, indicating an obligatory order of enzymatic processing. More than 90% of Francisella lipid A is free, in that it is not covalently linked to LPS (12).

Fig. 2.

Altered lipid A composition, lack of 4′-phosphatase activity, and polymyxin hypersensitivity of the F. novicida lpxF mutant. (A) Thin-layer chromatography of F. novicida lipids labeled uniformly with ³²P and extracted by using a two-phase Bligh–Dyer system (16, 37). Samples containing ≈10,000 cpm were spotted in each lane. The plate was developed with chloroform/pyridine/88% formic acid/water (50:50:16:5, vol/vol), and the lipids were detected with a PhosphorImager. Most of the lipid A found in F. novicida is not linked to LPS (12), possibly because of the presence of an unusual Kdo-cleaving activity (37). Therefore, most of the lipid A of F. novicida is extracted directly with chloroform/methanol, together with the glycerophospholipids (12). The structure of compound A4 is shown in Fig. 1C. It accumulates in the lpxF mutant, whereas A1 and A2 disappear. (B) Absence of 4′-phosphatase activity in membranes of XWK4. (C) Kanamycin resistance and polymyxin hypersensitivity of mutant XWK4.

Fig. 3.

NMR spectroscopy of lipid A isolated from the lpxF mutant of F. novicida. (A) ³¹P NMR spectrum of compound A4. The ³¹P NMR signals at 0.53 and −2.5 ppm indicate the presence of two phosphate groups, designated P-4′ and P-1 according to the numbering scheme shown in Fig. 1C and described in detail previously (12). The ³¹P NMR spectrum of compound A1 from the wild-type (data not shown) shows only a single peak near −1.3 ppm, consistent with the absence of P-4′. (B) ¹H NMR spectrum of component A4 in the anomeric and sugar proton regions. The relevant protons are labeled as in Fig. 1C (12). (C and D) Selective inverse decoupled difference ¹H NMR spectra of compound A4, implemented by selective irradiation of the individual phosphorus atoms (12, 18), provide unequivocal evidence that a 4′-monophosphate group (0.53 ppm ³¹P signal) is present (C) and that two sugar residues are attached to the anomeric 1-phosphate group (−2.5 ppm ³¹P signal) (D), as in compound A1 of wild-type cells (12). The full assignments of the NMR spectra of compound A4, which are based on two-dimensional analyses, are summarized in SI Table 1.

Fig. 4.

Loss of virulence in lpxF mutants of F. novicida. Three groups of 10 mice were injected into their right rear footpads with 10⁶ viable wild-type F. novicida U112, 10⁶ live vaccine strain (LVS), or 10⁶ lpxF XWK4 mutant F. novicida. Survival of the animals was recorded once a day. (A) Average weights of the surviving animals. (B) Percentage of surviving animals in each treatment group.

Fig. 5.

Enhanced recruitment of cytokines and neutrophils into mouse peritoneum injected with the lpxF mutant XWK4. In these experiments, 10⁶ viable bacteria were injected into each mouse peritoneum. After 4 h, the peritoneal fluid was withdrawn. (A) Cytokines tested were TNFα, macrophage inflammatory protein 2 (MIP2), MCP1, IL6, IL13, IL1β, and keratinocyte-derived chemokine. The cytokine levels were determined by the Elisa Tech Company (Aurora, CO). Six mice were used in each experiment; three were infected with U112 and three with XWK4. The experiment was repeated three times and the data averaged. (B) The percentage of neutrophils in the peritoneal fluids was determined. Six mice were used in each experiment; three were injected with U112 and three with XWK4. The experiment was repeated twice and the data averaged. (C) The titers of viable bacteria in the fluids were determined by serial dilution plating. The P value was <0.02 for all observed differences.