Expression Cloning and Periplasmic Orientation of the Francisella novicida Lipid A 4′-Phosphatase LpxF

Abstract

Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4-kinase, is present in Francisella, no 4-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4-phosphate residue from tetra-and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4-position. Expression of LpxF in wildtype E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria.Francisella tularensis is an intracellular Gram-negative pathogen that causes tularemia, a severe and often fatal pulmonary infection of humans and animals (1, 2). Francisella novicida U112 is an environmental isolate that is relatively easy to grow and is much less virulent (3). It is a model system for studying F. tularensis biochemistry. The lipopolysaccharide (LPS) 2 of F. tularensis and F. novicida shows low toxicity when compared with Escherichia coli LPS (4, 5). This phenomenon may be due to the unusual structure of the lipid A moiety of Francisella LPS (Fig. 1), which is tetraacylated and lacks the 4Ј-phosphate residue (6, 7). In contrast, E. coli lipid A (Fig. 1) is hexaacylated and is phosphorylated at both the 1-and 4Ј-positions (8, 9). E. coli lipid A is a potent activator of toll-like receptor 4 of the mammalian innate immune system (10, 11). The phosphate groups of lipid A are crucial for this bioactivity (12, 13). Lipid A variants lacking both phosphate groups are inactive. Monophosphorylated lipid A analogues retain some of the immunostimulatory properties of native lipid A, but they are not toxic and are often used as adjuvants (14 -17).Although they possess an orthologue of LpxK, the kinase that incorporates the lipid A 4Ј-phosphate group (18, 19), F. tularensis and related organisms synthesize lipid A molecules lacking the 4Ј-phosphate moiety (6, 7). Similar phosphate-deficient lipid A variants h...

Attenuated virulence of aFrancisellamutant lacking the lipid A 4′-phosphatase

“…Here, we demonstrate that inactivation of the periplasmic phosphatase, previously shown to be involved in Francisella lipid A 4Ј dephosphorylation in vitro (16), results in a mutant that retains the 4Ј-phosphate moiety in its lipid A in living cells (Figs. 1 and 3).…”

“…A significant proportion of the neutrophil bactericidal activity is attributed to the repertoire of secreted cationic antimicrobial peptides, such as the defensins (23). Because structural modifications that reduce the net negative charge of lipid A have been shown to alter susceptibility to cationic antimicrobial peptides (24)(25)(26), we compared the sensitivity of the mutant and wild-type with the cationic antimicrobial peptide polymyxin B (16,27). Employing a disk diffusion assay, we observed that wild-type F. novicida U112 bacteria are highly resistant to polymyxin (Fig.…”

“…We have recently discovered a selective lipid A 4Ј-phosphatase in F. novicida, designated LpxF (16). The active site of this phosphatase faces the periplasmic surface of the inner membrane (16). We hypothesized that F. novicida mutants lacking LpxF should synthesize lipid A molecules that retain the 4Ј-phosphate moiety and that LpxF might serve to enhance F. novicida virulence by conferring the capacity to avoid host immune recognition and/or killing by cationic antimicrobial peptides.…”

“…We have recently discovered a selective lipid A 4Ј-phosphatase in F. novicida, designated LpxF (16). The active site of this phosphatase faces the periplasmic surface of the inner membrane (16).…”

Automated Lipid A Structure Assignment from Hierarchical Tandem Mass Spectrometry Data

“…Yp strain KIM6+ was grown in Luria broth (pH 7.4) at 37°C with aeration and harvested in the late exponential phase referred to as Yp wild type (Yp_WT) [24]. Lipid A C-1 and C-4' phosphatase, LpxE and LpxF, respectively, have been expression cloned in Fn [25]. The individual plasmids with the structural genes of LpxE or LpxF and an ampicillin resistance gene were incorporated into KIM6+ cell via electroporation [11].…”