Structure and biosynthesis of free lipid A molecules that replace lipopolysaccharide in Francisella novicida

Materials and reagents

Glass backed 0.25 mm Silica Gel 60 thin layer chromatography plates were from E. Merck, Darmstadt, Germany. Chloroform, ammonium acetate, and sodium acetate were obtained from EMD Science, Gibbstown, NJ, while pyridine, methanol, and formic acid were from Mallinckrodt, Phillipsburg, NJ. Trypticase soy broth, yeast extract and tryptone were purchased from Difco, Detroit, MI. DEAE cellulose was purchased from Whatman, Florham Park, NJ. ³²P_i was purchased from NEN Life Science Products. Tri-Sil Reagent was from Pierce, Rockford, IL. The methanol-HCl kit was from Supelco, St. Louis, MO. D₂O, CD₃OD and CDCl₃ were from Sigma-Aldrich, St. Louis, MO. F. novicida U112 was obtained from Dr. F. Nano, University of Victoria, British Columbia, Canada. The F. tularensis LVS strain was purchased from the American Type Culture Collection, Rockville, MD.

Construction of a F. novicida mutant with a deletion in its arnT orthologue

The single orthologue of the arnT gene (27) present in the F. novicida genome (30) was amplified by PCR with 2 kb of flanking F. novicida genomic DNA. The forward primer (5′-CGTGCGGCCGCACTTATTACGACCTTCTTCGGTATTA-3′) was designed with a clamp region, a NotI restriction site (underlined), and a match to the coding strand of chromosomal DNA about 2 kb upstream of F. novicida arnT. The reverse PCR primer (5′-ACGCTCGAGATGTTTGATATTGTTTTTTATTAGCATATT-3′) was designed with a clamp region, a XhoI restriction site (underlined), and a match to the anti-coding strand of chromosomal DNA about 2kb downstream of F. novicida arnT. The PCR was performed using PfuTurbo polymerase and F. novicida genomic DNA as the template. Amplification was carried out in a 100 μl reaction mixture containing 200 ng template, 200 ng primers and 2 units of polymerase. The reaction was started at 94 °C for 2 min, followed by 30 cycles of denaturation (30 seconds at 94 °C), annealing (30 seconds at 58 °C), and extension (5 min at 72 °C). After the 30th cycle, a 10 min extension time was used. The reaction product was analyzed on a 1% agarose gel. The desired band was excised and gel-purified. The PCR product was then digested using XhoI and NotI, and ligated into the expression vector pWSK29 (31), which had been similarly digested. The ligation mixture was eletroporated into E. coli DY330 (32), and colonies were selected for those with the appropriate inserts on LB plates containing 100 μg/ml ampicillin. Plasmid pWSK29-5000F was isolated in this manner, and the insert was confirmed by DNA sequencing.

Next, the F. novicida arnT orthologue (30) in the plasmid pWSK29-5000F was replaced with a kanamycin resistance gene. Tn903 from plasmid pWKS130 was amplified by PCR. It was flanked on its 5′ and 3′ ends by 50 bp of the chromosomal DNA that is immediately upstream or downstream of F. novicida arnT. The forward and reverse primers are 5′-TAATTCCCGCTAAATTTATTCCATACTTTTGCCAGATAAATACTACTATCATGAGCCATATTCAACGGGAAACG-3′ and 5′-TAATCTAAAGATCATAAAAAAATAGTTAG-TTCTGTTTATTTTTAAATCAGTTAGAAAAACT-CATCAAGCATCAAATG-3′, respectively. The sequences belonging to the kanamycin resistance cassette are underlined. The PCR was performed using Pfu polymerase. Amplification was carried out in a 100 μl reaction mixture containing 100 ng of template, 250 ng primers and 2 units of Pfu polymerase. The reaction was started at 94 °C for 1 min, followed by 25 cycles of denaturation (45 seconds at 94 °C), annealing (45 seconds at 55 °C), and extension (60 seconds at 72 °C). After the 25th cycle, a 10 min extension time was used. The PCR product was resolved on a 1.0% agarose gel and purified. The purified PCR product was electroporated into E. coli DY330/pWSK29-5000F, which had been grown at 30 °C until A₆₀₀ reached 0.5 and then induced at 42 °C for 20 min. After electroporation, recombination occurred between the homologous sequences on the linear PCR product and the arnT gene on the plasmid, resulting in replacement of the arnT orthologue with the kanamycin resistance gene. The desired plasmid, designated pWSK29-5000K, was then isolated from kanamycin-resistant transformants, selected on LB agar containing 20 μg/ml kanamycin and 100 μg/ml ampicillin, and digested with ApaI and SacII. About 100 ng of the linear insert DNA was gel-purified, and the replacement of F. novicida arnT with the kanamycin resistance gene was verified by DNA sequencing.

The final step was to replace the arnT orthologue present in the genome of F. novicida with the kanamycin resistance gene. F. novicida U112 cells were grown in 100 ml of Chamberlain’s medium (33) to A₆₀₀ of 0.5. Cells were harvested by centrifugation and resuspended in 5 ml of transformation buffer (34). Next, 1 ml of the cell suspension was mixed with 100 ng of the linear insert DNA, prepared as described above, and shaken at 100 rpm at 37 °C for 30 min. Then 5 ml of Chamberlain’s medium was added, and the mixture was shaken at 37 °C for 2 hrs at 250 rpm. The cells were harvested and resuspended in 1 ml of TSB-C medium (3% trypticase soy broth and 0.1% cysteine). Approximately 100 μl of the cell suspension was spread onto a TSB-C plate containing 10 μg/ml kanamycin. Genomic DNA was isolated from a kanamycin-resistant transformant, and the replacement of the F. novicida arnT gene with the kanamycin resistance gene was confirmed by DNA sequencing.

Isolation of Phospholipids and Lipid A from ³²P Labeled Cells

F. novicida was grown in TSB-C medium, and E. coli was grown in LB broth (1% tryptone, 0.5% yeast extract and 1% NaCl). Typically, 20 ml cells, inoculated from an overnight culture to A₆₀₀ = 0.02, were grown in the presence of 5 μCi/ml ³²P_i to A₆₀₀ of 1.2. The cells were collected by centrifugation and washed with phosphate-buffered saline (35). The cell pellets were resuspended in 3 ml of a single-phase Bligh-Dyer mixture (26) consisting of chloroform, methanol and water (1:2:0.8, v/v/v), incubated at room temperature for 60 min, and centrifuged to remove insoluble debris. The supernatant (containing mostly glycerophospholipids and the free lipid A) was removed and converted to a two-phase Bligh-Dyer system (26) by adding chloroform and water to generate a mixture consisting of chloroform, methanol and water (2:2:1.8, v/v/v). The insoluble debris pellets from the initial extractions (containing intact LPS, proteins and nucleic acids) were resuspended in 3 ml of 12.5 mM sodium acetate, pH 4.5, with 1% SDS, heated at 100 °C for 30 min (36), cooled to room temperature and then converted to a two-phase Bligh-Dyer system by the addition of chloroform and methanol.

The solvent phases from both sets of extractions were individually mixed and separated by low-speed centrifugation. The upper phases were washed once with a fresh pre-equilibrated Bligh-Dyer lower phases. The appropriate lower phases were pooled and dried under a stream of nitrogen. The ³²P-labeled lipids were re-dissolved in chloroform and methanol (4:1, v/v) and spotted onto a Silica Gel 60 TLC plate (10, 000 cpm per lane), which was developed with chloroform, pyridine, 88% formic acid and water (50:50:16:5, v/v/v/v). After drying, the plates were exposed to a PhosphorImager Screen overnight, and the ³²P-labeled lipids were detected with a Molecular Dynamics Storm PhosphorImager.

Large Scale Purification of Free Lipid A from F. novicida U112

Two liters of F. novicida U112 cells were grown at 37 °C in TSB-C, harvested by centrifugation, and washed with phosphate-buffered saline. About 10 g wet cells were obtained. The cells were extracted for 1 hr at room temperature with 1.8 l of a single-phase Bligh-Dyer mixture and centrifuged to remove insoluble debris. The initial supernatant (containing most of the glycerophospholipids and free lipid A) was converted to a two-phase Bligh-Dyer system. The debris pellet (containing LPS) was washed once with a single-phase Bligh-Dyer mixture and then resuspended in 360 ml of 12.5 mM sodium acetate, pH 4.5, containing 1% SDS (36). The suspension was heated in a boiling water bath for 30 min to release covalently linked lipid A from intact LPS (36). The cooled suspension was converted to a two-phase Bligh-Dyer mixture. The lower phases from the initial supernatant and the hydrolyzed LPS were dried separately by rotary evaporation. About 260 mg lipids were obtained from the initial supernatant, and 4 mg of lipids were obtained from the hydrolyzed debris pellets.

The dried lipids from the initial supernatant and the debris pellets were dissolved in chloroform, methanol and water (2:3:1, v/v/v). Each sample was applied to a 4 ml DEAE-cellulose column in the acetate form equilibrated with the same solvent (36, 37). The run-through was saved. Each column was washed with 10 column volumes of chloroform, methanol and water (2:3:1, v/v/v). The various lipid components were then eluted stepwise with 5 column volumes each of chloroform, methanol and ammonium acetate (2:3:1, v/v/v) with ammonium acetate concentrations of 60 mM, 120 mM, 240 mM, 360 mM and 480 mM (36). Fractions equal to one column volume were collected, and 20 μl of each fraction was spotted onto a TLC plate to monitor the lipid A and phospholipid elution profile. The plates were developed in the solvent of chloroform, methanol, acetic acid and water (25:15:4:4 v/v/v/v), and the lipids were visualized by spraying the plates with 10% sulfuric acid in ethanol, followed by charring. All F. novicida lipid A species were found to emerge in the DEAE cellulose run-through together with phosphatidylethanolamine and phosphatidylcholine, as judged by TLC and ESI/MS. The run-through fractions were converted to two-phase Bligh-Dyer mixtures. The lower phases were recovered and dried by rotary evaporation. At this stage, 100 mg lipids were recovered from the initial supernatant, and 2 mg were obtained from the hydrolyzed debris pellets.

To purify the lipid A species further, preparative thin layer chromatography was employed. Lipids obtained from the run-through fractions of the DEAE column were dissolved in chloroform, methanol (4:1,v/v) and applied to a TLC plate, which was developed with chloroform, methanol, pyridine, acetic acid, water (25:10:5:4:3, v/v/v/v/v). While the plates were drying, the lipid A bands could be seen transiently as white zones. These bands were marked with a pencil and scraped off after the plates were dry. The silica chips were extracted with a single-phase Bligh-Dyer mixture for 1 hour at room temperature. The suspension was centrifuged. The supernatant was passed through a small column fitted with a small glass wool plug and converted into a two-phase Bligh-Dyer system. The two phases were separated by centrifugation, and the lower phase was dried. About 9.8 mg of lipid A component A1 and 1.4 mg of component A2 were purified from the initial supernatant, but only ∼ 0.1 mg of A1 was recovered from the hydrolyzed debris pellets.

Lipid A species from the arnT knockout mutant were isolated by the same procedure described above, except that the cells were grown at 30 °C rather than 37 °C, and the lipid A was retained by the DEAE cellulose column, given the loss of the galactosamine substituent in the mutant.

To remove the ester-linked 3-hydroxyacyl chains of A1 and A2, the purified compounds were treated with 10% aqueous triethylamine for 4 hours at room temperature. The partially deacylated lipids, designated HA1 and HA2, were extracted from the reaction mixture by the Bligh-Dyer method and purified by preparative thin layer chromatography.

MALDI/TOF MS

Matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF) mass spectra were acquired using an AXIMA-CFR from Kratos Analytical (Manchester, UK), equipped with a nitrogen laser (337 nm), 20 kV extraction voltage, and time delayed extraction. The samples were prepared for MALDI/TOF MS analysis by depositing 0.3 μl of the lipid sample dissolved in chloroform and methanol (4:1, v/v), followed by adding 0.3 μl of a saturated solution of 6-aza-2-thiothymine in 50% acetonitrile and 10% tribasic ammonium citrate (9:1, v/v) as the matrix. The samples were left to dry at room temperature before the mass spectra were acquired in linear mode. Each spectrum was the average of 100 laser shots.

Electrospray Ionization (ESI) MS and MS/MS Analysis

All MS/MS spectra were acquired on a QSTAR XL quadrupole time-of-flight tandem mass spectrometer (ABI/MDS-Sciex, Toronto, Canada) equipped with an ESI source. Lipid A samples were dissolved in chloroform and methanol (2:1, v/v) at about 25 μg/ml and subjected to ESI/MS in the negative ion mode (14). Nitrogen was used as collision gas for MS/MS experiments (14). Data acquisition and analysis were performed using the instrument’s Analyst QS software.

GC/MS Analysis

The purified lipid A species of F. novicida were hydrolyzed in acidic methanol, N-acetylated, and then converted to trimethylsilyl ethers. Mannose, galactose, glucosamine, galactosamine and glucose standards were processed in parallel with the lipid A samples. Typically, 0.5-1.0 mg sample was thoroughly dried in a Reacti-vial equipped with a Teflon-lined screw cap. Samples were hydrolyzed by adding 300 μl of 1 M HCl in methanol and heated at 80 °C for 15 hours. The reaction mixtures were then cooled, and the solvents were removed under a stream of nitrogen. Next, 200 μl of anhydrous methanol, 40 μl of pyridine, and 40 μl of acetic anhydride were added to the vial. The reaction mixtures were mixed and incubated overnight at room temperature. The solvents were evaporated under a stream of nitrogen. Finally, silylation of free OH groups was achieved by adding 200 μl of Tri-Sil reagent to the dried samples, mixing and incubating at room temperature for an hour. The samples were dried under a gentle stream of nitrogen, and re-dissolved in 100 μl of hexane and transferred to new vials for GC/MS analysis.

GC/MS was performed using a Finnigan Trace MS coupled with a Trace GC 2000 gas chromatography. The column is a 30 m RTX-5MS (0.25 mm internal diameter and 0.25 mm phase thickness) from Restek (Bellefonte, PA). The column oven temperature was held at 100 °C for 3 min, increased to 325 °C at a rate of 20 °C/min, and then held at 325 °C for 3 min. The injector was operated in the split mode (1:20 split), and the temperature of the injection port was kept at 200 °C. Helium was the carrier gas with a constant flow rate of 1 ml/min. The instrument was operated in the electron ionization mode with the electron energy set at 70 eV.

NMR spectroscopy

Typically, 1-2 mg samples of F. novicida lipid A were dissolved in 0.3 ml of CDCl₃, CD₃OD and D₂O (2:3:1, v/v/v) in a 3 mm NMR tube. Proton and carbon chemical shifts are reported relative to internal tetramethylsilane (TMS) at 0.00 ppm. The ²H signal of CD₃OD was used as a field frequency lock with the residual signal of CD₃OD serving as the secondary reference at 49.5 ppm for carbon spectra. ¹H NMR spectra were recorded at the Duke NMR Center on Varian Inova 800 or 600 NMR spectrometers, equipped with Varian cryogenic probes. ¹H NMR spectra at 800 MHz were obtained with a 7.2 kHz spectral window, a 67° pulse field angle (4.5 μsec), a 4.5 sec acquisition time, and a 1 sec relaxation delay. The spectra were digitized using 64k points to obtain a digital resolution of 0.225 Hz/pt. Two-dimensional NMR experiments (COSY, NOESY, TOCSY, HMQC and HMBC) were performed at 800 MHz as previously described (38-40). Zero-quantum correlation (ZQCOSY) 2D NMR was coded into Varian NMR software based on Mueller’s sequence (41). ¹H-decoupled ³¹P NMR spectra were recorded at 202.3 MHz on a Varian Inova 500 spectrometer with a spectral window of 12143.3 Hz digitized into 25,280 data points (digital resolution of 1Hz/point or ∼0.005 ppm/point), a 60° pulse flip angle (8 μs), and a 1.6-s repeat time. ³¹P chemical shifts were referenced to 85% H₃PO₄ at 0.00 ppm. Inverse decoupled difference spectra were recorded as ¹H-detected ³¹P-decoupled heteronuclear NMR experiments, as previously described (38, 40).

Free Lipid A Among the Phospholipids of F. novicida

F. novicida U112 and E. coli W3110 were labeled uniformly with ³²P_i until late log phase. Lipids were extracted directly from intact cells by the method of Bligh and Dyer, or after pH 4.5 hydrolysis at 100 °C of the intact LPS present in the Bligh-Dyer insoluble debris pellet, as previously described (36). Radioactive lipids were quantified by TLC and PhosphorImager analysis. E. coli phospholipids were recovered by the direct extraction (Fig. 2, lane 1), whereas E. coli lipid A was released from the insoluble debris pellet only after hydrolysis at pH 4.5 (36) (data not shown). Hydrolyzing the F. novicida debris pellet at pH 4.5 yielded very little lipid A (about 100 times less than from E. coli). However, several unknown radioactive substances were recovered together with the glycerophospholipids by direct Bligh-Dyer extraction of F. novicida cells (Fig. 2, lane 2). The rapidly-migrating lipids (Fig. 2, lane 2) are phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and phosphatidylcholine, as judged by ESI/MS/MS (not shown). The slowly-migrating unknowns, designated compounds A1 and A2 (Fig. 2, lane 2), were purified on a larger scale and confirmed as lipid A derivatives by MS and NMR spectroscopy (see below). A1 and A2 make up 15 - 20% of the total ³²P-labeled lipids recovered by direct extraction of intact F. novicida cells, as judged by PhosphorImager analysis. This is about the same ratio of lipid A to glycerophospholipids as is found in E. coli, except that E. coli lipid A must first be released from LPS by mild acid hydrolysis (11, 36). The fact that over 95 % of A1 and A2 is extracted directly from F. novicida with a single-phase Bligh-Dyer mixture demonstrates that F. novicida lipid A is not covalently linked to LPS. The amount of intact LPS present in F. novicida is low compared to E. coli, where it represents about 3.4 % of the dry weight (23). As in F. novicida, most of the lipid A in the F. tularensis LVS strain is in the free form (data not shown).

To purify larger amounts of A1 and A2, 2 l of F. novicida U112 were grown to late log phase. Lipids were extracted from intact cells with a single-phase Bligh-Dyer mixture or after pH 4.5 hydrolysis of the extracted, insoluble debris pellet. Portions of the crude lipids were separated on a TLC plate, which was sprayed with 10% sulfuric acid and charred on a hot plate. Consistent with the ³²P-labeling, almost all the lipids were recovered by direct extraction of the intact cells, and only a small amount was recovered after hydrolysis of the debris pellet. The pattern of lipids on the charred plate (not shown) was virtually the same as that determined by ³²P-labeling (Fig. 2, lane 2), showing that F. novicida does not synthesize lipid A species lacking phosphate groups, as is the case in Rhizobium etli and Rhizobium leguminosarum (39, 42, 43). Only compound A1 was observed amongst the lipids released by pH 4.5 hydrolysis of the debris pellet, indicating A1 as the sole hydrophobic anchor for the intact LPS in F. novicida cells.

The crude lipids extracted directly from the intact cells were subjected to anion exchange chromatography on a DEAE-cellulose column (36). A1 and A2 eluted together with phosphatidylethanolamine, lyso-phosphatidylethanolamine and phosphatidylcholine in the run-through, indicating that they possess no net negative charge. Compounds A1 and A2 were then further purified by preparative TLC (Fig. 3A). From 2 l of cells, about 9.8 mg of A1 and 1.4 mg of A2 were recovered by direct extraction, but only ∼ 0.1 mg of A1 was released from the Bligh-Dyer insoluble debris pellet by hydrolysis at pH 4.5.

Negative Ion MALDI/TOF MS of Compounds A1 and A2

A1 of F. novicida was analyzed by MALDI/TOF MS in the negative ion mode (Fig. 3B). The peaks at m/z 1665.4 and 1637.4 are interpreted as [M-H]^- molecular ions, which differ by 28 amu, i.e. two methylene units. The ion at m/z 1504.4 may be derived from the base peak ion at m/z 1665.4 by loss of a 161 amu moiety, possibly a hexosamine unit (22). The m/z 1383.3 ion is derived from the m/z 1665.4 ion by loss of a 3-hydroxystearoyl group.

The negative ion MALDI/TOF mass spectrum of A2 (Fig. 3C) shows a similar pattern as A1. The peaks at m/z 1827.5 and 1799.5 could be interpreted as molecular ions [M-H]^-, differing by 28 mass units. The peaks at m/z 1666.4 and 1545.7 might be derived from these parent ions by loss of a hexosamine or a 3-hydroxystearoyl moiety, respectively. All the peaks in the spectrum of A2 are 162 amu larger than the corresponding peaks of A1, suggesting that A2 contains an additional 162 amu substituent, possibly a hexose residue.

GC/MS Analysis of Compounds A1 and A2

The labile hexosamine unit, observed in the MALDI/TOF mass spectra of A1 and A2, could be attached to the 1-phosphate group, as proposed for lipid A of F. tularensis 1547-57 (Fig. 1D). To test this idea, A1 was hydrolyzed in 0.1 M HCl at 100 °C for 30 min. A hexosamine moiety attached to the 1-phosphate group of a lipid A molecule would be cleaved under these conditions. The hydrolysis mixture was then converted into a two-phase Bligh/Dyer system by the addition of chloroform and methanol. The phases were separated and dried. The materials in the dried upper and lower phases were hydrolyzed in acidic methanol, N-acetylated, converted to trimethylsilyl ethers and analyzed by GC/MS. Glucose, mannose, galactose, glucosamine and galactosamine standards were processed in parallel. The products recovered from the upper phase of the A1 hydrolysis mixture yielded peaks at 11.46 and 11.52 min, as did a galactosamine standard, confirming that the labile hexosamine unit of A1 is indeed galactosamine (data not shown). The products recovered from the lower phase yielded peaks at 10.59, 11.52 and 12.40 min (data not shown); their electron impact mass spectra were searched against the National Institute of Standards and Technology (NIST) library of mass spectra and identified as derivatives of palmitate, 3-hydroxypalmitate and 3-hydroxystearate, respectively. The presence of both 3-hydroxypalmitate and 3-hydroxystearate is consistent with the fatty acid chain length heterogeneity seen in the negative ion MALDI/TOF mass spectrum of A1 (Fig. 3B).

To characterize the additional hexose moiety present in A2, intact A1 and A2 were hydrolyzed in acidic methanol, N-acetylated, and converted to trimethylsilyl ethers, followed by GC/MS analysis (Fig. 4). Standards of glucose, mannose, galactose, glucosamine and galactosamine were processed in parallel. Peaks at 11.46 and 11.52 min were observed in the profiles of both A1 and A2, confirming that they both contain galactosamine. Peaks at 10.59, 11.52 and 12.40 min were observed with both A1 and A2 (Fig. 4A and B), the electron impact mass spectra of which matched with derivatives of palmitate, 3-hydroxypalmitate and 3-hydroxystearate, respectively. However, the peaks seen at 10.46 and 10.53 min in A2 (Fig. 4B) were not present in A1 (Fig. 4A), suggesting that they were derived from the additional hexose unit present in A2. Identical peaks at 10.46 and 10.53 min were seen with the glucose standard (Fig. 4C), but not with the other standards, showing that the additional hexose unit in A2 must be glucose.

ESI/MS/MS of Compounds A1 and A2

The molecular ions [M-H]^- of A1 and A2 (at m/z 1665.18 and 1827.23, respectively) were further analyzed by high resolution ESI/MS/MS (Fig. 5). The peaks at m/z 78.96 [PO₃]^- and 96.97 [H₂PO₄]^- confirm the presence of a phosphate group. The prominent peak at m/z 258.04, observed in both spectra (Fig. 5), has the ion mass expected for a phosphorylated hexosamine, suggesting the galactosamine moiety is connected to the phosphate group (Fig. 1D). Fragment ions derived from the parent ions by neutral loss of palmitic acid were observed at m/z 1408.96 for A1 and at m/z 1570.98 for A2 (Fig. 5), respectively. Fragment ions derived by neutral loss of both hydroxystearic acid and the hexosamine unit were observed at m/z 1203.83 for A1 and m/z 1365.89 for A2, and fragment ions arising from neutral loss of palmitic acid, hydroxystearic acid, and the hexosamine unit were observed at m/z 947.60 for A1 and m/z 1109.65 for A2. The further cleavage of an N-linked hydroxyacyl chain could generate the fragment ions at m/z 665.34 for A1 and m/z 827.40 for A2. Taken together with the GC/MS data (Fig. 4), the ESI/MS/MS spectra show that A1 and A2 are very similar in structure, both containing phosphate, galactosamine, palmitate and hydroxystearate residues. The phosphate and galactosamine units are connected to each other, as demonstrated by the peak at m/z 258.34.

Mild Alkaline Hydrolysis of A1 and A2

The ester-linked 3-hydroxyacyl chains of lipid A are very susceptible to mild alkaline hydrolysis (44). To determine the locations of the ester-linked fatty acids, A1 and A2 were treated with 10% aqueous triethylamine for 4 h at 25 °C. The products, HA1 and HA2, were purified and analyzed by negative ion MALDI/TOF MS. The molecular ions of HA1 and HA2 are each 282 amu smaller than those of A1 and A2, respectively (not shown), demonstrating that only one base-labile 3-hydroxystearoyl moiety is present in each compound. When analyzed by TLC in chloroform, pyridine, 88% formic acid and water (50:50:16:5, v/v/v/v), HA1 and HA2 migrate with Rfs that are similar to those of A1 and A2, respectively. These findings suggest that the base-labile 3-hydroxystearoyl moiety is attached to proximal glucosamine 3-position in both A1 and A2 (Fig. 1), as deacylation of the 3′-position usually causes a larger shift in the Rf than deacylation of the 3-position (44).

Positive Ion MALDI/TOF MS

Compounds A1, HA1, A2 and HA2 were analyzed by positive ion MALDI/TOF MS (Fig. 6). Parent ions, interpreted as [M+H]⁺, were observed at m/z 1667.0 for A1, m/z 1384.6 for HA1, m/z 1829.0 for A2 and m/z 1547.7 for HA2 (Fig. 6). Variable amounts of sodium adducts [M+Na]⁺ were also detected (not labeled in Fig. 6). A prominent B₁⁺ ion peak (45) near m/z 683, derived from the distal glucosamine unit, was present in the spectra of both A1 and HA1 (Fig. 6A and 6B), confirming that the ester-linked 3-hydroxystearoyl moiety is attached to the proximal glucosamine unit at position 3 in A1. Prominent B₁⁺ ions near m/z 845.0 were observed for both A2 and HA2 (Fig 6C and 6D), demonstrating that the base-labile 3-hydroxystearoyl acid moiety is connected to the 3-position in A2 as in A1. However, the difference in the masses of the B₁⁺ ions of A1 versus A2 or HA1 versus HA2 is 162 amu, showing that the additional glucose residue present in A2 is linked to the distal glucosamine unit.

Neutral loss of the galactosamine moiety from the 1-phosphate group of each of the four compounds gave rise to product ions mainly as sodium adducts at m/z 1527.8 for A1, m/z 1245.5 for HA1, m/z 1689.9 for A2, m/z 1408.7 for HA2 (Fig. 6). The B₂⁺ ions (45), which are derived from the parent ions by loss of the galactosamine 1-phosphate group, were observed at m/z 1407.8 for A1, m/z 1125.5 for HA1, m/z 1569.9 for A2, and m/z 1287.7 for HA2. The peaks at m/z 1125.4 in the A1 spectrum and at m/z 1287.5 in the A2 spectrum could arise by loss of the labile, ester-linked hydroxystearoyl moiety from the B₂⁺ ion of each compound. As in the negative ion spectra (Fig. 4), some fatty acid chain length heterogeneity was observed in the positive ion spectra, though less on the distal unit, as judged by the masses of the B₁⁺ ions (Fig. 6). The proposed structures of A1, HA1, A2 and HA2, which are based on the MS analysis together with the NMR data discussed below, are shown in Fig. 7.

¹H and ³¹P NMR Analysis of Compounds A1, A2, HA1 and HA2

The 800 MHz ¹H NMR spectra of compounds HA1, HA2, A1 and A2 dissolved in CDCl₃, CD₃OD and D₂O (2:3:1, v/v/v) reveal relatively well-resolved resonances in the sugar (3.2-5.8 ppm) (Fig. 8) and acyl chain regions (0.8-2.7 ppm) (shown for HA1 in Fig. 9), similar to previous NMR spectra for E. coli and R. leguminosarum lipid A in the same solvent (38-40). The numbering scheme for the relevant protons is shown in Fig. 7D. The full assignments of the NMR chemical shifts and coupling constants for HA1 and HA2 (Table 1) were made from 2D COSY, ZQCOSY, TOCSY, HMQC and HMBC spectra.

³¹P NMR spectroscopy at 202 MHz of HA1, HA2, A1 and A2 revealed a single ³¹P resonance around -2 ppm (not shown), indicating that each contains a single phosphorus atom. ¹H-observed, ³¹P-decoupled experiments (38, 40) were implemented to identify the linkage site of the phosphate group on the glucosamine disaccharide backbone. The difference ¹H NMR spectrum derived from selective on- and off-resonance decoupling of the -2.18 ppm ³¹P signal of HA1 revealed the simultaneous simplification of the H-1 and H-1⁰ signals (at 5.46 and 5.72 ppm respectively) into two doublets, and the H-2 and H-2⁰ resonances (at 3.90 and 3.49 ppm respectively) into two double-doublets (Fig. 8A). This indicates that the single phosphate group, present in HA1, bridges the proximal glucosamine and the capping galactosamine residue, consistent with the fragments ions seen by MS. Difference ¹H NMR spectra confirmed that the single phosphate moiety present in HA2, A1 and A2 likewise connected the proximal glucosamine and capping galactosamine residue (not shown).

The 1D ¹H NMR spectra of HA1 (Fig. 8B) and A1 (Fig. 8C) reveal three resolved anomeric resonances, consistent with the presence of three sugars. However, the NMR spectra of HA2 and A2 (Fig. 8D and E, respectively) show four resolved anomeric signals, consistent with the presence of the additional glucose moiety in A2 and HA2. The α CH₂ signals of HA1 (Fig. 9) and HA2 (not shown) integrate to six protons, while the α CH₂ resonances of A1 and A2 integrate to eight protons (not shown). The integration is consistent with the presence of three acyl chains in HA1 and HA2, and four acyl chains in A1 and A2, respectively (Fig. 7). All four lipids show one esterified β-hydroxymethine proton near 5.2 ppm (Fig. 8B, 8C, 8D and 8E), consistent with the presence of a single acyloxyacyl group (Fig. 7) (38, 39). The downfield shift of the H-3 signal to near 5.16 ppm in A1 and A2 (Fig. 8C and 8E) confirms the presence of an acyl chain at position 3 in these compounds (Fig. 7).

¹H-¹H COSY and NOESY Analysis

The anomeric 5.46 ppm double-doublet (H-1) and the 4.47 ppm doublet (H-1′) of compound HA1 are at similar shift positions to those of E. coli lipid A (38, 40). The low field position and double-doublet character of H-1 are typical of an α anomeric proton in a pyranose 1-phosphate (38-40). The anomeric double-doublet at 5.72 ppm (designated H-1⁰) (Figs. 8 and 9) is not seen in E. coli lipid A (38). H-1⁰ connects to six other sugar resonances (Table 1 and Fig. 9), consistent with the presence of a galactosamine residue. The low field position and double-doublet character of H-1⁰ indicates that the 1⁰-position is phosphorylated (Figs. 7-9). H-4′ of compound HA1 (3.33 ppm) (Table 1) resonates 0.8 ppm upfield of H-4′ of E. coli lipid A (4.17 ppm) (38), consistent with the proposal that the 4′-position of compound HA1 is not phosphorylated (Fig. 7). The presence of O-acyl chains of positions 3 and 3′ in E. coli lipid A (Fig. 1A) shifts the corresponding H-3 and H-3′ signals downfield to ∼5.2 ppm (38, 40). In contrast, the H-3 and H-3′ signals of compound HA1 appear in the sugar region at 3.72 and 3.46 ppm (Table 1), respectively, indicating that there is no O-acyl substituent at positions 3 and 3′ of HA1. This pattern is very similar in HA2 (Table 2).

The small J_1,2, J₁0_,20 and J_1”,2” couplings (3.2 - 3.7 Hz) (Table 1) provide further evidence that the proximal glucosamine unit, the galactosamine residue and the glucose residue of HA2 (Fig. 7) are in the α-anomeric configuration. Likewise, the proximal glucosamine and galactosamine units of HA1 are α-anomers. The large J_1′,2′ coupling (8.6 Hz) (Table 1) shows that the distal glucosamine unit of both HA1 and HA2 is in the β-configuration (Fig. 7). The NOE enhancements from H-1′ to H-6a and H-6b (and from H-1” to H6a′ and H6b′) in the NOESY spectrum of HA2 (Supplementary Fig. 1) provide independent evidence for the proposed 1′-6 and 1”-6′ glycosidic linkages in HA2 (Fig. 7D). The NOE enhancement from H-1′ to H6a and H6b is also seen with HA1 (data not shown). The NOE enhancement from H-1 to H-1⁰ in both HA2 (Supplementary Fig. 1) and HA1 (not shown) confirms the proposed monophosphodiester linkage between the proximal glucosamine and the capping galactosamine residues (Fig. 7C and D).

Two sets of α/β cross-peak pairs are seen in the COSY (Fig. 9) and TOCSY (not shown) spectra of F. novicida compound HA1, indicating the presence of only two β-hydroxyacyl chains (Fig. 7C). The cross-peak pairs near 2.3 ppm and 4.0 ppm (designated α2/β2) arise from the α-protons adjacent to the β-oxymethine of the unsubstituted β-hydroxyacyl chain. The other α/β cross-peak pairs near 2.5 ppm and 5.2 ppm (designated α2′/β2′) are considerably more downfield, consistent with a 2′ acyloxyacyl moiety in HA1 (Fig. 7C). Our results are consistent with the acylation patterns deduced from previous NMR studies of the lipid A obtained from LPS of the LVS strain of F. tularensis (18).

¹³C-NMR Analysis of HA1 and HA2

The HMQC spectra of HA1 and HA2 (Fig. 10) reveal the following key features: 1) The HMQC analysis confirms the presence of three anomeric protons in HA1 and four anomeric protons in HA2 (Fig. 10A and 10B). The C-1⁰, C-1 and C-1′ chemical shift positions support the proposed α-, α- and β-configurations respectively for the capping galactosamine, proximal glucosamine and distal glucosamine units in both HA1 and HA2 (46). 2) Nitrogen-substituted carbons of amino sugars usually resonate around 52 to 54 ppm (46). The HMQC spectra of compounds HA1 and HA2 (Fig. 10C and 10D) reveal three methine resonances at 54.5 ppm (C-2), 56.0 ppm (C-2′) and 52.4 ppm (C-2⁰), indicating that HA1 and HA2 contain three amino sugars (Fig. 7C and D). 3) The HMQC of HA1 (Fig. 10C) confirms the presence of a glycosylated C-6 (at 72 ppm) and a nonglycosylated C-6′ and C-6⁰ (signals at 61.3 and 62 ppm, respectively) (46-48). However, in the HMQC of HA2 (Fig. 10D), C6′ shifts from a nonglycosylated position to a glycosylated position at 66.7 ppm, in agreement with the NOESY data (Supplementary Fig. 1), which likewise indicates that the glucose moiety present in HA2 is linked to the 6′-position.

HA1 and HA2 gave excellent HMBC responses in both the sugar and acyl chain regions (Supplementary Fig. 2). Strong cross peaks from H-1” to C-6′ and from H-6a′ to C-1” were observed in the HMBC of compound HA2 (Supplementary Fig. 2B), thus confirming the attachment of the glucose unit to the 6′ position of HA2 (Fig. 7D). Linkage of a glucose residue to position 6′ is without precedent in lipid A biochemistry, as Kdo is normally attached at this important site.

Origin of the Galactosamine Group in Francisella Lipid A

d-galactosamine is similar in structure and charge to 4-amino-4-deoxy-l-arabinose (Fig. 1D). The latter is synthesized and attached to lipid A in polymyxin-resistant mutants of E. coli and Salmonella (49, 50). The inner membrane enzyme ArnT transfers the 4-amino-4-deoxy-l-arabinose residue from the donor substrate undecaprenyl phosphate-4-amino-4-deoxy-l-arabinose to the 1 and/or 4′ phosphate groups of lipid A (27, 28). We reasoned that a membrane protein with sequence similarity to ArnT might be responsible for the attachment of the galactosamine residue to lipid A in F. novicida. A deletion mutant of the single arnT orthologue present in F. novicida (30) synthesizes lipid A molecules lacking the galactosamine moiety found in the wild-type, as judged by the loss of the 161 amu substituent (Fig. 11A versus 11B). Loss of the galactosamine modification causes the cells to grow more slowly above 30 °C and is associated with a shift to shorter fatty acyl chains on lipid A (more of the molecular species at m/z 1476.014 versus m/z 1504.045) (Fig. 11B). Interestingly, the structure of lipid A present in this arnT deletion mutant of F. novicida is nearly identical to that reported by Phillips et al. for the LVS strain of F. tularensis (Fig. 1C) (22).