A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element
2007; Elsevier BV; Volume: 48; Issue: 11 Linguagem: Inglês
10.1194/jlr.m700193-jlr200
ISSN1539-7262
AutoresAlina Tîrşoaga, Asmaa El Hamidi, Malcolm B. Perry, Martine Caroff, А. В. Новиков,
Tópico(s)Salmonella and Campylobacter epidemiology
ResumoEndotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system. Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system. hexadecanoic acid dodecanoic acid tetradecanoic acid hydroxytetradecanoic acid glucosamine hydrofluoric acid lipopolysaccharide Endotoxins are lipopolysaccharides (LPSs), major components of the external membrane of Gram-negative bacteria. They may cause several pathophysiological symptoms, such as fever, septic shock, and death, but they are also able to elicit beneficial activities, such as the production of tumor necrosis factor, adjuvant, and radioprotection effects (1.Raetz C.R. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3285) Google Scholar, 2.Caroff M. Karibian D. Structure of bacterial lipopolysaccharides.Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (369) Google Scholar).LPS molecular architecture has three regions: a hydrophobic moiety, called lipid A, a core oligosaccharide, and a serospecific O-polysaccharide composed of repeating oligosaccharide units. Lipid A is embedded in the external bacterial membrane together with phospholipids and proteins. It is responsible for the major toxic and beneficial properties characteristic of bacterial endotoxins (2.Caroff M. Karibian D. Structure of bacterial lipopolysaccharides.Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (369) Google Scholar, 3.Alexander C. Rietschel E.T. Bacterial lipopolysaccharides and innate immunity.J. Endotoxin Res. 2001; 7: 167-202PubMed Google Scholar).Lipid A structure generally consists of a diglucosamine backbone substituted with varying numbers (usually four to seven) of ester- or amide-linked fatty acids. In most cases, phosphates, with and without other substituents, are linked to carbons at the C-1 and C-4′ positions of the lipid A disaccharide unit (2.Caroff M. Karibian D. Structure of bacterial lipopolysaccharides.Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (369) Google Scholar, 3.Alexander C. Rietschel E.T. Bacterial lipopolysaccharides and innate immunity.J. Endotoxin Res. 2001; 7: 167-202PubMed Google Scholar, 4.Trent M.S. Biosynthesis, transport, and modification of lipid A.Biochem. Cell Biol. 2004; 82: 71-86Crossref PubMed Scopus (82) Google Scholar). These and the number and chain lengths of fatty acids are highly important for the toxic effects of lipids A (5.Zahringer U. Salvetzki R. Wagner F. Lindner B. Ulmer A.J. Structural and biological characterisation of a novel tetra-acyl lipid A from Escherichia coli F515 lipopolysaccharide acting as endotoxin antagonist in human monocytes.J. Endotoxin Res. 2001; 7: 133-146Crossref PubMed Google Scholar). The addition of a single fatty acid can be responsible for an increase or decrease in bacterial virulence (6.Preston A. Maxim E. Toland E. Pishko E.J. Harvill E.T. Caroff M. Maskell D.J. Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract.Mol. Microbiol. 2003; 48: 725-736Crossref PubMed Scopus (72) Google Scholar).Lipid A classical structural analysis is a rather long and complicated process that includes the following main stages: LPS extraction from the bacteria, LPS purification, LPS acid hydrolysis to split the molecule into its hydrophobic and hydrophilic moieties, and lipid A extraction. This is followed by its characterization by different methods: MS, TLC, and the identification and localization of fatty acids, phosphate groups, and other substituents, if any, on the glucosamine backbone.Endotoxins can be isolated from Gram-negative bacteria by a variety of different methods (2.Caroff M. Karibian D. Structure of bacterial lipopolysaccharides.Carbohydr. Res. 2003; 338: 2431-2447Crossref PubMed Scopus (369) Google Scholar). Long and strong hydrolytic conditions, which are occasionally required to cleave the lipid A-polysaccharide bond, result in partial dephosphorylation and O-deacylation of lipid A (7.Karibian D. Deprun C. Caroff M. Use of plasma desorption mass spectrometry in structural analysis of endotoxins: effects on lipid A of different acid treatments.Prog. Clin. Biol. Res. 1995; 392: 103-111PubMed Google Scholar). Such modifications strongly diminish the biological activities of the molecule. Milder hydrolysis conditions, such as pH 4.4–4.5 in sodium acetate buffer, were shown to be efficient for lipid A liberation (8.Rosner M.R. Tang J. Barzilay I. Khorana H.G. Structure of the lipopolysaccharide from an Escherichia coli heptose-less mutant. I. Chemical degradations and identification of products.J. Biol. Chem. 1979; 254: 5906-5917Abstract Full Text PDF PubMed Google Scholar) and were usually improved by the addition of SDS when the hydrolysis kinetics were too slow or ineffective (9.Caroff M. Tacken A. Szabo L. Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin.Carbohydr. Res. 1988; 175: 273-282Crossref PubMed Scopus (199) Google Scholar). These hydrolytic processes are the conventional first steps for lipid A analysis after LPS isolation from the bacteria. An interesting approach using SDS-promoted hydrolysis of intact bacteria has been reported (10.Zhou Z. Lin S. Cotter R.J. Raetz C.R. Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Other new conditions for a quick extraction of lipid A directly from bacterial cells were developed recently (11.El Hamidi A. Tirsoaga A. Novikov A. Hussein A. Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization.J. Lipid Res. 2005; 46: 1773-1778Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar).We demonstrated previously that, because of steric hindrance, fatty acid ester linkages could be differentially hydrolyzed by alkaline treatment. Sequential alkaline deesterification conditions in combination with mass spectrometry can reveal the substitution positions of primary ester-linked fatty acids on the glycose residues as well as secondary ester-linked positions on the hydroxyl groups of other fatty acids, called acyloxyacyl acids. In mild conditions, the acyloxyacyl ester could be released without splitting the secondary ester linkage between the two fatty acids and characterized by GC-MS. These conditions have been used to determine the structures of many lipid A preparations, such as those of Bordetella (12.Caroff M. Brisson J.R. Martin A. Karibian D. Structure of the Bordetella pertussis 1414 endotoxin.FEBS Lett. 2000; 477: 8-14Crossref PubMed Scopus (80) Google Scholar, 13.Zarrouk H. Karibian D. Bodie S. Perry M.B. Richards J.C. Caroff M. Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution.J. Bacteriol. 1997; 179: 3756-3760Crossref PubMed Scopus (35) Google Scholar, 14.Aussel L. Brisson J.R. Perry M.B. Caroff M. Structure of the lipid A of Bordetella hinzii ATCC 51730.Rapid Commun. Mass Spectrom. 2000; 14: 595-599Crossref PubMed Scopus (25) Google Scholar, 15.Caroff M. Aussel L. Zarrouk H. Martin A. Richards J.C. Therisod H. Perry M.B. Karibian D. Structural variability and originality of the Bordetella endotoxins.J. Endotoxin Res. 2001; 7: 63-68Crossref PubMed Scopus (53) Google Scholar), Helicobacter (16.Therisod H. Monteiro M.A. Perry M.B. Caroff M. Helicobacter mustelae lipid A structure differs from that of Helicobacter pylori.FEBS Lett. 2001; 499: 1-5Crossref PubMed Scopus (16) Google Scholar), and Yersinia (17.Aussel L. Therisod H. Karibian D. Perry M.B. Bruneteau M. Caroff M. Novel variation of lipid A structures in strains of different Yersinia species.FEBS Lett. 2000; 465: 87-92Crossref PubMed Scopus (51) Google Scholar). We also demonstrated the importance of such a sequential release in the case of Yersinia lipid A structure previously erroneously described as being identical to that of Escherichia coli. In this special case, two pairs of fatty acids substituted different glycose positions (C-2′ and C-3′), leading to the same total molecular weight but introducing different structures. Therefore, it is recommended to consider this point with any lipid A having a molecular mass similar to that of any other well-known structure. Our first demonstration of selective conditions for the release of ester-linked fatty acids in lipid A was done in a 1 day step-wise use of alkali reagents (17.Aussel L. Therisod H. Karibian D. Perry M.B. Bruneteau M. Caroff M. Novel variation of lipid A structures in strains of different Yersinia species.FEBS Lett. 2000; 465: 87-92Crossref PubMed Scopus (51) Google Scholar): 10–15 min in 0.2 M NaOH for primary esters, and 1 h in hydrazine at 37°C for the secondary ester linkages. The two-step de-O-acylation strategy was also used by others (18.Silipo A. Lanzetta R. Amoresano A. Parrilli M. Molinaro A. Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of lipid A fatty acid distribution.J. Lipid Res. 2002; 43: 2188-2195Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) on lipids A isolated from LPS by SDS-promoted mild hydrolysis (9.Caroff M. Tacken A. Szabo L. Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin.Carbohydr. Res. 1988; 175: 273-282Crossref PubMed Scopus (199) Google Scholar). Ammonium hydroxide is frequently used in de-O-acylation, but it requires hydrolyses too long for our purpose.When the use of hydrazine was restricted for security reasons, we established new conditions, described here, that are also better adapted to our small lipid A samples isolated directly from bacteria.Citrobacter belongs to the Enterobacteriaceae group, and 11 species are known at this time. For the present study, three species were selected. They are C. freundii and C. sedlakii, two human pathogens afflicting particularly neonates, the elderly, and immunocompromised patients, and C. rodentium, a strict pathogen for mice (19.Vinogradov E. Conlan J.W. Perry M.B. Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobacter freundii and Citrobacter sedlakii.FEMS Microbiol. Lett. 2000; 190: 157-161Crossref PubMed Google Scholar, 20.Chart H. Willshaw G.A. Cheasty T. Rowe B. Structure and antigenic properties of Citrobacter freundii lipopolysaccharides.J. Appl. Bacteriol. 1993; 74: 583-587PubMed Google Scholar, 21.Kocharova N.A. Knirel Y.A. Kholodkova E.V. Stanislavsky E.S. Structure of the O-specific polysaccharide chain of Citrobacter freundii O28,1c lipopolysaccharide.Carbohydr. Res. 1995; 279: 327-330Crossref PubMed Scopus (4) Google Scholar, 22.MacLean L.L. Perry M.B. Structural studies on the O-polysaccharide of the lipopolysaccharide produced by Citrobacter rodentium (ATCC 51459).Eur. J. Biochem. 2001; 268: 5740-5746Crossref PubMed Scopus (13) Google Scholar). Apart from the latter, all of the Citrobacter species are opportunistic pathogens, especially in nosocomial infections.The Bordetella genus contains nine species. The lipid A structures of seven of them have been described (15, our unpublished results). These structures were notable for their peculiarly high variability among species and even strains. In B. bronchiseptica, lipid A structural variability has been attributed to relaxed enzyme specificity (6.Preston A. Maxim E. Toland E. Pishko E.J. Harvill E.T. Caroff M. Maskell D.J. Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract.Mol. Microbiol. 2003; 48: 725-736Crossref PubMed Scopus (72) Google Scholar, 13.Zarrouk H. Karibian D. Bodie S. Perry M.B. Richards J.C. Caroff M. Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution.J. Bacteriol. 1997; 179: 3756-3760Crossref PubMed Scopus (35) Google Scholar, 15.Caroff M. Aussel L. Zarrouk H. Martin A. Richards J.C. Therisod H. Perry M.B. Karibian D. Structural variability and originality of the Bordetella endotoxins.J. Endotoxin Res. 2001; 7: 63-68Crossref PubMed Scopus (53) Google Scholar). The lipids A of two human pathogens, B. pertussis and B. parapertussis, have been associated with their hypoacylation and short-chain fatty acids causing reduced endotoxicity (23.Cavaillon J.M. Haeffner-Cavaillon N. Characterization of the induction of human interleukin-1 by endotoxins.in: Paubert-Braquet M. Lipid Mediators in the Immunology of Shock. NATO Asi Series. Plenum Press, New York1987: 395-407Crossref Google Scholar, 24.Laude-Sharp M. Haeffner-Cavaillon N. Caroff M. Lantreibecq F. Pucineri C. Kazatchine M.D. Dissociation between the interleukin 1-inducing capacity and limulus reactivity of lipopolysaccahrides from Gram-negative bacteria.Cytokine. 1990; 2: 253-258Crossref PubMed Scopus (55) Google Scholar). Because of this high structural variability, the Bordetella strains presented a very suitable model for testing the new method.Here, we characterize three Citrobacter and two Bordetella lipids A by a new procedure involving direct extraction from cells. This method is especially convenient when only small amounts of bacteria, LPS, or lipid A are available. When applied to Bordetella strains as a routine test, the method led us to discover a new original lipid A structural element, increasing the LPS structural and biosynthetic originality and perhaps giving the bacteria a camouflage strategy to escape a host defense system.MATERIALS AND METHODSBacterial strainsBacterial strains used (all from the National research council of Canada, Ottawa, Canada) were C. sedlakii, C. freundii (ATCC 51541), C. rodentium (19.Vinogradov E. Conlan J.W. Perry M.B. Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobacter freundii and Citrobacter sedlakii.FEMS Microbiol. Lett. 2000; 190: 157-161Crossref PubMed Google Scholar, 20.Chart H. Willshaw G.A. Cheasty T. Rowe B. Structure and antigenic properties of Citrobacter freundii lipopolysaccharides.J. Appl. Bacteriol. 1993; 74: 583-587PubMed Google Scholar, 21.Kocharova N.A. Knirel Y.A. Kholodkova E.V. Stanislavsky E.S. Structure of the O-specific polysaccharide chain of Citrobacter freundii O28,1c lipopolysaccharide.Carbohydr. Res. 1995; 279: 327-330Crossref PubMed Scopus (4) Google Scholar, 22.MacLean L.L. Perry M.B. Structural studies on the O-polysaccharide of the lipopolysaccharide produced by Citrobacter rodentium (ATCC 51459).Eur. J. Biochem. 2001; 268: 5740-5746Crossref PubMed Scopus (13) Google Scholar), E. coli (strain 0119), and B. bronchiseptica (NRCC 4650).Bacterial growth conditionsCells were grown as described (13.Zarrouk H. Karibian D. Bodie S. Perry M.B. Richards J.C. Caroff M. Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution.J. Bacteriol. 1997; 179: 3756-3760Crossref PubMed Scopus (35) Google Scholar, 19.Vinogradov E. Conlan J.W. Perry M.B. Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobacter freundii and Citrobacter sedlakii.FEMS Microbiol. Lett. 2000; 190: 157-161Crossref PubMed Google Scholar, 20.Chart H. Willshaw G.A. Cheasty T. Rowe B. Structure and antigenic properties of Citrobacter freundii lipopolysaccharides.J. Appl. Bacteriol. 1993; 74: 583-587PubMed Google Scholar, 21.Kocharova N.A. Knirel Y.A. Kholodkova E.V. Stanislavsky E.S. Structure of the O-specific polysaccharide chain of Citrobacter freundii O28,1c lipopolysaccharide.Carbohydr. Res. 1995; 279: 327-330Crossref PubMed Scopus (4) Google Scholar, 22.MacLean L.L. Perry M.B. Structural studies on the O-polysaccharide of the lipopolysaccharide produced by Citrobacter rodentium (ATCC 51459).Eur. J. Biochem. 2001; 268: 5740-5746Crossref PubMed Scopus (13) Google Scholar) Briefly, Citrobacter cells were grown to late exponential phase in a brain-heart infusion (Difco) at 37°C under constant aeration in a New Brunswick 25 liter fermenter.B. bronchiseptica cells (two batches) were grown in 70 liter fermenters using a 3.7% brain-heart infusion containing 5% horse serum at 37°C and 200 rpm with aeration for 18 h (13.Zarrouk H. Karibian D. Bodie S. Perry M.B. Richards J.C. Caroff M. Structural characterization of the lipids A of three Bordetella bronchiseptica strains: variability of fatty acid substitution.J. Bacteriol. 1997; 179: 3756-3760Crossref PubMed Scopus (35) Google Scholar). All cells were killed with phenol (1% final concentration) before harvesting.LPS extraction conditionsThe wet bacteria were washed with 1% saline and were extracted by stirring with 50% aqueous phenol at 65°C and collected by centrifugation for 15 min (25.Westphal O. Jann K. Bacterial lipopolysaccharide. Extraction with phenol-water and further application of the procedure.Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar). The cooled extract was diluted with water (2 volumes), insoluble material was removed by centrifugation, and the cleared extract was dialyzed under tap water until free from phenol. The lyophilized retentate was dissolved in 0.02 M sodium acetate (pH 7), sequentially treated with RNase, DNase, and proteinase K, and cleared by centrifugation (105,000 g, 12 h, 4°C), and the precipitated LPS gel was dissolved in water and lyophilized (22.MacLean L.L. Perry M.B. Structural studies on the O-polysaccharide of the lipopolysaccharide produced by Citrobacter rodentium (ATCC 51459).Eur. J. Biochem. 2001; 268: 5740-5746Crossref PubMed Scopus (13) Google Scholar).Lipid A isolation from whole cells was described in detail previously (11.El Hamidi A. Tirsoaga A. Novikov A. Hussein A. Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization.J. Lipid Res. 2005; 46: 1773-1778Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Briefly, lyophilized bacterial cells (10 mg) were suspended in 400 μl of isobutyric acid-1 M ammonium hydroxide mixture (5:3, v/v) and were kept for 2 h at 100°C in a screw-cap test tube under magnetic stirring. The suspension was cooled in ice water and centrifuged (2,000 g for 15 min). The supernatant was diluted with the same volume of water and lyophilized. The lyophilized sample was then twice washed with 400 μl of methanol and centrifuged (2,000 g for 15 min). Finally, the lipid A was extracted from the pellet in 100 to 200 μl of a mixture of chloroform, methanol, and water (3:1.5:0.25, v/v/v). For 1 mg samples, 100 μl of the solvent mixtures was used at each step.Sequential liberation of ester-linked fatty acids by mild alkali treatmentThis method was first developed with a relatively homogeneous E. coli lipid A. The following reagents were tested to define convenient two-step liberation of ester-linked fatty acids: methylamine, dimethylamine, ethylamine, diethylamine, triethylamine, and ammonium hydroxide, at various concentrations. Different temperatures (37, 50, and 60°C) were also tested and followed by kinetics, and the products were monitored by TLC and MALDI-MS.The following conditions were then selected for the first-step liberation of primary ester-linked fatty acids. Lipid A (50 μg) was suspended (1 mg/ml) in 35% ammonium hydroxide and stirred for 5 h at 50°C. To liberate the secondary ester-linked fatty acids, the resulting lipid A (50 μg) was suspended in 50 μl of 41% methylamine and stirred for 5 h at 37°C. The resulting samples were dried under a stream of nitrogen, and the residues were taken up in a mixture of chloroform, methanol, and water (3:1.5:0.25, v/v/v) and followed by TLC and MALDI-MS analyses.Methylamine and ethylamine were similarly efficient at liberating ester-linked fatty acids. We chose to use the amine with the smallest alkyl moiety. In addition, diethylamine produced some degradation products. This was not surprising, because secondary amines are known to be more strongly basic.Micro quantities of lipid A isolated directly from the bacterial cells were used for deesterification. Fifty to 100 μl aliquots of the chloroform-methanol-water extracts were transferred to Eppendorf® tubes and dried with a stream of nitrogen before treatment. To define volumes of the solutions necessary for the treatment, the total mass of the isolated lipid A was estimated to be ∼1% of the initial mass of the bacterial sample.Hydrolysis procedures used to liberate lipid AAcetic acid hydrolysisLPS was suspended in 2% acetic acid (5 mg/ml) and kept for 2 h at 100°C under stirring. Acid was removed under vacuum, and the residue, suspended in water (5 mg/ml), was ultracentrifuged (45 min, 300,000 g, 4°C). The pellet containing lipid A was lyophilized, and lipid A was extracted with chloroform-methanol-water extraction mixture (3:1.5:0.25, v/v/v).SDS-promoted hydrolysisLPS was dispersed at a concentration of 5 mg/ml in 20 mM sodium acetate-acetic acid buffer (pH 4.5) containing 1% SDS and hydrolyzed at 100°C for 1 h. After removal of SDS with acidified ethanol, lipid A was isolated as described previously (9.Caroff M. Tacken A. Szabo L. Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin.Carbohydr. Res. 1988; 175: 273-282Crossref PubMed Scopus (199) Google Scholar).Hydrolysis procedures used for liberation of the lipid A glycosidic phosphateHydrochloric acid hydrolysisLPS or lipid A was suspended in 0.1 M HCl at a concentration of 5 mg/ml and kept for 15 mn at 100°C under stirring (7.Karibian D. Deprun C. Caroff M. Use of plasma desorption mass spectrometry in structural analysis of endotoxins: effects on lipid A of different acid treatments.Prog. Clin. Biol. Res. 1995; 392: 103-111PubMed Google Scholar). The acid was neutralized with a 0.1 M NaOH solution and ultracentrifuged. The pellet containing the dephosphorylated lipid A was lyophilized, and the lipid A was extracted as above.Hydrofluoric acid treatmentLPS or lipid A was suspended at 5 mg/ml in hydrofluoric acid (HF) and kept at 4°C under stirring for 48 h. After solvent removal under a stream of nitrogen under a hood, the residue was taken up in water and lyophilized before extraction.MALDI-MSAnalyses were performed on a PerSeptive Voyager-DE STR time-of-flight mass spectrometer (Applied Biosystems) at the Institut de biochimie et biophysique moléculaire et cellulaire, Université de Paris Sud. The analysis of the small lipid A samples used was done in linear mode with delayed extraction. Both negative- and positive-ion spectra were recorded. The ion-accelerating voltage was set at 20 kV. Dihydroxybenzoic acid (Sigma Chemical Co., St. Louis, MO) was used as a matrix. A few microliters of lipid A solution (1 μg/μl) in the extraction mixture was desalted with a few grains of ion-exchange resin [Dowex 50W-X8 (H+)], either in an Eppendorf® tube or for small samples in a single surface droplet on Parafilm®. A 1 μl aliquot of the solution was deposited on the target and covered with the same volume of the matrix dissolved at 10 mg/ml in the same solvent. Different analyte-matrix ratios were tested when necessary. B. pertussis or E. coli highly purified lipids A were used as external standards for mass calibration.Thin-layer chromatographyChromatography was performed on aluminum-backed silica TLC plates (Merck), and compounds were visualized by charring at 145°C after spraying with 10% sulfuric acid in ethanol. Mixtures of isobutyric acid and 1 M ammonium hydroxide were used for migration of oligosaccharides (3:5, v/v) and LPSs (5:3, v/v) (26.Caroff M.G. Karibian D. Several uses for isobutyric acid-ammonium hydroxide solvent in endotoxin analysis.Appl. Environ. Microbiol. 1990; 56: 1957-1959Crossref PubMed Google Scholar). The solvent used for lipid A migration was a mixture of chloroform, methanol, water, and triethylamine (3:1.5:0.25: 0.1, v/v/v/v) (9.Caroff M. Tacken A. Szabo L. Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin.Carbohydr. Res. 1988; 175: 273-282Crossref PubMed Scopus (199) Google Scholar).RESULTS AND DISCUSSIONThe need for a rapid method for analyzing lipid A structures on small bacterial samples initiated our search for new lipid A isolation methods (11.El Hamidi A. Tirsoaga A. Novikov A. Hussein A. Caroff M. Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization.J. Lipid Res. 2005; 46: 1773-1778Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In the present work, we applied selective mild alkaline treatments sequentially liberating fatty acids as well as acid-dephosphorylating treatments to micro quantities of Citrobacter and Bordetella lipids A. The latter, which were isolated by microhydrolysis of bacteria, could thus be further characterized in 1 day experiments, as shown schematically in Fig. 1 .Full-scale analysis of Citrobacter lipids AComparison of direct microhydrolysis of bacteria with conventional hydrolytic methodsWe compared the lipid A preparations obtained by direct microhydrolysis of whole Citrobacter bacterial cells with those obtained by conventional hydrolytic methods applied to phenol-extracted endotoxins of Citrobacter. Figure 2 shows negative-ion MALDI mass spectra of C. sedlakii lipid A obtained by direct microhydrolysis of bacteria (Fig. 2A), mild SDS-promoted pH 4.5 hydrolysis of the LPS, 1 h at 100°C (Fig. 2B), 2% acetic acid hydrolysis of the LPS, 2 h at 100°C (Fig. 2C), 0.1 M HCl hydrolysis of the LPS, 10 min at 100°C (Fig. 2D), and HF treatment (48 h) of lipid A isolated by direct microhydrolysis of the bacteria (Fig. 2E).Fig. 2.Negative-ion MALDI mass spectra of C. sedlakii lipid A obtained by various methods. A: Hydrolysis of bacterial cells in a mixture of isobutyric acid and 1 M ammonium hydroxide (5:3, v/v) for 2 h at 100°C. B: SDS-promoted hydrolysis at pH 4.5 for 1 h at 100°C. C: Hydrolysis of lipopolysaccharide (LPS) in 2% acetic acid for 2 h at 100°C. D: Hydrolysis of LPS in 0.1 M HCl for 10 min at 100°C. E: A 48% hydrofluoric acid (HF) lipid A hydrolysis at 4°C for 48 h. −P, minus phosphate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly, the direct microhydrolysis performed on bacteria (Fig. 2A) shows peaks for the four main molecular species having four to seven fatty acid structures as well preserved as, if not better than, those obtained by the SDS-promoted mild hydrolysis (Fig. 2B). The quality of the spectrum is comparable with a good signal-to-noise ratio for molecular ion peaks and a negligible level of dephosphorylation. The latter point is a great advantage for good structural and biological practice. Few lipid A preparations
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