Complex Carbohydrate Research Center, The University of Georgia, 220 Riverbend Road, Athens, GA 30602, USA1
Departments of Medicine and Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30303, and Department of Veterans Affairs Medical Center, Atlanta, GA, USA2
Author for correspondence: Russell W. Carlson. Tel: +1 706 542 4439. Fax: +1 706 542 4412. e-mail: rcarlson{at}ccrc.uga.edu
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ABSTRACT |
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Keywords: Neisseria meningitidis, Neisseria gonorrhoeae, membranes, phospholipids
Abbreviations: DPG, cardiolipin; FAB, fast atom bombardment; LPA, lysophosphatidic acid; PA, phosphatidic acid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol
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INTRODUCTION |
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In Gram-negative bacteria, such as Escherichia coli and Neisseria spp., the outer membrane has an asymmetrical organization in which the outer leaflet is primarily composed of LPS and proteins and the inner leaflet contains phospholipids (Nikaido, 1996 ). The role of phospholipids in maintaining the integrity of the outer membrane has been demonstrated. Perturbation of outer-membrane biogenesis in E. coli by mutations which decrease lipid A synthesis (Karow et al., 1992
) and suppress outer-membrane protein assembly (Kloser et al., 1998
) results in elevated levels of phospholipid synthesis and incorporation into the outer membrane. Since both LPS and phospholipid pathways share common fatty acid precursors, this effect may be mediated by the shift in the flow of these precursors from one pathway to the other. In Proteus mirabilis, it has been noted that LPS mutants contain an increased level of unsaturated fatty acid content which may compensate for fluidity changes in the outer membrane (Rottem et al., 1978
). Additionally, the export and trimerization of the major porins of the outer membrane rely upon a tight association with intact LPS, and these proteins do not localize to the outer membrane of E. coli mutants expressing LPS cores deficient in heptose (Sen & Nikaido, 1991
). Interestingly, the relative abundance of membrane phospholipids also significantly affects the functional activities of outer-membrane proteins (Senff et al., 1976
; Wolf-Watz et al., 1975
).
Earlier studies conducted in the 1960s and 1970s using TLC indicated that the major phospholipid component of membranes isolated from N. gonorrhoeae consisted largely of phosphatidylethanolamine (PE) with varying amounts of phosphatidylglycerol (PG), cardiolipin (DPG) and lysophosphatidylethanolamine (LPE) (Guymon et al., 1978 ; Sud & Feingold, 1976
; Senff et al., 1976
; Wolf-Watz et al., 1975
; Lewis et al., 1968
; Moss et al., 1970
). Subsequently, it was shown that the LPE found in gonococcal membranes was due to the degradation of PE by phospholipase A (Cacciapuoti et al., 1978
, 1979
). Lauric (C12:0), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids were the major fatty acids attached to gonococcal phospholipid head groups (Guymon et al., 1978
; Sud & Feingold, 1976
; Wolf-Watz et al., 1975
; Wilkinson, 1988
; Moss et al., 1970
; Lewis et al., 1968
). The total cellular fatty acids and extractable cellular lipids of N. meningitidis isolates were subsequently found to be similar to those of gonococci (Lewis et al., 1968
; Moss et al., 1970
; Wilkinson, 1988
). However, the precise structures of N. meningitidis phospholipids, including their fatty acylation patterns, have not been examined in detail.
This study is the first to detail by MS the complete structure of the phospholipids of both N. meningitidis and N. gonorrhoeae. In addition, a series of meningococcal capsule and LOS mutants were studied to determine whether changes in the expression of these surface structures affected the phospholipid profile. Considerable heterogeneity was observed in the N. meningitidis phospholipid fatty acylation patterns when compared to the phospholipids from various enteric bacterial species. This heterogeneity is likely to be a result of novel genetic mechanisms of neisserial phospholipid assembly and regulation.
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METHODS |
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Extraction of phospholipids.
The phospholipids were extracted from total membranes isolated from each strain using a modified method of Radin (1969) . Glassware was rinsed with chloroform, then air-dried and Teflon-lined caps were used to prevent contamination with plastics. The membranes were suspended in the polyallomer tubes by the addition of solvent (2 vols chloroform to 1 vol. methanol) in a 17:1 (v/w) ratio of solvent to pellet and stirred constantly using micro-stir bars for 30 min at room temperature. The suspension was transferred to Corex glass tubes and the debris was collected by 15 min centrifugation at 10000 g in a JA-17 rotor (Beckman) at 4 °C. The supernatant was collected and the pellet was again extracted with solvent as before. The two supernatants were combined and 2 vols Folch upper phase reagent (chloroform/methanol/water containing 0·74% potassium chloride; 3:48:47, by vol.) was added. The mixture was briefly vortexed to form a single phase. The phases were separated by low speed 1 min centrifugation in a clinical centrifuge. The upper phase and the precipitate at the interface were discarded. The lower phase was extracted with the Folch upper phase reagent at least three more times or until there was no precipitate at the interface. After the final extraction, the lower organic phase was transferred to a glass vial and the solvent evaporated under a stream of nitrogen. The dried phospholipids were stored at -70 °C under a nitrogen atmosphere.
Fatty acid analysis.
Total fatty acids were released by methanolysis of phospholipid with methanolic 1 M HCl at 80 °C for 4 h and were trimethylsilylated. The resulting fatty acid methyl esters were analysed by GLC-MS (York et al., 1985 ). Trimethylsilylation allows one to observe any hydroxy fatty acyl residues, if present. The double bond location of unsaturated fatty acids was determined by the preparation and GLC-MS analysis of dimethyl disulfide fatty acid methyl esters as described by Yruela et al. (1990)
.
MS.
Fast atom bombardment (FAB)-MS was performed in negative-ion mode with a JEOL SX/SX 102A tandem mass spectrometer, which was operated at 10 kV accelerating potential. Ions were produced by FAB with xenon, using a JEOL FAB gun operated at 6 kV in a conventional FAB ion source. Spectra acquired for the first MS are averaged profile data of three scans as recorded by a JEOL XMS data system. These spectra were acquired from 1002000 m/z at a rate that would scan the mass range from 0 to 2500 in 1 min. A filtering rate of 100 Hz was used in acquiring these spectra. The samples were dissolved in chloroform/methanol (2:1, v/v) solution and 1 µl aliquots were mixed with an equal volume of the FAB matrix, m-nitrobenzyl alcohol, on an MS probe tip. After permitting solvent evaporation, the probe was placed into the sample port of the mass spectrometer. The MS/MS spectrum was obtained by using a linked scan (B/E constant) at a rate that would scan the mass range from 5 to 2400 in 1 min with a 300 Hz filter.
Quantification of phospholipids by FAB-MS.
Dipalmitoyl PE, PG and phosphatidic acid (PA) were purchased from Sigma. Two series of 1 ml solutions of these phospholipids were prepared. One series consisted of four different solutions varying in both PE and PG (10, 30, 60 and 90 µg) but with each containing a constant 100 µg amount of PA as an internal standard. The second series varied in both PA and PG (10, 30, 60 and 90 µg) with a constant 100 µg of PE as an internal standard. Each solution was analysed by FAB-MS as described above. The data were used to (a) determine if the intensity of the molecular ion for each phospholipid was proportional to the amount of that phospholipid, and (b) determine the FAB-MS response of PE relative to PG and of PA relative to PG. Analysis of the first series of solutions allowed the correlation of the molecular ion intensity with mass for both PG and PE, and also allowed the calculation of the FAB-MS response factor of PE relative to PG. The second series of solutions allowed such correlation for both PA and PG, and also the calculation of the FAB-MS response factor of PA relative to PG. Using these normalized response factors, the relative amounts of the various phospholipids for each membrane preparation were calculated, assuming that all of the various PE, PG and PA molecules have the same, or very similar, FAB-MS responses to those of the respective dipalmitoyl PE, PG and PA standards. This approach overcomes the discrepancies in phospholipid quantification that were noted by Smith et al. (1995) where a comparison of MS techniques and conventional methods of analysis were shown to disagree.
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RESULTS |
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The total fatty acid composition of phospholipids from wild-type N. meningitidis and N. gonorrhoeae and meningococcal mutant strains were also analysed by methanolysis followed by trimethylsilylation and analysis by GLC-MS. The results of the GLC-MS analysis (Table 3) show that the major fatty acids observed by this method are consistent with the major fatty acids observed by FAB-MS analysis (Table 2
). The C12:0 fatty acid was observed in both the FAB-MS and GLC-MS analyses, but the high volatility of its methyl ester prevented accurate quantification by the GLC-MS procedure. However, the FAB-MS spectra clearly show that C12:0 was present in the phospholipids of all meningococcal strains examined (6·510%) and a lesser amount (3·3%) in N. gonorrhoeae FA 1090.
The phospholipids of N. gonorrhoeae FA 1090 contained significantly less shorter-chain fatty acids (e.g. C12:0, C14:0 and C14:1) compared to the N. meningitidis strains (Figs 1 and 2
, Tables 2
and 3
). The phospholipids from the three N. meningitidis isolates, NMB, F8229 and F8239, had similar fatty acid profiles. However, N. meningitidis 6940 had a fatty acid profile resembling that of N. gonorrhoeae FA 1090. Both 6940 and FA 1090 contained 1·53-fold more C18:1 and a corresponding 1·53-fold decrease of C14:0 when compared to the meningococcal isolates NMB, F8229 and F8239. No significant differences were observed in the fatty acid profiles of the capsule and LOS mutants compared to that of the parent strain, NMB.
Phospholipid compositions
The relative mass percentage of each phospholipid was determined from the molecular ion intensities of the various phospholipid molecules. To determine if ion intensity was proportional to the amount of a particular phospholipid, the molecular ion intensities were measured for known amounts of dipalmitoyl PA, PG and PE by varying the level of two of the phospholipids while holding the amount of the third phospholipid constant. Under these conditions, the phospholipid that was held constant acted as an internal standard. In the first series, PG and PA were varied while PE was the constant internal standard. Fig. 3(a) shows that both the PG/PE and PA/PE molecular ion intensity ratios were proportional to the amount of PG and PA, respectively. Likewise, Fig. 3(b)
shows that the PG/PA and PE/PA molecular ion intensity ratios were proportional to the amounts of PG and PE, respectively. By calculating the (PA/PE)/(PG/PE) and (PE/PA)/(PG/PA) values for each concentration from Fig. 3(a)
and (b)
, respectively, the response of PA to PG and of PE to PG were determined to be 0·37±0·06 and 0·24±0·04, respectively. Thus, the amount of each PA molecule can be normalized to PG by dividing its molecular ion intensity by 0·37 and the amount of each PE molecule can be normalized to PG by dividing its molecular ion intensity by 0·24.
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A major difference between neisserial phospholipids and those from Gram-negative enteric bacterial species is the abundance of the short-chain fatty acyl substituents in the former (see review by Wilkinson, 1988 ), a result confirmed by this study. This increase in shorter-chain fatty acyl groups was reflected in the proportion of PE and PG molecules which contain C12:0, C14:0 or C14:1. The relative percentage of total PE and total PG which contain shorter-chain fatty acyl groups (i.e. fatty acyl chains in which the number of carbons for both fatty acids total 30 or less) varies from 21 to 47% for PE and from 24 to 66% for PG, while those same values for E. coli are 8·1 and 4·4% for PE and PG, respectively (calculated from the phospholipid data reported for E. coli in the review by Wilkinson, 1988
). It is clear from these results that, in comparison to E. coli, all of the neisserial phospholipids are greatly enriched with molecules that contain shorter-chain fatty acyl substituents.
Isobaric phospholipid molecules and the positions of fatty acyl substituents
The structures of the various phospholipids were further characterized via tandem MS/MS analysis of single molecular ions which resulted in characteristic formation of fragment ions during collision-induced dissociation (CID). In addition, the relative intensities of the acyl carboxylate fragment ions from tandem MS/MS analysis indicate their positions in the phospholipid (Jensen et al., 1987 ; Cole & Enke, 1991
); i.e. at the sn-1 or sn-2 positions. The carboxylate fragment ion from the fatty acyl component at sn-2 will form in preference to that at the sn-1 position and will, therefore, have the greatest intensity (Jensen et al., 1987
; Cole & Enke, 1991
). Fig. 4
shows the fragment ion spectrum obtained by tandem MS/MS analysis of a phospholipid from strain NMB. The structural basis for these assignments is also given in Fig. 4
. These data are consistent with the m/z 688 ion being due to two isobaric PE molecules; one major PE component with C16:1 (m/z 253) and C16:0 (m/z 255) as the acyl substituents and a second minor component with C18:1 (m/z 281) and C14:0 (m/z 227) as the acyl substituents. Similarly, tandem MS/MS analysis of m/z 719 (spectrum not shown) suggests that this ion is due to two isobaric PG structures, the major component with C16:0 and C16:1 as the acyl substituents and a minor component substituted with C14:0 and C18:1. The MS/MS spectra of other major phospholipids having masses of 693, 665 and 635 were also obtained and the resulting data indicate that they have the structures given in Table 5
. According to these results, every phospholipid examined by tandem MS/MS contained a saturated fatty acyl substituent at the sn-1 position and contained either a saturated or unsaturated fatty acyl substituent at the sn-2 position.
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DISCUSSION |
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The results of the present study both support and are at variance with results of previous studies in gonococci. The major classes of phospholipids present in meningococci and gonococci were PE (6577%), followed by PG (1424%), confirming other work (Sud & Feingold, 1976 ; Wolf-Watz et al., 1975
; Senff et al., 1976
; Guymon et al., 1978
; Lewis et al., 1968
; Moss et al., 1970
). The present study also confirms that pathogenic Neisseria spp. contain significant levels of short-chain fatty acids, C12:0, C14:1 and in particular C14:0, which account for one-fifth of the total fatty acid composition of some meningococcal strains. However, in contrast to the earlier reports, we detected significant levels of PA (711%) and very low levels of DPG in all strains. The reason for the contrast between our results and those previously reported is not known; however, the contrast in results could reflect real strain differences in phospholipid profiles or differences in the FAB-MS technique used in our work versus the TLC procedures used in the earlier work. Variability in amounts of PE, PG and PA was observed between the different N. meningitidis strains, including the LOS mutants of strain NMB and N. gonorrhoeae. The biological significance, if any, of these differences in overall phospholipid distribution requires further study, but illustrates the variability of meningococcal and gonococcal surface constituents.
Detailed structural analysis of the phospholipids of N. meningitidis and N. gonorrhoeae has not been previously reported (e.g. the fatty acylation patterns of the various phospholipids). The FAB-MS spectra of the phospholipid species produced by both meningococci and gonococci revealed a diverse array of acyl chains attached to each type of phospholipid head group. Tandem MS/MS was used to identify the acyl chain attachments at the sn-1 and sn-2 positions of the major phospholipids. This analysis confirmed that neisserial phospholipids contain a saturated fatty acyl substituent at sn-1 and either a saturated or unsaturated fatty acyl substituent at sn-2, verifying the diversity of these structures. The major phospholipids of E. coli have an asymmetric distribution with the sn-1 position of the glycerol phosphate backbone containing the saturated fatty acid C16:0, while sn-2 is occupied by unsaturated fatty acids C16:1 or C18:1 (Rock et al., 1996 ; Wilkinson, 1988
). Minor phospholipids from E. coli have C14:0, C16:1 or C18:1 in the sn-1 position (see review by Wilkinson, 1988
). The predominant phospholipids of N. meningitidis NMB, PG(32:1) (28% of total PG) and PE(32:1) (29% of total PE), have a similar asymmetrical distribution of saturated (C16:0 or C14:0) and unsaturated (C16:1 or C18:1) acyl chains at the sn-1 and sn-2 positions, respectively, of the glycerol phosphate backbone. A major difference between the neisserial phospholipids and those from E. coli, is that the former have a much greater level of short-chain fatty acid substituents. This greater diversity of short-chain fatty acyl substituents is consistent with our previous report (Shih et al., 1998
) showing that strain NMB contains at least two lysophosphatidic acid (LPA) acyltransferases which catalyse the transfer from acyl-ACP to the sn-2 position of the glycerol phosphate backbone. In comparison, E. coli contains only one LPA acyltransferase (Coleman, 1992
).
In two of the isolates, N. meningitidis 6940 and N. gonorrhoeae FA 1090, the amount of C14:0 was significantly decreased (1·53-fold) with a concomitant increase in C18:1 content compared to the other strains examined. Although Wolf-Watz et al. (1975) described multiple isolates of N. gonorrhoeae with high and low amounts of C18:1, this feature has not been previously reported for meningococcal isolates. In E. coli, the unsaturated fatty acids, palmitoleic acid (C16:1) and cis-vaccenic acid (C18:1), are synthesized by a branched pathway which diverges from that of the saturated fatty acids (Rock et al., 1996
). A 3-hydroxydecanoyl-ACP dehydrase encoded by fabA introduces a trans double bond into the acyl chain of ß-hydroxy-decanoyl-ACP, which is then isomerized to cis-3-decanoyl-ACP. This compound serves as the substrate for further elongation cycles catalysed by the ß-ketoacyl-ACP synthetases (FabB and FabF) which extends the acyl chain to form palmitoleoyl-ACP (C16:1
9) and cis-vaccenoyl-ACP (C18:1
11). Therefore, in E. coli, C18:1 content increases at the expense of C16:1 primarily due to the activity of the temperature-regulated FabF (Rock et al., 1996
). However, we found the relative content of C16:1 did not vary significantly among the different meningococcal isolates with high or low amounts of C18:1, nor was the C18:1 content of meningococcal phospholipids temperature-regulated (data not shown). Instead, compared to the other neisserial phospholipids in this report, the phospholipids from N. meningitidis 6940 and N. gonorrhoeae FA 1090 appear to be increased in C18:1 at the expense of C14:0 and not at the expense of C16:1 as is the case for E. coli, suggesting that the enzymic activities of the meningococcal condensing enzymes will be different from those of E. coli.
The recent completion of the genome databases of N. meningitidis Z2491 (N. meningitidis sequencing group at the Sanger Center; http://www.sanger.ac.uk) and N. gonorrhoeae FA 1090 (Gonococcal Genome Sequencing Project; http://www.genome.ou.edu) has provided some insight into the genetic basis of the fatty acid biosynthesis pathway in these bacteria. The current model of fatty acid synthesis in E. coli requires two essential condensing enzymes (FabH and FabB), a ß-ketoacyl reductase (FabG), two ß-hydroxyacyl-ACP dehydrases (FabA and FabZ) and an enoyl-ACP reductase (FabI). Although homologues for these enzymes were found in the N. meningitidis Z2491 genome, only a single dehydrase homologue was present, presumably FabZ, since this enzyme is essential for the fatty acid elongation cycle. The absence of a FabA homologue in Neisseria spp. is not unique. A search for clusters of orthologous groups of proteins (COGs) in the complete genomes of 18 organisms (see Entrez genomes at http://www.ncbi.nlm.nih.gov) revealed that only three, E. coli, Haemophilus influenzae and Bacillus subtilis, contain two dehydrases. Six species, including Rickettsia, Chlamydia, Synechocystis, Aquifex, Thermotoga and Helicobacter species, contained a single E. coli dehydrase homologue, while the remainder did not contain any COGs related to the E. coli dehydrase. Therefore, it appears that the production of unsaturated fatty acids in these species, including N. meningitidis and N. gonorrhoeae, may proceed via different biochemical pathways to that of E. coli.
The variability of phospholipid structures suggests that outer-membrane biogenesis of meningococci and gonococci may differ from that of E. coli. As noted above, E. coli has one essential LPA acyl transferase, while at least two are found in N. meningitidis. The synthesis of lipid A is also essential for cell viability in E. coli (Nikaido, 1996 ; Raetz, 1996
). In contrast, a meningococcal mutant has been reported to be without lipid A and remains viable (Steeghs et al., 1998
). Examination of the outer-membrane profile of this meningococcal lipid-A-deficient mutant did not reveal any major alterations in outer-membrane protein expression, although the amount of PE containing shorter acyl chains was increased (Van der Ley et al., 1998
). Therefore, the ability of meningococci and gonococci to produce variable phospholipids containing short acyl chains may allow such mutants to functionally replace the absent lipid A and remain viable.
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ACKNOWLEDGEMENTS |
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Received 6 March 2000;
revised 11 May 2000;
accepted 18 May 2000.