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W1974565437 abstract "A new class of outer membrane lipid (OML) was isolated from the oral spirochete Treponema denticolastrain ATCC 33521 using a phenol/chloroform/light petroleum procedure normally applied for lipopolysaccharide extraction. In addition to chemical analysis, Fourier transform infrared (FTIR) spectroscopy was applied to compare the biophysical properties of OML with lipopolysaccharides (LPS) and lipoteichoic acids (LTA). Isolated OML fractions represent 1.4% of the total dry cell weight, are about 4 kDa in size, and contain 6% amino sugars, 8% neutral sugars, 14% phosphate, 35% carbazol-positive compounds, and 11% fatty acids (containing iso- and anteiso-fatty acyl chains). Rare for outer membrane lipids, OML contains no significant amount of 3-deoxy-d-manno-octulosonic acids, heptoses, and β-hydroxy fatty acids. The fatty acyl chain composition, being similar to that of the cytoplasmic membrane, is quite heterogeneous with anteiso-pentadecanoic acid (12%), palmitic acid (51%), and iso-palmitic acid (19%) as the predominant fatty acids present. Findings of a glycerol-hexose unit and two glycerol-hexadecanoic acid fragments indicate a glycolipid membrane anchor typically found in LTA. There was also no evidence for the presence of a sphingosine-based lipid structure. The results of FTIR measurements strongly suggest that the reconstituted lipid forms normal bilayer structures (vesicles) expressing a high membrane state of order with a distinct phase transition as typical for isolated LPS. However, in contrast to LPS, OML of T. denticola has a lowerTm near 22 °C and a lower cooperativity of the phase transition. The results suggest a different kind of permeation barrier that is built up by this particular OML ofT. denticola, which is quite different from LPS normally essential for Gram-negative bacteria. A new class of outer membrane lipid (OML) was isolated from the oral spirochete Treponema denticolastrain ATCC 33521 using a phenol/chloroform/light petroleum procedure normally applied for lipopolysaccharide extraction. In addition to chemical analysis, Fourier transform infrared (FTIR) spectroscopy was applied to compare the biophysical properties of OML with lipopolysaccharides (LPS) and lipoteichoic acids (LTA). Isolated OML fractions represent 1.4% of the total dry cell weight, are about 4 kDa in size, and contain 6% amino sugars, 8% neutral sugars, 14% phosphate, 35% carbazol-positive compounds, and 11% fatty acids (containing iso- and anteiso-fatty acyl chains). Rare for outer membrane lipids, OML contains no significant amount of 3-deoxy-d-manno-octulosonic acids, heptoses, and β-hydroxy fatty acids. The fatty acyl chain composition, being similar to that of the cytoplasmic membrane, is quite heterogeneous with anteiso-pentadecanoic acid (12%), palmitic acid (51%), and iso-palmitic acid (19%) as the predominant fatty acids present. Findings of a glycerol-hexose unit and two glycerol-hexadecanoic acid fragments indicate a glycolipid membrane anchor typically found in LTA. There was also no evidence for the presence of a sphingosine-based lipid structure. The results of FTIR measurements strongly suggest that the reconstituted lipid forms normal bilayer structures (vesicles) expressing a high membrane state of order with a distinct phase transition as typical for isolated LPS. However, in contrast to LPS, OML of T. denticola has a lowerTm near 22 °C and a lower cooperativity of the phase transition. The results suggest a different kind of permeation barrier that is built up by this particular OML ofT. denticola, which is quite different from LPS normally essential for Gram-negative bacteria. Treponema denticola is an oral anaerobic spirochete with typical helical morphology (1Westergaard J. Fiehn N.-E. APMIS Sect. B. 1987; 95: 49-55Google Scholar). These bacteria have been implicated in the induction of peridontitis (2Listgarten M.A. Levin S.L. J. Clin. Peridontol. 1981; 8: 122-127Crossref PubMed Scopus (163) Google Scholar, 3Loesche W.J. Laughon B.E. Genco R.J. Mergenhagen S.E. Proceedings Host-parasite Interactions in Periodontal Diseases. American Society of Microbiology, Washington, D.C.1982: 62-75Google Scholar). They are composed of a so-called protoplasmic cylinder, in which the cytoplasm is surrounded by a cytoplasmic membrane with a thin murein network on top of it. This cell unit is surrounded by an additional membrane, the outer sheath, an ultra structure similar to the outer membrane of Gram-negative bacteria (4Wolf V. Lange R. Wecke J. Arch. Microbiol. 1993; 160: 206-213PubMed Google Scholar). Axial flagellae are located inside the outer sheath and are wound directly around the protoplasmic cylinder. The outer sheath of some other spirochetes shows major differences in composition and properties as compared with typical outer membranes of Gram-negative bacteria (5Takayama K. Rothenberg R.J. Barbour A.G. Infect. Immun. 1987; 55: 2311-2313Crossref PubMed Google Scholar, 6Hindersson P. Thomas D. Stamm L. Penn C. Norris S. Joens L.A. Res. Microbiol. 1992; 143: 629-639Crossref PubMed Scopus (15) Google Scholar). In addition to the lipids, transmembrane channel proteins are further components within the membrane (7Haapasalo M. Müller K.-H. Uitto V.-J. Leung W.K. McBride B.C. Infect. Immun. 1992; 60: 2058-2065Crossref PubMed Google Scholar). They are characterized by the largest porin channel size observed to date (three times larger than in Escherichia coli) and are able to form hexagonally, self-organized arrays within the membrane (8Egli C. Leung W.K. Müller K.H. Hancock R.E. McBride B.C. Infect. Immun. 1993; 61: 1694-1699Crossref PubMed Google Scholar). This reduces the effectivity of the normal permeation barrier function significantly as compared with that of E. coli. The biophysical membrane properties are also different, as the outer sheath of T. denticola has no continuous and direct contact to the protoplasmic cylinder and is, therefore, able to form spherical bodies, morphological structures that contain more than one treponema cell surrounded by only one common outer sheath (suggested as a possible survival strategy in vivo (4Wolf V. Lange R. Wecke J. Arch. Microbiol. 1993; 160: 206-213PubMed Google Scholar, 9Wecke J. Wolf V. Fath S. Bernimoulin S.-P. Oral Microbiol. Immunol. 1995; 10: 278-283Crossref PubMed Scopus (10) Google Scholar, 10Wolf V. Ultrastrukturelle und Immunologische Charakterisierung von Treponema denticula-Isolation.Ph.D. thesis. Free University, Berlin, Germany1993Google Scholar)). The capability of the outer sheath to form cell-independent structures such as spherical bodies leads to the expectation that the structural properties differ from those of lipopolysaccharides. Only a limited amount of information is available on the macromolecular composition and structure of the outer sheath of oral spirochetes, and knowledge of the lipids is particularly inconsistent. Fourier transform infrared (FTIR) 1The abbreviations used are: FTIR, Fourier transform infrared; OML521, outer sheath lipid from strain T. denticola ATCC 33521; HVPE, high voltage paper electrophoresis; LPS, lipopolysaccharide; OML521HF, HF-degraded outer sheath lipid; GlcN, 2-amino-2-deoxy-d-glucose; Kdo, 3-deoxy-d-manno-2-octulosonic acid; Hep, heptose; PAGE, polyacrylamide gel electrophoresis; HF, hydrofluoric acid; LTA, lipoteichoic acid. spectroscopy has frequently been applied to characterize different kinds of lipid structures. FTIR can provide information about chemical composition, state of order, and overall bilayer organization structure (11Mantsch H.H. McElhaney R.N. Chem. Phys. Lipids. 1991; 57: 213-226Crossref PubMed Scopus (468) Google Scholar, 12Naumann D. Schultz C.P. Sabisch A. Kastowsky M. Labischinski H. J. Mol. Struct. 1989; 214: 246-313Crossref Scopus (75) Google Scholar, 13Schultz C.P. Naumann D. FEBS Lett. 1991; 294: 43-46Crossref PubMed Scopus (27) Google Scholar). The spectral comparison between isolated complex lipids can profitably be used to determine the character of the lipid (e.g.primary structure). Structural data on the thermotropic phase behavior of isolated and reconstituted lipids can indicate biophysical properties that may affect the natural membrane environment (e.g. high or low membrane fluidity) (14Schultz C.P. Untersuchungen zur Membran-Organisation Lebender Bakterien unter Besonderer Berücksichtigung des Thermotropen Phasenverhaltens Rekonstituierter Lipopolysaccharid-Doppelschichten: Eine Fourier Transform Infrarot-spektroskopishe Studie (FT-IR) an Lipopolysacchariden und LPS-Mutanten von Salmonella minnesota und Salmonella typhimurium.Ph.D. thesis. Free University, Berlin, Germany1993Google Scholar). Lipopolysaccharides were either obtained from Sigma or donated by Dr. Brade (Borstel, Germany). Lipoteichoic acids were obtained from Sigma, and some samples were donated by Dr. Fischer (Erlangen-Nürnberg, Germany). T. denticola strain ATCC 33521 was obtained from the American Type Culture Collection and grown anaerobically (6% H2, 10% CO2, 84% N2) as described earlier (4Wolf V. Lange R. Wecke J. Arch. Microbiol. 1993; 160: 206-213PubMed Google Scholar). For optimal growth, isobutyric acid, dl-2-methylbutyric acid, isovaleric acid, and valeric acid were added to the medium. All steps of preparation for electron microscopy (embedding procedures, ultrathin sectioning) were performed as described previously (15Wecke J. Franz M. Giesbrecht P. APMIS. 1990; 98: 71-81Crossref PubMed Scopus (9) Google Scholar). Chemical extraction of the outer sheath lipid OML521 from T. denticola ATCC 33521 was performed using a simple phenol/water procedure or a modified phenol/chloroform/light petroleum procedure for lipopolysaccharides (16Brade H. Galanos C. Eur. J. Biochem. 1982; 122: 233-237Crossref PubMed Scopus (69) Google Scholar). The SDS-polyacrylamide gel (PAGE) analysis was carried out on 14 and 18% (w/v) gels following the method developed by Laemmli (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar). The staining was performed in ammoniacal silver nitrate solution after fixation in 40% ethanol, 5% acetic acid and oxidation with additional 0.7% periodate. Phosphorus was determined according to Lowry et al. (18Lowry O.H. Roberts N. Leiner K. Wu M. Farr L. J. Biol. Chem. 1954; 207: 1-17Abstract Full Text PDF PubMed Google Scholar) and HexN after strong overnight hydrolysis according to Strominger et al. (19Strominger J. Park J.T. Thompson R. J. Biol. Chem. 1959; 234: 3263-3267Abstract Full Text PDF PubMed Google Scholar). To estimate normal and substituted amino sugars, an automatic amino acid analyzer was used (Chromakon 500, Kontron, Germany). The neutral sugars were determined as their alditol acetates according to Sawardeker et al. (20Sawardeker J.S. Sloneker J.H. Jeanes A. Anal. Chem. 1965; 37: 1602-1604Crossref Scopus (1487) Google Scholar). The sugar and fatty acid analysis was performed by gas-liquid chromatography on a Varian aerograph (model 3700) or by combined gas-liquid chromatography/mass spectrometry (MS) on a Hewlett-Packard instrument (model 5985) (see Ref. 21Kawahara K. Brade H. Rietschel E.T. Zähringer U. Eur. J. Biochem. 1987; 163: 489-495Crossref PubMed Scopus (94) Google Scholar for further details). The lipid extracts OML521 of the outer sheath, freeze dried at pH 7.0, were reconstituted in double distilled water with a final concentration of 5 mg/ml. The samples were pulse-sonicated at room temperature three times, directly before preparation. For analytical purposes, 25-μl drops of lipid suspension were placed on an infrared-transparent ZnSe window and dried down as films under mild vacuum conditions (60 mbar). The dried film experiments were performed on an automated sample wheel with 16 sampling positions (22Helm D. Naumann D. Microbiol. Lett. 1995; 126: 75-80Crossref Google Scholar). Spectra of dried samples were obtained by co-adding 256 interferograms at 2 cm−1 spectral resolution on a Bruker IFS-25/B spectrometer. Measurements of the lipid order parameter were performed on a Bruker IFS-66 spectrometer using a concentrated lipid suspension in D2O (∼100 mg/ml gel pellet) placed between two CaF2 windows with a pathlength of 50 μm and placed into a temperature cell holder (12Naumann D. Schultz C.P. Sabisch A. Kastowsky M. Labischinski H. J. Mol. Struct. 1989; 214: 246-313Crossref Scopus (75) Google Scholar, 13Schultz C.P. Naumann D. FEBS Lett. 1991; 294: 43-46Crossref PubMed Scopus (27) Google Scholar, 14Schultz C.P. Untersuchungen zur Membran-Organisation Lebender Bakterien unter Besonderer Berücksichtigung des Thermotropen Phasenverhaltens Rekonstituierter Lipopolysaccharid-Doppelschichten: Eine Fourier Transform Infrarot-spektroskopishe Studie (FT-IR) an Lipopolysacchariden und LPS-Mutanten von Salmonella minnesota und Salmonella typhimurium.Ph.D. thesis. Free University, Berlin, Germany1993Google Scholar). A linear temperature gradient was applied from 2 to 80 °C at a heating rate of 0.2 °C/min. Spectral analysis of the temperature measurements was performed, applying a center of gravity algorithm (14Schultz C.P. Untersuchungen zur Membran-Organisation Lebender Bakterien unter Besonderer Berücksichtigung des Thermotropen Phasenverhaltens Rekonstituierter Lipopolysaccharid-Doppelschichten: Eine Fourier Transform Infrarot-spektroskopishe Studie (FT-IR) an Lipopolysacchariden und LPS-Mutanten von Salmonella minnesota und Salmonella typhimurium.Ph.D. thesis. Free University, Berlin, Germany1993Google Scholar) to evaluate the accurate band position of the symmetric stretching vibration of methylene groups around 2850 cm−1 (functioning as a marker for membrane order). All spectra were automatically subtracted for remaining water vapor bands (23Fabian H. Schultz C.P. Naumann D. Landt O. Hahn U. Saenger W. J. Mol. Biol. 1993; 232: 967-981Crossref PubMed Scopus (139) Google Scholar). The lipid analysis of treponemes published in the past decade suggests that structure and function of the outer sheath of spirochetes is different to that of the outer membrane of Gram-negative bacteria, such as those from the family of Enterobacteriaceae. Some articles have reported that LPS can be found in treponemes (e.g. Refs. 24Beck G. Habicht G.S. Benach J.L. Coleman J.L. J. Infect. Dis. 1985; 152: 108-117Crossref PubMed Scopus (53) Google Scholar, 25Greer J.M. Wannemuehler M.J. Infect. Immun. 1989; 57: 717-723Crossref PubMed Google Scholar, 26Wannemuehler M.J. Hubbard R.D. Greer J.M. Infect. Immun. 1988; 56: 3032-3039Crossref PubMed Google Scholar). Other authors classified these structures as lipo-oligosaccharides because of their similarities to LPS without having the key components Kdo and β-hydroxy fatty acids (e.g. Refs. 5Takayama K. Rothenberg R.J. Barbour A.G. Infect. Immun. 1987; 55: 2311-2313Crossref PubMed Google Scholar, 27Brondz I. Fiehn N.E. Olsen I. Sjöström M. APMIS. 1991; 99: 567-575Crossref PubMed Scopus (7) Google Scholar, 28Livesley M.A. Thompson I.P. Gern L. Nuttall P.A. J. Gen. Microbiol. 1993; 139: 2197-2201Crossref PubMed Scopus (16) Google Scholar, 29Yotis W. Keene J. Hoerman K. Simonson L.G. J. Periodontal Res. 1993; 28: 387-395Crossref PubMed Google Scholar). Even in Borrelia burgdorferi, unusual LPS-like compounds were discovered, suggesting that there is probably no LPS present in the genusBorrelia (30Cinco M. Banfi E. Balanzin D. Godeas C. Panfili E. FEMS Microbiol. Immun. 1991; 76: 33-38Crossref Google Scholar). The electron microscopical analysis of single T. denticola cells clearly indicates a typical bilayer structure not only for the cytoplasmic membrane but also for the outer sheath (see Fig. 1 a, showing a small segment of the helical form). The unstained (hydrophic) inner part of the bilayers indicates a similar thickness for both membranes. The outer sheath is generally positioned further from the cytoplasmic membrane than is usual for Gram-negative bacteria and also demonstrates great variation in separation along its length. Fig. 1 b shows a section directly through the body of a single cell and demonstrates the perfect cylindrical shape of the cell (protoplasmic cylinder). The outer sheath tightly covers the cell body, including its axial flagellae. The darker deposits on top of the outer sheath could indicate the existence of polymeric material such as is present in LPS with its O-antigenic structure (31Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar). Fig. 2 a shows the typical SDS-PAGE electrophoretic pattern of a lipid fraction phenol/chloroform/light petroleum-extracted from dried cells of T. denticola strain ATCC 33521 (lanes 1–5), directly compared with well known lipopolysaccharide samples isolated from enterobacteriaceae (lanes A–D). In contrast to protein standards, the LPS lanes (A–D) perfectly describe the molecular weight distribution of complex lipids such as LPS, which is essential to approximate the correct size of the newly isolated lipid. The wild-type LPS in lane A (densitogram shown in Fig. 2 b) fromPseudomonas aeruginosa F2 indicates a typical band pattern starting with RaLPS, the complete core structure (32Holst O. Brade H. Carbohydr. Res. 1993; 245: 159-163Crossref PubMed Scopus (7) Google Scholar), followed by multiple repeating units with a size of 0.534 kDa (33Dmitriev A. Knirel Y.A. Kocharova N.A. Kochetkov N.K. Stanislavsky E.S. Mashilova G.M. Eur. J. Biochem. 1980; 106: 643-651Crossref PubMed Scopus (43) Google Scholar). Lanes B–D represent LPS (from strains Salmonella helsinkii777, Salmonella minnesota R60, and E. coli F515) with enzyme defects in their LPS biosynthesis, in which lane B shows only RaLPS and one repeating unit, lane C pure RaLPS, and lane D ReLPS having only lipid A and Kdo (32Holst O. Brade H. Carbohydr. Res. 1993; 245: 159-163Crossref PubMed Scopus (7) Google Scholar). The isolated lipid fraction OML521 (lanes 1–5 in Fig. 2 a and densitograms in Fig. 2 c) seems to contain only one major molecular weight population around 4 kDa with little variability (lane 1). Only a small number of molecules (<10%) have a size greater than 4 kDa and are organized into two different populations of structures: (i) one with only an additional large unit of approximately 2 kDa in size and (ii) others with an additional pattern-like structure of a smaller unit size (about 0.7 kDa each). Even in very concentrated lanes of OML521, no repeating structure has been observed between 4 and 6 kDa, suggesting a well defined core-like structure essential for the connection with repeating units such as in LPS. The lipid fraction with the largest detectable molecular size had a molecular mass of approximately 10.2 kDa. Biochemical studies of isolated outer sheath (34Yotis W.W. Sharma V.K. Gopalsami C. Chegini S. McNulty J. Hoerman K. Keene J. Simonson L.G. J. Clin. Microbiol. 1991; 29: 1397-1406Crossref PubMed Google Scholar, 35Yotis W.W. Macaluso F. Gopalsami C. J. Basic Microbiol. 1995; 35: 255-268Crossref PubMed Scopus (12) Google Scholar) indicate the possibility of larger sized lipids with up to 21 kDa for the same strain, but their extraction pretreatment (adding MgCl2) induced a separation in aggregable and nonaggregable moieties, which could have enhanced minor parts of very large and perhaps different lipid molecules (such as the enterobacterial common antigen present inEnterobacteriaceae). Others suggest that an LPS-like structure exists in a different strain of T. denticola (ATCC 33520) on account of their findings of proteinase-indigestible low molecular weight molecules in silver-stained gels (36Cockayne A. Sanger R. Ivic A. Strugnell R.A. MacDougall J.H. Russell R.R.B. Penn C.W. J. Gen. Microbiol. 1989; 135: 3209-3218PubMed Google Scholar). An interesting observation in our study is the actual size of OML521, which is around the same size as RaLPS, a structure essential to the assembly of perfectly active porin-trimers in normal outer membranes (37Sen K. Nikaido H. J. Bacteriol. 1991; 173: 926-928Crossref PubMed Google Scholar). The discovery of LPS-like structures in different genera of spirochetes and the highly variable chemical description of these lipid fractions led us to conduct a more detailed chemical analysis on the outer sheath lipid composition of T. denticola. The phenol/chloroform/light petroleum extraction, developed for LPS isolations, indicated that 1.4% (possibly more) of the dry cell weight is due to outer sheath lipids, which is in good agreement with normal LPS isolations fromEnterobacteriaceae. This would also be a quite sufficient amount of lipid to cover the outer leaflet of the outer sheath. OML521 consists of 4.5% (252 nmol/mg lipid) Glc (partially phosphorylated in 6-position according to high voltage paper electrophoresis (HVPE) and amino acid analysis), 2.7% (151 nmol/mg lipid) Gal, 0.5% (26 nmol/mg lipid) Man, and no detectable quantity of Hep, which is normally essential for LPS structures with a 4-kDa size. Two types of amino sugars are present having a relatively high proportion of about 1.7% (93 mmol/mg lipid) GlcN and 4.8% (268 nmol/mg lipid) GalN of the total lipid weight. The thiobarbituric acid assay (used for detecting Kdo) indicates only traces of stained material, and HVPE clearly demonstrates that no Kdo is present in OML521. The isolated lipid is very rich in phosphate (13.6% by molybdate assay) and contains an enormous amount of 35.3% carbazole-positive (CA) compounds. HVPE experiments clearly show that the source for the CA-positive reaction is not a typical uronic acid such as GlcA or GalA, which would normally be stained by this method. Despite the negatively charged GlcA and GalA, the uronic acid compound found in HVPE, is positively charged overall and seems to be a special sugar, containing additional amino components. The large quantity of this particular compound suggests that most of the lipid core structure is dominated by a positively charged molecular structure, partly compensated by phosphate groups. In addition, we also found other small components in detectable quantities, e.g. alanine, citric acid, and glycerol, which can vary the overall charge of the OML glycolipid structure. The membrane anchor itself has quite a different fatty acid composition compared with that of normal lipopolysaccharides (see TableI) and is missing 3-hydroxy fatty acids (e.g. myristic acid in many Salmonellae) essential to the manufacturing of lipid A-like structures. Only recently has it been reported that the T. denticola strain FM contains iso- and anteiso-hydroxy fatty acids, but their quantity is still too low to create a functioning lipid A structure (38Dahle U.R. Tronsstad L. Olsen I. Endod. Dent. Traumatol. 1996; 12: 202-205Crossref PubMed Scopus (13) Google Scholar). At 5.5% (214 nmol/mg lipid), palmitic acid is the major fatty acid in OML521 (also dominant in isolated phospholipids from the same strain, data not shown) and represents half of all fatty acids present in this lipid fraction. The high content of iso- and anteiso-fatty acids in outer sheath and cytoplasmic membrane indicates that the adjustment of membrane fluidity in T. denticola is similar to Gram-positive bacteria such as Staphylococcus aureus. Many Gram-negative bacteria can change their membrane fluidity by modifying the quantity of double bond-carrying fatty acids using enzymes within the membrane, whereas many Gram-positive cells manipulate the quantity of iso- and anteiso-fatty acids to normal saturated chains, which requires complete synthesis of new fatty acids. Also very interesting is that relative to the molecular mass of about 4 kDa, the overall proportion of fatty acids in OML521 is unexpectedly low (10.7%), as LPS structures of this size normally contain more than 20% fatty acids. This all suggests that the membrane anchor may be significantly smaller and might possibly consist of a phospholipid-like or glycerolipid-like structure containing only two fatty acids instead of six in lipid A.Table IFatty acids (FA) found in outer sheath lipid OML521 and in total cell membrane extracts of T. denticola ATCC 33521Identified constituentLipidLipid structureTotal cell FA extractnmol/mg%%All fatty acids—10.69100n-C12:0TraceTraceTracen-C12:0 (2-OH)———n-C12:0 (3-OH)———n-C13:0——Tracei-C14:02.360.053.42n-C14:014.950.3411.40Di-Me-ac i-C14:0NDND—Di-Me-ac n-C14:0NDND—n-C14:0(2-OH)———n-C14:0(3-OH)———ai-C15:054.081.3114.46n-C15:022.220.549.96Di-Me-ac C15:0NDND—i-C16:078.302.014.15n-C16:0213.835.4824.13n-C16:1——6.11n-C16:0 (2-OH)———n-C16:0 (3-OH)———ai-C17:018.220.49Tracen-C17:04.510.12Tracen-C18:012.290.353.50n-C18:1——14.14n-C18:2——5.60ai-C19:0TraceTrace3.13ai-C19:0TraceTraceTraceThe abbreviations used are: Di-Me-ac, dimethyl-acetate; i-, iso-; ai-, anteiso-; ND, not determined; —, not found. Open table in a new tab The abbreviations used are: Di-Me-ac, dimethyl-acetate; i-, iso-; ai-, anteiso-; ND, not determined; —, not found. Phospholipid-like membrane anchors of complex polymeric lipids are well known and can be found in the outer sheath of the cytoplasmic membranes of Gram-positive bacteria, the lipoteichoic acids (LTA) (39Fischer W. Rosel P. Koch H.U. J. Bacteriol. 1981; 146: 467-475Crossref PubMed Google Scholar, 40Labischinski H. Naumann D. Fischer W. Eur. J. Biochem. 1991; 202: 1269-1274Crossref PubMed Scopus (31) Google Scholar). This molecule is simply composed of a phospholipid (functioning as membrane anchor) and a long chain of repeating units, which consists of alternating phosphate and glycerol residues. The isolated fatty acid composition was consistently very similar to that of the bacterial phospholipids located in the cytoplasmic membrane. Chemical similarities between parts of LTA and OML521 can be simply demonstrated by comparing infrared spectra of both lipids to that of LPS (see Fig. 3). Fig. 3, A and B (showing spectra of two fractions of OML521 differing only in molecular weight distribution), clearly indicates a polymeric lipid structure showing some spectral similarities to both lipid polymers LTA and LPS (spectra Cand D), respectively. The range between 700 and 1300 cm−1 seems to be quite similar to LTA, exhibiting a spectral character that suggests a polymeric phosphorylated backbone structure (see spectral range 1). Other characteristic bands are those marked 2 and 3, representing the symmetric and asymmetric deformation vibration bands of methyl groups at 1377 cm−1 and at 1455 cm−1, respectively. Even LTA, a polymeric lipid extensively substituted by alanine, does not show comparably strong methyl bands in this region, which is an indication of a relatively large proportion of methylated structures present in OML521. These bands also seem to correlate to the polymeric parts of the isolated lipid, indicated by increased intensities in spectra of fractions containing larger sized OML521 components (compare spectrumA with B). Similar observations can be made for increased relative intensities of the bands 4 and 5 (at 1575 and 1657 cm−1) possibly representing the amide II and I bands of the secondary amide functional groups. Absorption band 6 reflects carbonyl stretching vibrations of esters and/or carbonic acid compounds, which differ in spectra of fractions containing more high or low molecular components. The comparison of both OML521 spectra in the region 2800–3000 cm−1 indicates that in spectrumA the lipid part (see bands 7 and 8) is more dominant than in spectrum B, correctly evidencing the relatively larger lipid structure. Finally, the absence of protein in the isolated lipid fractions (proved by amino acid analysis, SDS-PAGE, proteinase degradation, and FTIR spectroscopy) suggests that the specific increase in intensity of bands 2–6 in spectrum B may simply reflect a higher degree of N- and O-acetylation in addition to an overall higher content of sugars, similar to that found for the multiple substitution of LTA with alanine. One major difference between LPS and LTA is their propensity to form lamellar bilayer vesicles (LPS) or micelles (LTA). Isolated LPS can form perfectly assembled bilayer structures when reconstituted in water (12Naumann D. Schultz C.P. Sabisch A. Kastowsky M. Labischinski H. J. Mol. Struct. 1989; 214: 246-313Crossref Scopus (75) Google Scholar). By contrast, LTA can only form micellar structures and shows no tendency to undergo vesiculation (40Labischinski H. Naumann D. Fischer W. Eur. J. Biochem. 1991; 202: 1269-1274Crossref PubMed Scopus (31) Google Scholar,41Fischer W. Markwitz S. Labischinski H. Eur. J. Biochem. 1997; 244: 913-917Crossref PubMed Scopus (20) Google Scholar). It is, therefore, very interesting to gain more insight into the biophysical membrane behavior of OML521 to achieve a better understanding of how the outer sheath membrane of spirochetes can cover the cell (in vivo) without being closely connected to the cell wall. For this reason, we undertook experiments to test the thermotropic phase behavior of reconstituted membranes of OML521, LPS, and LTA (see Fig. 4). It is well established that FTIR spectroscopy can provide information on organization and structure of various lipid bilayers and isolated membranes and is a very sensitive means to determining structural and dynamic properties of lipids (11Mantsch H.H. McElhaney R.N. Chem. Phys. Lipids. 1991; 57: 213-226Crossref PubMed Scopus (468) Google Scholar, 12Naumann D. Schultz C.P. Sabisch A. Kastowsky M. Labischinski H. J. Mol. Struct. 1989; 214: 246-313Crossref Scopus (75) Google Scholar). The temperature profiles (Fig. 4) demonstrate two typical differences usually observed between bilayer-forming LPS and micelle-type organized LTA: (i) higher frequency values of the >CH2 symmetric stretching band (2 cm−1 higher than RaLPS at any given temperature value), indicating much less ordered acyl chains in LTA micelles, and (ii) absence of any kind of phase transition in LTA micelles typical for non-lamellar arrangements. The temperature profile of OML521 indicates strong evidence for a well ordered membrane that undergoes a two-state phase transition similar to isolated LPS. However, in contrast to RaLPS (see circles in Fig. 4), which is similar in molecular size to the main compound of OML521 (see squares in Fig. 4), the phase transition seems to be significantly broader than for LPS (Δ23 °C versus Δ15 °C) and induced at much lower temperatures Tm (22.5 versus35.5 °C). The slightly higher frequency of the >CH2symmetric stretching band also indicates that OML521 is not as perfectly well ordered as LPS when reconstituted in water after extraction. Interestingly, the fatty acid composition of OML521 is quite similar to that of LTA, which was isolated from the cytoplasmic membrane of Gram-positive S. aureus (data not shown). Compared with OML521 and LPS, the biophysical properties of LTA allow a single molecule only a small degree of change within a micelle as a function of temperature and can only be modified to a certain extent by reducing alanine substitution in the polymeric part (triangles versus black triangles in Fig. 4). The missing d-alanine substituents and the higher number of negative charges (stretching phosphate-glycerol chain of the LTA molecule) decrease the required space within a micelle at the hydrophobic surface and therefore lead to a slightly better acyl chain packing. The fatty acid composition, the missing Hep and Kdo, and the high levels of phosphate and a carbazole-positive component are all properties that place the treponemal OML521 chemically very close to LTA, whereas the biophysical behavior of OML521 clearly indicates that this lipid has membrane properties similar to LPS. The relatively small proportion of fatty acids compared with the rest of the OML521 components also indicates that OML521 may not contain a typical lipid A structure. To examine the chemical nature of the lipid component, OML521 was treated with hydrofluoric acid (HF). In this manner, we expected to find either a complete hydrolysis of the molecule, if the basic structure followed LTA architecture, or an almost unaltered OML521, if it had more of an LPS architecture. The basis of this experiment is that HF removes all phosphates from the OML521 structure, which breaks the molecule down in smaller fragments if phosphodiesters are connecting parts of the molecule (such as in LTA). The comparison of lane G with lane H in Fig. 5 confirms the idea of a basic core lipid due to the observation that the HF treatment can only reduce the lipid size of OML521. A structure as small as 2.3 kDa of mass (57% of the original) still remains after treatment with HF and dialysis against water. This experiment seems to validate the existence of a non-phosphate-mediated connection between the core and the lipid part in OML521 similar to that in LPS (see model in Fig. 5). In case of LTA, HF treatment removes the repeating units completely, and only a small core and the lipid anchor remain (see model in Fig. 5). Because the phosphates can be completely removed from OML521, they should only be connected to the boundary of the remaining (quite large) lipid molecule forming a negatively charged phosphate shell. The missing pattern of the repeating units in OML521HF suggests a more LTA-like structure of the polymeric molecule parts. The chain could simply be connected by one phosphate or even composed of a phosphate-containing repeating unit (see both models in Fig. 5). In addition, the fatty acid analysis of OML521HF indicates nearly the same pattern as seen for the original OML521, and the two LPS characterizing sugars Hep and Kdo (which could have been masked by phosphates) were also not present in the dephosphorylated form. Because we did not find any sphingosine derivatives after hydrolysis, it can also be concluded that the membrane anchor of T. denticola does not contain any sphingolipid structures,e.g. the outer membrane lipids of Sphingomonas paucimobilis (42Kawahara K. Seydel U. Matsuura M. Danbara H. Rietschel E.T. Zähringer U. FEBS Lett. 1991; 292: 107-110Crossref PubMed Scopus (130) Google Scholar, 43Kawasaki S. Moriguchi R. Sekiya K. Nakai T. Ono E. Kume K. Kawahara K. J. Bacteriol. 1994; 176: 284-290Crossref PubMed Scopus (140) Google Scholar). Instead, we found and identified three molecular fragments, indicating evidence for a membrane anchor structurally similar to those in LTA (44Ratledge C. Wilkinson S.G. Microbial Lipids. 1. Academic Press, New York1988Google Scholar) of Gram-positive bacteria (see Fig. 6). By referring to library components, the 3 EI mass spectra indicate a possible hexosyl-diacyl-glycerol lipid in which all three glycerol positions are connected either to fatty acids or sugars and are therefore not available for phosphates (excluding a simple phospholipid structure). In addition, the hexadecanoic acid representing 50% of all fatty acids in the T. denticola lipid anchor forms two isomeric structures with glycerol:one in the second and the other one in the third position of the glycerol (see fragments II and III in Fig. 6). All these findings suggest a new type of glycolipid structure that differs significantly from that of LPS. As in Gram-negative bacteria, this novel structure is the determining factor of the stability, flexibility, and functionality of the outer sheath of T. denticola. Most important, the absence of structural components essential for LPS (Hep, Kdo, and β-hydroxy fatty acids), the polymeric lipid character and the relatively high membrane order together with a well expressed phase behavior, provides strong evidence for a possibly new lipid structure, which may also be directly responsible for variations in immunological host response. The specific structure of OML521 seems to simulate well known cellular surface antigens, which allows Treponema cells to invade the host tissue undetected (without triggering the body's defense mechanism) and may be responsible for their extremely long survival rates. It is expected that a better understanding of the structure and function of this particular outer sheath could provide new strategies on how to reduce the long survival rates of Treponema cells in human tissues and how to act against them more effectively using antibiotics or more specific monoclonal antibodies. It may be possible thatTreponema (or all spirochetes) represent a second Gram-negative-like class of bacteria containing only a non-lipopolysaccharide structure in the outer membrane, e.g.the sphingolipids in S. paucimobilis (42Kawahara K. Seydel U. Matsuura M. Danbara H. Rietschel E.T. Zähringer U. FEBS Lett. 1991; 292: 107-110Crossref PubMed Scopus (130) Google Scholar, 43Kawasaki S. Moriguchi R. Sekiya K. Nakai T. Ono E. Kume K. Kawahara K. J. Bacteriol. 1994; 176: 284-290Crossref PubMed Scopus (140) Google Scholar). We gratefully acknowledge the skillful technical assistance of Sabine Barten, Stefanie Pautz, and Herrmann Moll. We thank Dr. Wolfgang Fischer for the supply of isolated, purified, and chemically well characterized lipoteichoic acid samples." @default.
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W1974565437 date "1998-06-01" @default.
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W1974565437 title "Evidence for a New Type of Outer Membrane Lipid in Oral Spirochete Treponema denticola" @default.
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W1974565437 doi "https://doi.org/10.1074/jbc.273.25.15661" @default.
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