Novel Bacterial Polar Lipids Containing Ether-linked Alkyl Chains, the Structures and Biological Properties of the Four Major Glycolipids from Propionibacterium propionicum PCM 2431 (ATCC 14157T)*

Mariola PasciakDagger , Otto Holst§, Buko Lindner, Halina MordarskaDagger , and Andrzej GamianDagger ||

From the Dagger  Institute of Immunology and Experimental Therapy, the Polish Academy of Sciences, Weigla 12, Wrocław PL-53-114, Poland and the Divisions of § Structural Biochemistry and  Biophysics, Borstel Research Center, Parkallee 22, Borstel D-23845, Germany

Received for publication, June 17, 2002, and in revised form, November 5, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Propionibacterium propionicum belongs to the "acnes group" of propionibacteria, which is currently considered as clinically important because of its growing potential in infections, in particular with those connected with immune system dysfunctions. Propionibacteria are thought to be actinomycete-like microorganisms and may still cause diagnostic difficulties. The chloroform-methanol extracts of the cell mass of P. propionicum (type strain) gave in TLC analysis the characteristic glycolipid profile containing four major glycolipids, labeled G1 through G4. These polar lipids were found to be useful chemotaxonomic markers to differentiate P. propionicum from other cutaneous propionibacteria, in particular from strains of the acnes group. Glycolipids G1-G4 were isolated and purified using gel-permeation chromatography, TLC, and high performance liquid chromatography, and their structures were elucidated by compositional and methylation analyses, specific chemical degradations, MALDI-TOF mass spectrometry, and 1H NMR and 13C NMR spectroscopy, including HMBC, TOCSY, HMQC, and NOESY experiments. Glycolipids G2 and G3 possess as backbone alpha -D-Glcp-(1 right-arrow 3)-alpha -D-Glcp-(1 right-arrow 1)-Gro (Gro, glycerol), in which position O-2 of the glycerol residue is acylated by a fatty acid (mainly C15:0) while O-3 is substituted by an alkyl ether chain. In glycolipid G3, an additional fatty acyl chain was linked to O-6 of the terminal glucose residue. Glycolipid G4 was structurally related to G2 but devoid of one glucose residue. Glycolipid G1 was isolated in small amounts, and its structure was therefore deduced from MALDI-TOF-MS experiments alone, which revealed that it possessed the structure of G2 but was lacking one fatty acid residue. In studies on the biological properties of P. propionicum glycolipids, the anti-P. propionicum rabbit antisera reacted in dot enzyme-immunoblotting test with G2 and G3. Glycolipid G3 was able to induce the delayed type of hypersensitivity. The results indicated that these novel ether linkage-containing polar glycolipids are immunogenic and possibly active in hypersensitivity, and thus, in pathogenesis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Propionibacteria represent a heterogeneous group of anaerobic or microaerophilic Gram-positive microorganisms with the characteristic feature of producing propionic acid. Generally, on the basis of their habitat, members of the genus are divided into two groups (1). So-called classic propionibacteria are not pathogenic and, like Propionibacterium freudenreichii, P. thoenii, P. jensenii, P. acidipropionici, and P. cyclohexanicum originate mainly from dairy products like cheese, milk products, fermented olives, or orange juice but have also been isolated from soil, silage, or other organic matter of natural environments. The second group, the so-called skin or cutaneous propionibacteria, or acnes group, is considered to consist of pathogenic strains, including species like P. acnes, P. avidum, P. granulosum, P. lymphophilum, P. propionicum, and P. innocum. They belong to the endo- and exogenous bacterial flora of human mucosa and skin and are among the indigenous oral flora and intestinal tract, occurring sometimes as opportunistic pathogens. Also, they are found in cervicovaginal secretions of healthy women as well as in secretions of uninfected conjunctiva and cornea. Besides the typical skin sickness, such as acne vulgaris or actinomycosis-like diseases caused by P. acnes or P. propionicum, respectively (2, 3), propionibacteria are found more recently to be involved in endocarditis, arthritis, rheumatism, endophthalmitis (4-6) and many other nonspecific, single or mixed, systemic, or disseminated, primary or secondary, opportunistic infections (7-10). Most of them can be dangerous for high risk patients suffering from serious devastating illnesses, including tumors, tuberculosis, AIDS, organ and tissue transplantation, or foreign body implantation joining to deeply immunosuppression. Interestingly, some of these bacteria were used recently as immunostimulators, serving a protective role against virus, parasite, and bacterial infections or as antitumor adjuvants.

Diagnostic difficulties are caused by specific nutritional conditions, microaerophilic preferences, and prolonged time of incubation for cultivation as well as polymorphic growth, which causes a different Gram staining. The classic as well as the opportunistic propionibacteria may still cause confusion not only in taxonomy but also in microbiological and clinical diagnoses. Thus, alternative techniques for the differentiation of the species are still needed. The modern era of microorganism systematics, proposed for Actinobacteria classis novum, including Propionibacterium (11), has been based on a so-called polyphasic strategy. Its origin has three dominant sources: first, the establishment of chemotaxonomy, i.e. structural constituents such as peptydoglycan, fatty acids, polar lipids, isoprenoid chinones, cytochromes, and the base composition of DNA (12-16), all of which are important at each level of bacterial relationship; second, DNA-DNA reassociation of closely related organisms at the species level; and third, 16 S rRNA and rDNA analyses of strains at all levels of relatedness (17), including PCR (18) and/or PCR/restriction fragment length polymorphism, which has rarely been used so far for the identification of the classic and skin propionibacteria (19, 20).

It is worth emphasizing that a clear correlation exists between the chemotaxonomic and genotaxonomic characteristics of microorganisms, especially with regard to their lipid composition, mainly for the polar lipids and 16 S rRNA-DNA hybridization homology groups (21, 22). Therefore, some major glycolipids appear to be useful for the rapid recognition of bacterial isolates at the genus or species level (23-28).

P. propionicum was initially classified as Actinomyces propionicus. However, owing to the production of propionic acid, the presence of LL-diaminopimelic acid and glycine in its peptidoglycan, this species was included in another taxon, i.e. Arachnia propionica. Then, further studies revealed metabolic and genetic relationships with Propionibacterium, which resulted in its reclassification to P. propionicum. The studied strain belonged to serotype 1 and was isolated from an advanced periodontal lesion. The identification of these bacteria by applying molecular biological techniques is questionable because of the coexistence of numerous endogenous bacterial floras. The analysis of the glycolipid profile is a promising method of choice for the identification of these bacteria and the diagnosis of infections caused by them.

This work describes a study of the structures and biological properties of glycolipids isolated from P. propionicum. Structurally uncommon polar glycolipids, which occur in P. propionicum, are of taxonomic value and allow us to distinguish this taxon from other propionibacteria as well as from all microaerophilic actinobacteria (25, 29). The characteristic glycolipid profile of P. propionicum contains four major glycolipids possessing novel structures, which, together with their biological properties, are reported here.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Microorganisms and Growth Conditions-- The object of this investigation was the type strain P. propionicum PCM 2431 (ATCC 14157T). Other strains were also used in comparative experiments, namely P. acidipropionici PCM 2434 (NBIMCC 1371), P. acnes PCM 2400T (ATCC 6919T), P. avidum PCM 2467 (CIP 103261T, ATCC 25577), P. freudenreichii PCM 2435 (NBIMCC 1207T, ATCC 9614), and P. granulosum PCM 2462 (ATCC 25564T). Bacteria were cultivated anaerobically in an atmosphere containing CO2 (4-10%) in flasks with thioglycollate broth (Difco)/trypticase soy broth (BioMerieux) (1:1, v/v) at 37 °C for 5-7 days. The cultures were checked for purity then heat-killed, harvested by centrifugation, and washed with saline.

Thin-layer Chromatography-- Samples (4 µl from 50 mg/ml solutions) were applied to pre-coated TLC plates (Silica Gel 60, Merck) and developed using solvent system I (chloroform-methanol-water, 65:25:4, v/v). In the case of two-dimensional TLC, solvent system I was used in the first dimension and chloroform-acetic acid-methanol-water (80:15:12:4, v/v, solvent system II) was used in the second. Also, solvent system I was applied in the first dimension, and then chloroform-acetone-methanol-water (50:60:2.5:3, v/v, solvent system III) was applied in the second dimension. Several specific spray reagents were used to develop the chromatograms, namely vanillin, orcinol, ninhydrin, and Dittmer and Lester reagent (25).

Extraction of Lipids-- The wet cell mass was extracted twice by stirring with chloroform-methanol (2:1, v/v, 15 ml/g wet mass) at 37 °C for 12 h. The combined extracts were evaporated by rotary evaporation and then suspended in chloroform-methanol-water (5:5:4.5, v/v) to remove non-lipid material (30). Finally, the dried crude extract was dissolved in CHCl3 (50 mg/ml) and monitored by TLC for a specific glycolipid pattern as described before (25). This material was kept under N2 in the freezer before further fractionation.

Separation and Purification of Glycolipids-- The crude lipid extract (500 mg) was fractionated on a column (1.8 × 45 cm) of activated Silica Gel 60 (Merck, 70-230 mesh) eluted successively with the solvents chloroform (4× 100 ml), acetone (5× 100 ml), and methanol (4× 100 ml). The eluates were monitored for polar lipids on silica gel TLC-plates using solvent system I and vanillin reagent. The acetone-soluble fractions that contained glycolipids were further fractionated on a column (1.2 × 35 cm) of Silica Gel 60 (Merck, 200-300 mesh) with gradually increased concentrations of methanol in chloroform (0, 5, 10, 15, 20, and 30 vol%) (31). Fractions of 50 ml were collected. The eluates were monitored by TLC as before. Those glycolipid fractions that contain similarly migrating material were combined and further purified (up to 2-3 times, depending on the degree of contamination) by preparative TLC (28). Briefly, 30-40 mg of a sample per Silica Gel F 254 (Merck) plate was developed in chloroform-acetic acid-methanol-water (80:15:12:4, v/v) for the G1 and G2 pair of glycolipids and in chloroform-acetone-methanol-water (50:60:2.5:3, v/v) for the G3 and G4 fractions. Glycolipid bands were detected with a UV lamp and eluted from silica gel with different chloroform-methanol solvents (2:1, 1:1, and 1:2, v/v). The final purification of fraction G3 was performed by high-performance liquid chromatography (HPLC)1 on a Waters chromatography system equipped with an M600E pump and an M996 photodiode array detector, which were controlled by the software Millennium 2010. A column (4 × 250 mm) of Nucleosil 100-5 was eluted with a linearly increasing gradient of 5-15 vol% methanol in chloroform. Fractions were collected every 30 s, over 20 min, and the degree of purification was monitored by two-dimensional TLC.

Analytical Methods-- The total neutral sugar content was determined by the phenol/sulfuric acid method (32). Glucose was determined colorimetrically (33). The sugar composition was identified by gas-liquid chromatography/mass spectrometry (GLC/MS) after acidic hydrolysis of the lipids (34). For fatty acid analysis, aliquots (0.2 mg) of the purified glycolipids were dissolved in 0.5 ml of 1 M methanolic HCl and incubated at 80 °C for 1 h (35). After drying in a stream of nitrogen, the liberated fatty acid methyl esters were directly analyzed by GLC/MS. In another experiment, the methanolized and dried sample was treated with 100 µl of bis(trimethylsilyl)trifluoracetamide (Sigma) at 65 °C for 4 h, and the obtained trimethylsilyl (TMS) derivatives were identified by GLC/MS. Glycerol was determined in acetate form by GLC/MS (34), using the program with lower temperature starting from 100 °C, and the integrated area of the peak was counted using the factor calculated from the sample of glycerol with glucose, separately run as a standard mixture.

Methylation Analysis-- The glycolipid fraction (0.3 mg) was methylated according to Hakomori (36) and purified on a SepPak C18 cartridge (37). The product was hydrolyzed with HCl in 80% CH3COOH (0.6 M, 80 °C, 18 h), reduced with NaBD4, and acetylated. Partially methylated alditol acetates were analyzed by GLC/MS. The position of the O-acylation of the sugars was established by methylation analysis according to Prehm (38). Here, the glycolipid (0.3 mg) was solubilized in trimethyl phosphate (0.9 ml) and ultrasonicated with 2,6-di-(tert-butyl)pyridine (0.15 ml) and methyl trifluoromethanesulfonate (0.15 ml) at 50 °C for 2 h. The product was extracted twice with chloroform-water (1:1, v/v), and the organic layer, after drying, was methylated again according to Hakomori (36) using deuterated methyl iodide. The methylated product was hydrolyzed with HCl in 80% CH3COOH (0.6 M, 80 °C, 18 h), reduced with NaBD4, acetylated, and analyzed by GLC/MS.

Determination of the Absolute Configuration of the Sugar Constituents-- The glycolipid (0.4 mg) was hydrolyzed with 1 M HCl at 100 °C for 4 h. D-Glucose was quantified from the dried neutralized sample by enzymatic procedure with D-glucose oxidase (39).

Mass Spectrometry-- GLC/MS was performed on a Hewlett-Packard 5971A gas-liquid chromatograph-mass spectrometer equipped with an HP-1 glass capillary column (0.2 mm × 12 m). A temperature program of 150-270 °C at 8 °C/min was applied.

MALDI-TOF mass spectra of native glycolipids were recorded with a Bruker-Reflex II spectrometer (Bruker-Franzen Analytik, Bremen, Germany) in both linear and reflection TOF configuration at an acceleration voltage of 20 kV and delayed ion extraction. The glycolipids were dissolved in chloroform-methanol (2:1, v/v, 1 µg/ml). One microliter of sample was then mixed with 1 µl of 0.5 M matrix solution of 2,5-dihydroxybenzoic acid (Aldrich) in methanol, and aliquots of 0.5 µl were deposited on a metallic sample holder and analyzed immediately after drying in a stream of air. The mass spectra shown are the sum of at least 50 laser shots and were calibrated externally with glycolipids of known chemical structure.

High resolution mass spectrometric analyses were performed in the positive ion mode using an electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer (APEX II, Bruker Daltonics, Billerica, MA) equipped with a 7-tesla actively shielded magnet and an Apollo ion source. Samples were dissolved in a 30:30 mixture of 2-propanol and 6 mM ammonium acetate, at a concentration of ~10 ng/µl, and sprayed at a flow rate of 2 µl/min. Broad-band mass spectra (1 M acquisition size) were acquired using standard experimental sequences as provided by the manufacturer.

NMR Spectroscopy-- The NMR spectra of glycolipids were recorded from solutions in CDCl3/CD3OD (2:1, v/v, 0.5 ml). Measurements were achieved at 27 °C relative to tetramethylsilane (delta H = 0.0) and CDCl3 (delta C = 77.0). For structural assignments, one- and two-dimensional 1H NMR spectra were recorded with a Bruker DRX 600 spectrometer (operating frequency, 600 MHz). 13C NMR spectra were recorded with a Bruker AMX-360 spectrometer (operating frequency, 90 MHz). The correlation (COSY), the heteronuclear multiple bond correlation (HMBC), and the total correlation spectroscopy (TOCSY) data were recorded using standard Bruker software. The heteronuclear multiple quantum coherence (HMQC) spectrum was measured in the 1H-detected mode via multiple quantum coherence with proton decoupling in the 13C domain. Nuclear Overhauser enhancement spectroscopy (NOESY) was measured using data sets (t1 × t2) of 2048 × 512 points, and 48 scans were acquired. A mixing time of 300 ms was employed.

Preparation of Rabbit Immune Sera-- The sera tested in the study were obtained from rabbits immunized with P. propionicum strain PCM 2431 (ATCC 14157T). Additionally, some control sera were used from rabbit individuals, before treatment. Rabbits (5-6 months old) were immunized twice a week with a cell mass of P. propionicum according to the schedule used previously (40) with a small modification. The cells were previously disintegrated in an X-Press (AB BIOX), lyophilized, and suspended in phosphate-buffered saline (PBS). The following antigenic doses were applied: 100 µg of dry cell mass/ml for the first subcutaneous injection and doubly increasing amounts (200, 400, 800, 1600, 3200, and 6400 µg/ml) for the succeeding intravenous injections. The antisera were checked by conventional passive hemagglutination test (PHT) using sheep erythrocytes coated with P. propionicum cellular antigens prepared earlier for immunization. Because of poor antigenicity of propionibacteria, the immunization was usually prolonged by two additional injections with the highest dose of cells (6400 µg/ml). After the last injection (7-10 days) the titers of antisera were verified by PHT, then rabbits were bled. The separated antisera were decomplemented (56 °C, 30 min) and stored at -20 °C.

Delayed-type Hypersensitivity-- Delayed-type hypersensitivity reaction was induced at the 2- and 4-week time points of the rabbit immunization experiment. Glycolipid suspensions in PBS (1000, 100 µg/ml) were prepared by sonication in ultrasonic water bath (37 °C, 3 min), and 0.1-ml doses were injected intracutaneously in the clipped paravertebral region of immunized animals. For control purposes 0.1 ml of bacterial cell suspension (1 mg/ml) or PBS was injected.

Dot-EIA-- A cellulose nitrate membrane (Micro Filtration Systems, Dublin, CA, pore size 0.45 µm) and the method employed by Papa et al. (41) (called here dot-EIA) were used with a small modification. Briefly, glycolipid antigens (dissolved in chloroform) were placed on nitrocellulose strips in an equal volume (1 µl) with decreasing concentrations (1000, 500, 100, 50, and 10 ng). Nitrocellulose was dipped in 0.2% casein in Tris-buffered saline, pH 7.5 (TBS) (with gentle shaking) for 2 h and rinsed twice with TBS. Then strips were incubated with specific antiserum diluted in TBS at 37 °C for 12 h. After the strips were washed in TBS (3×), the anti-rabbit IgG/horseradish peroxidase conjugate (DAKO) was used. The membranes were then incubated in the substrate 0.05% 4-chloro-1-naphtol (Sigma) at 20-22 °C, and washing with water stopped the reaction.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation and Characterization of the Glycolipids-- The extraction of 106 g of wet bacterial cell mass of P. propionicum, obtained from 35 liters of culture medium, yielded 1.5 g of a crude lipid extract. Smaller amounts (10-15 mg) of chloroform-methanol extracts were obtained for comparative purposes from 2-3 g of wet cell mass batches of six other representative species of propionibacteria.

Thin-layer chromatography of the polar lipids from the Propionibacterium strains revealed that P. propionicum contained rather high amounts of its major glycolipids, resulting in a chromatographic pattern of two pairs with two compounds each (Fig. 1). Extracts of P. acidipropionici and P. freudenreichii contained single components as major glycolipids, possessing different mobility than those obtained from P. propionicum. The other strains tested were devoid of major glycolipids.


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Fig. 1.   TLC chromatogram of lipid extracts from some clinically important and classical propionibacteria. 1, P. acnes PCM 2400T; 2, P. avidum CIP 103261T; 3, P. granulosum ATCC 25564T; 4, P. propionicum ATCC 14157T; 5, P. acidipropionici NBIMCC 1371; 6, P. freudenreichii NBIMCC 1207T. The TLC was developed in chloroform-methanol-water (65:25:4, v/v), and for the detection of lipids orcinol reagent and heating at 120 °C were applied. G1-G4, glycolipids of P. propionicum; G, major glycolipid of classic propionibacteria studied.

The characteristic glycolipids G1-G4 of P. propionicum migrated in TLC as the two pairs G1+G2 and G3+G4, respectively. Re-chromatography of both groups applying various two-dimensional TLC developing systems did not improve the separation of their components. However, the glycolipids of P. propionicum could still be isolated and purified for chemical analysis and structural studies. From the crude lipid extract subjected to column chromatography, fractions were obtained by successive elution with chloroform, acetone, and methanol (Fig. 2). These eluates contained neutral lipids, glycolipids, and phospholipids, respectively, in yields of 15.1%, 38.8%, and 46.1% of the crude lipid extract. The acetone-soluble eluates (containing glycolipids) were separated into the two pairs G3, G4 and G1, G2, utilizing successively 5% MeOH in CHCl3 and 10-20% MeOH in CHCl3, respectively, which could finally be purified by preparative TLC. From isolated G4, traces of contaminating G3 were removed by HPLC. The purity of glycolipids was confirmed by two-dimensional TLC in two pairs of solvents, by MALDI-TOF MS and HPLC.


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Fig. 2.   TLC of P. propionicum lipid fractions from column chromatography eluted subsequently with chloroform, acetone, and methanol. Fractions: 1-4, neutral lipids (NL); 5-9, glycolipids (GL); 10-13, phospholipids (PH). The TLC was developed in chloroform-methanol-water (65:25:4, v/v), and for the detection of lipids vanillin reagent and heating at 120 °C were applied.

The chemical characteristics of the glycolipids (RF values, yields, and quantitative sugar and fatty acids compositions) are presented in Table I. Glucose, glycerol (in non-stoichiometric amounts), and fatty acids were detected as components, but neither amino sugars nor phosphorus could be found. The composition of the fatty acids (branched iso- and anteiso-C15:0 as well as linear fatty acids n-C15:0, n-C16:0, and n-C18:0) varied in G1-G4. After methanolysis and TMS derivatization of glycolipids G2-G4, 1-O-pentadecyl-2,3-di-O-TMS-glycerol, and less amounts of 1-O-hexadecyl-2,3-di-O-TMS-glycerol were identified (Fig. 3). The presence of glucose was proven by GLC/MS and a specific colorimetric method, which distinguishes glucose from ketoses and other aldoses (33). The enzymatic procedure used for the determination of the glucose configuration revealed that in all glycolipids glucose (Glc) possessed the D configuration.

                              
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Table I
The chemical composition of major glycolipids from P. propionicum PCM 2431 (ATCC 14157T)


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Fig. 3.   Identification of glycerol ethers prepared from glycolipid G3 of P. propionicum. A, separation by GLC of TMS derivatives of 1-O-pentadecyl glycerol (P) and 1-O-hexadecyl glycerol (H); B, the mass spectrum of P.

Methylation Analyses-- Methylation analyses, according to Hakomori (36), performed on G1-G3 resulted in two derivatives in equal proportions, namely 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylglucitol, identifying one terminal and one 3-substituted Glc residues. From glycolipid G4, only the first derivative was obtained, identifying one terminal Glc residue. Applying non-alkaline conditions for the methylation of the glycolipids (38), the fatty acyl substituent was not cleaved. Re-methylation of the product with deuterated methyl iodide according to Hakomori (36) resulted in the case of glycolipid G3 in a stoichiometric incorporation of a deuteriomethyl group at C-6 of the terminal Glc residue, yielding 1,5-di-O-acetyl-6-O-deuteriomethyl-2,3,4-tri-O-methylglucitol.

MALDI-TOF Mass Spectrometry-- Positive-ion MALDI-TOF mass spectra of the native glycolipids G1-G4 (Fig. 4, A-D) revealed two (G1, Fig. 4A) or one (G2-G4, Fig. 4, B-D) prominent groups of quasimolecular ions, each expressing the heterogeneity in the length of the aliphatic chains (Delta m/z 14). The most abundant peak at m/z 663.2 in the mass spectrum of G1 corresponds, in agreement with the compositional analyses, to a sodium adduct ion [M+Na]+ consisting of a dihexose unit condensed with glycerol and one C15 saturated fatty acid (calculated m/zcal, 663.356). The second group of peaks, centered around m/z 887.4, of G1 can be attributed to molecules with one additional fatty acid residue. Considering the results of the compositional analysis and the NMR data (see below) the quasimolecular ion peaks observed in the spectrum of G2 (Fig. 4B) can be attributed to molecules consisting of two Hex, one Gro, one acyl, and one akyl chains. Glycolipid G3 differed from G2 by the addition of another, C15 saturated fatty acyl chain, as indicated by the group of ion peaks around m/z 1097.3. Interestingly, the spectrum of glycolipid G4 contained a major ion at m/z 711.3, which was 162 mass units lower than the quasimolecular ion in the spectrum of G2, indicating that glycolipid G4 contained only one hexose residue.


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Fig. 4.   Positive-ion MALDI-TOF mass spectra of glycolipids of P. propionicum. A, G1; B, G2; C, G3; and D, G4.

Due to the limited mass resolution and accuracy of the MALDI-reflection-TOF MS, it could not be concluded unequivocally if, e.g., the mass peak at m/z 887.4 originated from a quasimolecular ion [M+Na]+ consisting of two Hex, one Gro, and two C15:0 acyl residues (m/zcal, 887.5702) or of two Hex, one Gro, one C15:0 acyl, and one C16:0 alkyl residues (m/zcal, 887.6066), because the calculated masses differ from each other by only 0.037 mass units. Therefore, high resolution ESI-FT-ICR mass spectra were recorded. The extended parts of the spectra, showing the respective isotopic peaks obtained from G1 and G2 (Fig. 5, A and B), clearly demonstrate that the molecule in G1 exclusively carries two C15:0 acyl chains (measured m/z, 887.571). Instead, in G2 two molecular species could be detected. The most abundant monoisotopic peak at m/z 887.607 originated from a glycolipid carrying one C15:0 acyl chain and one C16:0 alkyl chain, the peak of minor intensity at m/z 887.570 from a glycolipid carrying two C15:0 fatty acid residues. For molecules with a shorter aliphatic chain the same accurate mass (m/z 873.592) was determined for G1 and G2 (data not shown) proving that this glycolipid species carries one C15:0 acyl chain and one C15:0 alkyl chain (m/zcal, 873.5917). Respective mass spectrometric analyses of G3 and G4 (data not shown) confirmed the interpretation given above. All quasimolecular ion peaks detected for G3 at around m/z 1097 carried one alkyl and two acyl chains and that from G4 at around m/z 711 each carried one alkyl chain and one acyl chain.


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Fig. 5.   Parts of high resolution positive ion ESI-FT-ICR mass spectra of glycolipids G1 (A) and G2 (B) showing the isotopic peaks of one molecular species.

NMR Spectroscopy-- The structures of the glycolipids G2-G4 were established by 1H and 13C NMR spectroscopy. Chemical shifts were assigned utilizing COSY, TOCSY, NOESY, HMQC, and HMBC experiments. Anomeric configurations were assigned on the basis of the chemical shifts observed and of J1,2 values, which were determined from the one-dimensional 1H spectra. The data are presented in Fig. 6 and in Tables II and III. The anomeric regions of the 1H NMR spectra of G2 and G3 contained three signals and that of G4 contained two signals, representing two (G2 and G3) and one (G4) alpha -glucose residues A and B (see Fig. 7). Their identification was made possible by the complete assignment of all signals. The sugar residues were pyranoses. The third signal (G2, 5.18 ppm; G3, 5.19 ppm; G4, 5.18 ppm) originated in all three substances from H-2 of the glycerol moiety, indicating the substitution of O-2 of glycerol by an acyl chain. Furthermore, the 1H NMR spectrum contained signals of fatty acid alkyl chains, which could be assigned tentatively (Table III).


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Fig. 6.   Sections of COSY spectra of glycolipids G2 (A), G3 (B), G4 (C). Monosaccharide units A and B are as shown in Fig. 7. Gro, glycerol.

                              
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Table II
1H and 13C NMR chemical shifts of glycolipids G2, G3, and G4 from P. propionicum PCM 2431 (ATCC 14157T)
Assignments of the sugar and glycerol signals.

                              
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Table III
1H and 13C NMR chemical shifts of glycolipids G2, G3, and G4 from P. propionicum PCM 2431 (ATCC 14157T)
Fatty acid alkyl signals (tentative assignments).


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Fig. 7.   Structures of glycolipids G2-G4.

The 13C NMR chemical shifts were assigned by an HMQC experiment, using the interpreted 1H NMR spectrum. The anomeric regions of the spectra of G2 and G3 contained two signals and that of G4 contained one signal, representing two (G2 and G3) and one (G4) alpha -glucose residues A and B (Table II). In the spectra of G2 and G3, low field-shifted signals of C-3 (G2, 84.79 ppm; G3, 85.34 ppm) indicated the substitution at O-3 of residue B in both glycolipids. Residue A was a terminal alpha -D-Glcp in G2 and G3, but in G3 it was substituted at O-6 as indicated by downfield-shifted C-6 (64.00 ppm) and H-6a,b (4.434 and 4.234 ppm) signals.

The sequence of constituents was determined using NOE and HMBC data. Contacts between anomeric and trans-glycosidic protons and carbons were observed for both glucose residues. In the NOESY spectra of G2 and G3, an inter-residual NOE contact was observed between H-1 of Glcp A and H-3 of Glcp B. This linkage was confirmed by HMBC contacts (Fig. 8) between proton A-1 and carbon B-3, and proton B-3 and carbon A-1, thus establishing the presence of an alpha -D-Glcp-(1right-arrow3)-alpha -D-Glcp disaccharide in G2 and G3. In both glycolipids, residue B was linked to O-1 of glycerol, as indicated by HMBC contacts between proton B-1 and C-1 of glycerol, and protons H-1a,b of glycerol and carbon B-1. In the HMBC spectrum of G2, one fatty acid residue was identified by its C-1 signal at ~174 ppm, which gave a contact to H-2 of glycerol. Thus, the alkyl ether chain, as identified by its CH2-O-CH2 signals at 3.44 ppm (1H) and 72.20 ppm (13C), was linked to O-3 of glycerol. In the HMBC spectrum of G3, two fatty acid residues were identified by their C-1 signals at ~174.8 and ~175 ppm, the first of which gave an HMBC contact from C-1 to H-2 of glycerol and the second from its C-1 to H-6a,b of glucose A. Again, the alkyl ether chain, as identified by its CH2-O-CH2 signals at 3.45 (1H) and 72.26 ppm (13C), was linked to O-3 of glycerol. In the NOESY spectrum of G4, an inter-residual NOE contact was observed between H-1 of Glcp B and H-1b of glycerol. This linkage was confirmed by HMBC contacts between proton B-1 and C-1 of glycerol, and H-1a,b of glycerol and carbon B-1, thus establishing that the Glcp residue is linked to O-1 of glycerol. In the HMBC spectrum of G4 (Fig. 8), one fatty acid residue was identified by its C-1 signal at ~175 ppm, which gave a contact to H-2 of glycerol. Thus, the alkyl ether chain, as identified by its CH2-O-CH2 signals at 3.45 (1H) and 72.16 ppm (13C), was linked to O-3 of glycerol. Taken together, the structures of glycolipids G2-G4 of P. propionicum PCM 2431 (ATCC 14157T) are as shown in Fig. 7.


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Fig. 8.   Sections of HMBC spectra of glycolipids G2 (A), G3 (B), and G4 (C). Monosaccharide units A and B are as shown in Fig. 7. Gro, glycerol; FA, fatty acid. Cross-peak X in C originated from a contaminant.

Immunological Study-- When the rabbits were immunized with the cell mass suspension (Cm) of P. propionicum strain PCM 2431 (ATCC 14157T), the titers of anti-glycolipid sera measured by the passive hemagglutination test were 1/1024 after 4 weeks and reached 1/2048 after 5 weeks. The polyclonal rabbit antisera obtained against P. propionicum reacted in the simple dot-EIA test with all glycolipid antigens studied (Table IV). A strong positive reactivity was observed for the sera diluted 1/100 with decreasing concentrations of the glycolipid markers up to 5 ng for G2 and 10 ng for G3, respectively. Control sera from healthy non-immunized animals were negative.

                              
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Table IV
The reactivity in the dot-EIA test of glycolipids G1-G4 from P. propionicum PCM 2431 (ATCC 14157T)
The titers are expressed as the antigen dilution reciprocal averaged from three experiments in reaction with rabbit polyclonal antisera against P. propionicum ATCC 14157T and serum from non-immunized rabbit.

The delayed-type of skin hypersensitivity reaction developed with glycolipids G1-G4 as well as with Cm and PBS (positive and negative controls, respectively) was measured at 48 h after immunization of rabbits, and the results are presented in Table V. The positive reactions at 2 weeks after immunization were obtained only for G3 and Cm (with a dose of 100 µg). After next 2 weeks this reaction was stronger. The positive reactions with G1, G2, and G4 antigens were not observed until 4 weeks after the immunization, and no reaction had been developed with PBS at any time of testing.

                              
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Table V
The induction of delayed hypersensitivity by glycolipids from P. propionicum PCM 2431 (ATCC 14157T) in rabbits injected with these bacteria


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For the first time, actinomycetal glycolipids have been isolated and characterized that possess an ether-linked alkyl chain in their lipid moiety, i.e. the herein described glucolipids from P. propionicum strain PCM 2431 (ATCC 14157T). Propionibacteria are pleomorphic Gram-positive microorganisms producing propionic acid in the fermentation process. They have many common phenotypic and chemotaxonomic properties rendering them difficult for classification and identification. In contrast to the classic propionibacteria originally classified in this genus, other species have been combined in a second group of skin propionibacteria, which are assigned as pathogenic. These microorganisms are currently considered as being clinically important because of their growing potential in infections connected very often with immune system dysfunctions. Propionibacteria are considered as actinomycete-like microorganisms, because in the initial phase of cultivation cells are found that morphologically resemble Actinomyces.

Propionibacteria are the etiological factors of acute or chronic, suppurative or localized, actinomycete-like infections of lungs. The etiology of these infections is difficult to determine, especially when mixed infections are present due to synergistic interactions of these microorganisms with facultative pathogens. Secondary infections with propionibacteria are severe due to their specific affinity to the central nervous system tissue causing meningitis. Propionibacterium acnes, except of its etiology in common acne, is known to develop the Kawasaki disease in children, which leads to coronary disease (42). P. lymphophilum has been isolated from urinary tract infections and lymphatic nodes of patients with Hodgkin's disease (1), whereas P. avidum is often found in chronic sinusitis, especially purulent. P. granulosum, the second most frequent, occurring in skin and in acne lesions, is occasionally found, for example in cases of bacteremia in patients with immunodeficiency (43), endocarditis (44), or post-operative ophthalmitis (45).

Amphipatic polar lipids of microorganisms are basic structural constituents of cell plasma membranes, but they have been also found as specific major lipids from bacterial wall envelopes (46). Phospholipids are admittedly known as the most common polar lipids (47), but glycolipids also fall into this category and considerable interest has been focused on their implications in bacterial systematic as useful chemotaxonomic markers (24-27, 48-56).

The novel polar lipids of P. propionicum, being sugar derivatives of glycerol ethers, are clearly distinguished members of this species from others that are phylogenetically related and generally from all actinoform organisms. As far as we know ether-containing polar lipids are rare (57), being mainly confined to the Archaea domain (synonym Archaebacteria (58)), i.e. organisms living in extreme environmental conditions such as obligate anaerobes; thermo-, halo-, acido-, and alkalophilic organisms; or methanogenic bacteria. Their lipid ethers seem to be the most invariant and unique aspects from both biological and taxonomic points of view. In fact, diether structures are dominant in halophiles, methanogens, thermoacidophiles, and thermophilic anaerobes; however, tetraethers are restricted to the thermoacidophiles, thermophilic anaerobes, and some methanogenes, and no tetraether has been detected in the halophilic archaeal group (59). Identification of individual ether-containing polar lipids was based on data generated by Kates (60), Kushwaha et al. (61), De Rosa et al. (62, 63), Ross et al. (64), Nishihara (65, 66), Kamekura (59), Kamekura and Kates (67, 68), and Trincone et al. (69). The ether-containing lipids structurally similar to platelet-activating factor were found in the cell membrane of Mycoplasma fermentans (70). Some specific glycosyl ethers, revealed in facultative anaerobic and opportunistic pathogens of P. propionicum (25), and their unique chemical structures presented in this report are interesting not only as taxonomic or etiological markers of this species but also, as a whole, as a structurally novel type of alkylglycerols. Because of their ether bonds, they possibly represent an important step in the phylogenetic process of these bacteria.

    ACKNOWLEDGEMENTS

We thank Regina Engel, Sylvia Düpow, Helga Lüthje, and Kamila Tercka for technical assistance.

    FOOTNOTES

* This work was supported by the Polish Committee for Scientific Research (Grant 4-PO5A-073-19) and by the Deutsche Forschungsgemeinschaft (Grant LI-448/1-1 to B. L. and O. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 48-71-337-1172; Fax: 48-71-337-1382; E-mail: gamian@immuno.iitd.pan.wroc.pl.

Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M206013200

    ABBREVIATIONS

The abbreviations used are: HPLC, high performance liquid chromatography; GLC/MS, gas-liquid chromatography/mass spectrometry; TMS, trimethylsilyl; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ESI, electrospray ionization; FT-ICR, Fourier-transform ion cyclotron resonance; HMBC, heteronuclear multiple bond correlation; TOCSY, total correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; PBS, phosphate-buffered saline; PHT, passive hemagglutination test; TBS, Tris-buffered saline; Hex, hexose; Gro, glycerol; Cm, cell mass suspension; EIA, enzyme immunosorbent assay; Glcp, glucopyranose.

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