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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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 (
H = 0.0) and CDCl3
(
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 |
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.
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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.
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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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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.
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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 (
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.
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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.
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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)
-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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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)
-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
-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
-D-Glcp-(1
3)-
-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.
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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.
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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
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DISCUSSION |
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.