Evidence for a New Type of Outer Membrane Lipid in Oral
Spirochete Treponema denticola
FUNCTIONING PERMEATION BARRIER WITHOUT
LIPOPOLYSACCHARIDES*
Christian P.
Schultz
§,
Violetta
Wolf
¶,
Robert
Lange¶,
Erich
Mertens
,
Jörg
Wecke
,
Dieter
Naumann
, and
Ulrich
Zähringer
From the
Robert Koch-Institut, D-13353
Berlin, Nordufer 20, ¶ Immanuel Krankenhaus, D-14109
Berlin, Koenigstrasse 63, and
Forschungszentrum Borstel,
Zentrum für Medizin und Biowissenschaften, D-23845
Borstel, Parkallee 1-40, Germany
 |
ABSTRACT |
A new class of outer membrane lipid (OML) was
isolated from the oral spirochete Treponema denticola
strain 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
-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 lower
Tm 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 of
T. denticola, which is quite different from LPS normally
essential for Gram-negative bacteria.
 |
INTRODUCTION |
Treponema denticola is an oral anaerobic spirochete
with typical helical morphology (1). These bacteria have been
implicated in the induction of peridontitis (2, 3). 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 (4). 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 (5, 6). In addition to the lipids, transmembrane
channel proteins are further components within the membrane (7). 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 (8).
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 (4, 9, 10)).
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)1 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 (11-13).
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) (14).
 |
EXPERIMENTAL PROCEDURES |
Reference Compounds and Bacterial
Growth--
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 (4). For optimal growth, isobutyric acid, DL-2-methylbutyric acid, isovaleric acid,
and valeric acid were added to the medium.
Electron Microscopy--
All steps of preparation for electron
microscopy (embedding procedures, ultrathin sectioning) were performed
as described previously (15).
Chemical OML Extraction and Analysis--
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
(16). The SDS-polyacrylamide gel (PAGE) analysis was carried out on 14 and 18% (w/v) gels following the method developed by Laemmli (17). 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. (18) and HexN after strong overnight hydrolysis according to
Strominger et al. (19). 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. (20). 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. 21 for further details).
FTIR Measurements--
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 (22). 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 (12-14). 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 (14) 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 (23).
 |
RESULTS AND DISCUSSION |
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. 24-26). Other authors classified these
structures as lipo-oligosaccharides because of their similarities to
LPS without having the key components Kdo and
-hydroxy fatty acids
(e.g. Refs. 5, 27-29). Even in Borrelia
burgdorferi, unusual LPS-like compounds were discovered,
suggesting that there is probably no LPS present in the genus
Borrelia (30).
Outer Sheath Morphology--
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. 1a,
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.
1b 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 (31).

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Fig. 1.
Ultrathin sections of T. denticola
ATCC 33521. a, longitudinal section of a single cell
presenting typical Gram-negative features such as a double-membrane
structure with outer sheath (OS), cytoplasmic membrane
(CM), and protoplasmic cylinder (PC).
b, cross-section of a single cell showing flagellae within
the cell; the outer sheath (OS) surrounds the axial
filaments (AF).
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Approximation of Molecular Size--
Fig.
2a 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. 2b) from
Pseudomonas aeruginosa F2 indicates a typical band pattern
starting with RaLPS, the complete core structure (32), followed by
multiple repeating units with a size of 0.534 kDa (33). Lanes
B-D represent LPS (from strains Salmonella helsinkii
777, 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 (32).
The isolated lipid fraction OML521 (lanes 1-5 in Fig.
2a and densitograms in Fig. 2c) 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 (34, 35) 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 in
Enterobacteriaceae). 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 (36). 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 (37).

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Fig. 2.
Electrophoretic analysis of OML521 of
T. denticola ATCC 33521. a, SDS-PAGE silver
nitrate-stained calibrated with known LPS isolates. Reference lanes
are: A, wild-type LPS (Pseudomonas aeruginosa
F2); B, S/R-LPS (S. helsinkii 777); C,
RaLPS (S. minnesota R60); D, ReLPS (E. coli F515). The OML521 lanes are 1-5 representing 2, 4, 6, 8, and 10 µg of outer sheath lipid (T. denticola
ATCC 33521). b, gel profile densitometer scans of LPS
reference lanes A-D and in c of OML521
lanes 1-5. Arrows in a indicate
molecular weight approximated from the well known LPS standards. The
small arrows in b and c indicate the
pattern of repeating units, and the large arrows mark gel
positions of RaLPS, ReLPS, and the main fraction of OML521.
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Chemical Analysis--
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 from
Enterobacteriaceae. 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 Table
I) 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 (38). 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.
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Table I
Fatty acids (FA) found in outer sheath lipid OML521 and in total
cell membrane extracts of T. denticola ATCC 33521
The abbreviations used are: Di-Me-ac, dimethyl-acetate; i-, iso-; ai-,
anteiso-; ND, not determined; , not found.
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Spectral Characterization--
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) (39, 40). 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 C
and 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 spectrum
A 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 spectrum
A 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.

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Fig. 3.
Infrared spectra of large polymeric membrane
lipids isolated from Gram-positive and Gram-negative bacteria.
A, lower molecular weight fraction OMLL of outer
sheath lipid from T. denticola ATCC 33521. B,
higher molecular weight fraction OMLH of outer sheath lipid
from T. denticola ATCC 33521. C, LTA from
S. aureus H-LTA269. D, wild-type LPS from
S. minnesota SF1111. 1 (1', 1''), skeleton
vibrations dominated by phosphate and sugar; 2 (2', 2''),
syCH3; 3 (3', 3''), asCH3; 4 (4', 4''), amide II;
5 (5', 5''), amide I; 6 (6', 6''), C=O ester;
7 (7', 7''), syCH2; 8 (8', 8''), asCH2; 9 (9', 9''),
asCH3; 10 (10', 10''), OH and NH
stretching vibrations. Numbered bands are also described in
the text.
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Membrane Properties--
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 (12). By contrast, LTA can only form
micellar structures and shows no tendency to undergo vesiculation (40,
41). 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 (11, 12). 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 (
23 °C versus
15 °C) and induced at much lower
temperatures Tm (22.5 versus
35.5 °C). The slightly higher frequency of the >CH2
symmetric 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.

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Fig. 4.
Frequency/temperature profiles of
reconstituted outer sheath lipid OML521 from T. denticola
(squares), RaLPS from S. minnesota
(circles), and LTA269 from S. aureus
(triangles). Tm
indicates phase transition temperature, and indicates the
transition interval. The shaded area emphasizes the changes
between LTA substituted with many D-alanines
(triangles) and the same LTA free of any
D-alanine substitution (black triangles). The
frequency values were determined using the >CH2 symmetric
stretching vibration band near 2852 cm 1 as a monitor
(order/disorder).
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Structural Proposal--
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.

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Fig. 5.
Electrophoretic analysis of the HF hydrolysis
of OML521 of T. denticola ATCC 33521 and proposed model
representation of the outer sheath lipid. a, SDS-PAGE silver
nitrate-stained calibrated with known LPS isolates. Reference lanes
are: A, wild-type LPS (P. aeruginosa F2);
B-D, RaLPS, RbLPS, and RcLPS (S. minnesota R60,
R345, and R5); E, RdLPS (Shigella flexneri 3A);
F, S/R-LPS (S. helsinkii 777); G,
OML521 as isolated; and H, the HF-hydrolyzed
OML521HF after dialysis and pH adjustment to 7 (T. denticola ATCC 33521). b, simplified models of OML521
compared with known structures of LPS and LTA. The LPS structure is
assembled of a large membrane anchor lipid A (LA), an inner (Re, Rd)
and outer core (Rc, Rb, Ra), and many repeating units (of three sugars
with a combined mass of about 500 Daltons for P. aeruginosa
F2). The structure of LTA contains a simple glycerol-based lipid
connected with a few core sugars, which carry a chain of many small
repeating units (of phosphate-glycerol with alanine side chain
substitutions for S. aureus LTA269). The proposed OML521
structure suggests the same structural elements: membrane lipid, core
structure, and repeating units. The model of OML521HF
considers the results of the HF hydrolysis, which creates a smaller
molecule of the same lipid composition but without any repeating units.
This indicates a phosphate shell surrounding the core structure and a
phosphate-mediated repeating unit system similar to LTA. Black
circles, phosphates; shaded ellipsoids in LTA,
D-alanine; open rounded squares in LTA,
glycerols.
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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 (42, 43). Instead, we found and identified three
molecular fragments, indicating evidence for a membrane anchor
structurally similar to those in LTA (44) 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).

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Fig. 6.
EI mass spectra (I-III) of three structures
of the HF-hydrolyzed OML521 of T. denticola ATCC
33521. The molecular drawings on the right side indicate the main
fragments of hexose, glycerol, and hexadecanoic acid. The different
positions of substitution in the two glycerol-hexadecanoic acids are
evident from the different intensities of m/z = 239.
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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
-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 that
Treponema (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 (42, 43).
 |
ACKNOWLEDGEMENTS |
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.
 |
FOOTNOTES |
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant We 1414/1.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: National Research
Council of Canada, Institute for Biodiagnostics, 435 Ellice Avenue,
Winnipeg, Manitoba, R3B 1Y6, Canada. Tel.: 204-984-5396; Fax:
204-984-5472; E-mail: schultz{at}ibd.nrc.ca or
chris.schultz{at}nrc.ca.
1
The 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.
 |
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