Download Isolation and purification of cell wall polysaccharide of Bacillus

FEMS Microbiology Letters 82 (1991) 257-262
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
ADONIS 037810979100384B
257
FEMSLE 04579
Isolation and purification of cell wall polysaccharide
of Bacillus anthracis ( A Sterne)
F.S. E k w u n i f e l, j. Singh
2,
K.G. T a y l o r 2 a n d R.J. D o y l e 2
l Department of Natural Sciences, Unit,ersity of Maryland, Eastern Shore, Maryland, and 2 UniL'ersity of Louisrille, School of Medicine,
Louiscille, Kentucky, U.S.A.
Received 19 February 1991
Revision received 22 May 1991
Accepted 27 May 1991
Key words: Cell wall polysaccharide; HF-extracted polysaccharide; Ethanolic precipitation;
Bacillus anthracis
1. S U M M A R Y
A polysaccharide fraction was isolated from
sodium-dodecyl-sulfate (SDS) treated cell walls of
Bacillus anthrac& (A Sterne) by hydrofluoric acid
(HF) hydrolysis and ethanolic precipitation. The
polysaccharide fraction was subsequently purified
by several washings with absolute ethanol. Purity
of the isolated polysaccharide was tested using
the anthrone assay and amino acid analyzer. The
molecular mass of the polysaccharide fraction as
determined by gel filtration chromatography was
about 12000 Da. Preliminary analyses of the
polysaccharide was done using thin layer chromatography and amino acid analyzer, and results
obtained from these analyses were further confirmed by gas liquid chromatography and ~3CNMR spectroscopy. Results showed that the
Correspondence to: F.S. Ekwunife, Department of Natural
Sciences, University of Maryland, Eastern Shore, Princess
Anne, MD 21853, U.S.A.
polysaccharide moiety contained galactose, Nacetylglucosamine, and N-acetylmannosamine in
an approximate molar ratio of 3 : 2 : 1. This moiety
was devoid of muramic acid, alanine, diaminopimelic acid, glutamic acid, and lipid, thus
indicating that the isolated polysaccharide was of
pure quality.
2. I N T R O D U C T I O N
Evidence for the presence of a polysaccharide
material as part of the cell wall of Bacillus anthracis is well documented [1-5]. Several attempts have been made by researchers in the past
to isolate the polysaccharide fraction [2-5]. However, these attempts were met with little or no
success since isolation was done mainly with culture filtrates of the cell and not from cell wall
fraction. This usually yielded polysaccharides that
were contaminated with other cellular components such as membrane components. It is known
that bacterial wall polymers such as teichoic acid,
258
and polysaccharides such as teichuronic acid are
covalently linked to glycan chains of peptidoglycan via the 6-phosphorylated muramic acid
residue [6,7]. It is also known that when such cell
walls are treated with hydrofluoric acid (HF) for
short periods, the hydrofluoric acid causes specific cleavage of phosphodiester bonds, without
affecting acid-labile glycosidic bonds [6]. With
these facts in mind, it was decided in this study to
attempt to isolate and purify the B. anthracis
polysaccharide from pure cell wall fraction using
hydrofluoric acid hydrolysis and ethanolic precipitation. This approach hopefully will yield a
polysaccharide polymer that is essentially devoid
of other cell wall contaminants and at the same
time remain as homogeneous as possible. A pure
polymer is necessary for proper sequencing or
characterization of the polysaccharide.
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacterial strain
Bacillus anthracis (A Sterne) was supplied by
the United States Army Medical Research Institute of Infectious Diseases ( U S A M R I I D ) stock
collection (Fort Detrick, MD). Cells were then
maintained on A K sporulation agar slants (BBL,
Cockeysville, MD).
3.2. Preparation of cell walls"
Bacillus anthracis (a Sterne) cells were grown
overnight in a 20-ml Penassay broth (PAB)
medium contained in nephelometers. About 5 - 6
ml of cell culture were then transferred to 1-1.2 1
PAB in 2.8-1 baffled flasks. The transferred cells
were then grown to late exponential phase (200250 Klett units), harvested by centrifugation and
washed 2 - 3 times in cold distilled water. The
cells were disrupted with a sonicator (Sonics and
Materials, Inc.) and cell walls obtained as previously described by Doyle et al. [8], and Brown [9].
The cell wall preparation was purified by sequential extraction in hot 1% ( w / v ) sodium dodecyl
sulfate (SDS), and finally by 3 - 4 washes in water
[10]. The walls were then stored in a freeze-dried
form.
3.3. Isolation and purification of polysaccharide
from cell wall
100 mg of freeze-dried cell wall were treated
in 20 ml 4 8 - 5 1 % hydrofluoric acid (HF; reagent
grade) at 4°C for 18 h [6,7]. The preparation was
then centrifuged at 14000 r.p.m, for 10 min. 1 ml
of supernatant was mixed with 5 ml absolute
ethanol and allowed to stand in the cold for 30
min. The mixture was centrifuged as above, and
the precipitate washed 2 - 3 times with absolute
ethanol. The final product (polysaccharide) was
left at room temperature for 30 min in order for
the alcohol to evaporate and was then dissolved
in 1-2 ml cold distilled water. The dissolved
material was freeze-dried and stored in a desiccator in the cold. Polysaccharide samples were subsequently prepared under various hydrolysis conditions (24 h, 30 h, HF-hydrolysis) and under
different methods of precipitation (ethanolic precipitation and dialysis), in order to determine the
optimal conditions for isolation.
3.4. Testing the purity of isolated polysaccharide
The polysaccharide was usually analyzed with
an amino acid analyzer (Dionex D-300) to check
for any contamination by cell wall amino acids.
Samples were in turn assayed for the presence of
lipids [11] and for phosphorus [12]. Finally, the
amount of neutral sugar in the samples were
determined using the anthrone method [13]. A
consistent micromolar amount of hexose per milligram polysaccharide from one batch of polysaccharide to another gave a good indication that
the isolated polysaccharide materials were pure
and homogeneous.
3.5. Analytical methods
3.5.1. Preliminary analyses. Preliminary analyses of the polysaccharide samples were carried
out using a thin layer chromatography method of
Rebers and Wessman [14], and amino acid analyzer (Dionex D-300) equipped with a ninhydrin
detection system and Dionex CP-3 programmer.
Samples (2 mg) for the amino acid analyzer were
first acetylated as described by Niedermeier [15],
and then hydrolyzed in 4 M HC1 for 8 h at 100°C
in sealed tubes. Sugar standards were similarly
treated and run with each set of samples.
259
3.5.2. Confirmatory analyses. Confirmatory
analyses of the polysaccharide samples were done
using gas liquid chromatography (GLC) and 13CN M R spectroscopy. Samples for GLC were prepared by suspending 1 mg of polysaccharide in
250 pA 2M trifluoroacetic acid and heated at
120°C for 1 h. The samples were cooled and acid
removed under a flow of air and by co-distillation
with isopropyl alcohol (2 × 250 ~1). The residue
was N-acetylated by treatment with a solution of
m e t h a n o l / p y r i d i n e / a c e t i c anhydride [5 : 1 : 1:
v / v ) for 20 rain at room temperature. The solvents were removed under air and the residue
subsequently treated with 300 /zl M H C I /
methanol for 16 h at 80°C, re-N-acetylated and
further methanolyzed at 100°C for 16 h. The
residue was N-acetylated and silylated with Tri-zil
(Pierce) at 80°C for 20 min. The derivatized
monosaccharides (in the form of TMS ethers of
methyl glycosides) were extracted into hexane
and then analyzed.
All 13C-NMR spectra were recorded at 75
MHz on a Varian XL-300 instrument. An acquisition time of 0.2 sec was used with a pulse width
( P W ) = 3.0, line broadening ( L B ) = 6.0, relaxation delay (D~) = 0.2 as other important parameters. Usually, 30000-50000 acquisitions were
made.
Table 1
Composition of hydrofluoric-acid extracted polysaccharide
Component
Amount
(~zmol/mg
pCHO)
Molar
ratio
Galactose
N-Acetylglucosamine
N-Acetylmannosamine
Total hexose
2.02
1.37
0.69
2.02
2.92 (3)
1.98 (2)
1.00 (1)
A m o u n t s are expressed in Fzmol of component per mg of
polysaccharide (PCHO). Each amount represents an average
of 10 polysaccharide samples. Molar ratios (ratio of galactose
or N-acetylglucosamine to N-acetylmannosamine) are also
indicated.
These polysaccharides also contain mainly galactose and N-acetylglucosamine and have considerably more nitrogen than corresponds to glucosamine content (approximately 60-73% of the
total nitrogen is accounted for by glucosamine).
The HF-extracted polysaccharide, on the other
hand, is made up of galactose, N-acetylglucosamine and N-acetylmannosamine, and as such, the
additional nitrogen unaccounted for in the previously isolated polysaccharides may be due to the
N-acetylmannosamine. The preliminary results
4. R E S U L T S AND DISCUSSION
Analyses of the HF-extracted polysaccharide
show that it is made up of galactose, N-acetylglucosamine and N-acetylmannosamine in an approximate molar ratio of 3 : 2 : 1 (Table 1, Fig. 1).
The polysaccharide is free of cell wall amino acid
contaminants, such as muramic acid, diaminiopimelic acid, alanine, and glutamic acid; phosphorus and lipids are also absent. The molecular
mass as estimated by gel filtration chromatography is about 12000. The HF-extracted polysaccharide is isolated from a sodium-dodecyl-sulfate
(SDS) treated cell wall, and this accounts for the
absence of the cell wall contaminants. All previously isolated B. anthracis polysaccharides usually contain traces of cell wall amino acids [2-5],
indicating the presence of cell wall contaminants.
Fig.
Lane
Lane
dard;
1. Thin layer chromatography of P C H O hydrolyzate.
1: Mixture of standards; Lane 2: P C H O hydrolyzate;
3: Glucosamine standard; Lane 4: Mannosamine stanLane 5: Galactose standard: Lane 6: Galactosamine
standard; Lane 7: Galacturonic acid standard.
260
{,. ? 7
L .--:
--ttl~
FUC
•- -
+
,
+
.....
'
,
----
13.71 Gal
' •. 2"7"
14.82' Gal
~
" 1 8 . I 0 Gal
~
,
•. 7 ,
>
16.09 Gal
z':
:'=+
.:5
2 :-: , ~" ?
19.41 M a n N a c
20.74 M a n N a c
=+
21.17
~
?
22,
.
2-+
: - z . ?'~
~.+
.
G'IcN.~
.
.
23.03 G I c N J ~
23¢/~
24.69 GIcNAc
'-
~-
=
....
-
"~" ~+"
--
28.42
Inositol
26.10 OIcNJru=
Fig. 2, Gas liquid chromatogram of TMS ethers of methyl glycosides of the HF-extracted polysaccharide. Column: 15m DB-I, initial
temperature: 140°C/3 rain, program rate l: 4°C/rain to 180°C, program rate 2: ]0°C/min to 240°C, Ara: Arabinose; Rha:
Rhamnose; Fuc: Fucose; Xyl: Xylose; Gal: Galactose; ManNAc: N-acetylmannosamine; GLcNAc: N-acetylglucosamine.
261
obtained using amino acid analyzer (Table 1) and
by thin layer chromatography (Fig. 1) were subsequently confirmed by gas liquid chromatography
(GLC) (Fig. 2) and ~3C-NMR spectroscopy (Fig.
3). The GLC clearly shows the absence of HF-labile 6-deoxysugars such as rhamnose and fucose,
as well as the absence of pentoses such as arabinose and xylose. It is known that when cell walls
are treated with hydrofluoric acid for short periods, (up to 30 h), the acid causes specific cleavage
of phosphodiester bonds without affecting acidlabile glycosidic bonds [6,7]. The hydrolysis condition we employed in this study (18 h HF-hydrolysis in the cold, 0-4°C) obviously would not have
affected any of the glycosidic bonds of the
polysaccharide. Indeed our study shows that 18 h
HF-hydrolysis is optimal for isolation, and
ethanolic precipitation as opposed to dialysis gives
maximum yields of the polysaccharide. Fig. 3
shows an expanded form of the ~3C-NMR spectrum of anomeric (C-l) carbon atoms of the HFextracted polysaccharide. Anomeric carbons with
glycosidic linkages in the alpha configurations
usually have signals (resonances) in the range
between 99 and 102 ppm, while those with /3
configurations have signals between 102 and 105
ppm [16-18]. Also coupling constants (IJc-H) of
about 170 Hz are displayed by a pyranosides,
whereas fi pyranosides display coupling constants
of about 160 Hz [19,20]. Based on these standard
references, the 13C-NMR spectrum of the
anomeric carbons of the HF-polysaccharide indicates the presence of: 2 beta galactose residues
with chemical shifts of 105.8 and 105.3 ppm, and
1jc-H of 160 and 159 Hz respectively, 1 alpha
galactose residue with a chemical shift of 101.4
ppm and ~Jc-H of 175 Hz, 1 alpha N-acetylglucosamine residue, with a chemical shift of 99.1
ppm and ~Jc-H of 171 Hz, 1 alpha or beta Nacetylglucosamine residue with a chemical shift of
102.9 ppm and ~Jc-H of 172 Hz, 1 alpha or beta
N-acetylmannosamine residue with a chemical
shift of 100.9 ppm and :Jc-H of 164 Hz. The
13C-NMR-spectrum, therefore, confirms that the
number of anomeric carbon atoms (six) is consistent with a molar ratio of 3 galactose:2 Nacetylgtucosamine : 1 N-acetylmannosamine. The
method of isolation and purification outlined in
this study is relatively simple, inexpensive and
less time-consuming. Above all, the method of
hydrolysis is specific enough to yield pure
polysaccharide samples that are consistent in their
chemical compositions, thus indicating homogeneity of the samples. The constancy of the
C~,13)
v
105.3 105.8
(/~) ~
102.9
101.4 100.9
Chemicel shift (ppm)
(ct)
99.1
Fig. 3. ]3C-NMR spectrum of anomeric (C-l) carbon atoms of the HF-extracted polysaccharide.Solvent: D20; temperature: room
temperature; chemical shifts expressed as parts per million (ppm).
262
chemical c o m p o s i t i o n s is shown by the fact that
the total a m o u n t of hexose per milligram of
polysaccharide is the same as that of galactose for
each sample (Table 1). Successful isolation of a
pure polysaccharide from B. anthracis will ultimately lead to p r o p e r c h a r a c t e r i z a t i o n of the
polymer. I n d e e d , we are c u r r e n t l y e n g a g e d in the
s e q u e n c i n g or structural analysis of the polysaccharide, the results of which will be p u b l i s h e d in
the n e a r future. T h e overall study of the B.
anthracis cell wall polysaccharide will certainly
shed some light o n the biological role or roles of
this ancillary p o l y m e r in the native cell wall of the
organism. F o r example, it is k n o w n that attachm e n t or a d h e r e n c e of b a c t e r i a to their host is
crucial for successful c o l o n i z a t i o n of the host by
the invading o r g a n i s m [21-23]. It is also k n o w n
that a wide r a n g e of such cell to cell i n t e r a c t i o n s
is m e d i a t e d by c a r b o h y d r a t e s on the cell surfaces
of either the m i c r o o r g a n i s m s or their hosts
[21,24-27]. It is therefore likely that the polysaccharide moiety of B. anthracis c o n t r i b u t e s towards the b i n d i n g of the o r g a n i s m to its target
host cells. I n addition, i m m u n o l o g i c a l cross-reactivity has b e e n shown to occur b e t w e e n B. anthracis a n d type X I V p n e u m o c o c c u s due to their
c o m m o n galactose a n d N - a c e t y l g l u c o s a m i n e antigens [28-32]. T h e s e shared a n t i g e n s may have
significance in b o t h serodiagnosis of these
p a t h o g e n s as well as in p a t h o g e n e s i s a n d p r e v e n tion of their infections.
ACKNOWLEDGEMENTS
T h e a u t h o r t h a n k s A n n H a for help in p r e p a r ing the m a n u s c r i p t . This work was s u p p o r t e d by
the U n i t e d States A r m y Medical R e s e a r c h Institute of Infectious Diseases.
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