Vol. 263, No. 23, Issue of August 15, PP. 11291-11295,1988
Printed in U.S.A.
T H EJ O U R N A L OF BIOLOGICAL
CHEMISTRY
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of an Extracellular Cross-reactive Polysaccharide from
Pseudomonas aeruginosa Immunotype 4*
(Received for publication, December 8, 1987)
Nina A. Kocharova, YuriyA. Knirel, AlexanderS. Shashkov, NikolayK. Kochetkov,
and GeraldB. Pier$
From the N . D. Zelinsky Institute of Organic Chemistry, Academyof Sciences of the Union of Soviet Socialist Republics,
Moscow B-334, Unionof Soviet Socialist Republics and the Chunning Laboratory, Departmentof Medicine,
Brighum and Women’s Hospital, Harvard Medical School, Boston. Massachusetts02115
al. (2) have described five closely related variants of the most
common serotype of P. aeruginosa isolated from infected
patients. Therefore, understanding the structure and distribution of polysaccharide antigens of P. aeruginosa willbe
important for designing vaccines and passive immunoglobulin
therapies to augment treatment of infected patients. In this
-3)-8-~-GlcP-(1-3)-8-~-ManP-(l-3)-8-~-ManP-(l-3)-a-L-Rh~-(l2
report we describe a small molecular mass (-6.5 kDa) neutral
1
polysaccharide isolated from a clinical strain of P. aeruginosa
a-D-ManP
immunotype 4 which bound antibody reactive with the LPS
of
the Fisher immunotype strains of P. aeruginosa.
where Rha is rhamnose. The structure
was determined
using acid hydrolysis, solvolysis with anhydrous hyMATERIALS AND METHODS AND RESULTS’
drogen fluoride, methylationanalysis, and ‘H and 13C
nuclearmagneticresonancespectroscopyincluding
P. aeruginosa Strains-An isolate from a patient with P.
nuclearOverhauserenhancementexperiments.The
aeruginosa bacteremia determined to be a Fisher immunotype
polysaccharide bound antibody raised to the lipopoly- 4 strain by agglutination was used to prepare the polysacchasaccharide of the seven P . aeruginosa Fisher-Devlin ride antigen. Fisher immunotype strains 1-7 (ATCC 27312immunotype strains. Inhibition assays demonstrated 27318) were also used for evaluation of polysaccharide prothe presence of a serologically similar polysaccharide duction. Several clinical isolates of mucoid (M) and nonmuin supernatants of thesestrains. Affinity-purifiedan- coid (NM) P.aeruginosa were used in the colony blot assay.
tibody to the polysaccharide bound to lipopolysacchaAntisera to P. aeruginosa LPS-Antisera to purified LPS
ride and whole cells of the immunotype strains of P .
aeruginosa in a Western immunoblot and colony blot and alkali-treated (0.1 M NaOH, 56 “C, 2 h) LPS from the
assay, respectively. This polysaccharide seems to con- seven Fisher immunotype strains was prepared as described
tain an antigenic determinant present in the core of (3).
Purification and Characterization of LPS and Polysacchathe P . aeruginosa lipopolysaccharide ormay represent
another minor polysaccharide substituent on the lipo- ride-Lipopolysaccharide from the seven Fisher immunotype
strains was preparedas described (3) from bacterial cells
0 side chain.
polysaccharide in addition to the
grown for 18 h at 37 “C. The neutral polysaccharide was
prepared from culturesupernatants of the clinical isolate
using the same methodology described for preparation of high
Differentiation of strains of Pseudomonas aeruginosa by molecular weight polysaccharides (3). The neutral polysacimmunologic methods most often relies upon heat-stable sur- charide was recovered in the late fractions eluting from a 5.0
face antigens (1).These antigens are almost invariably the X 95-cm Sephacryl S-300 column and purified further on a
polysaccharide portion of the lipopolysaccharide (LPS).’ column of Fractogel TSK HW 40 in 1%acetic acid. Elution
Careful characterization of these structures is crucial to de- profiles were recorded with a Knauer differential refractomtermining the overall diversity of P. aeruginosa serotypes. eter. Biochemical analysis was as described (3), and size was
More importantly, the LPS polysaccharide side chain is a determined on a 1.6 x 90-cm column of Sephacryl S-300 in
critical target for protective antibodies. Because of the high 0.02 M Tris, 0.15 M NaC1, pH 7.4, using dextran molecular
specificity of these antibodies, slight variations in the chemi- weight markersasstandards.
The koa elution value was
cal structure of the LPS side chains could allow some strains calculated for dextrans with average molecular masses of 2.5,
to infect hosts otherwise thought to be protected by the 10, 40, 70, and 110 kDa. Plotting the log,, of the molecular
presence of type-specific antibodies. For example, Knirel et mass uersus km yielded a correlation coefficient of -0.998.
Chemical and Mass Determination of the Neutral Polysac*This workwas supported in part by Grant A122535 from the
charide-Chemical
analysis of the components of the neutral
National Institutes of Health. The costs of publication of this article
were defrayed in part by the payment of page charges. This article polysaccharide are shown in Table1. Most of the components
must therefore be hereby marked “aduertisement” in accordance with were determined to be either hexose or deoxyhexose with little
A neutral small molecular mass(-6.5 kDa) polysaccharide comprising a pentasaccharide repeat unit
was
isolatedfromculturesupernatantsof
Pseudomonas
aeruginosa immunotype 4. The polysaccharide had a
pentasaccharide repeating unitas follows
18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom reprint requests should be addressed Channing Laboratory, 180 Longwood Ave., Boston, MA 02115.
The abbreviations used are: LPS, lipopolysaccharide; NOE, nuclear Overhauser enhancement; KDO, 2-keto-3-deoxyoctulosonic
acid.
* Portions of this paper (including “Materials and Methods,” part
of “Results,” Tables 2-6, and Figs. 3 and 4) are presented in miniprint
at the end of this paper. Miniprint is easily read with the aid of a
standard magnifying glass. Full size photocopies are included in the
microfilm edition of the Journal that
is available from Waverly Press.
11291
P. aeruginosa Cross-reactive Neutral Polysaccharide
11292
contaminating protein, nucleic acid, or LPS components.
Determination of the molecular mass by liquid chromatography indicated an average mass of approximately 6.5 kDa.
Polysaccharide-"H
Structural Analysis of theNeutral
NMR spectra were run on a BrukerWM-250 instrument with
samples dissolved in DzO at 50 "C, using acetone (SH 2.23
ppm) as an internal standard. Selective spin-decoupling experiments were performed in the difference mode. To increase
the selectivity a decoupling power 5-10 times less than that
necessary for full spin decoupling was used. As a result, only
the initial view of signals of protons coupled with the irradiated one clearly appeared in the difference spectra. Nuclear
Overhauser enhancement (NOE) measurements were carried
out in the difference mode as described (6) at 40 and 80 "C,
with a relaxation delay of 4 s and a build-up NOE of 0.5 s.
NMR spectra were run on a Bruker AM-300 instrument
with samples in DzOat 50 "C for the polysaccharide and 30 "C
for the oligosaccharides, using methanol (6c 50.15 ppm) as an
internal standard. Optical rotations were measured with an
EPO-1 polarimeter in water at 20 "C. Combined gas-liquid
chromatography/mass spectrometry was performed on a Varian MAT 111instrument using a column of OV-1 on Diatomite CQ (100-120 mesh).
Acid hydrolysis was performed with 2 M sulfuric acid
hydrolysate was neutralized with barium
(100 "C, 4 h), and the
carbonate. Monosaccharides were identified with a Technicon
sugar analyzer. Methylation analysis was performed as described (7), and the methylated polysaccharide was isolated
by dialysis and gel filtration. Methylated oligosaccharide (11)
was isolated using a cartridge of silica gel Cls (Sep-Pak) as
described (8).
Solvolysis with anhydrous hydrogen fluoride was carried
out for 15 min at -40 "C in a Teflon vessel. The solvolysate
precipitate
was poured into ethercooled to -78 "C (9), and the
was collected on a metal filter, washed with cold ether, dissolved in water, and separated by gel filtration on TSK HW
40 to give oligosaccharide (I) (33% yield). Oligosaccharide (I)
TABLE1
Chemical analysis of the components of the P. aeruginosa
neutral Dolvsacchuride
Component
Percent of
total
Hexose
81"
Deoxyhexose
18*
Protein
C0.1'
Nucleic acid
C0.lC
Lipids
C0.lC
C0.1'
KDO
a Determined by phenol-sulfuric test.
Determined by para-nitrophenylhydrazinemethod.
Lower limit of detection.
was reduced with an excess of sodium borohydride in water
(20 "C, 1h), acidified with glacial acetic acid, and theresulting
oligosaccharide (11) isolated by gel filtration.
According to the 13C NMR spectrum (Fig. 1) the neutral
polysaccharide had a regular repeating pentasaccharide structure (thespectrum contained five signals for anomeric carbons
between 97.8 and 102.3 ppm). One of the monosaccharides
was a 6-deoxy sugar (signal for its methyl group at 17.7 ppm)
while the other four were hexoses (four signals for hydroxymethyl groups between 62.0 and 62.2 ppm). Acid hydrolysis
of the polysaccharide yielded glucose, mannose, and rhamnose
in a ratio of -1:3:1 as identified in the sugar analyzer. Oxidation of the hydrolysate with D-glucose oxidase proved the
D configuration of glucose.
Methylation analysis of the polysaccharide yielded 2,4-di2,4,6-tri0-methylrhamnose, 2,3,4,6-tetra-O-methylmannose,
0-methylmannose, 2,4,6-tri-O-methylglucose,and 4,6-di-0methylmannose, which wereidentified as theiralditol acetates
by gas-liquid chromatography/mass spectrometry. Hence the
polysaccharide had a branched structure, with one of the
mannose residues as theterminal monosaccharide in theside
chain, a second residue of mannose substituted a t positions 2
and 3 as thebranch point inthe backbone, and theremaining
three monosaccharides (mannose, glucose, andrhamnose)
monosubstituted a t position 3. The methylation data also
revealed that all of the monosaccharide residues occur in the
pyranosidic form.
The polysaccharide was next subjected to solvolysis with
anhydrous hydrogen fluoride, and theproducts were separated
by gel filtration on TSK HW40 to give oligosaccharide (I) as
the main product and two products of higher molecular weight
in much lower amounts. The 'H and 13C NMR data, as well
as acid hydrolysis, showed that (I) contained all five of the
constituent sugars of the polysaccharide and likely represents
the repeating unit. The 13C NMR spectrum showed that the
reducing end of (I) contained a residue of rhamnose (6c 94.8
ppm, C-la,p). The two other products had the same monosaccharide composition and appeared to be a decasaccharide
(two repeat units) and the undegraded polysaccharide. Thus
the solvolysis selectively cleaved the polysaccharide at the
rhamnosidic linkages.
Sodium borohydride reduction converted (I) into oligosaccharide (11) with the rhamnitol residue at the reducing ter~
ppm, C1 and 20.2 ppm, C6). Methylation
minus ( 6 63.8
analysis of (11) showed it to have a branched structure, with
the mannose and glucose residues being the terminal monosaccharides of the two chains.
The "H NMR spectrum of (11) (Fig. 2) was interpreted by
sequential selective decoupling experiments (Table 2). The
values of the chemical shifts of the signals of H-1 (10) and
the coupling constants Jl,z (11) showed that the residue of
P. aeruginosa Cross-reactive Neutral Polysaccharide
11293
FIG. 2. ‘H NMR spectrum of the
neutral polysaccharide.
1.
4.6
5-4
4.9
glucose and 2 residues of mannose are /?-linked (6 4.68, 4.82,
7.9,<1, and <1 Hz, respectively), while the
and 4.89 ppm; J1,*
third residue of mannose is a-linked (6 5.43 ppm; J1,*1.8 Hz)
(10). The NOE arising in (11) on sequential preirradiation of
the anomeric protons of all four monosaccharide residues
(Table 3) enabled us to determine the sequence of the sugars
and their mode of substitution (12). Preirradiation of H-1 of
the residue of P-glucose resulted in the enhancement of the
signal for H-3 of one of the /?-mannose residues. Preirradiation of H-1 of the a-mannose residue caused NOE at H-2of
the same @-mannoseresidue indicating it to be the branch
point of the oligosaccharide, substituted at position 3 by the
0-glucose and atposition 2 by the a-mannose. Further preirradiation of H-1 of this disubstituted residue of @-mannose
resulted in the enhancement of the signals for H-2 and H-3
of the second residue of /?-mannose,which is characteristic of
a @l,3-linked mannobiose residue (cf. e.g. Ref. 13). Finally
preirradiation of H-1 on the monosubstituted /3-mannose
residue caused a marked NOE at H-3 of the rhamnitol residue.
These data enabled us to determine unambiguously the structure of oligosaccharide (11) and oligosaccharide (I), which
differs from (11) by the presence of the rhamnose (Rha)
residue in place of rhamnitol (Rhaol).
effect (4.6ppm) at C-1 of the @-D-glucopyranoseresidue favors
the D configuration of the 0-mannose residue glycosylated by
the former a t position 3. Similarly, the small effect (2.9 ppm)
at c-1 of this first residue of 0-D-mannopyranose indicates
that the adjacent residue of /?-mannopyranose which is glycosylated by the firstresidue of P-mannopyranose at position
3 also has the D configuration. In contrast, a relatively large
effect (7.4 ppm) at C-1 of the second residue of P-mannopyranose proves that the residue of rhamnopyranose which is
glycosylated at position 3 has the L configuration.
The absolute configuration of the a-mannopyranose residue
was determined by the calculation of the specific optical
rotation of the polysaccharide using Klyne’s rule (16). The D
configuration for this monosaccharide was assigned, since the
calculation gave a value practically coincident with the observed one (Table 5).
Thus the dataindicate that the neutral extracellular polysaccharide from this clinical isolate of P. aeruginosa immunotype 4 has the following structure, where Rha is rhamnose.
-3)-P-~-GlcP-(1-3)-P-~-ManP-(l-3)-8-D-ManP-(l-3)-~-~-RhaP
n
L
1
a-D-ManP
P - D - G ~ ~ P - ( ~ - ~ ) - P - D - M ~ ~ P - ( ~ - ~ ) - P - D - M ~ ~ P - ( ~ - ~ ) - L - R ~DISCUSSION
~P
2
1
a-D-ManP
(1)
A neutral polysaccharide having thestructure depicted
above was isolated from culture supernatants of a clinical
isolate of P. aeruginosa. This neutral polysaccharide bound
p-D-GlcP-(l-3)-p-D-ManP-(l-3)-8-D-ManP-(l-3)-~-Rhaol
antibody raised to the LPS and alkali-treated LPS from the
2
Fisher-Devlin immunotype strains, and a serologically cross1
reactive small molecular mass antigen was identified in cula-D-ManP
ture supernatants of six of seven of thesestrains. When
The signals in the 13C NMR spectrum of (11) were assigned antibodies raised to alkali-treated LPS were affinity-purified
by using the selective heteronuclear 13C[lH]double resonance, on a column of immobilized neutral polysaccharide, they also
and then the
NMR spectra of oligosaccharide (I) and the bound to LPS in an immunoblot, as well as whole cells of P.
polysaccharide were interpreted by comparison with the data aeruginosa in a colony blot assay, including cells from strains
for (11) (Table 4). The values of the coupling constants ’Jc,H with a rough LPS. Thus it appears that the neutral polysacfor all anomeric carbons, which are also listed in Table 4, charide contains an antigenic determinant also found in the
were determined from the gated decoupling
NMR spec- P. aeruginosa LPS. The neutral polysaccharide or a subunit
trum of the polysaccharide. These values confirmed the con- of it may be present in the LPS as a structure in the outer
figurations of the glycosidic linkages of the glucose and man- core linking the inner core containing the 2-keto-3-deoxynose residues and showed the rhamnose residue to be a-linked octolusonic acid (KDO), heptose, and hexosamine residues
( l J ~ . l . ~ 172
-I
HZ)(15).
with the type-specific polysaccharide side chain or may be a
Analysis of the effects of glycosidation on the chemical separate polysaccharide substituent on the LPS in addition
shifts of the signals for C-1 in the 13CNMR spectrum of the to the0-specific side chain.
polysaccharide allowed determination of the absolute configWe are only able to recover this neutral polysaccharide
urations of the 1,3-linked residues (14). The relatively small from supernatants of P. aeruginosa that have been in culture
11294
P. aeruginosa Cross-reactive Neutral Polysaccharide
for >72 h, even though logarithmic growth ceased after 18 h.
High molecular weight polysaccharides are also best recovered
from cultures >72 h of age, and these antigens clearly represent high molecular mass (>lo0 kDa) forms of theLPS
polysaccharide side chain. When side chains areisolated from
intact LPS the largest molecular mass is only 20 kDa (17).
These data are similar to the findings of Cadieux e t al. (18)
that P. aeruginosa continues active synthesis of lipopolysaccharide units after cessation of logarithmic growth.
Numerous investigators have studied the core portion of
the P. aeruginosa LPS (19-21). In general there appears to be
KDO, heptose, hexosamine, rhamnose, and glucose present.
Mannose appears as a minor component in only one report
(21). Since our neutral polysaccharide was mannose-rich, it
raises the question of its true relatedness to theP. ueruginosa
LPS core. One possibility is that the neutral polysaccharide
cross-reacts serologically with a structurally distinct portion
of the core. Another explanation is that a mannose-rich
portion of the core has not been identified because it makes
up only a minor portion of the total sugars in LPS. Hancock
e t al. (22) have shown that only a small proportion of the
total LPS molecules obtained from P. ueruginosa contain 0
side chains. Thus if the neutral polysaccharide or a subunit
of it is a bridge linking the inner core containing KDO,
heptose, hexosamine, glucose, and rhamnose, as suggested by
Horton et al. (17), with the 0 polysaccharide side chains, it
may be present as only a minor component of P. ueruginosa
LPS. The serologic reaction of our neutral polysaccharide
with antibodies to P. ueruginosa LPS should provide a basis
for a more detailed analysis of the structure of the P. ueruginosa LPS in order to confirm or deny the presence of a
mannose-rich region with a structure similar or identical to
the neutralpolysaccharide reported here.
In summary, we have isolated a neutralpolysaccharide from
supernatants of a strain of P. aeruginosa immunotype 4 and
determined its structure. Serologicallyrelated smallmolecular
weight material was found in supernatants of six of seven
heterologous strains of P. ueruginosa. Affinity-purified antibody to theneutral polysaccharide reacted with the LPSfrom
multiple strains of P. aeruginosa in an immunoblot and to
whole cells in a colony blot assay. A corresponding structure
to the neutral polysaccharide has not been identified in the
P. aeruginosa LPS by other investigators, but our serologic
data suggest its existence. If a mannose-rich structure is found
to be a component of the P. ueruginosa LPS, then the
isolation
and characterization of this neutral polysaccharide will have
provided an important clue to the overall structure of this
LPS molecule.
REFERENCES
and Farmer, J. J., 111 (1979) in Pseudomonas
aeruginosa, Clinical Manifestations of Infection and Current
Therapy (Doggett, R. G., ed) pp. 90-134, Academic Press,
Orlando, FL
2. Knirel, Y.A., Vinogradov, E. V., Shashkov, A. S., Dmitriev, B.
A., Kochetkov, N. K., Stanislavsky, E. S., and Mashilova, G .
M. (1985) Eur. J. Biochem. 150,541-550
3. Pier, G. B., Sidberry, H. F., Zolyomi, S., and Sadoff, J. C. (1978)
Infect. Immun. 2 2 , 908-918
4. Pier, G. B., DesJardins, D., Aguilar, T., Barnard, M., and Speert,
D. P.(1986) J. Clin. Microbiol. 24, 189-196
5. Blake, M. S., Johnston, K. H., Russell-Jones, G. J., and Gotschlich, E. C. (1984) Anal. Biochern. 1 3 6 , 175-179
6. Wagner, G., and Wuthrich, K. (1979) J. Magn. Reson. 3 3 , 675680
7. Conrad, H.E. (1971) in Methods in Carbohydrate Chemistry
(Whistler, R.L., and Be Miller, J. N., eds), Vol. 6,pp. 361-364,
Academic, New York
8. Mort, A. J., Parker, S., and Mao-Sung-Kuo, H. (1983) Anal.
Biochem. 133,380-384
9. Mort, A. J., Utille, J. P., Torri, G., and Perlin, A. S. (1983)
Carbohydr. Res. 1 2 1 , 221-232
10. Bebault, G. M., Choy, Y.H., Dutton, G . G. S., Funnel], N.,
Stephen, A.M., and Yang,M. T. (1973) J. Bacteriol. 1 1 3 ,
1345-1347
11. Altona, C., and Haasnoot, C. A. (1980) J . Org. Magn. Reson. 1 3 ,
417-429
12. Keller, R. M., and Wuthrich, K. (1981) Bid. Magn. Reson. 3 , l 52
13. Shashkov, A. S., Knirel, Y. A., Tanatar, N. V., and Kochetkov,
N. K. (1986) Carbohydr. Res. 1 4 6 , 346-349
14. Bock, K., and Pedersen, C. (1974) J. Chern. SOC.Perkin Trans.
II,293-297
15. Kochetkov, N. K., Chizhov, 0. S., and Shashkov, A. S. (1984)
Carbohydr. Res. 133,173-185
16. Klyne, W. (1950) Biochem. J. 47, xli-xlii
17. Horton, D., Riley, D.A., Samreth, S., and Schweitzer, M.G.
(1983) in Bacterial Lipopolysaccharides: Structure,Synthesis
and Biological Activities. ACS Symposium Series, No. 231 (Anderson, L., and Unger, F. M., eds) pp. 21-47, American Chemical Society, New York
18. Cadieux, J. E., Kuzio, J., Milazzo, F. H., and Kropinski, A.M.
(1983) J. Bacteriol. 155,817-825
19. Rowe, P. S. N., and Meadow, P. M. (1983) Eur. J. Biochem. 1 3 2 ,
329-337
20. Wilkinson, S. G., and Galbraith, L. (1975) Eur. J. Biochem. 5 2 ,
331-343
21. Fensom, A. H., and Meadow, P. M. (1970) FEBS Lett. 9,81-84
22. Hancock, R. E. W., Mutharia, L. M., Chan, L., Darveau, R. P.,
Speert, D. P., and Pier, G . B. (1983) Infect. Immunol. 42,170177
1. Brokopp, C.D.,
P. aeruginosa Cross-reactive Neutral Polysaccharide
11295
Supplementary material to: Structure o f an € x t n c r l l u l a r .
Cross Reactive Polysaccharide from PI.udaonlr lwab9.a
I m m o t y p e 4.
Ulna A. Kocharwa. Vurly A. Knlrel.
Alexander 5 . Shashkov. Hlkolry 1. Kochttkov and Gerald 8.
Table 4 .
COnpOvnd and
Data forI3C-lllR
c-l
101.7
101.4
97.8
14.2
71.2
74.2
77.2
71.5
70.8
67.9
80.5
101.9
68.8
63.8
72.3
82.2
79.5
66.8
66.4
0ligosa~ch.rlde (I)
GlCb
101.9
n.M
101.5
.2,3)nm6
97.9
.3)Mrn$
-3)Rhm
102.2
Polysaccharide
.3)GlC$
74.7
-
e.
61.9
77.3
73.6
71.4
77.7
69.5
72.9
62.0
62.2
62.2
62.1
62.2
62.3
20.2
77.3
71.6
80.7
70.8
68.1
66.9
82.4
66.3
80.4
82.7
72.5
72.3
83.8
69.4
77.2
62.0
67.9
73.5
62.1
62.3
17.9
17.9
71.5 71.3
.2,3)Hanb
(1731
97.8
74.5
80.6
66.9
77.4
62.1
W.8
82.5
66.3
77.7
62.2
71.5
17.780.5
70.0
72.3
%g;
!!:I(173)
6.D.glucopyrmosldc I81 -34.2
e-D.mmnopyranoside 181 t79.2
6-D.mnnopyranoslde 181 -49
a-L-rhamnOpyrmOsldC (91 47.2
Methyl
Methyl
nethyl
lethyl
e.
77.2
73.5
77.3
77.7
67.9
(160)
101.5
-3)Rhm
e.
101.7
75.5
[Hz11 reslduc
C-6
Ham
-3)Manp
Antibodies t o theneutralpolysaccharide
In antisera ralsed t o alkali-treated
tPS from Flsher
INnotypes
1.3 and 5 were recovered vrlng affinity
chrcutoclrwhy
techniques. These m t l b o d t c s bound well totheneutralpolysaccharlde
I n an €LISA plate.
By I n u n o b l o t analysis these antlbodles bound t o both the 0-polysaccharide contalnlng
ladders and fast . o v l n g core c ~ l p o n m t s of f l v eo f
six r t n l n r of
&walWIi LVS
evaluated by t h i s procedure ( f i g 3). The spacing o f the ladder l l k e 0 sldechalnshele was
ldent>cal 10 t h a t observed when antisera t o the 0 sldc Chalns i s employed i n an Imunoblot
(not s h a ) . I n a colony b l o t a s s l y t h e a f f l n l t y p w l f i e d
antibodies bound s p e c l f l c l l l y t o
stlains. but not other b a c t e r l a l splclcs (Flg 41, except f ocr e l l s
Of
i m n o t y p e 6. The non-reactlve Innunotype 3 LPS ( F l g 3) and i r y n o t y p e 6 cells (Fig 4 1 did
react w i t h the a f f l n l t yp w l f l e da n t l b o d l e r
*hen they r r e used a t I 3 fold
higher
ThlsCOnflnLdthe
cross rractlre natule Of IntlbQdy t o the
LOnCPntratlOn (not s h a ) .
neutral polysaccharide w n g isolated LPS and whole cells of
74.5
71.3
74.2
68.8
72.0
72.2
94.8
94.8
.3)Rhap
sputn.
s h l f t s I n p p : (coupling constants
IJc.H
C-2
C-3
(-4
C-5
Ch"lCal
Polysaccharide
observed value
calculated for
.a-D-mannopyranosc
a-L-mannopynnose
194
194
-66.3
,153.6
-95.1
178
-119.6
194
.29.0 (c 21
-222.5
794
-529.7
794
-28.0
-66.6
-.
LF5
A l k tanl ti e d L Q S
Yhole cells
1
2.8'
2
3
4
5
6
1
1.6
7.4
3.9
2.0
9.0
8.6
2.0
2.0
2.9
5.0
1.9
8.6
8.5
1.4
10.9
4.9
7.5
7.8
6.5
9.7
....................................................................................
1
61C
Man
H-l
H-2
H-3
:3.56
H-4
3.43-3.52
H-5
H-6 (2 H)
3.43-3.52
H.1
H-2
H-3
H-4
H.5
5.43
4.10
H-6
H-6'
-2.3)Man H - l
4.89
H-2
H-3
4.43
4.04
H-4
3.79
3.47
H-5
d
dd
dd
t
ddd
dd
dd
b
d
dd
t
H-6 (2 H)
3.90-4.03
H-l
4.82
.
.
.90-4.03
dd
dd
ddd
dd
dd
H-5
3.95
3.74
3.85
4.12
3.64
3.79
H-6 (3 H)
1.33
q
:
H-l
H-I'
H-2
H-3
H-4
"-
b
.45
-3)Rhaolb
€ L I S indexcalculated by d l v i d i n g mean o p t i c a ld e n r l t yo f
pre-llunltien serum by mean O p t l C I l density Of 1:lOO
3.90-4.03
3.99
3.73
4.22
3.87
3.82
-31M.n
d
dd
1
7 6 5 4 3 1
m
.
.
Fbs
,
M1
TT3T2 l
Table 3.
nsldws
the
PC
J5
T4
Huclear Overhauser m h a n c m n t s In OllgOsaccharlde (11) a r l s l n g on
p r e - i r r a d i a t l o n of H - l a
vrc-lrradlated
Muclew
rcrldue
Overhauser e n h a n c a n t s a t the protons
5.3
Of
5.5
3.6 5.6 9.0 5.5 7.2
7.3 4.5 7.37.4
T5
T7
T6
NW
1:lOO d l l u t i o n o f
d l l u t l oonf
~011