- Email: [email protected]
An unusual acidic polysaccharide produced by a rough strain of Escherichia coli
Vol. 61, No. 1,1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ANuNmJALlAClD1cPOLYSAammD
EPIFCOUlEDBYAFQUGM
P.W. Taylor Deparhnentof~cine,CharingCrossHaspital~cdlS~~l,~PdLace Road, Lcndcm, W.6., England. Received
August
28,1974
Z3LMWlY. - Lipqolysacchariae andanacidicpolysaccharidewre extracted withjjLwl-waterfranamughstrainofEs&erichiacoli (LP1092). The plysacxhari& portian of lipoplysac&aride antained galactme, glucose, Dnnwnoheptose,sndLl~~of~~~an~~uallyhi~ WlYpropotionoffdeoxyIhnmwo&ulmnic acid: thispolysaccharidewas shmntorepresenttbeca@etecoliR2oXe. Theacidicpolysaccharide, ~icfi~~~asaKanti~,ccntainedlargeanountsofa2-keto-3-deoxy sugar acid. Q1colorimtricand&ranatogra@cevidemethisacidappeared tobe3-deoxyDamm-c&uloscnic acid.
It
is well
established
that
mugh
of Gramnegative
strains
bacteria
~,in~~,susoeptibletothebactericidal~~ofnormalh~ senrm (1).
'Iheincubaticnoflog~e~~of~~Escheri~acoli
strainsinsenrmusudllyresultsina~~icplintheviablec;olmttoless thanl%
of the inoculmsisewithincme
hour (unpklishedcbservation).
An
E.colistrainisolatedfrcmacaseofurinarytractinfectioninthis Departmntappearedtobe
anexceptiontothis
rule.
ated in 3.5% w/v NaCl and in 0.2% w/v acriflavine ically pattern
rough colonies
onnutiientagarplates.
to rough-specific
ba&erioPhages
its sensitivity
lipcpolysacdmride
assayed by a technigue
thisdelayedkillingeffectis (6).
In additim,
frm
a
R2 core stzuct-
StrainLP1092waskilledbyserunbutornlyafteradelayof
l-2 hours (Fig. 1) ken
strains
agglutin-
and prcducedmxpholog-
(Table 1) was that eqected
roughE.mlistrainpossessingacarplete ure (2,3,4).
StrainIJ?1092
I@xemer,
described
previously
(5);
characteristicofcertainsaootbE.coli in cmtrasttiF576
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
(R2) oells
liveLP1092
cells
did
BIOCHEMICAL
Vol. 61, No. 1,1974
Bacterio@age sensitivity* FU, R2 and Ft3 lipoplysaccharlde
(2,3).
+, anflwnt
StrainNo. IJ?1092
*
lysis;
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pattsms core
ofE.coli
pmbtype
LPlO92 andtheE.coli strains 0fSdmidtetal
-, no lysis.
ctxe Type +
?R2
+
F470
Rl
--++++++
F576
R2
+
Ix53
R3
-++++-++
+
BrlO
-1
6SR
C21
T3
T4
+
+
+
-
+
+
I-
+
+
-
+
+
deemhed by applying dmps of @age antaining mlcntosurface-inoculatednutsientagarplates. s=liedbyDr. G. SMdtandDr. R. Wilkinson.
mtely 108 p.f.u./ Pha~werekindly
1000
I 2
loo
: z 2 10
0
1
2
3
TIME(h)
Fig.
1.
not agglutinate
'Ibexes&mseofE.cxXIiIP1092 to five saqles of fresh nomalhunanserm&tainedfmndifferentinvididuals. Washed log&asebacteria (1x106) in lml oftris [email protected] perfarmed after 0,1,2 and 3h incubation at 3 in rabbit
or F576 cells,
aMmugh
unhea~LP1092
cells.
antiseraprepamd they did agglufzinate
againstheated in antisera
~eresultss~~~thatE.coli~1092,dlthoufP
149
(loO°C; lh) Ip1092 prepared against
deiznninedhy s
5120
<20
F576
**
160
5120
00
Ll?1092 0.25
0.17
Malmose
Ratio
1.00
1.11
Galactose
t-mar
1.18
1.36
Gluco6e
**
1.05
2.58
3+koxy-D-imnmcctulosonic acid.
LP1092 and F576.
W&am104 series Gas-ChranQ.
1.00
1.00
sL-glycem-P
fmn E.ccli
gas-liquid chmnatogr@yof alditilacetates using aPye fittedwithcolunns axtaining 3%ECNSS-Mon100/1201nesh
320
anti.F653
antiF576
Passive haernagg1utlnatioxl*
antiE470
serolcgicsl
TABLF, 2:
BIOCHEMICAL
Vol. 61, No. 1,1974
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
thecellsurfaceautigensofthis
rough,mi~tsynthesiseaK-antigen; strainwereUmxfore
investigated
IPlO92
cultivated
cellswem
water (7).
Water-soluble
further. innutrientbroth
amdextractedwith@enol-
polysaccharideswereprecipitatedinooldethauol
~fractionatedwith~tav~acoordingtoprocedureIIIofweSt~~ Jauu (7).
lko major ribmucleic
olysaccharide G-3/g),
(25ng/gdry
acid-free
cellmass)
fractims,
audthe
one representing
lipair
other acidicpolysaccharide
were -.
The results
ofpassivehaenagglutinationnts
lipopolysaocharide neutral
fmnIJ?lC#2
contained
sugaraqcsitimofLp1092
by gas - liquid hydrolysis
(2) amfinnE?dthat the K2 core (Table 2).
audF576
lipc@ysaccharides,
chramtogra@yofalditolacetates
inO.lNHCl
(lOO°C;
ainedslnallanKnmtsof Daamoheplxxe
48h),wereverysimilar.
The mount
asdetmmined
of sugars releasedby
(8,9)
mannose in additionto
(Table 2).
Also, the
Eothpolymm
galactose,gluoose
of 2-ketc~3-deoxy
cont-
andI.-glym
sugar acid (assunedto
be3-deoxy-~cacid)inthepreparationswasda~bythe thicbarbiturate
reacticm
octuloscmicacid, acid (L!),
(10,ll);
tie mum&m
pnapamdbytbe
cmknsatimof
was used as a standard.
acid
hexurrmic
fran rough strains
hoi+mer,
in the thicbarbiturati
ccntained
(Table 2).
colorimtrically
for the presence
acid (14) and hexDsannine (15) residwas;
test
(10, 11) with a)tnax of 547rrm (Fig.2);
(11),N-aoety~~~icaciddidnat~~this
The acidicpolysaccharide
a~peamdto
consistof
90-100% 2-keto-3-deoxy
subjectedto&scendingpaperchranatogra@y 151
as test.
The acidicpol~~i~was~~lysedin0.1NH2s04
audhydmlysates
mdoxalacetic
TheuuhydrolysedpreIzeraticmdidreactverystmmgly,
also foundbyEllmod
acid.
of E.coli
examizd
(13), neurminic
nmaweredetected.
D-arabinose
uloscmicacidthanisnomallyfoundin
The acidicpolysacdmridewas of
of 3-&oxy-Imkmno-
LPlC@2 lipopolysac&aride
gmaaterammtsof3Aeoxy-kmm-oct lipopolysaccharides
salt
(lm°C;
sugar 3Omin)
inbutan-2-01+
Vol. 61, No. 1,1974
BIOCHEMICAL
I 450
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
500
550
t
WAVELENGTH
Fig. 2.
AbcQt.i~spectra franE.coliLP1092, octuhxnicacid,
for (a) 3O)qacidicpolysaazharids md (b) 3Cjq 34eoxy-D-mnnointhethidmrbituratereactim.
TABLE 3: Ideactivityoflipqplysaccfiarideandacidicpolysaccharidefran E.aAiWlO92 in mtiserara.bedagainstheab?d ("F?'antisera) (“RK”
antisera)
Lao92
and live
adls.
Passivehelagghltinatim* “R”
Preparation
“lx”
antisera
antisera
Lipopolysacdmride
640
10240
ZIcidicpolysa~
< 20
5120
acetic
acid + water (8+1+l,vb)
v/d ;
dmmaWgramwaxedevelapedacmnAingtiAnder=(16).
sys-,
ee hydrolysate
and butan-
+ pyriw
+ O.lN HCl (5+3+2,
ChrcmatograFhed asasinglepinkthicbaxbiturate
reactivespotwithanidenticdt~i~tyto~i~trreatedsrmplesofsynthetiC
152
Inboth
BIOCHEMICAL
Vol. 61, No. 1,1974
3-~Pmamo-octulosonicacid. sugar acids were mt 3-deoq-D-m results
Althou& available,
therefore,
fmnE.coliIP1092
standards of other 2-keW3-deoxy
theymaybemadilydifferet-~tiatedf~~~~~
acid in the solvent
ulosanic
suggest,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
thatthemajor
systems used (11).
oqmnentof
is +deoxy-IF maum-o&uloscmic
hamagglutinaticm
tests;
sheepredcells
acidicpolysaccharide
acid.
frunIP1092behaveda.s
Acidicplysaccharide
aK-antigeninpassive
coatedwith
acidicpolysaccharide
readedinrabbitantiseraraisedagainstLiveLP1092 preparedwithheatedcells able to non-specifically
(Table 3). inhibit
These
cellsbutnotinantisera In addition,
acidicpolysaccharidswas
the agglutinaticm
redce11seruminthesystmofGlynnandHaward
of sheep red oell.5 by anti(17);
thisabilityischar-
acteristicofmauyK-antigenpreparations. K-antigens
areproducedbyalargenmberofE.colistrains,andthe
majorityexancinedhaTRbeenpolysa~arideswithacidicpropertiesdue ic acid
thepzsenceofhexumn @ms#ate
(20) residues.
(18), N-acetyl-neuraminic
lIhere appears tobeno
~~,ofacidicpolysaccfiarides~~~~~D acid.
Therelativelylarge
report
to acid
(19) or glycerol
in the literature, -CtUlOSOniC
mmntsofthe
sugaracidassociatedwiththe
~papolysa~~couldresultfran~~ti~withacidicpolysa~aride, oritmi~tsu~tthatthereis~ilecrreeofLinkagebetkRenthepo1~. ~rstudieswillbe~saryinordertodetenninewhetherornotthe acidicpolysaccharide
is reqxmsible
forthedelayedsenmkillingeffect,
aluloughitisinterestingtonoteulatitdoesnotappeartoaff~~ &age sensitivity
1. 2. 3. 4.
of E.coli
LP1092.
I&an-, R.J. (1967) Win. I&v. Micrcbiol. 2& 443-466. Schmidt, G., Jam, B. aud Jam, K. .(1969) Eurap. J. Biocimn. lo, 501-510. SchmLdt, G., Frarme, I. and Mayer, H. (1970) Euop. J. Biochm. 2, 357-366. Schmidt, G. Janu, B. and Jam, K. (1974) FXuq. J. Biochrm. 42, 303-309. 153
Vol. 61, No. 1,1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10. 11. 12.
Taylor, P.W. &berts, A.P. and GakFerP.E. (1972) I4zd. Lab. Techml. 2, 272-279. Taylor, P.W. (1974) J. Clin. Path. (in press). West@al,O.andJann,K. (1965)&hzdsinCbchydrateCbernistry, 2, 83-91. Sawardeker, J.S., Slmeker, J.H. and Jeans, A. (1965) Anal. C&m. ~,1602-1604. Holnq T., Lindbeq, A.A., Garegg, P.J. and Qm, T. (19681, J. Gen. Micmbiol. 52, 45-54. Qnkin, M.A. and Ashmll, G. (1960) Nature I&, 155-156. Ellwood, D.C. (1970), J. Gen. Microbial. 60, 373-380. Hershberger, C., Davis, M. and Binkley, S.B. (1968) J. Biol. Clmn.
13, 14.
Dia, Barry,
15.
Struninger,
5. 6. 7. 8. 9.
243, 2, C!bn. 16. 17. 18. 19. 20.
1585-1588. Z. (l947),
G.T., Abbott,
J. Biol. Chem.167, 189-198. V. and Tsai, T. (19621, J. Gen. Micrdzhol.
335-352. 234,
J.L.,
Park, J.T. and Tharpson R.E. (19591, J. Biol.
3263-3268.
zmdemon, P.J. (1966) J. Chranatq. 21, 163-164. Glynn, A.A. and How&, C.J. (1970) Imnmology 18, 331-346. Jann, K. (1965) Colloquiun der gsellscfiaft fiir physiolcgische chemie, &p 144-157, Springer Verlag, Berlin. FhzLsman, A-P., Senwiratne, E.C.A. and Hawkhs, D.C. (1967) Biochem. J. 102, %P-9P. Jann, B., Jann, K., Sdxnidt, G., OrskoV, I. and Orskov, F. (1970) Eump. J. Biodwn. 15, 29-39.
154
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ANuNmJALlAClD1cPOLYSAammD
EPIFCOUlEDBYAFQUGM
P.W. Taylor Deparhnentof~cine,CharingCrossHaspital~cdlS~~l,~PdLace Road, Lcndcm, W.6., England. Received
August
28,1974
Z3LMWlY. - Lipqolysacchariae andanacidicpolysaccharidewre extracted withjjLwl-waterfranamughstrainofEs&erichiacoli (LP1092). The plysacxhari& portian of lipoplysac&aride antained galactme, glucose, Dnnwnoheptose,sndLl~~of~~~an~~uallyhi~ WlYpropotionoffdeoxyIhnmwo&ulmnic acid: thispolysaccharidewas shmntorepresenttbeca@etecoliR2oXe. Theacidicpolysaccharide, ~icfi~~~asaKanti~,ccntainedlargeanountsofa2-keto-3-deoxy sugar acid. Q1colorimtricand&ranatogra@cevidemethisacidappeared tobe3-deoxyDamm-c&uloscnic acid.
It
is well
established
that
mugh
of Gramnegative
strains
bacteria
~,in~~,susoeptibletothebactericidal~~ofnormalh~ senrm (1).
'Iheincubaticnoflog~e~~of~~Escheri~acoli
strainsinsenrmusudllyresultsina~~icplintheviablec;olmttoless thanl%
of the inoculmsisewithincme
hour (unpklishedcbservation).
An
E.colistrainisolatedfrcmacaseofurinarytractinfectioninthis Departmntappearedtobe
anexceptiontothis
rule.
ated in 3.5% w/v NaCl and in 0.2% w/v acriflavine ically pattern
rough colonies
onnutiientagarplates.
to rough-specific
ba&erioPhages
its sensitivity
lipcpolysacdmride
assayed by a technigue
thisdelayedkillingeffectis (6).
In additim,
frm
a
R2 core stzuct-
StrainLP1092waskilledbyserunbutornlyafteradelayof
l-2 hours (Fig. 1) ken
strains
agglutin-
and prcducedmxpholog-
(Table 1) was that eqected
roughE.mlistrainpossessingacarplete ure (2,3,4).
StrainIJ?1092
I@xemer,
described
previously
(5);
characteristicofcertainsaootbE.coli in cmtrasttiF576
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
148
(R2) oells
liveLP1092
cells
did
BIOCHEMICAL
Vol. 61, No. 1,1974
Bacterio@age sensitivity* FU, R2 and Ft3 lipoplysaccharlde
(2,3).
+, anflwnt
StrainNo. IJ?1092
*
lysis;
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
pattsms core
ofE.coli
pmbtype
LPlO92 andtheE.coli strains 0fSdmidtetal
-, no lysis.
ctxe Type +
?R2
+
F470
Rl
--++++++
F576
R2
+
Ix53
R3
-++++-++
+
BrlO
-1
6SR
C21
T3
T4
+
+
+
-
+
+
I-
+
+
-
+
+
deemhed by applying dmps of @age antaining mlcntosurface-inoculatednutsientagarplates. s=liedbyDr. G. SMdtandDr. R. Wilkinson.
mtely 108 p.f.u./ Pha~werekindly
1000
I 2
loo
: z 2 10
0
1
2
3
TIME(h)
Fig.
1.
not agglutinate
'Ibexes&mseofE.cxXIiIP1092 to five saqles of fresh nomalhunanserm&tainedfmndifferentinvididuals. Washed log&asebacteria (1x106) in lml oftris [email protected] perfarmed after 0,1,2 and 3h incubation at 3 in rabbit
or F576 cells,
aMmugh
unhea~LP1092
cells.
antiseraprepamd they did agglufzinate
againstheated in antisera
~eresultss~~~thatE.coli~1092,dlthoufP
149
(loO°C; lh) Ip1092 prepared against
deiznninedhy s
5120
<20
F576
**
160
5120
00
Ll?1092 0.25
0.17
Malmose
Ratio
1.00
1.11
Galactose
t-mar
1.18
1.36
Gluco6e
**
1.05
2.58
3+koxy-D-imnmcctulosonic acid.
LP1092 and F576.
W&am104 series Gas-ChranQ.
1.00
1.00
sL-glycem-P
fmn E.ccli
gas-liquid chmnatogr@yof alditilacetates using aPye fittedwithcolunns axtaining 3%ECNSS-Mon100/1201nesh
320
anti.F653
antiF576
Passive haernagg1utlnatioxl*
antiE470
serolcgicsl
TABLF, 2:
BIOCHEMICAL
Vol. 61, No. 1,1974
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
thecellsurfaceautigensofthis
rough,mi~tsynthesiseaK-antigen; strainwereUmxfore
investigated
IPlO92
cultivated
cellswem
water (7).
Water-soluble
further. innutrientbroth
amdextractedwith@enol-
polysaccharideswereprecipitatedinooldethauol
~fractionatedwith~tav~acoordingtoprocedureIIIofweSt~~ Jauu (7).
lko major ribmucleic
olysaccharide G-3/g),
(25ng/gdry
acid-free
cellmass)
fractims,
audthe
one representing
lipair
other acidicpolysaccharide
were -.
The results
ofpassivehaenagglutinationnts
lipopolysaocharide neutral
fmnIJ?lC#2
contained
sugaraqcsitimofLp1092
by gas - liquid hydrolysis
(2) amfinnE?dthat the K2 core (Table 2).
audF576
lipc@ysaccharides,
chramtogra@yofalditolacetates
inO.lNHCl
(lOO°C;
ainedslnallanKnmtsof Daamoheplxxe
48h),wereverysimilar.
The mount
asdetmmined
of sugars releasedby
(8,9)
mannose in additionto
(Table 2).
Also, the
Eothpolymm
galactose,gluoose
of 2-ketc~3-deoxy
cont-
andI.-glym
sugar acid (assunedto
be3-deoxy-~cacid)inthepreparationswasda~bythe thicbarbiturate
reacticm
octuloscmicacid, acid (L!),
(10,ll);
tie mum&m
pnapamdbytbe
cmknsatimof
was used as a standard.
acid
hexurrmic
fran rough strains
hoi+mer,
in the thicbarbiturati
ccntained
(Table 2).
colorimtrically
for the presence
acid (14) and hexDsannine (15) residwas;
test
(10, 11) with a)tnax of 547rrm (Fig.2);
(11),N-aoety~~~icaciddidnat~~this
The acidicpolysaccharide
a~peamdto
consistof
90-100% 2-keto-3-deoxy
subjectedto&scendingpaperchranatogra@y 151
as test.
The acidicpol~~i~was~~lysedin0.1NH2s04
audhydmlysates
mdoxalacetic
TheuuhydrolysedpreIzeraticmdidreactverystmmgly,
also foundbyEllmod
acid.
of E.coli
examizd
(13), neurminic
nmaweredetected.
D-arabinose
uloscmicacidthanisnomallyfoundin
The acidicpolysacdmridewas of
of 3-&oxy-Imkmno-
LPlC@2 lipopolysac&aride
gmaaterammtsof3Aeoxy-kmm-oct lipopolysaccharides
salt
(lm°C;
sugar 3Omin)
inbutan-2-01+
Vol. 61, No. 1,1974
BIOCHEMICAL
I 450
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
500
550
t
WAVELENGTH
Fig. 2.
AbcQt.i~spectra franE.coliLP1092, octuhxnicacid,
for (a) 3O)qacidicpolysaazharids md (b) 3Cjq 34eoxy-D-mnnointhethidmrbituratereactim.
TABLE 3: Ideactivityoflipqplysaccfiarideandacidicpolysaccharidefran E.aAiWlO92 in mtiserara.bedagainstheab?d ("F?'antisera) (“RK”
antisera)
Lao92
and live
adls.
Passivehelagghltinatim* “R”
Preparation
“lx”
antisera
antisera
Lipopolysacdmride
640
10240
ZIcidicpolysa~
< 20
5120
acetic
acid + water (8+1+l,vb)
v/d ;
dmmaWgramwaxedevelapedacmnAingtiAnder=(16).
sys-,
ee hydrolysate
and butan-
+ pyriw
+ O.lN HCl (5+3+2,
ChrcmatograFhed asasinglepinkthicbaxbiturate
reactivespotwithanidenticdt~i~tyto~i~trreatedsrmplesofsynthetiC
152
Inboth
BIOCHEMICAL
Vol. 61, No. 1,1974
3-~Pmamo-octulosonicacid. sugar acids were mt 3-deoq-D-m results
Althou& available,
therefore,
fmnE.coliIP1092
standards of other 2-keW3-deoxy
theymaybemadilydifferet-~tiatedf~~~~~
acid in the solvent
ulosanic
suggest,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
thatthemajor
systems used (11).
oqmnentof
is +deoxy-IF maum-o&uloscmic
hamagglutinaticm
tests;
sheepredcells
acidicpolysaccharide
acid.
frunIP1092behaveda.s
Acidicplysaccharide
aK-antigeninpassive
coatedwith
acidicpolysaccharide
readedinrabbitantiseraraisedagainstLiveLP1092 preparedwithheatedcells able to non-specifically
(Table 3). inhibit
These
cellsbutnotinantisera In addition,
acidicpolysaccharidswas
the agglutinaticm
redce11seruminthesystmofGlynnandHaward
of sheep red oell.5 by anti(17);
thisabilityischar-
acteristicofmauyK-antigenpreparations. K-antigens
areproducedbyalargenmberofE.colistrains,andthe
majorityexancinedhaTRbeenpolysa~arideswithacidicpropertiesdue ic acid
thepzsenceofhexumn @ms#ate
(20) residues.
(18), N-acetyl-neuraminic
lIhere appears tobeno
~~,ofacidicpolysaccfiarides~~~~~D acid.
Therelativelylarge
report
to acid
(19) or glycerol
in the literature, -CtUlOSOniC
mmntsofthe
sugaracidassociatedwiththe
~papolysa~~couldresultfran~~ti~withacidicpolysa~aride, oritmi~tsu~tthatthereis~ilecrreeofLinkagebetkRenthepo1~. ~rstudieswillbe~saryinordertodetenninewhetherornotthe acidicpolysaccharide
is reqxmsible
forthedelayedsenmkillingeffect,
aluloughitisinterestingtonoteulatitdoesnotappeartoaff~~ &age sensitivity
1. 2. 3. 4.
of E.coli
LP1092.
I&an-, R.J. (1967) Win. I&v. Micrcbiol. 2& 443-466. Schmidt, G., Jam, B. aud Jam, K. .(1969) Eurap. J. Biocimn. lo, 501-510. SchmLdt, G., Frarme, I. and Mayer, H. (1970) Euop. J. Biochm. 2, 357-366. Schmidt, G. Janu, B. and Jam, K. (1974) FXuq. J. Biochrm. 42, 303-309. 153
Vol. 61, No. 1,1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10. 11. 12.
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