The Vibrio cholerae O139 Serogroup Antigen Includes an O-Antigen Capsule and Lipopolysaccharide Virulence Determinants

Abstract

Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.

Full text

Proc.
Natd.
Acad.
Sci.
USA
Vol.
91,
pp.
11388-11392,
November
1994
Microbiology
The
Vibrio
chokrae
0139
serogroup
antigen
includes
an
0-antigen
capsule
and
lipopolysaccharide
virulence
determinants
/cpuea
dnl
loa
tlverse
ICR)
MATTHEW
K.
WALDOR*,
RITA
COLWELLt,
AND
JOHN
J.
MEKALANOS*
*Depatent
of
Microbiology
and
Molecular
Genetics,
Harvard
Medical
School,
200
Longwood
Avenue,
Boston,
MA
02115;
and
tUniversity
of
Maryland
Biotechnology
Institute,
4321
Hartwick
Road,
College
Park,
MD
20741
Communicated
by
Emil
C.
Gotschlich,
August
10,
1994
ABSTRACT
Vibno
cholera
serogroup
0139
emergd
on
the
India
subcontinent
In
October
1992
to
become
the
first
non-01
V.
cholra.
serou
document
to
cause
epidemic
cholera.
APlt
related
to
V.
cholra.
El
Tor
01
strains,
0139
strains
have
unique
surface
structures
that
indude
a
capsular
surface
layer
and
lpopolysaccharide
(LPS).
Immu-
noblot
analysis
of
either
whole-cell
lysates
or
LPS
preparations
revdead
three
electrophoretic
forms
of
the
0139
antigen:
two
slowly
migrating
form
and
one
rapidly
m
ig
form
that
appeared
identical
to
0139
LPS.
Al
three
forms
of
the
antigen
shared
an
epitope
defined
by
an
0139-specific
monoclonal
antibody.
A
serum-sensitive
ted
mutawas
iso-
lated
that
lacks
only
the
slow
miting
forms.
The
slow
m
ingat
forms
did
not
stain
with
silver
whereas
the
rapidly
migrating
form
did,
that
the
former
might
stitute
highly
polymerized
O-antigen
side-cain
oules
that
were
not
covalently
bound
to
core
polysaccharide
and
lipid
A
(an
"0-antigen
capsule").
A
single
insertion
uled
in
the
loss
of
mnraivity
of
both
the
LPS
and
the
O-atigen
c
e,
Implying
that
there
are
genes
common
to
the
biosyn-
theds
of
both
these
m
am
.
The
0139
LPS
and
O-antgen
capsule
were
both
Iportant
for
colnzation
f
the
small
inetne
of
the
newborn
mouse
and
for
serum
resistance,
demonstrating
that
both
of
these
forms
of
the
0139
serogroup
antigen
are
vrudence
factors.
Cholera
is
a
severe
and
sometimes
lethal
secretory
diarrheal
disease
caused
by
the
Gram
negative
bacterium
Vibrio
chol-
erae.
Historically,
only
the
01
serogroup
of
V.
cholerae
has
been
associated
with
cholera
epidemics.
However,
in
early
1993
a
dramatic
shift
in
the
cause
of
cholera
was
observed
with
the
emergence
of
a
major
cholera
epidemic
in
India
and
Bangladesh
caused
by
a
non-Ol
serogroup
(1,
2).
This
non-O1
serogroup
was
named
V.
cholerae
0139
synonym
Bengal
(3).
Epidemiologic
data
from
the
large
0139
epidemics
in
Bangladesh
and
India
strongly
suggest
that
immunity
to
V.
cholerae
01
does
not
protect
against
0139
infection
and
thus
support
the
notion
that
the
0
antigen
of
lipopolysaccharide
(LPS)
is
the
major
protective
antigen
of
V.
cholerae
(4,
5).
The
01
serogroup
of
V.
cholerae
is
divided
into
two
biotypes-classical
and
El
Tor.
Many
laboratories
have
shown
that
0139
strains
are
very
similar
to
El
Tor
01
strains
(4,
6-11).
However,
0139
strains
belong
to
a
distinct
sero-
group
defined
by
monoclonal
antibodies
and
polyclonal
an-
tisera
that
only
recognize
0139
strains
(3,
12).
In
V.
cholerae
01,
the
chemical
basis
for
the
serogroup-defining
antigen
lies
in
the
0
side
chain
of
LPS
(13).
Biochemical
characterization
of
the
LPS
from
0139
strains
has
demonstrated
that
0139
LPS
differs
from
01
LPS
in
that
it
has
a
short
O-side-chain
length
and
different
sugar
composition
(13-15).
Electron
microscopic
studies
have
demonstrated
that
0139
strains
are
encapsulated
(10,
15).
The
chemical
composition
of
a
capsular
polysaccharide
has
been
reported
(15),
but
it
is
not
clear
whether
this
material
corresponds
to
an
0139-
specific
antigen
and/or
the
capsular
surface
layer
seen
in
the
electron
microscopic
studies.
Also,
it
is
not
known
whether
the
capsule
or
the
0
antigen
is
important
for
intestinal
colonization
and
virulence
of
0139
strains.
In
the
current
study,
we
have
isolated
and
characterized
mutant
strains
with
different
forms
of
the
0139
antigen.
Immunoblot
analyses
revealed
three
electrophoretic
forms
of
the
0139
antigen.
Our
analysis
suggests
that
one
of
these
electrophoretic
forms
corresponds
to
LPS
and
the
other
two
correspond
to
an
0-antigen-related
capsular
polysaccharide.
Both
the
O-antigen
capsule
and
the
LPS
0
side
chains
were
shown
to
play
an
important
role
in
serum
resistance
and
in
colonization
of
the
infant
mouse
small
intestine.
MATERIALS
AND
METHODS
Bacterial
Strains
and
Antisera.
Bacterial
strains
were
main-
tained
at
-700C
in
LB
broth
(16)
containing
20%6
(vol/vol)
glycerol.
The
phenotypes
of
the
V.
cholerae
strains
used
in
this
study
are
listed
in
Table
1.
Formalin-treated
cells
of
the
0139
type
strain
M045
were
used
to
raise
a
polyclonal
antiserum
in
New
Zealand
White
rabbits.
Monoclonal
anti-
body
2D12
was
raised
against
the
0139
strain
SG24
and
is
a
mouse
IgG2a
(12).
Transposon
Mutagenesis
and
Selection
of
0139-Negative
Mutants.
TnSlac
(17)
was
used
to
mutagenize
the
0139
strain
Bengal-2,
a
prototype
vaccine
derivative
of
MOlO
(11).
Tn5lac
mutagenesis
of
Bengal-2
was
performed
with
plasmid
pMW1,
in
a
manner
similar
to
that
described
for
TnphoA
mutagenesis
of
V.
cholerae
01
strains
with
plasmid
pRT291
(18).
TnSlac
insertion
mutants
of
Bengal-2
that
were
0139-
negative
were
enriched
from
pools
of
transposon
mutants
by
agglutination
of
the
0139-positive
cells
with
polyclonal
anti-
0139
antisera.
Nonagglutinating
cells
were
purified
and
retested
by
slide
agglutination
with
the
anti-0139
antisera.
Southern
blot
analysis
was
used
to
confirm
that
each
0139-
negative
strain
contained
a
single
transposon
insertion.
Biocemial
and
munobt
Analyses.
V.
cholerae
strains
were
grown
in
LB
broth
overnight
at
3rC
on
a
roller
incubator.
To
prepare
whole-cell
lysates,
cells
were
collected
by
centrifugation,
resuspended
in
sample
buffer,
and
boiled
for
5
min.
LPS
was
prepared
from
the
same
cultures
by
using
the
hot
phenol/water
method
as
modified
by
Slauch
et
al.
(19).
Samples
were
electrophoresed
in
15%
polyacrylamide
gels
in
the
presence
of
SDS
(SDS/PAGE)
and
either
stained
by
the
Bio-Rad
silver
staining
protocol
or
electrotransferred
to
nitrocellulose
for
Western
blot
analysis
(7).
V.
cholerae
Abbreviations:
LPS,
lipopolysaccharide;
RM,
rapid
migrating;
MM,
medium
migrating;
SM,
slow
migrating.
11388
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Nati.
Acad.
Sci.
USA
91
(1994)
11389
Table
1.
Serum
resistance
and
intestinal
colonization
of
opaque
and
translucent
strains
Serum
resistance,
Colonization
Strain
Relevant
phenotype
%
survival
ratio
MO10
0139
opaque
wild
type
100
2.0
M010-T4
Spontaneous
translucent
variant
of
MO10
2
0.07
MO10-OpR3
Spontaneous
opaque
revertant
of
MO10-T4
100
2.0
AI-1837
Opaque
wild
type
100
33.6
1837-T3
Spontaneous
translucent
variant
of
AI-1837
0.8
0.06
Bengal-2
Opaque
vaccine
derivative
of
MO10
100
0.9
Bengal-2R1
0139-negative
TnSlac
derivative
of
Bengal-2
0.1
0.01
Bengal-2R2
0139-negative
Tnlac
derivative
of
Bengal-2
0.08
0.002
0395
01
wild
type
90
1.0
0395R-1
01-negative
Tn5lac
derivative
of
0395
0.1
0.003
Serum
resistance
was
calculated
as
100
x
the
ratio
of
the
number
of
colony-forming
units
found
after
cells
were
incubated
in
guinea
pig
serum
divided
by
the
number
of
colony-forming
units
found
after
cells
were
incubated
in
heat-inactivated
guinea
pig
serum.
The
colonization
ratio
is
the
mean
ratio
of
the
number
of
blue
(the
lacZ-positive
test
strain)
to
white
(the
AIacZ
LAC-1
strain)
colony-forming
units
found
in
small
intestinal
homogenates
after
20
hr
of
in
vivo
growth.
There
were
seven
mice
per
group.
chromosomal
DNA
was
purified
and
Southern
blot
analysis
was
carried
out
as
described
(7).
Serum
Reiance
and
Mouse
Colnation
Assays.
For
serum
bacteriocidal
assays,
cells
were
grown
at
370C
to
nidlogarithmic
phase
in
LB
broth,
washed,
and
mixed
1:1
with
16%
(vol/vol)
guinea
pig
serum
or
with
16%
(vol/vol)
heat-inactivated
guinea
pig
serum
in
phosphate-buffered
sa-
line.
Approximately
1
x
106
cells
were
used
in.
each
assay.
After
incubation
at
370C
for
1
hr.
LB
broth
at
40C
was
added
to
each
mixture
and
then
viable
cells
were
enumerated
by
plating
on
L
agar.
Identical
serum
bacteriocidal
assays
were
performed
using
pooled
human
sera
at
a
final
concentration
of
30%6.
Colonization
of
the
infant
mouse
small
intestine
was
as-
sessed
by
competition
assays
using
1:1
mixtures
of
test
strains
with
the
0395
AlacZ
strain
LAC-1,
as
described
(7).
nsertloal
Inactivation
of
IcX.
Inverse
polymerase
chain
reaction
(PCR)
(20)
was
used
to
amplify
DNA
sequences
adjacent
to
the
right
end
of
TnSlac
in
the
0139-negative
insertion
mutant
Bengal-2R1.
Chromosomal
DNA
from
Ben-
gal-2R1
was
digested
with
Taq
I
and
ligated
under
dilute
conditions
to
favor
intramolecular
ligation.
The
ligated
DNA
was
then
used
as
a
template
for
PCR.
The
two
primers
that
were
used
for
the
PCR
are
complementary
to
bp
84-65
(5'-ACCATGTTAGGAGGTCCT-3',
primer
2)
and
bp
231-
250
(5'-CCATCTCATCAGAGGGTAGT-3',
primer
1)
of
the
right
end
of
TnSlac.
The
product
of
the
PCR
was
subcloned
to
yield
plasmid
pCRII-I3.
The
cloned
insert
in
pCRII-I3
was
subcloned
into
the
EcoRI
site
of
pGP704
(21),
a
plasmid
that
is
unable
to
replicate
in
V.
cholerae.
The
resultant
plasmid
pMW704-I3
was
grown
in
Escherichia
coli
SMlOApir
(21)
and
then
transferred
into
0139
strain
MO10
by
conjugation.
The
integration
of
pMW704-I3
into
the
homologous
locus
rfcX
in
MO10
was
confirmed
by
Southern
blot
analysis.
RESULTS
Isolaion
of
Spontaeous
and
TnSlac
Mutant
Strains.
Initial
streaks
of
some
0139
clinical
isolates
show
a
mixture
of
opaque
and
translucent
colonies
(10).
This
phase
variation
in
colony
morphology
has
been
observed
and
studied
in
non-Ol
V.
cholerae
strains
(22)
and
in
Vibrio
vulnificus
(23).
In
non-Ol
strains
and
in
V.
vulneflcus,
opaque
colonies
are
encapsulated
and
translucent
colonies
are
nonencapsulated
(22,
23).
Johnson
et
al.
(10)
have
shown
that
opaque
0139
strains
are
encapsulated,
but,
to
our
knowledge,
no
analysis
of
translucent
colonies
has
been
reported.
We
began
our
analysis
of
0139
antigen
by
characterizing
spontaneous
translucent
variants
of
opaque
0139
strains.
A
single
colony
from
a
translucent
sector
of
the
opaque
0139
clinical
isolate
MO10
was
isolated
and
designated
MO1-T4.
A
spontaneous
opaque
revertant
of
MO10-T4
was
isolated
and
designated
MO10-OpR3.
A
similar
translucent
variant
of
the
opaque
clinical
isolate
AI-1837
was
desig
e
1837-T3.
Both
of
these
translucent
variants
were
0139positive
in
slide
agutination
tests
with
0139
typing
sera.
To
identify
the
genes
encoding
the
biosynthesis
of
the
0139
antigen,
we
constructed
a
pool of
transposon
insertion
mu-
tants
in
the
opaque
strain
Bengal-2,
a
prototype
live
attenu-
ated
vaccine
derivative
of
MO10
(11).
The
transposon
TnSlac
(17)
was
used
to
mutagenize
Bengal-2,
and
TnSlac
insertion
mutations
that
rendered
Bengal-2
0139-negative
were
en-
riched
for
by
agglutination
of
0139-positive
cells
with
an
anti-0139
antisera.
Two
such
0139-negative
TnSlac
insertion
mutants
were
designated
Bengal-2R1
and
Bengal-2R2
and
used
for
subsequent
analyses.
Southern
blot
analysis
was
used
to
confirm
that
these
two
strains
each
contained
a
single
TnSlac
insertion
(data
not
shown).
Multple
Elophoretic
Forms
o
the
0139
Antge.
Im-
munoblot
analysis
was
used
to
detect
changes
in
the
0139
serogroup
antigen
in
the
spontaneous
translucent
colony
morphology
mutants
and
the
TnSlac
0139-negative
insertion
mutant
strains.
Both
a
polyclonal
anti-0139
antisera
and
an
anti-0139
monoclonal
antibody,
2D12
(12),
were
used
to
analyze
whole
cell
lysates
of
these
strains
and
the
V.
cholerae
01
strain
0395
and
its
01-negative
derivative
of
0395-R1.
The
anti-0139
polyclonal
antisera
defined
0139-specific
bands
that
were
not
seen
in
the
01
strain
0395,
or
in
the
0139-negative
TnSlac
insertion
mutant
strains
Bengal-2R1
and
Bengal-2R2
(Fig.
1A).
In
all
four
of
the
opaque
0139
strains,
MO10,
MO1O-OpR3,
Bengal-2,
and
AI-1837,
three
0139-specific
electrophoretic
forms
of
the
0139
antigen
were
observed:
a
rapidly
migating
(RM)
form,
a
medium
migrating
(MM)
form
that
was
observed
as
a
series
of
fine
bands,
and
a
slowly
miring
(SM)
form
that
migrated
into
the
stacking
gel
but
not
into
the
running
gel
(Fig.
1A).
In
MO1O-T4,
the
spontaneous
translucent
variant
of
MO10,
the
SM
and
MM
forms
of
the
antigen
were
not
observed
and
only
the
RM
form
of
the
0139
antigen
was
detected
(Fig.
1A).
The
SM
and
MM
forms
of
the
antigen
were
also
missing
in
the
other
sponta-
neous
translucent
mutant
strain
1837-T3.
The
RM
form
of
0139
antigen
was
seen
in
1837-T3,
but
only
in
overloaded
and
over-developed
immunoblots
(data
not
shown).
Thus
immu-
noblots
with
anti-0139
polyclonal
antisera
of
whole
cell
lysates
revealed
that
there
are
three
electrophoretic
forms
of
the
0139
antigen
of
which
two,
the
apparently
higher
mo-
lecular
mass
forms,
are
absent
in
translucent
strains.
The
absence
of
the
SM
and
MM
forms
of
the
0139
antigen
in
translucent
strains
was
more
apparent
when
the
anti-0139
monoclonal
antibody
was
used
in
immunoblots
of
whole
cell
Microbiology:
Waldor
et
al.

Proc.
Nati.
Acad.
Sci.
USA
91
(1994)
1
2
3
4
_5
6
7
8
9
III
I)
1
2
3
4
5-
6
7
8
9
Ate
I2
3
4
5
6
7
8
9
1
0
Pill'"''
SN-1
SM{l
S
j
I
M-DC
1
RXm
{
RM{
FIG.
1.
Electrophoretic
forms
of
the
0139
serogroup
antigen
detected
in
unoblots
of
whole
cell
lysates
(A
and
B)
or
LPS
preparations
(C)
of
the
indicated
strains
with
either
anti-0139
polyclonal
antisera
(A)
or
anti-0139
monoclonal
antibodies
(B
and
C).
C
was
developed
with
a
chemiluminescent
substrate
(7).
D
is
a
silver-stained
SDS/PAGE
gel
of
LPS
from
the
indicated
strains.
The
three
electrophoretic
forms
of
the
0139
antigen,
SM,
MM,
and
RM,
are
indicated.
The
Bio-Rad
prestained
molecular
mass
markers
with
apparent
molecular
masses
of
107
kDa,
80
kDa,
49.5
kDa,
32.5
kDa,
27.5
kDa,
and
18.5
kDa
are
shown
in
unlabeled
lanes.
lysates
(Fig.
1B).
However,
the
monoclonal
antibody
did
not
define
an
epitope
unique
to
the
SM
and
MM
forms
of
0139.
The
RM
form
of
the
antigen
was
reactive
with
the
monoclonal
antibody
in
immunoblots
of
whole
cell
lysates
and
LPS
when
the
blots
were
developed
with
a
more
sensitive
chemilumi-
nescent
detection
method
(see
below).
Also
immunoelectron
microscopic
studies
with
this
monoclonal
antibody
have
shown
that
this
antibody
recognizes
the
surface
of
MO10
cells,
suggesting
that
the
SM
and
MM
forms
of
the
0139
antigen
are
surface-localized
(data
not
shown).
LPS
preparations
were
made
from
the
same
10
strains.
Immunoblot
analysis
of
SDS/PAGE
gels
of
these
LPS
prep-
arations
with
the
anti-0139
monoclonal
antibody
revealed
the
same
three
electrophoretic
forms
of
the
0139
antigen
in
the
opaque
strains
that
were
observed
in
immunoblots
of
whole
cell
lysates
(Fig.
1A
and
C).
Also,
as
in
immunoblots
of
whole
cell
lysates,
the
SM
and
MM
forms
were
not
detected
in
MO10-T4;
only
the
RM
form
was
apparent
in
this
translucent
strain
(Fig.
1C).
Since
the
monoclonal
antibody
reacted
with
all
three
electrophoretic
forms
of
the
0139
antigen,
the
RM,
MM,
and
SM
forms
of
the
antigen
must
share
an
epitope.
When
duplicate
SDS/PAGE
gels
of
LPS
preparations
were
stained
with
silver,
the
translucent
variant
MO10-T4
ap-
peared
identical
to
the
opaque
strains
MO10,
MO10-OpR3,
Bengal-2,
and
AI-1837
and
different
from
the
01
strain
0395,
which
is
known
to
express
LPS
molecules
with
relatively
long
0
side
chains
(13)
(Fig.
iD).
The
two
0139-negative
TnSlac
insertion
mutant
strains
Bengal-2R1
and
Bengal-2R2
lacked
the
major
silver staining
band
seen
in
MO10-T4
and
the
opaque
0139
strains.
In
contrast,
the
major
silver
staining
band
in
these
two
0139-negative
strains
comigrates
with
the
band
seen
in
the
01-negative
derivative
of
0395,
0395R-1,
which
we
have
shown
carries
a
TnSlac
insertion
in
rJbT
(13)
(data
not
shown).
This
comigrating
band
in
LPS
preparations
of
01-
and
0139-negative
strains
is
also
seen
in
the
wild-type
01
and
0139
strains
and
apparently
corresponds
to
the
core
polysaccharide
attached
to
lipid
A.
Thus,
the
0139-specific
silver
staining
band
seen
in
MO10,
MO1O-T4,
M010-OpR3,
Bengal-2,
and
AI-1837
corresponds
to
the
0139
LPS
mole-
cules
containing
an
0
side
chain
and
comigrates
with
the
RM
form
of
the
antigen
observed
in
immunoblots
(Fig.
1
C
andD).
This
is
consistent
with
the
observation
by
Hisatsune
et
aL
(14)
that
0139
strains
have
relatively
short
0
side
chains
in
their
LPS
compared
with
the
01
strains
(e.g.,
Fig.
iD,
lane
10).
A
striking
feature
of
silver-stained
gels
of
the
0139
LPS
preparations
is
the
relative
absence
of
silver
staining
material
where
the
SM
and
MM
antigen
forms
migrate.
Moreover,
suspensions
of
MO10,
MO10-T4,
and
Bengal-2R1
in
india
ink,
followed
by
air-drying
and
counterstaining
with
crystal
violet,
produced
smears
that
differentiated
these
strains
under
light
microscopy;
strain
MO10
cells
frequently
dis-
played
translucent
halos
whereas
MO10-T4
and
Bengal-2R1
did
not,
suggesting
that
a
capsular
layer
was
absent
in
the
latter
two
strains.
Therefore,
we
propose
that
the
SM
and
MM
species
are
capsular
forms
of
0139
0
antigen
that
are
present
in
LPS
preparations.
This
O-antigen
capsule
is
ap-
parently
not
covalently
linked
to
the
LPS
core
and
lipid
A
since
SM
and
MM
species
do
not
significantly
stain
with
silver.
Inser[on
Inactivation
of
rfcX.
TnSlac
insertion
mutants
Bengal-2R1
and
Bengal-2R2
are
deficient
in
the
production
of
all
three
electrophoretic
forms
of
the
0139
antigen
when
analyzed
with
anti-0139
polyclonal
antisera
or
monoclonal
antibodies
(Fig.
1
A
and
C).
To
establish
that
this
phenotype
was
linked
to
the
transposon
insertion
in
one
of
these
two
strains,
we
devised
a
site-directed
mutational
strategy
out-
lined
in
Fig.
2.
Inverse
PCR
(20)
was
used
to
isolate
a
DNA
ticX
Bengal
-
2
I
Tn5lac
Mutagenesis
KnR
Taql
Taq1
_I
1
2
I
inverse
PCR
Fragment
2R1
E2l\&&
I
subclone
into
pGP704
~~~~~bla
(
pMW704-I3
EcoRI
EdooF
I
I1&111
1
-
_
MOlO
I
X
bla
MO10
-
13
FiG.
2.
Schema
for
cloning
the
chromosomal
DNA
adjacent
to
the
right
end
of
the
Tn5lac
insertion
in
Bengal-2R1
and
the
insertional
inactivation
of
the
homologous
locus
in
M010,
rfcX,
with
this
cloned
fragment.
The
open
box
represents
transposon
DNA
and
the
hatched
box
represents
chromosomal
DNA.
Arrows
1
and
2
represent
the
PCR
primers
that
are
complementary
to
bp
231-250
and
bp
84-65,
respectively,
of
the
right
end
of
TnSlac.
A
l
RN{
R\I{
.sM-
IM-C
5
B
I
{
1;
B
11
.,
NNI
NI
IN,
.-I
11
11.390
Microbiology:
WaIdor
et
A
nab_--

Proc.
Natl.
Acad.
Sci.
USA
91
(1994)
11391
fragment
immediately
adjacent
to
the
right
end
of
the
TnSlac
insertion
present
in
strain
Bengal-2R1.
This
450-bp
PCR
product
was
then
subcloned
into
the
suicide
plasmid
pGP704
(21).
The
resultant
plasmid
pMW704-I3
was
transferred
into
M010
by
conjugation
and
integrated
into
the
chromosome
by
homologous
recombination
occurring
via
the
cloned
PCR
fragment.
A
representative
transconjugate,
designated
M010-13,
was
0139-negative
when
tested
by
slide
aggluti-
nation,
and an
immunoblot
analysis
of
a
whole
cell
lysate
of
M010-13
with
the
anti-0139
polyclonal
antisera
demon-
strated
that
this
strain,
like
Bengal-2R1,
lacked
all
three
electrophoretic
forms
of
the
0139
antigen
(Fig.
3A).
The
insertion
of
fragment
2R1
into
the
homologous
locus
in
M010,
which
was
designated
ifcX,
was
confirmed
by
South-
ern
blot
analysis
(Fig.
3B).
Thus,
MO10-13
reproduces
the
0139-negative
phenotype
of
Bengal-2R1
and
establishes
link-
age
between
the
TnSlac
insertion
in
Bengal-2R1
and
the
strain's
0139-negative
phenotype.
Se
Ri
ance
ad
Itesnal
Colonation
of
Wid-Type
and
Mutant
strains.
The
pathogenic
significance
of
the
0139
O-antigen
capsule
and
LPS
0
side
chains
was
investigated
by
comparing
the
serum
resistance
and
intestinal
colonization
properties
of
mutant
and
wild-type
strains.
The
spontaneous
translucent
strains
MO10-T4
and
1837-T3
exhibited
markedly
increased
serum
sensitivity
compared
with
encapsulated
0139
strains
(Table
1).
The
0-antigen-capsule-negative
O-side-chain-negative
strains
Bengal-2R1
and
Bengal-2R2
were
even
more
sensitive
to
serum
than
MO10-T4
(Table
1).
When
human
sera
was
used
to
assess
serum
resistance,
MO10-T4
and
Bengal-2R1
were
three
orders
of
magnitude
and
greater
than
five
orders
of
magnitude
more
serum-
sensitive
than
wild-type
0139
strains,
respectively.
These
results
indicate
that
the
LPS
0
side
chains
and
the
O-antigen
capsule
are
important
for
conferring
serum
resistance.
Intestinal
colonization
was
assessed
in
a
competition
assay
using
the
perorally
infected
CD-1
suckling
mouse.
In
this
assay,
suckling
mice
were
coinfected
with
approximately
equal
numbers
of
two
strains
of
V.
cholerae.
One
strain
(LAC-1)
was
a
AlacZ
derivative
of
the
01
strain
0395
(7);
the
other
strain
was
one
of
the
wild-type
or
mutant
lacZ-positive
0139
strains
being
studied.
After
in
vivo
growth,
homoge-
nates
of
the
small
intestine
were
plated
and
the
ratio
of
"</;
ix'l
fI]]f
l
Sw..
IV.."
I
V/.
..~
..~
//,
LacZ-positive
colonies
to
LacZ-negative
colonies
was
deter-
mined.
MO10
colonized
the
suckling
mouse
small
intestine
2-fold
better
than
the
01
strain
LAC-1
(Table
1).
MO10-T4
was
significantly
reduced
in
its
ability
to
colonize
the
mouse
small
intestine,
whereas
its
opaque
revertant,
MO10-OpR3,
had
wild-type
colonization
properties
(Table
1).
Thus,
the
capsular
form
of
the
0139
0
antigen
that
is
present
in
MO10
and
MO10-OpR3,
but
not
in
MO10-T4,
is
important
for
the
colonization
of
the
small
intestine.
The
two
0139-negative
strains
Bengal-2R1
and
Bengal-2R2
like
the
01-negative
strain
0395R-1
were
severely
attenuated
in
vivo
and
had
virtually
no
capacity
to
colonize
the
infant
mouse
small
intestine
(Table
1).
Thus,
the
expression
of
the
RM
form
of
the
0139
antigen,
which
is
0139
LPS
with
its
short
0
side
chain
and
which
distinguishes
Bengal-2R1
and
Bengal-2R2
from
MO1O-T4,
also
contributes
significantly
to
intestinal
colonization
and
serum
resistance.
DISCUSSION
By
analogy
to
our
understanding
of
immunity
to
V.
chokerae
01
infection,
the
mucosal
immune
response
directed against
the
0139
serogroup
antigen
is
likely
to
be
the
determinant
of
protective
immunity
to V.
cholerae
0139
infection.
A
genetic
and
immunologic
understanding
of
the
0139
serogroup
anti-
gen
will
aid
in
constructing
vaccines
against
0139
infection
and
will
help
to
explain
the
evolution
of
this
serogroup.
Based
on
our
analysis
of
wild-type
and
mutant
0139
strains,
we
propose
a
model
for
the
0139
serogroup
antigen
(Fig.
4).
Encapsulated
opaque
strains
have
two
forms
of
the
serogroup
antigen-an
0-antigen
capsule
and
LPS.
The
0-antigen
capsule
corresponds
to
the
two
slowly
migrating
forms
of
the
antigen
observed
in
immunoblots
(the
SM
and
MM
forms
of
the
0139
antigen).
The
LPS
corresponds
to
the
RM
form
of
the
antigen.
Immunoblots
with
the
anti-0139
monoclonal
antibody
demonstrated
that
there
is
a
shared
epitope
in
the
0-antigen
capsule
and
the
LPS
(represented
by
the
circle
in
Fig.
4).
Since
there
was
virtually
no
silver
staining
of
the
SM
and
MM
forms
of
the
antigen,
the
0-antigen
capsule
is
not
bound
to
the
LPS
core
and
lipid
A.
Strain
mo1
C
LPS
O-antigen
Capsule
.
/7
!:,
...
.
ii
[None
.
/
BENGAL-2R1
X
\
...FIY
...-..
R~~~
l
{
*~~
~
FIG.
3.
(A)
Immunoblot
of
whole
cell
lysates
of
the
indicated
strains
with
an
anti-0139
polyclonal
antisera.
MO10-3,
which
con-
tains
the
insertional
inactivation
of
rfcX
with
pMW704-I3,
lacks
all
three
forms
of
the
0139
antigen.
(B)
Southern
blot
of
chromosomal
DNA
from
M010
or
MO10-13.
The
single
EcoRI
and
Hind1l
fiagments
that
hybridize
with
labeled
fragment
2R1
in
MO10
are
lost
in
MO10-3
and
two
new
junction
fragments
are
seen
for
each
enzyme
in
this
strain.
IN
KEY
*
I.
FIG.
4.
Schematic
representation
of
a
model
for
the
0139
sero-
group
antigen.
n
oelf.
Nn
o
n
it
hficrobiology:
WaIdor
et
al.
.10
..!!:N
K
':
P.
.1
II-%
-%
0
4
-)
-i
jr.
\
W
.,.,
'.'
'..
....
AL
,Q;7
ll.l
"N"
-,,
I'.*-
'IN
SN
I
L

Proc.
Nadl.
Acad.
Sci.
USA
91
(1994)
Translucent
strains
like
MO10-T4
lack
the
0-antigen
capsule,
lose
the
SM
and
MM
forms
of
the
antigen
on
immunoblots,
and
become
more
serum-sensitive.
All
translucent
strains
are
not
identical;
for
example,
1837-T3
appears
similar
to
rough
0139-negative
strains
on
silver-stained
LPS
gels
and
only
expresses
very
small
amounts
of
the
RM
form
of
the
antigen
in
immunoblots.
0139-negative
mutant
strains,
such
as
Bengal-2R1,
lack
both
the
LPS-associated
0
side
chains
and
the
0-antigen
capsule.
Insertional
inactivation
of
the
rfcX
locus
in
M010
with
the
cloned
junctional
fragment
from
Bengal-2R1
(frag-
ment
2R1)
established
that
the
0139-negative
phenotype
of
this
insertion
mutant
is
indeed
associated
with
the
TnSlac
insertion
in
Bengal-2R1.
That
the
rfcX
locus
presumably
defines
one
of
the
genes
required
for
0139-antigen
synthesis
is
further
supported
by
our
observation
that
fragment
2R1
defines
an
0139-specific
DNA
sequence
(24).
Since
the
single
transposon
insertion
led
to
the
loss
of
both
the
0-antigen
capsule
and
the
0
side
chain
of
LPS
in
Bengal-2R1,
there
must
be
genes
common
to
the
biosynthesis
of
these
structures
or,
alternatively,
the
assembly
or
expression
of
these
two
macromolecules
is
biosynthetically
coordinated.
We
have
defined
the
SM
and
MM
forms
of
the
0139
antigen
as
an
0-antigen
capsule.
This
is
based
on
the
finding
that
these
two
slowly
migrating
forms
of
the
antigen
exhibit
little
if
any
staining
with
silver
in
LPS
gels
and
that,
by
analogy
to
other
encapsulated
vibrios
(22,
23),
opaque
0139
strains
produce
these
forms
of
the
antigen
whereas
translucent
strains
do
not.
Also,
similar
to
encapsulated
vibrios
(22,
23),
opaque
0139
strains
show
enhanced
serum
resistance
and
seem
to
exhibit
a
capsular
surface
layer
when
stained
with
polycationic
ferritin
(10)
or
ruthenium
hexamine
trichloride
(15)
and
examined
with
electron
microscopy.
Our
data
sug-
gest
that
the
SM
and
MM
forms
of
the
0139
antigen
represent
highly
polymerized
0
side
chains
that
are
not
linked
to
core
oligosaccharide
and
lipid
A.
These
forms
of
the
0139
antigen
are,
therefore,
analogous
in
structure
to
certain
0-side-chain-
related
capsules
found,
for
example,
in
E.
coli
K30
(group
I)
(25)
or
in
E.
coli
0111
(26).
The
SM
and
MM
forms
of
the
0139
antigen
might
correspond
to
the
capsular
polysaccha-
ride
described
by
Weintraub
et
al.
(15)
or
could
be
distinct
from
this
polysaccharide
material.
It
is
interesting
that
while
Weintraub
et
al.
(15)
concluded
that
0139
capsular
polysac-
charide
contained
a
3,6-dideoxyhexose,
Hisatsune
et
al.
(14)
concluded
that
this
sugar
was
a
major
component
of
0139
LPS.
The
use
of
the
mutants
we
have
described
in
this
report
should
aid
in
the
clarification
of
these
conflicting
results.
Both
the
O-antigen
capsule
and
LPS-associated
0
side
chains
of
V.
cholerae
0139
proved
to
be
virulence
factors.
Thus,
colonization
of
the
intestine
by
MO10-T4
was
consid-
erably
reduced
relative
to
wild-type
MO10,
and
the
0-anti-
gen-capsule-negative
and
LPS-0-side-chain-negative
strains
Bengal-2R1
and
Bengal-2R2
were
essentially
unable
to
col-
onize
the
intestine.
What
is
the
mechanism
of
the
attenuation
of
these
strains?
Is
the
0139
O-antigen
capsule
and/or
the
LPS-associated
0
side
chain
important
for
adherence
to
the
small
intestine
or
does
the
loss
of
these
antigens
interfere
with
the
assembly
of
other
cell
surface
virulence
determinants?
Alternatively,
either
or
both
of
these
structures
may
be
important
for
the
bacterium's
resistance
to
a
complement-
like
activity
previously
proposed
to
be
present
in
the
gastro-
intestinal
tract
(27).
We
are
grateful
to
Drs.
Y.
Takeda,
G.
B.
Nair,
A.
Huq,
and
T.
Shimada
for
the
gift
of
the
clinical
isolates
of
V.
cholerae
0139
and
anti-0139
antisera.
We
thank
Dr.
Dale
Kaiser
for
the
gift
of
TnSlac.
We
are
also
grateful
to
Drs.
H.
Kimsey
and
C.
Gardel
for
working
out
the
conditions
for
inverse
PCR.
Finally,
many
colleagues
offerd
valuable
advice
and
criticism
to
our
experiments
and
manuscript
includig
Drs.
J.
Slauch,
G.
Pier,
A.
Camilli,
E.
Rubin,
andJ.
Tobias.
This
work
was
supported
by
National
Institutes
of
Health
Grant
AI18045.
M.K.W.
is
a
postdoctoral
fellow
of
the
Howard
Hughes
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Waldor
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aL