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Infection and Immunity, November 1999, p. 5930-5937, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Characterization of the Locus Encoding Biosynthesis of
the Lipopolysaccharide O Antigen of Escherichia coli
Serotype O113
Adrienne W.
Paton and
James C.
Paton*
Molecular Microbiology Unit, Women's and
Children's Hospital, North Adelaide, S.A. 5006, Australia
Received 19 April 1999/Returned for modification 16 July
1999/Accepted 30 August 1999
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ABSTRACT |
Shiga toxigenic Escherichia coli (STEC) strains are a
diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, eae-positive
STEC strains, particularly those belonging to serogroups O157 and O111,
appear to have greater virulence for humans. However, in spite of being eae negative, STEC strains belonging to serogroup O113 have
frequently been associated with cases of severe STEC disease, including
hemolytic-uremic syndrome (HUS). Western blot analysis with
convalescent-phase serum from a patient with HUS caused by an O113:H21
STEC strain indicated that human immune responses were directed
principally against lipopolysaccharide O antigen. Accordingly, the
serum was used to isolate a clone expressing O113 O antigen from a
cosmid library of O113:H21 DNA constructed in E. coli K-12.
Sequence analysis indicated that the O113 O-antigen biosynthesis
(rfb) locus contains a cluster of nine genes which may be
cotranscribed. Comparison with sequence databases identified candidate
genes for four glycosyl transferases, an O-acetyl
transferase, an O-unit flippase, and an O-antigen polymerase, as well
as copies of galE and gnd. Two additional,
separately transcribed genes downstream of the O113 rfb
region were predicted to encode enzymes involved in synthesis of
activated sugar precursors, one of which (designated wbnF)
was essential for O113 O-antigen synthesis, and so is clearly a part of
the O113 rfb locus. Interestingly, expression of O113 O
antigen by E. coli K-12 significantly increased in vitro
adherence to both HEp-2 and Henle 407 cells.
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INTRODUCTION |
Shiga toxigenic Escherichia
coli (STEC) strains are an important cause of gastrointestinal
disease in humans, particularly since such infections may result in
life-threatening sequelae, such as hemolytic-uremic syndrome (HUS)
(19, 30, 38). It has been recognized for a number of years
that STEC strains causing human disease may belong to a broad range of
O serogroups (19). However, a subset of these (particularly
O157 and O111) appear to be responsible for the majority of serious
cases (those complicated by HUS) (12, 19, 38). These STEC
strains have the capacity to produce attaching-and-effacing (A/E)
lesions on intestinal mucosa, a property encoded by a pathogenicity
island termed the locus for enterocyte effacement (LEE) (7,
9). LEE encodes proteins with a range of functions, including a
type III secretion system, various secreted effector proteins and their
chaperons, the outer membrane protein intimin (the eae gene
product), which mediates intimate attachment to the enterocyte cell
surface, as well as the receptor for intimin (Tir) which is
translocated into the plasma membrane of the enterocyte (6,
21). However, production of intimin is not essential for
pathogenesis, because a significant minority of sporadic cases of HUS
are caused by eae-negative STEC strains (38). One
of the more common eae-negative STEC serogroups associated
with human disease is O113 (particularly serotype O113:H21) (19,
38). Indeed, 2 of the 12 STEC strains originally isolated from
patients with HUS by Karmali et al. (20) belonged to this serotype. An O113:H21 STEC strain was also responsible for a recent cluster of three HUS cases in Adelaide, South Australia
(37); this was the first report of an apparent outbreak of
HUS caused by an eae-negative STEC strain. Collectively,
these findings indicate that an investigation of O113 STEC virulence
factors may be warranted.
In previous studies, we have used Western immunoblot analysis and
convalescent-phase HUS patient sera to examine the serological response
to infection with eae-positive STEC strains belonging to
serogroups O111 and O157 (32, 47). Antibody responses to intimin and Tir were detected, and convalescent-phase serum was also
used to isolate clones containing a portion of the LEE from a cosmid
library of O111 STEC DNA constructed in E. coli K-12 (47). However, the strongest immune response was directed
against the lipopolysaccharide (LPS) O antigen. In the nonimmune host, LPS is believed to contribute to virulence by shielding the infecting organism from the bactericidal effects of serum (17, 39,
46). However, antibodies directed against LPS are likely to be
highly protective (17), and anti-LPS seroconversion probably
contributes to the sometimes rapid elimination of the causative STEC
strain from the patient's gut during the course of HUS. Indeed, an
O157-specific O-antigen-protein conjugate vaccine is currently being
developed for prevention of infections caused by this STEC serogroup
(22).
In the present study, we used Western immunoblot analysis to examine
the antibody response of a patient with HUS due to an O113:H21 STEC
strain. The convalescent-phase serum was also used to screen a cosmid
gene bank of O113:H21 STEC DNA constructed in E. coli K-12,
resulting in the isolation and characterization of the locus encoding
biosynthesis of the O113 O antigen. The effect of expression of O113 O
antigen on adherence of E. coli K-12 to epithelial cells was
also investigated.
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MATERIALS AND METHODS |
Bacterial strains and cloning vectors.
The O113:H21 STEC
strain 98NK2 was isolated from a patient with HUS at the Women's and
Children's Hospital, South Australia, and has been described elsewhere
(37). E. coli K-12 strains DH1 and JM109 have
been described previously (13, 50). The cosmid vector pHC79
has also been described previously (16), and the phagemid
pBC SK, which encodes chloramphenicol resistance, was obtained from
Stratagene, La Jolla, Calif. All E. coli strains were
routinely grown in Luria-Bertani (LB) medium (27) with or
without 1.5% Bacto-Agar (Difco Laboratories, Detroit, Mich.). Where
appropriate, ampicillin and chloramphenicol were added to growth media
at concentrations of 50 and 25 µg/ml, respectively.
Western blot analysis.
Crude lysates of STEC or other
E. coli strains were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as described by
Laemmli (24), and antigens were electrophoretically
transferred onto nitrocellulose filters, as described by Towbin et al.
(45). Filters were probed with convalescent-phase serum from
the HUS patient from whom the O113:H21 STEC 98NK2 had been isolated
(kindly provided by K. F. Jureidini and P. Henning, Renal Unit,
Women's and Children's Hospital, North Adelaide, South Australia)
(used at a dilution of 1:3,000), followed by goat anti-human immunoglobulin G (IgG) conjugated to alkaline phosphatase (Bio-Rad Laboratories, Hercules, Calif.). Alternatively, filters were probed with absorbed polyclonal rabbit E. coli O113-specific
antiserum (obtained from the Institute of Medical and Veterinary
Science, Adelaide, South Australia) (used at a dilution of 1:3,000),
followed by goat anti-rabbit IgG-alkaline phosphatase conjugate
(Bio-Rad). Immunoreactive bands were visualized with a chromogenic
substrate (4-nitro blue tetrazolium and X-phosphate).
Construction of cosmid gene bank.
High-molecular-weight
chromosomal DNA was extracted as described previously (34)
from STEC 98NK2 and was digested partially with Sau3A1 in
order to optimize the yield of fragments in the size range 35 to 40 kb.
This DNA was ligated with a fivefold molar excess of pHC79 DNA, which
had been digested with BamHI. Ligated DNA was packaged into
lambda heads by using a Packagene kit (Promega Biotec, Madison, Wis.)
and transfected into E. coli DH1, which had been grown in LB
medium plus 2% maltose. The cells were then plated onto LB agar
supplemented with ampicillin, and after incubation, clones were stored
in LB medium plus ampicillin plus 15% glycerol in microtiter plates at
70°C.
Screening of cosmid clones by immunoblotting.
Cosmid clones
were grown overnight at 37°C in 200 µl of LB medium plus ampicillin
in microtiter plates and then spotted onto a nitrocellulose filter.
Filters were then blocked, reacted with HUS patient serum (diluted
1:3,000), and then developed as described for Western blots above.
DNA sequencing.
Nested deletions of STEC DNA cloned into pBC
SK were constructed by the method of Henikoff (14) with an
Erase-a-base kit (Promega). This DNA was transformed into E. coli JM109, and the resulting plasmid DNA was characterized by
restriction analysis. Double-stranded template DNA for sequencing was
prepared as recommended in the Applied Biosystems sequencing manual.
The sequence of both strands was then determined by using dye-labelled
terminator chemistry on an Applied Biosystems model 373A automated DNA
sequencer. The sequence was analyzed with DNASIS and PROSIS version 7.0 software (Hitachi Software Engineering, South San Francisco, Calif.).
Comparison with sequence databases was carried out with the program
BLASTX version 2.0 (1).
Cell culture and bacterial adherence assays.
The capacity of
E. coli K-12 derivatives to adhere to either Henle 407 or
HEp-2 cells was assessed essentially as described previously for
adherence of STEC to Henle 407 cells (35). Briefly, Henle
407 or HEp-2 cells were grown in Dulbecco's modified Eagle's medium
buffered with 20 mM HEPES and supplemented with 10% fetal calf serum,
2 mM L-glutamine, 50 IU of penicillin per ml, and 50 µg
of streptomycin per ml. For bacterial adherence assays, cells were
seeded into 24-well tissue culture trays and grown for 24 h before
use, at which time the monolayer had attained confluence. The
monolayers were washed twice with phosphate-buffered saline (pH 7.5)
immediately prior to infection with bacteria.
For adherence assays, E. coli cells were grown overnight at
37°C in LB broth, and diluted to a density of 2 × 104 CFU/ml (confirmed by viable count) in the tissue
culture medium described above supplemented with 50 µg of ampicillin
per ml. Washed Henle 407 or HEp-2 monolayers were then infected with
1-ml aliquots of bacterial suspension. After incubation at 37°C for 3.5 h, the culture medium was removed, and the monolayers were washed four times with phosphate-buffered saline to remove nonadherent bacteria. The cell monolayers were then detached from the plate by
treatment with 100 µl of 0.25% trypsin-0.02% EDTA. Cells were then
lysed by addition of 400 µl of 0.025% Triton X-100, and 50-µl aliquots (and serial 10-fold dilutions thereof) were plated on LB agar
to determine the total number of adherent bacteria. Assays were
performed in quadruplicate, and the significance of differences between
mean adherence values was analyzed with the unpaired Student's t test (two tailed).
Nucleotide sequence accession number.
The nucleotide
sequence of the segment of 98NK2 DNA described in this study has been
deposited with GenBank under accession no. AF172324.
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RESULTS AND DISCUSSION |
Western blot analysis of O113:H21 STEC and screening of cosmid gene
bank using convalescent-phase HUS patient serum.
As a first step
in characterization of O113 STEC antigens which might be involved in
pathogenesis, a Western blot of STEC 98NK2 was probed with
convalescent-phase serum from the HUS patient from whom this strain was
isolated (used at a dilution of 1:3,000) (Fig.
1A, lane 1). The serum reacted
principally with a smear of material migrating between the sizes of 50 and 150 kDa and with a discrete species of approximately 30 kDa. A
similar labelling pattern was observed when two other O113:H21 STEC
strains in our collection (97MW1 and MW10) were probed with the same
serum or when 98NK2 lysates were probed with convalescent-phase sera
from two other HUS patients with O113 STEC infection (results not
presented). Predigestion of the 98NK2 lysate with proteinase K had no
obvious impact on the intensity of labelling of the slower-migrating
material, but removed the 30-kDa species completely (result not shown). This suggested that the higher-molecular-weight smear of immunoreactive material was nonproteinaceous, possibly LPS O antigen.
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FIG. 1.
Western immunoblot analysis. Lysates of O113:H21 STEC
strain 98NK2 (lane 1), or E. coli DH1 carrying pJCP590 (lane
2), pJCP591 (lane 3), pJCP592 (lane 4), pJCP593 (lane 5), or pHC79
(lane 6) were separated by SDS-PAGE, electroblotted, and probed with
convalescent-phase HUS patient serum (A) or rabbit anti-E.
coli O113 typing serum (B). The mobility of protein size markers
is also indicated.
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The convalescent-phase serum was then used to screen a 700-clone gene
bank of 98NK2 DNA constructed in
E. coli K-12 DH1 with
cosmid pHC79, as described in Materials and Methods. Lysates from
clones exhibiting immunoreactivity above background by dot
immunoblotting
were then retested by Western blotting. One of these was
confirmed
as positive and yielded a smear of high-molecular-weight
immunoreactive
material similar to the putative LPS seen in lysates of
98NK2
(Fig.
1A, lane 2). The recombinant cosmid from this clone
(designated
pJCP590) was found to contain an insert of approximately 35 kb
of 98NK2 DNA. A series of deletion derivatives of pJCP590 were
then
generated by digestion with either
SalI,
EcoRI,
or
HindIII,
followed by religation. These derivatives,
designated pJCP591,
pJCP592, and pJCP593, respectively, were then
transformed into
E. coli DH1. Unique
EcoRI,
HindIII, and
SalI sites are located
at
nucleotide positions 0, 31, and 649, respectively, in pHC79,
whereas
the 98NK2 DNA had been cloned into the
BamHI site at
position
374. Thus, pJCP591 retains 98NK2 DNA from the opposite end of
the original insert from that retained by the other two derivatives.
Western blot analysis indicated that lysates of
E. coli
DH1(pJCP591)
contained high-molecular-weight material that reacted with
the
convalescent-phase HUS patient serum, similar to that seen in
lysates of
E. coli DH1(pJCP590) (Fig.
1A, lane 3). This was
not
seen in lysates of
E. coli DH1(pJCP592), DH1(pJCP593),
or DH1(pHC79),
which contained only weakly immunoreactive smaller
species common
to all strains (Fig.
1A, lanes 4 to 6). To confirm the
identity
of the high-molecular-weight immunoreactive material, Western
blots of the various strains were probed with rabbit anti-O113
typing
serum (Fig.
1B). This serum labelled high-molecular-weight
material in
the lysates of 98NK2,
E. coli DH1(pJCP590), and
DH1(pJCP591),
but not any of the other strains. A culture of
E. coli DH1(pJCP591)
was also sent to a reference laboratory at the
Institute of Medical
and Veterinary Science, Adelaide, South Australia,
and was confirmed
as belonging to serogroup O113 on the basis of tube
agglutination
tests (result not shown). Thus, the 98NK2 DNA insert in
pJCP591,
which was approximately 14 kb in size, as judged by
restriction
analysis, was sufficient to direct biosynthesis of O113 LPS
O
antigen by
E. coli DH1.
DNA sequence analysis.
In order to determine the DNA sequence
of the O113 O-antigen biosynthesis (rfb) locus, a series of
EcoRI and HindIII restriction fragments from
pJCP591 were subcloned into pBC SK and transformed into E. coli JM109 (Fig. 2). None of the
subclones, designated pJCP594 to pJCP598, were capable of directing
biosynthesis of O113 O antigen (result not shown). The various
subclones were then subjected to sequence analysis, as described in
Materials and Methods. Where subclones did not overlap (that is,
between pJCP595 and pJCP596 and between pJCP596 and pJCP597), the
sequence across the junction was determined with custom-designed
oligonucleotide primers by using pJCP591 DNA as the template.
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FIG. 2.
Map of the region of 98NK2 DNA cloned in pJCP591,
showing the locations of the various subclones used to determine the
DNA sequence. A, AatII; E, EcoRI; H,
HindIII; Sal, SalI; Sau, Sau3A.
The locations and direction of transcription of the various ORFs are
shown as shaded pointed boxes below the map, and genes are named
according to Reeves et al. (40). The locations of three
putative transcription terminators are also indicated. The open box
above the map indicates the region of the insert of pJCP591 deleted in
pJCP599.
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The positions of open reading frames (ORFs) within the compiled
14,263-bp sequence of the 98NK2 DNA insert in pJCP591 are
shown in Fig.
2. Examination of the DNA sequence indicated that
it contained 11 complete and 2 partial ORFs, and some features
of the predicted protein
products are listed in Table
1.
BLASTX analysis indicated that the partial ORF at the 5' end of the
pJCP591 insert encoded a protein with 100% identity to
the last 262 amino acids (aa) of GalF of
E. coli K-12. GalF is
not
involved in O-antigen biosynthesis in
E. coli, but is
believed
to regulate cellular levels of uridine diphosphoglucose
(UDP-Glc)
by interacting with the UDP-Glc pyrophosphorylase GalU
(
28).
However,
galF is known to be located just
upstream of the
rfb promoter region in
E. coli O7
and K-12, as well as in a number
of enteric microorganisms, including
Salmonella typhimurium LT2,
Salmonella enterica,
Shigella dysenteriae, and
Shigella flexneri (
28). The
galF gene was followed by a stem-loop
structure (
G =
29.5 kcal/mol). Downstream of this,
a copy of the JUMPstart
sequence is located. This is a 39-bp element
present just upstream
of a number of polysaccharide gene clusters
(
15).
The JUMPstart sequence is followed by a cluster of nine genes
exhibiting features common to other
rfb loci, and these have
been named in accordance with the system proposed by Reeves et
al.
(
40). Each of the genes is preceded by a putative ribosome
binding site, and most are tightly linked. Indeed, the genes named
wbnB,
wbnC,
wbnD, and
wbnE
(Fig.
2) overlap each other by from
1 to 4 nucleotides (nt), and the
only significant intergenic gaps
are located between
wbnE
and
galE (34 nt) and between
galE and
gnd (59 nt). These intergenic regions did not contain any
potential
stem-loop structures. However, a potential stem-loop
transcription
terminator (
G =
24.6 kcal/mol) was
located immediately downstream
of
gnd. E. coli rfb loci are
typically flanked by the JUMPstart
element and
gnd, and Wang
and Reeves (
48) have recently exploited
this to amplify the
rfb region of
E. coli O157 by long-range PCR.
Interestingly, with the exception of
gnd, all of the genes
in
the putative O113
rfb cluster have very low G+C contents.
Indeed,
for six of the eight genes, the G+C content is <30%. This is
markedly
lower than that of the flanking
E. coli O113 DNA
sequences and
that of the
E. coli genome as a whole,
suggesting that these
rfb genes have been acquired from
another bacterial
species.
Predicted functions of proteins encoded by the major O113
rfb operon.
The structure of the E. coli
O113 O-antigen chemical repeat unit has been shown to consist of a
tetrasaccharide backbone with an additional galactose (Gal) side chain
(31) and is shown in Fig. 3.
The repeat unit also contains O-acetyl groups, but the position of these has not been determined. On the basis of this structure and the known mechanism of biosynthesis of other O-antigen polysaccharides (49), we predicted that the O113
rfb locus should contain genes encoding a total of five
glycosyl transferases required for assembly of the oligosaccharide
repeat unit, including one which initiates synthesis by linking the
first sugar to a lipid carrier undecaprenyl phosphate (UndP) on the
cytoplasmic face of the membrane. We would also expect to find at least
one acetyl transferase, a repeat unit transporter (flippase) for
translocation of the completed repeat unit across the cell membrane,
and a polymerase to link repeat units together to form
high-molecular-weight O antigen. We therefore conducted BLASTX analyses
to identify candidates for these functions, the results of which are
summarized in Table 1.
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FIG. 3.
Structure of the oligosaccharide repeat unit of E. coli O113 O antigen, as determined by Parolis and Parolis
(31). The position of O-acetyl groups was not
determined. Sugars are abbreviated as follows: Gal, galactose; GalA,
galacturonic acid; GlcNAc, N-acetyl glucosamine; and GalNAc,
N-acetyl galactosamine.
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The first gene in the major O113
rfb operon has been
designated
wzx, because it encodes a predicted protein with
similarities
to several putative polysaccharide repeat unit
transporters (flippases),
including WzxC from the colanic acid
biosynthesis locus of
E. coli K-12 (
41). Wzx from
E. coli O113 is predicted to be a highly
hydrophobic
integral membrane protein; hydrophobicity analysis
conducted according
to the method of Kyte and Doolittle (
23)
yielded a
hydrophobicity index of 0.75 and indicated the presence
of multiple
potential membrane-spanning domains (result not
presented).
The third ORF in the major O113
rfb operon encodes another
highly hydrophobic protein (hydrophobicity index = 0.89) with 12
potential membrane-spanning domains (result not presented). This
is a
typical feature of polysaccharide polymerases, and the protein
shows a
modest, but significant degree of similarity to the O-antigen
polymerase of
Salmonella choleraesuis (
25) (Table
1). Thus,
this gene (designated
wzy) is presumed to encode
the O113 O-antigen
polymerase. Polysaccharide polymerases from
different bacteria
typically exhibit only minimal sequence similarity
to each other,
because they must be specific for both the
oligosaccharide repeat
unit itself and the type of glycosidic linkage
joining the repeat
units.
The predicted products of the genes designated
wbnA,
wbnB,
wbnD, and
wbnE (see Fig.
2 for
location) all exhibit significant
sequence similarity to glycosyl
transferases from a variety of
gram-negative and gram-positive
microorganisms (Table
1). However,
in all of these cases, the
specificity of the transferase is either
unknown, or the degree of
similarity is insufficient to assign
substrate specificity to the
putative O113 transferase with any
confidence. None of the other O113
ORFs cloned in pJCP591 encode
proteins with similarity to known
glycosyl transferases, and so
there appears to be one transferase gene
too few to synthesize
the O113 repeat unit. The gene most likely to be
missing is that
which encodes the transferase linking the first sugar
to UndP.
The enzyme responsible for this reaction would need to be
capable
of interaction with membrane lipid and would therefore be
expected
to contain hydrophobic membrane-spanning anchorage domains.
However,
none of the protein products of
wbnA,
wbnB,
wbnD, or
wbnE contain
such
regions. A similar situation was observed by Wang and Reeves
(
48) in the
rfb locus of
E. coli O157,
which contained three
rather than the predicted four putative glycosyl
transferase genes.
They explained this apparent deficiency by proposing
that WecA
(formerly Rfe), which initiates synthesis of enterobacterial
common
antigen by transferring
N-acetyl glucosamine (GlcNAc)
phosphate
to UndP, could also initiate O-unit synthesis by transferring
N-acetyl galactosamine (GalNAc) phosphate, as previously
shown
for
Yersinia enterocolitica serotype O:8
(
51). Similarly, WecA
could also initiate O113 repeat unit
synthesis by transferring
GlcNAc phosphate (or possibly GalNAc
phosphate) to
UndP.
The predicted product of
wbnC is a small protein (20.2 kDa)
with significant similarity to members of the CysE-LacA-LpxA-NodL
family of acetyl transferases from a wide range of bacteria (Table
1).
Thus, WbnC is likely to be responsible for O acetylation
of the O113
O-antigen polysaccharide, although the precise sugar(s)
modified is yet
to be determined. O acetylation is also likely
to be responsible for
the fact that the O113 O antigen migrates
as a smear on SDS-PAGE (Fig.
1). Many O antigens migrate as a
ladder-like pattern of discrete bands,
each corresponding to a
polysaccharide containing a different number of
oligosaccharide
repeat units. O acetylation, however, is often not
stoichiometric,
and so for O antigens with such modifications,
polysaccharides
containing a given number (
n) of repeat
units will vary in the
extent to which each is O acetylated. Thus, the
molecular weight
will vary to the point where the mobility on SDS-PAGE
overlaps
that of polysaccharides containing either
n 1 or
n + 1 repeat
units.
The eighth ORF in the major O113
rfb operon encodes a
protein with a very high degree of similarity to GalE proteins
(UDP-Glc-4-epimerases)
from a large number of gram-negative and
gram-positive bacteria,
the most closely related being that from
Y. enterocolitica (57%
identity and 73% similarity over
335 aa) (
52). These enzymes
convert UDP-Glc to UDP-Gal, and
since the latter is required by
E. coli for a number of
purposes, a housekeeping copy of
galE is located elsewhere
in the chromosome (
41). Thus, one might
not have expected to
find an additional copy of
galE in the O-antigen
biosynthesis locus of
E. coli O113. However, the
housekeeping
galE gene in
E. coli is subject to
repression by Glc, and so under
normal growth conditions, cellular
levels of UDP-Gal may be low.
Such levels may become limiting in
E. coli O113, because its O
antigen is rich in Gal. Thus,
the presence of an additional
galE gene, which may or may
not be subject to catabolite repression,
may be necessary to ensure
adequate supplies of the activated
precursor.
The final gene in the major O113
rfb operon is
gnd, which as stated previously, is located at the 3' end of
numerous
rfb loci
(
48). The product of this
particular copy of
gnd is 99% identical
to several other
E. coli Gnd proteins for which sequences have
been deposited
with GenBank (Table
1). Gnd is a 6-phosphogluconate
dehydrogenase, but
there is no evidence that this enzyme functions
in O-antigen
biosynthesis.
Predicted functions of proteins encoded by other genes in
pJCP591.
Downstream of the major O113 rfb operon is a
copy of ugd, which encodes UDP-glucose dehydrogenase. This
gene is unlikely to be cotranscribed with the O113 rfb
locus, because, as mentioned previously, there is a strong
transcription terminator between gnd and ugd. The
O113 Ugd protein exhibits 91% identity and 95% similarity to Ugd
encoded by the his region of E. coli O8:K40 (2). UDP-Glc dehydrogenase converts UDP-Glc to
UDP-glucuronic acid (UDP-GlcA), a precursor of UDP-galacturonic acid
(UDP-GalA), which is one of the activated sugars required for O113
O-antigen biosynthesis. Thus, it is possible that this gene does
actually function in O113 O-antigen biosynthesis. However, it is not
possible to test this hypothesis by deletion mutagenesis of
ugd in pJCP591, because E. coli K-12 contains
another copy of ugd, the product of which exhibits 82%
identity and 91% similarity (accession no. P76373).
Conversion of UDP-GlcA to UDP-GalA would require the appropriate
epimerase, and it is possible that the enzyme encoded by
the
galE homologue in the O113
rfb locus could
fulfill this function.
Interestingly, however, downstream of
ugd, but on the opposite
DNA strand, is a gene which we have
designated
wbnF. A putative
transcription terminator
sequence (
G =
29.2 kcal/mol) is also
located
between
ugd and
wbnF (Fig.
2). The predicted
product of
wbnF has significant homology to a variety of
nucleotide sugar
epimerases (Table
1). The most closely related protein
is that
encoded by a gene designated
orf2, which is located
in an analogous
position downstream of the
rfb region of
E. coli O111 (
3).
This gene is also transcribed
in the opposite direction from the
rfb locus, but it is not
known whether it is essential for synthesis
of O111 O antigen. Thus,
WbnF might be involved in synthesis of
UDP-GalA; a possible alternative
function could be conversion
of UDP-GlcNAc to UDP-GalNAc, because the
latter is also a precursor
required for assembly of the O113 O-antigen
repeat unit. BLAST
analysis indicated that the
E. coli K-12
genome does not contain
a homologue of
wbnF, and so its role
in O113 O-antigen biosynthesis
was examined by deleting the distal
portion of the insert of pJCP591
by restriction with
AatII,
followed by religation. This removes
the DNA between the
AatII cleavage site at nucleotide position
13,004 in the
pJCP591 insert (within
wbnF) and that in the vector
approximately 450 bp downstream from the original cloning site
(Fig.
2).
E. coli DH1 transformed with the religated deletion
derivative (designated pJCP599) did not synthesize O113 O antigen,
as
judged by Western blot analysis with the O113-specific rabbit
antiserum
(Fig.
4) and was not agglutinated by the
same serum.
Thus, notwithstanding the uncertainty as to its precise
function,
wbnF is essential for O113 O-antigen biosynthesis
in
E. coli K-12;
in spite of the fact that it is transcribed
separately from the
major O113
rfb operon, it should be
considered part of the O113
rfb locus.
View larger version (59K):
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|
FIG. 4.
Western immunoblot analysis. Lysates of E. coli DH1 carrying pHC79 (lane 1), pJCP599 (lane 2), or pJCP591
(lane 3) were separated by SDS-PAGE, electroblotted, and probed with
rabbit anti-E. coli O113 serum. The mobility of protein size
markers is also indicated.
|
|
The final partial ORF in the insert of pJCP591 is the 5' end of
wzz. The predicted product has a very high degree of
identity
to the first 91 aa of Wzz proteins (polysaccharide chain
length
determinants) from various
E. coli strains,
S. dysenteriae, and
S. flexneri. The sequence of the
remainder of the O113
wzz gene
was determined by direct
analysis of PCR products obtained with
primers based on that published
for
wzz from
E. coli O8:K40 (
2).
The
complete O113 Wzz was 94% identical to the homologue from
E. coli O8:K40 and 99% identical to that from an
E. coli
O157
strain (
11) (result not
presented).
Clearly, a complete copy of the O113
wzz gene is not
essential for expression of O113 O antigen in
E. coli K-12
DH1(pJCP591),
and presumably the chain length regulation function is
carried
out by the
E. coli K-12 Wzz protein. However, the
Western blot
shown in Fig.
1 (lanes 2 and 3) suggests that the mean
chain length
of O113 O antigen expressed by
E. coli K-12
carrying either pJCP590
or pJCP591 may be slightly higher than that
expressed by the wild-type
O113 strain 98NK2 (lane 1). Franco et al.
(
11) have recently
reported that amino acid sequence
variation in the Wzz proteins
from
E. coli strains belonging
to different serogroups is directly
responsible for variations in modal
chain length of O antigen.
A final feature worthy of mention is that
examination of the noncoding
O113 DNA between
wbnF and
wzz indicates that the region from nt
13737 to the
wzz initiation codon (nt 13991) has 88% nucleotide
sequence
identity with
E. coli O8:K40, including the last 106
bp of
the O8:K40
ugd gene. Thus, it appears that the 3' end of
ugd has been duplicated in
E. coli O113, perhaps
as a consequence
of a recombination event which resulted in insertion
of
wbnF;
a homologue of this gene is not present in
E. coli O8:K40.
Effect of expression of O113 O antigen on adherence to epithelial
cells.
The O113 O antigen contains GalA, which is negatively
charged at physiological pH. Thus, E. coli expressing this
O-antigen type could have a significant net surface charge that could
influence its capacity to interact with other structures, such as the
surface of host cells during establishment of gut infection. To examine this possibility, the capacity of E. coli DH1(pJCP591) and
DH1(pHC79) to adhere to either HEp-2 or Henle 407 cell monolayers was
assessed, as described in Materials and Methods. At an initial dose of
2 × 104 CFU, the mean adherence ± standard
error of E. coli DH1(pHC79) to Henle 407 cells after
3.5 h of incubation was (3.05 ± 0.42) × 104 CFU per well. This represented approximately 3.1% of
the total number of bacteria present in the culture medium at the end
of the assay. In contrast, the adherence of E. coli
DH1(pJCP591) was (1.26 ± 0.13) × 105 CFU per
well. Thus, expression of O113 O antigen increased adherence of
E. coli DH1 to Henle 407 cells approximately fourfold
(P < 0.001). A similar result was obtained with HEp-2
cells. Total adherence of E. coli DH1(pHC79) to HEp-2 cell
monolayers after 3.5 h of incubation was (2.32 ± 0.38) × 104 CFU per well, compared with (4.80 ± 0.94) × 104 CFU per well for E. coli DH1(pJCP591)
(P < 0.05). This enhancement of adherence due to
expression of O113 O antigen contrasts with our previously reported
finding that expression of O111 O antigen by E. coli K-12
had no effect on adherence to Henle 407 cells under the same
experimental conditions as those used in the present study
(36). O111 O antigen is a neutral polysaccharide, and so it
is conceivable that net negative charge facilitates interaction between
E. coli and the epithelial cell surface. However, analysis of the effects of additional O-antigen types is required before a
definitive conclusion can be drawn. Interestingly, two previous studies
have examined the role of O antigen in adherence of O157:H7 STEC by
using TnphoA mutagenesis to construct strains deficient in
O-antigen biosynthesis. In both studies, such mutants were found to be
hyperadherent to HEp-2 cells in vitro (4, 5).
Conclusions.
This study demonstrates that O antigen is one of
the principal targets of the immune responses of HUS patients infected
with STEC belonging to serotype O113. Moreover, among a cosmid library of O113 STEC DNA constructed in E. coli K-12, a clone
expressing O antigen was the only one to react strongly with
convalescent-phase HUS patient serum. A deletion derivative of this
cosmid (pJCP591) with a 14,263-bp O113 DNA insert was sufficient to
direct biosynthesis of O113 O antigen in E. coli K-12.
Sequence analysis indicated that this region contains a cluster of nine
closely linked genes, including gnd at the 3' end and with
the JUMPstart sequence (15) at the 5' end, as shown for
rfb regions from other E. coli serogroups (48). Examination of the Bacterial Polysaccharide Gene
Database (2a) indicates that to date only 7 of the 165 other
E. coli O-antigen biosynthesis loci have been sequenced
(either partially or in full). The O113 rfb region includes
candidate genes for four glycosyl transferases, an O-acetyl
transferase, an O-unit flippase, and an O-antigen polymerase, as well
as a copy of galE. Assuming that WecA is capable of
initiating O-antigen synthesis, as discussed above, these gene products
are theoretically sufficient to assemble, export, and polymerize the
O113 O-antigen repeat unit. Interestingly, two additional, separately
transcribed genes downstream of the O113 rfb region were
predicted to encode enzymes involved in synthesis of activated sugar
precursors. One of these (wbnF) is on the complementary DNA
strand, and deletion analysis indicated that it is essential for O113
O-antigen synthesis. WbnF has significant homology with nucleotide
sugar epimerases from a variety of bacteria and may therefore be
involved in synthesis of either UDP-GalA or UDP-GalNAc, both of which
are present in the O113 repeat unit. Interestingly, however, the
closest homologue of WbnF (85% identity) is encoded by a gene
(orf2) located in a similar position and orientation
downstream of the rfb region of E. coli O111.
This serotype does not contain either of the sugars mentioned above in
its O-antigen repeat unit. However, it is not known whether
orf2 is essential for synthesis of O111 O antigen. Thus, it
remains a possibility that orf2 is a remnant from an
ancestral rfb locus, which has since undergone a
serogroup-converting recombination event and that its retention in
E. coli O111 has no functional significance. Such
recombination events are likely to have played an important role in the
evolution of the 166 distinct E. coli O serogroups.
The sequence data generated in this study also provide an opportunity
to develop PCR assays for serogroup-specific portions
of the O113
rfb locus. We have previously described a multiplex
PCR
assay for such regions from
E. coli O111 and O157
(
33),
which are the most common STEC types responsible for
HUS in Australia
(
38). We routinely use this assay, in
combination with another
specific for the genes encoding Shiga toxins 1 and 2, intimin,
and a plasmid-encoded hemolysin, for direct detection
and characterization
of STEC in crude fecal culture extracts
(
33). STEC strains belonging
to serogroup O113 were among
the first STEC types to be associated
with HUS in the landmark studies
of Karmali et al. (
20), and
over the last 5 years have been
the third most prevalent STEC
strains associated with cases of HUS in
South Australia. Thus,
inclusion of a pair of primers specific for O113
in the
rfb multiplex
assay is likely to be a useful adjunct
in the diagnosis of STEC
disease and in epidemiological studies. A
portion of the O113
wzy gene would be the preferred target
for such an assay, because
it showed the lowest degree of deduced amino
acid sequence homology
with any genes deposited with GenBank. Moreover,
the fact that
wzy encodes the putative O-antigen polymerase,
which must exhibit
absolute specificity for both the oligosaccharide
repeat unit
and the type of glycosidic linkage formed during
polymerization,
renders existence of homologous sequences in other
organisms extremely
unlikely.
The finding that expression of O113 O antigen significantly enhanced
adherence of
E. coli K-12 to epithelial cells of human
origin was unexpected, given the results of studies with O157
and O111
strains discussed previously (
4,
5,
36). However,
the fact
that in patients with HUS caused by O113 STEC, the major
host immune
response is directed against O antigen suggests an
important role in
the host-pathogen interaction. Unlike O111 and
O157 STEC, O113 STEC
strains are LEE negative and so lack the
capacity to form A/E lesions
on enterocytes (
8). Thus, the
molecular mechanisms whereby
these bacteria interact with and
attach to intestinal mucosa may be
fundamentally different. The
isolation of the O113
rfb
region in the present study will facilitate
future studies of the
direct or indirect contribution of O antigen
to intestinal colonization
by
STEC.
| |
ACKNOWLEDGMENTS |
We are grateful to Renato Morona and Peter Reeves for numerous
helpful discussions and to Paul Henning and Fred Jureidini for
providing convalescent-phase HUS patient sera.
This work was supported by grants from the National Health and Medical
Research Council of Australia and the Women's and Children's Hospital Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Microbiology Unit, Women's and Children's Hospital, North Adelaide,
S.A. 5006, Australia. Phone: 61-8-8204 6302. Fax: 61-8-8204 6051. E-mail: patonj{at}wch.sa.gov.au.
Editor:
J. T. Barbieri
| |
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Infection and Immunity, November 1999, p. 5930-5937, Vol. 67, No. 11
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
