Escherichia Coli: The Structures of O-Polysaccharide Antigens

The structures of Escherichia coli O-polysaccharide antigens
Roland Stenutz1, Andrej Weintraub2 & Göran Widmalm1

1
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University; and 2Karolinska Institutet, Department of Laboratory Medicine,
Division of Clinical Bacteriology, Karolinska University Hospital, Stockholm, Sweden
Correspondence: Andrej Weintraub, Abstract

Karolinska Institutet, Department of
Laboratory Medicine, Division of Clinical
Escherichia coli is usually a non-pathogenic member of the human colonic flora.
Bacteriology, F82, Karolinska University However, certain strains have acquired virulence factors and may cause a variety of
Hospital, Huddinge, S-14186 Stockholm, infections in humans and in animals. There are three clinical syndromes caused by
Sweden. Tel.: 146 858 587831; fax: 146 871 E. coli: (i) sepsis/meningitis; (ii) urinary tract infection and (iii) diarrhoea.
13918; e-mail: andrej.weintraub@ki.se Furthermore the E. coli causing diarrhoea is divided into different ‘pathotypes’
depending on the type of disease, i.e. (i) enterotoxigenic; (ii) enteropathogenic;
Received 31 August 2005; revised 15 November (iii) enteroinvasive; (iv) enterohaemorrhagic; (v) enteroaggregative and (vi)
2005; accepted 21 November 2005.
diffusely adherent. The serotyping of E. coli based on the somatic (O), flagellar
First published online 9 February 2006.
(H) and capsular polysaccharide antigens (K) is used in epidemiology. The
doi:10.1111/j.1574-6976.2006.00016.x
different antigens may be unique for a particular serogroup or antigenic
determinants may be shared, resulting in cross-reactions with other serogroups of
Editor: Simon Cutting E. coli or even with other members of the family Enterobacteriacea. To establish
the uniqueness of a particular serogroup or to identify the presence of common
Keywords epitopes, a database of the structures of O-antigenic polysaccharides has
Enterobacteriacea, serotype, O-antigen, been created. The E. coli database (ECODAB) contains structures, nuclear
structure, NMR, database. magnetic resonance chemical shifts and to some extent cross-reactivity relation-
ships. All fields are searchable. A ranking is produced based on similarity,
which facilitates rapid identification of strains that are difficult to serotype
(if known) based on classical agglutinating methods. In addition, results pertinent
to the biosynthesis of the repeating units of O-antigens are discussed. The
ECODAB is accessible to the scientific community at http://www.casper.organ.
su.se/ECODAB/.
plasmid DNA encoding enterotoxins or invasion factors

Introduction
become virulent. Among the E. coli causing intestinal
Escherichia coli is the type species of the genus Escherichia diseases, there are six well-described pathotypes: entero-
that contains mostly motile Gram-negative bacilli that fall pathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC),
within the family Enterobacteriaceae. It is the predominant enteroinvasive E. coli (EIEC), enterohaemorrhagic E. coli
facultative anaerobe of the human colonic flora. The organ- (EHEC), enteroaggregative E. coli (EAEC) and diffusely
ism typically colonizes the infant gastro-intestinal tract adherent E. coli (DAEC) (Nataro & Kaper, 1998). These
within hours after birth, and E. coli and the host derive pathotypes have virulence attributes that help bacteria to
mutual benefit for the rest of the host’s life (Kaper et al., cause diseases by different mechanisms.
2004). However, several E. coli clones have acquired specific
virulence factors which increase their ability to adapt to new
niches and allow them to cause a broad spectrum of diseases.
Enteric/diarrhoeal Escherichia coli
Three general clinical syndromes can result from infection
Enteropathogenic Escherichia coli (EPEC)
with pathogenic E. coli strains: enteric/diarrhoeal disease;
urinary tract infection; and sepsis/meningitis (Nataro & Enteropathogenic Escherichia coli was the first pathotype of
Kaper, 1998). As long as these bacteria do not acquire Escherichia coli to be described. Large outbreaks of infant
genetic elements encoding for virulence factors, they remain diarrhoea in UK led Bray, in 1945, to describe a group of
benign commensals. Strains that acquire bacteriophage or serologically distinct E. coli strains that were isolated from

c 2006 Federation of European Microbiological Societies FEMS Microbiol Rev 30 (2006) 382–403
Published by Blackwell Publishing Ltd. All rights reserved
Escherichia coli O-polysaccharide antigens 383
children with diarrhoea but not from healthy children with the pathogenic mechanism of ETEC enterotoxins.
(Kaper et al., 2004). The hallmark of infections due to EPEC ETEC diarrhoea may be mild, brief, and self-limiting or
is the attaching-and-effacing histopathology, which can be may be as severe as that seen in V. cholerae infection (Levine
observed in intestinal biopsy specimens from patients or et al., 1977; Wolf, 1997). The percentage of ETEC in children
infected animals (Nataro & Kaper, 1998). The most pre- with diarrhoea varies from 10% to 30% (Albert et al., 1992;
valent serogroups within this group of E. coli are: O18ac, Mangia et al., 1993; Hoque et al., 1994; Flores Abuxapqui
O20, O25, O26, O44, O55, O86, O91, O111, O114, O119, et al., 1999). Several studies suggest that 20–60% of travellers
O125ac, O126, O127, O128, O142 and O158 (Nataro & from developed countries experience diarrhoea when visit-
Kaper, 1998). ing the areas where ETEC infection is endemic; 20–40% of
Enteropathogenic Escherichia coli infection is primarily a the cases are due to ETEC (Black, 1990; Arduino & DuPont,
disease of infants younger than 2 years (Nataro & Kaper, 1993; DuPont & Ericsson, 1993). The most common ETEC
1998). EPEC primarily causes acute diarrhoea, although serogroups are: O6, O8, O11, O15, O20, O25, O27, O78,
many cases of persistent EPEC diarrhoea have been reported O128, O148, O149, O159 and O173.
(Nataro & Kaper, 1998; Scaletsky et al., 1996). In addition to
watery diarrhoea, vomiting and low-grade fever are com- Enteroinvasive Escherichia coli (EIEC)
mon symptoms of EPEC infection. EPEC plays a more
Enteroinvasive Escherichia coli is a pathogenic form of E. coli
important role in developing countries where it is the
that can cause dysentery (Nataro & Kaper, 1998). EIEC
foremost cause of diarrhoea. Many case-control studies have
strains are biochemically, genetically and pathogenically
found EPEC to be more frequently isolated from children
closely related to Shigella spp. The precise pathogenic
with diarrhoea than from the controls. Studies in Brazil,
scheme of EIEC has yet to be elucidated. However, patho-
Mexico, and South Africa have shown that 30–40% of infant
genesis studies of EIEC suggest that its pathogenic features
diarrhoea can be attributed to EPEC (Robins-Browne et al.,
are virtually identical to those of Shigella spp. (Goldberg &
1980; Cravioto et al., 1988, 1990; Gomes et al., 1989, 1991).
Sansonetti, 1993; Parsot & Sansonetti, 1996). Genes neces-
Recently, the pathogenesis of EPEC has been reviewed from
sary for invasiveness are carried on a 120-MDa plasmid in
the historical point of view and although the pathotype has
Shigella sonnei and a 140-MDa plasmid in other Shigella
been described in the 1940s, the exact mechanism of the
species and in EIEC (Baudry et al., 1987; Small & Falkow,
disease is not completely understood (Chen & Frankel,
1988; Sasakawa et al., 1992). EIEC penetrates the intestinal
2005).
mucosa, predominantly that lining the large intestine, to
cause inflammation and mucosal ulceration that are char-
Enterotoxigenic Escherichia coli (ETEC) acteristic of bacillary dysentery.
The most severe manifestation of infection with Shigella
Enterotoxigenic Escherichia coli is a common cause of
spp. and EIEC is bacillary dysentery, a syndrome character-
infectious diarrhoea (Black, 1993), especially in tropical
ized by frequent small-volume stools with blood and mucus.
climates, where uncontaminated water is not readily avail-
The disease is responsible for a substantial proportion of
able. Most of the illnesses, in terms of both numbers of cases
acute diarrhoeal diseases worldwide. However, most persons
and severity of symptoms, occur in infants and young
infected with Shigella spp. or EIEC experience watery
children after weaning. This pathogen may express heat-
diarrhoea that may or may not be followed by dysentery
labile and/or heat-stable toxins. Heat-labiles are a class of
(Snyder et al., 1984; Nataro et al., 1998; Taylor et al., 1988).
enterotoxins that are closely related in structure and func-
In most cases, EIEC elicits watery diarrhoea that is indis-
tion to cholera enterotoxin, which is expressed by Vibrio
tinguishable from that caused by other E. coli pathotypes
cholerae O1 and O139 (Sixma et al., 1993). The genes
(Nataro et al., 1998). EIEC can cause outbreaks of gastro-
encoding heat-labile and heat-stable toxins are carried on
enteritis. In sporadic cases, EIEC may be misidentified as
plasmids. ETEC colonizes the surface of the small bowel
Shigella spp. or non-pathogenic E. coli strains. EIEC out-
mucosa and elaborates enterotoxins, which give rise to
breaks are usually food-borne or waterborne (Nataro et al.,
intestinal secretion. Colonization is mediated by one or
1998). The most common EIEC serogroups are: O28ac,
more proteinaceous fimbrial or fimbrillar adhesins termed
O29, O112ac, O124, O136, O143, O144, O152, O159, O164
colonization factor antigens (CFA) (Kaper et al., 2004). A
and O167.
single plasmid often carries a toxin and CFA, for example,
heat-stable toxin and CFA/I (Reis et al., 1980; McConnell
Enterohaemorrhagic Escherichia coli (EHEC)
et al., 1981; Murray et al., 1983), heat-labile and heat-stable
toxins and CFA/II (Penaranda et al., 1983; Smith et al., Enterohaemorrhagic Escherichia coli is an etiological agent
1983), and heat-stable toxin and CFA/IV (Thomas et al., of diarrhoea with life-threatening complications. EHEC
1987). The clinical features of ETEC diarrhoea are consistent belongs to a group of E. coli called VTEC (‘verotoxigenic
FEMS Microbiol Rev 30 (2006) 382–403

c2006 Federation of European Microbiological Societies
384 R. Stenutz et al.
E. coli’ or ‘Vero cytotoxin-producing E. coli’) or STEC years of age, all with diarrhoea or acute diarrhoea, have
(‘Shiga toxin-producing E. coli’), formerly SLTEC (‘Shiga- shown a significant difference in the EAEC prevalence
like toxin producing E. coli’). It is believed that this compared to the controls (Nataro et al., 1987; Cravioto
pathotype adheres to the colon and distal small intestine; et al., 1991; Bhatnagar et al., 1993; Bouzari et al., 1994;
however, typical lesions have not been demonstrated (Kehl, Gonzalez et al., 1997). The increasing number of such
2002). The best-characterized adherence phenotype is the reports and the rising proportion of diarrhoeal cases in
intimate or attaching and effacing adherence mediated by which EAEC is implicated suggest that this pathotype is an
the eaeA gene. STEC isolates that possess the eaeA gene are important emerging agent of paediatric diarrhoea. The
capable of producing diarrhoea. However, the pathological serogroups that have been identified within the EAEC group
lesions associated with haemorrhagic colitis and haemor- are O3, O7, O15, O44, O77, O86, O111, O126 and O127.
rhagic uremic syndrome are due to the action of Shiga toxin
(Stx) with endothelial cells. The term ‘enterohaemorrhagic
Diffusely adherent Escherichia coli (DAEC)
E. coli’ (EHEC) was originally coined to denote strains that
cause haemorrhagic colitis and haemorrhagic uremic syn- Diffusely adherent Escherichia coli is a category of E. coli that
drome, express Stx, cause attaching-and-effacing lesions on produces a diffuse adherence in the HEp-2 cell assay (Nataro
epithelial cells, and possess a c. 60-MDa plasmid (Levine & et al., 1998). Little is known about the pathogenesis of
Edelman, 1984; Levine, 1987). Thus, EHEC denotes a subset DAEC. A surface of fimbria that mediates diffuse adherence
of STEC. Whereas not all STEC strains are believed to be phenotype has been cloned and characterized (Bilge et al.,
pathogens, all EHEC strains by the above definition are 1993a, b, 1989; Kerneis et al., 1991). The gene encoding the
considered to be pathogens. EHEC can cause nonbloody fimbria can be found on either the bacterial chromosome or
diarrhoea, bloody diarrhoea, and haemorrhagic uremic a plasmid. Few epidemiological and clinical studies have
syndrome in all age groups, but the young and the elderly been carried out to be able to describe adequately the
are the most susceptible. The most notorious E. coli serotype epidemiology and clinical aspect of diarrhoea caused by
associated with EHEC is O157:H7, which has been the cause DAEC. In one study, the patients with DAEC had watery
of several large outbreaks of disease in North America, diarrhoea without blood and faecal leukocytes (Poitrineau
Europe and Japan (Boyce et al., 1995; Grimm et al., 1995; et al., 1995). The association of DAEC with diarrhoea has
Kaper, 1998; Ozeki et al., 2003; Ezawa et al., 2004). The most been shown in some studies (Giron et al., 1991; Jallat et al.,
common EHEC serogroups are: O4, O5, O16, O26, O46, 1993; Levine et al., 1993) but not in others (Gunzburg et al.,
O48, O55, O91, O98, O111ab, O113, O117, O118, O119, 1993; Germani et al., 1996; Scaletsky et al., 2002).
O125, O126, O128, O145, O157 and O172. Recently, several
new EHEC serogroups have been described: O176, O177,
O178, O179, O180 and O181 (Scheutz et al., 2004). In
Urinary tract infections
addition, many of the EHEC serogroups are also identified
Uropathogenic Escherichia coli (UPEC)
as EPEC.
The urinary tract is among the most common sites of
bacterial infection and Escherichia coli is by far the most
Enteroaggregative Escherichia coli (EAEC)
common infecting agent at this site. The subset of E. coli that
Enteroaggregative Escherichia coli is defined as E. coli that do causes uncomplicated cystitis and acute pyelonephritis is
not secrete heat-labile or heat-stable enterotoxins and ad- distinct from the commensal E. coli strains that make up
here to HEp-2 cells in an aggregative pattern (Nataro & most of the E. coli populating the lower colon of humans.
Kaper, 1998; Nataro et al., 1998). The basic strategy of EAEC E. coli from a small number of O serogroups – O4, O6, O14,
seems to comprise colonization of the intestinal mucosa, O22, O75 and O83 – cause 75% of these urinary tract
probably predominantly that of the colon, followed by infections. Furthermore, they have phenotypes that are
secretion of enterotoxins and cytotoxins (Nataro et al., epidemiologically associated with cystitis and acute pyelo-
1998). Studies on human intestinal specimens indicate that nephritis in the normal urinary tract. Clonal groups and
EAEC induces mild, but significant, mucosal damage (Hicks epidemic strains that are associated with urinary tract
et al., 1996). The clinical features of EAEC diarrhoea are infections have been identified (Phillips et al., 1988; Manges
increasingly well defined in outbreaks, sporadic cases and et al., 2001). Although many urinary tract infection isolates
the volunteer model. A growing number of studies have seem to be clonal, there is no single phenotypic profile that
supported the association of EAEC with diarrhoea in devel- causes urinary tract infections. Specific adhesins, including
oping countries, most prominently in association with P (Pap), type 1 and other fimbriae, seem to aid in coloniza-
persistent diarrhoea (Bhan et al., 1989a–c; Fang et al., 1995; tion (Phillips et al., 1988; Nowicki et al., 1989; Johnson,
Lima et al., 1992). Previous studies in children less than 5 1991; Manges et al., 2001).

Sepsis/meningitis 2002). Each O antigen defines a serogroup. E. coli of specific

serogroups can be associated with certain clinical syndromes
Meningitis/sepsis associated Escherichia coli (Nataro & Kaper, 1998; Campos et al., 2004). A specific
(MNEC) combination of O and H antigens defines the ‘serotype’ of
an isolate. One pathotype can comprise several serogroups
This Echerichia coli pathotype is the most common cause of
and one serogroup may belong to several pathotypes and
Gram-negative neonatal meningitis, with a case fatality rate
even to non-pathogenic E. coli (Nataro & Kaper, 1998;
of 15–40% and severe neurological defects in many of the
Campos et al., 2004). Due to the limited sensitivity and
survivors (Unhanand et al., 1993; Dawson et al., 1999). A
specificity, and the various combinations of antigens, ser-
majority (80%) of the E. coli strains that cause meningitis
otyping is tedious and expensive and is performed reliably
possess the K1 capsular polysaccharide.
only by a small number of reference laboratories.
Among the most useful methods to diagnose different
Other potential Escherichia coli pathotypes pathotypes of E. coli are phenotypic assays, which are based
Several other potential E. coli pathotypes have been de- on the virulence characteristics. Of them, the HEp-2 adher-
scribed, but none of these is as well established as the ence assay is useful to identify the adherence patterns of
pathotypes described above. Among the most intriguing of diarrhoeagenic E. coli. It remains the ‘gold standard’ for the
these potential pathogens are strains of E. coli that are diagnosis of EAEC and DAEC (Vial et al., 1990; Nataro
associated with Crohn’s disease and are known as adherent- et al., 1998; Donnenberg & Nataro, 1995). Identification of
invasive E. coli (Darfeuille-Michaud, 2002). An inflamma- ETEC has relied on the detection of heat-labile and/or heat-
tory process and necrosis of the intestinal epithelium are stable enterotoxins. The classical phenotypic assay for EIEC
characteristics of necrotizing enterocolitis (NEC), an im- identification is the Sereny (guinea pig keratoconjunctivitis)
portant cause of mortality and long-term morbidity in pre- test, which correlates with the ability of the strain to invade
term infants. Necrotoxic E. coli (NTEC) have been asso- epithelial cells and spread from cell to cell (Kopecko, 1994).
ciated with disease in both humans and animals (De Rycke Molecular genetic methods remain the most popular and
et al., 1999). The relationships among the NEC-associated most reliable techniques for differentiating pathogenic
strains, NTEC and strains associated with Crohn’s disease strains from non-pathogenic members. The assays are based
have not yet been clearly established. A poorly characterized on nucleic acid probes and PCR and have been extensively
subset of E. coli infections outside the gastrointestinal or used. The advantages of PCR include its high sensitivity in
urinary tract is a group implicated in intra-abdominal detection of target templates and both rapid and reliable
infections, including abscesses, wounds, appendicitis and results due to its high specificity (Schultsz et al., 1994;
peritonitis. Ramotar et al., 1995; Stacy-Phipps et al., 1995; Kai et al.,
2000; Dutta et al., 2001; Pulz et al., 2003; Gioffre et al.,
2004).
Typing of Escherichia coli
There have been several available assays to identify different
categories of diarrhoeagenic Escherichia coli. Isolation and
Shigellae
identification of E. coli based on the biochemical properties Shigellae are Gram-negative, non-motile, facultative anaero-
are widely used in most microbiological laboratories as they bic rods. Shigella are differentiated from the closely related
do not require sophisticated equipment or complicated E. coli on the basis of pathogenicity, physiology (failure to
protocols. E. coli can be easily recovered from clinical ferment lactose or decarboxylate lysine) and serology (Sa-
samples on general or selective media at 37 1C under aerobic muel, 1996). The genus is divided into four species with
conditions. E. coli are usually identified by biochemical multiple serotypes: Shigella dysenteriae (12 serotypes), Shi-
reactions. In general, the different pathotypes cannot be gella flexneri (6 serotypes), Shigella boydii (18 serotypes) and
identified based on biochemical criteria alone, as in most S. sonnei (1 serotype) (Samuel, 1996). Shigella enterotoxin 1
cases they are indistinguishable from non-pathogenic E. coli. (ShET1) is found in S. flexneri 2a, but it is only occasionally
In addition to the biochemical tests, serology is com- found in other serotypes. In contrast, ShET2 is more wide-
monly used. It is based on Kauffmann’s scheme for the spread and detectable in 80% of Shigella representing all
serologic classification of E. coli, which is extensively four species. Shigella dysenteriae serotype 1 expresses Shiga
reviewed in (Orskov & Orskov, 1984; Ewing, 1986). Sero- toxin, an extremely potent, ricin-like cytotoxin that inhibits
typing E. coli is performed on the basis of their O (somatic), protein synthesis in susceptible mammalian cells. This toxin
H (flagellar), and K (capsular) surface antigen profile. More also has enterotoxic activity in rabbit ileal loops, but its
than 180 O, 60 H, and 80 K antigens have been proposed role in human diarrhoea is unclear. Shiga toxin is associated
(Whitfield & Roberts, 1999; Robins-Browne & Hartland, with haemorrhagic uremic syndrome, a complication of

infections with S. dysenteriae serotype 1. Closely related Structural determination of O-antigens

toxins are expressed by EHEC strains including the poten- from strains that are difficult to type or of
tially lethal, food-borne O157:H7 serotype (Samuel, 1996). nontyped strains
The four Shigella species cause varying degrees of dysen-
tery, characterized by fever, abdominal cramps and diar- The serotyping of clinical isolates of Escherichia coli is under
rhoea containing blood and mucous. Shigellosis is endemic constant development and usually it is possible to identify
in developing countries where sanitation is poor. In devel- the isolated strains. In some cases, however, it is not possible
oped countries, single-source, food or water-borne out- properly to characterize the strain with available monospe-
breaks occur sporadically, and pockets of endemic cific polyclonal antisera, either due to auto agglutination or
shigellosis can be found in institutions and in remote areas because the isolated E. coli strain is novel and appropriate
with substandard sanitary facilities. Isolation and identifica- antisera have not been raised. Under such circumstances it is
tion of Shigella spp. is usually based on culture, biochemical of great interest to have a procedure that rapidly could
tests, and serotyping. Molecular methods can be used to indicate, independently of immunological tests, the serotype
determine some target genes. of the isolated strain.
Since immunochemical tests require cultivation of the
strain, we obtain sufficient material for analysis by other
Lipopolysaccharide methods.
Lipopolysaccharide (LPS), also known as endotoxin, is Nuclear magnetic resonance spectroscopy is a powerful
anchored in the outer membrane of the Gram-negative tool that is used for studying biomolecules, including
bacterium. It consists of three parts: lipid A, which is the bacterial polysaccharides. In structural studies of these
toxic component; the core region, which can be divided into polysaccharides, NMR signals from the polymers may be
an inner and an outer part; and finally the O-antigen observed from live bacteria preparations or of the extracted
polysaccharide, which is specific for each serogroup (Fig. 1) LPS. In the structural determination of the O-antigen
(Brade et al., 1999). The sugar residues in lipid A and the polysaccharide part of an LPS, the O-polysaccharide is often
core region are decorated to a varying extent with phosphate released from the lipid A part by treatment with dilute acid
groups or phosphodiester-linked derivatives, which ensures and purified by gel permeation chromatography. These steps
microheterogeneity in each strain. The lipid A part is highly are laborious and should be omitted, in particular, if only
conserved in Escherichia coli. The core, however, contains typing of the strain is required.
five different basic structures, denoted R1 to R4 and K12. As it is easy to perform the phenol-water extraction from
The O-polysaccharide is linked to a sugar in the outer core. the cultivated bacterial isolates to obtain LPS, we focus on a
The O-antigen usually consists of 10–25 repeating units procedure that rapidly can identify the most probable O-
containing two to seven sugar residues. Thus, the molecular antigenic formula from a crude LPS preparation. A 1H NMR
mass of the LPS present in smooth strains will be up to spectrum of the LPS in D2O can be obtained in a few
25 kDa. minutes. Such a spectrum contains a number of character-
The present scheme of E. coli O-antigens comprises O1 to istic signals, even though most of them are not resolved (Fig.
O181. The following O groups have been removed: O31, 2). To utilize the information contained in a 1H NMR
O47, O67, O72, O93, O94 and O122. The O93 strain, spectrum, a database with the structures of the E. coli O-
however, will probably be re-introduced (Scheutz et al., antigen polysaccharides was implemented. Each structure
2004). Escherichia coli strain 73-1 has been typed as E. coli has published NMR data associated with it as well as
O73:K :H33 and strain 62D1 was suggested to belong to described cross-reactivity when present. Links to the origi-
the genus Erwinia herbicola (Scheutz, 2004). In several cases nal publications are also provided in the web-based imple-
the O-antigens of E. coli are identical or nearly identical to mentation. In the following approach we often enter sugar
those of other bacteria (Table 1). components of the O-polysaccharide as some or all can be
determined in a few hours by chemical derivatization and
analysis with gas-liquid chromatography/mass spectrometry
(GLC-MS), high performance liquid chromatography
(HPLC) or electrophoresis techniques from a hydrolysate of
the polymer, where the choice of technique for practical
reasons is the one used in each investigator’s laboratory.
Fig. 1. Schematic structure of an enterobacterial lipopolysaccharide
We will now exemplify the approach by analysis of E. coli
molecule. The lipids are depicted by curved lines and the sugar residues isolates that were not possible to serotype. Two clinical
are as follows: GlcN (’), Kdo (.), heptose (m), hexose (^), and O- isolates of E. coli from children with diarrhoea in León,
antigen components (), most commonly hexose. Nicaragua, termed strains 97RN and 121RN, showed

Table 1. Escherichia coli O-antigens identical or nearly identical to the strains. Thus, the procedure rapidly revealed the ser-
other bacterial polysaccharides ogroup of these two strains and no further structural
Serogroup Identical to Ref. investigation was necessary.
O8 Klebsiella pneumoniae O5 Jansson et al. (1985)
Serratia marcescens S3255 Aucken & Pitt (1991)
Biosynthesis considerations
O9 Hafnia alvei PCM 1223 Katzenellenbogen et al.
(2001) The biosynthesis of an LPS molecule and its transport to the
Klebsiella pneumoniae O3 Prehm et al. (1976) outer membrane of Gram-negative bacteria depend on
O18 Serratia marcescens O8 Oxley & Wilkinson (1986)
several complex events taking place at different locations in
O21 Hafnia alvei O39 Staaf et al. (1999a)
the bacterium (Raetz & Whitfield, 2002; Samuel & Reeves,
O35 Salmonella enterica O62 Rundlöf et al. (1998)
O55 Salmonella enterica O50 Kenne et al. (1983b) 2003). For the synthesis of the O-chain part, two of the three
O58 Shigella dysenteriae type 5 Dmitriev et al. (1977) reported pathways are present in Escherichia coli, namely, the
O97 Yersinia enterocolitica O5,27 Perry & MacLean (1987) Wzy-polymerase-dependent pathway present in most cases
O98 Yersinia enterocolitica Marsden et al. (1994) and typical for heteropolysaccharides and the ABC-trans-
O11,24 porter-dependent pathway, typical for homopolymers. Once
O104 Escherichia coli K9 Gamian et al. (1992)
the nucleotide sugars have been synthesized they can be
O105 Shigella boydii type 11 L’vov et al. (1991)
incorporated into the growing O-chain. In the Wzy-depen-
O111 Salmonella enterica O:35 Kenne et al. (1983b)
O121 Shigella dysenteriae type 7 Parolis et al. (1997) dent pathway a glycosyl-1-phosphoryl residue is transferred
O124 Shigella dysenteriae type 3 Dmitriev et al. (1976) to an undecaprenyl phosphate acceptor to form an undeca-
O143 Shigella boydii type 8 Landersjö et al. (1996) prenyl-PP-sugar intermediate. Subsequent transfer of addi-
O147 Shigella flexneri type 6 Hygge Blackeman et al. tional sugars to this acceptor results in an undecaprenyl-PP-
(1998) oligosaccharide intermediate in which the sequence of
O157 Citrobacter sedlakii NRCC Vinogradov et al. (2000)
sugars is related to the biological repeating unit to be formed
46070
in the O-chain. Translocation of this intermediate occurs
Citrobacter freundii F90 Bettelheim et al. (1993)
Citrobacter freundii OCU158 Nishiuchi et al. (2000, from the cytoplasmic side of the membrane to the periplas-
2002) mic side in a Wzx-dependent process. The Wzy-dependent
Salmonella enterica O30 Bundle et al. (1986) polymerization of the O-antigen occurs at the reducing end
of the nascent chain being formed, meaning that the O-
chain on the undecaprenol-PP carrier is transferred to the
identical 1H NMR spectra (cf. Fig. 2) and contained glucose,
most recently synthesized undecaprenol-PP-oligosacchar-
galactose and glucosamine according to GLC analysis. These
ide. The extent of polymerization, i.e. the chain-length
sugar components together with selected 1H NMR data were
modality, is determined by the Wzz product. The action of
entered to the web-based search interface, which then selects
the Wzy-polymerase from a linear undecaprenol-PP-oligo-
a best fit to the records in the database. The results of this
saccharide to produce a branched structure with a side-
search gave a close match to the O-antigen structure of
chain offers several possibilities just at this step to produce
E. coli O21 (and E. coli strain 105). Further inspection and
different structures with regard to anomeric configuration,
comparison of NMR data confirmed the identity between
linkage position and sugar residue.
The ABC-transporter pathway utilizes the b-D-GlcNAc-
PP-undecaprenol entity as a primer for the chain elongation
taking place on the cytoplasmic side of the membrane. In E.
coli O9, a homopolymer of mannose, an adaptor (a-D-Man)
is (1 ! 3)-linked to the N-acetylglucosamine residue. Sub-
sequent chain growth occurs by processive glycosyl transfer
to the non-reducing terminus. In E. coli O8, also a homo-
polymer of mannose, the O-chain is terminated by a 3-O-
methyl-D-Man residue. Although the sugars are added one
by one, sodium dodecyl sulphate-polyacrylamide electro-
phoresis (SDS-PAGE) analysis of these LPS molecules reveal
distributions of distinct bands. It is therefore reasonable to
describe, also in this case, the repeating units of the O-chain
in the context of biological repeating units. The undecapre-
Fig. 2. 1H nuclear magnetic resonance spectrum of the lipopolysacchar- nyl-linked polymer depends on Wzm and Wzt for transfer
ide from Escherichia coli strain 97RN in D2O solution. to the periplasmic face of the membrane. For both these

pathways the O-chain-PP-undecaprenyl entity is ligated to Table 2. Abundance of glycosyl residues in Escherichia coli O-antigens
the Lipid A-core acceptor and subsequently translated to the Anomers Sidechains
outer membrane.
Sugar a1b a b Anyw Terminalz Internal‰
In Shigella flexneri the O-antigens have different structures
as a result of acquisition of genetic material from bacter- Colp 1 1 0z 6 13 0
iophages via transduction (Lerouge & Vanderleyden, 2001). L-Fucp 3 3 o 1z 10 13 8
L-FucpNAc 2 2 0
The glucosyl residues, present as side-chains in the repeating
D-Glcp 11 7 3 32 25 38
unit, are proposed to be transferred to the growing D-GlcpA 2 o1 2 2 4 0
O-antigen chain on the periplasmic side of the membrane. D-GlcpNAc 19 7 13 8 0 15
A similar pathway could be possible for some of the E. coli D-Galf 2 o1 2 2 4 0
O-antigens, as indicated by their substituent sugars and their D-Galp 12 6 7 18 13 23
location within the repeating unit of the polymer (vide infra). D-GalpA 2 1 1
D-GalpNAc 13 8 5
We also note that a gene has been identified for a
D-GalpNAcA 1 1 0
glucosylphosphate transferase, which then is responsible for
D-Manp 10 6 4 4 0 2
the formation of the phosphodiester-linked glycosyl residue Neu5Ac 1 1 0
within the repeating unit of the O-antigen of E. coli O172 D-Quip3NAc 1 o1 1
(Guo et al., 2004). Thus, this finding indicates that the D-Quip4NAc 1 0 1
‘phospho-sugar’ is transferred en bloc in the biosynthesis. L-Rhap 12 10 2 12 21 4
D-Ribf 1 0 1 2 4 0
L-6dTalp 1 1 0 2 4 0
O-antigen repeating units: characteristics Otherk 5 3 2 4 8 4
and statistics of the structures In percent of 361 residues.
w
In humans, only a handful of different sugar residues are Abundance in any sidechain. Percent of 50 residues.
z
utilized in most glycoconjugates such as glycolipids and Abundance in sidechains in which the glycosyl group is directly linked to
a branch-point residue, which itself is adjacent to and substituted by a
glycoproteins (Varki et al., 1999). In bacteria, however, a
2-acetamido-D-hexose. Percent of 24 residues.
large number of different sugars are found and the O- ‰
Abundance in side chains not included in footnote z. Percent of 26
antigens of Escherichia coli contain a great variety of them residues.
(Table 2). In addition, a number of unusual sugars are found z
0, not found; o 1, less than 1%.
in these polymers (Scheme 1), including pentoses, deoxy- k
All remaining residues that occur fewer than three times.
hexoses, lactyl substituted hexoses, heptoses and nonuloses.
The number of sugar residues in the O-antigen repeating The O-antigens synthesized by the ABC-transporter-
unit ranges from two to seven and the topology of the dependent pathway (see above) or herein tentatively as-
repeats may be described as linear, branched or double signed to that pathway are homopolymers or have only two
branched. We have analysed the topology based on the sugar residues in the backbone of the repeating unit (Table
number of sugar residues in the backbone (Table 3). By far, 4). In 1994 it was shown that in E. coli O7 (Table 5), having a
the most common topology contains four sugars in the Wzy-dependent pathway, the repeating unit of the O-anti-
backbone being linear or containing a single terminal gen had an N-acetylglucosamine residue at its reducing end
residue in the side-chain. The 3- and 5-residue backbones (Alexander & Valvano, 1994) and the authors proposed that
are also common, whereas the 2- and 6-residue backbones this pattern should also be found in other O-antigen
are only present in a few cases. structures. By arranging the E. coli O-antigen structures
Each sugar residue is found in either the a- or the b- hitherto determined (Tables 5 and 6) with the D-GlcNAc
configuration at the anomeric centre. The common sugars residue at the reducing end one readily observes that this
(including ring form) of E. coli O-antigens, viz., D-Glcp, D- pattern is quite reasonable. In cases when D-GlcNAc is not
GlcpNAc, D-Galp, D-GalpNAc, D-Manp, and L-Rhap are all present in the polymer, D-GalNAc takes its place, in agree-
found with both anomeric configurations. Other sugars, e.g. ment with the observation that WecA can transfer either of
L-FucpNAc or D-Quip4NAc, have hitherto only been found the N-acetylhexosamine sugars (Marolda et al., 2004). In
in one of the anomeric configurations, namely the a- or the several of the O-antigens both amino sugars are components
b-configuration, respectively. Some of the sugars in the side- of the repeating unit. In just two strains, D-FucNAc has been
chains are present only as nonterminal residues, e.g. D- found and is expected to be the sugar at the reducing end of
GlcpNAc, whereas others are only found at a terminal the repeating unit. As noted above, one of these, strain 62D1,
position in the biological repeating unit, e.g. Colp. The was recently identified as a non-E. coli species. In all but a
unusual groups are then highly accessible and consequently few cases it is possible to identify that the amino sugar at the
specific for that particular E. coli serogroup. reducing end is 3-substituted. The other cases being the

HO OH OH OH
O O O
OH HO
OH
OH
OH
OH OH CH3 OH
D-ribofuranose D-fucofuranose D-threo-pentulofuranose
OH OH NHCOCH3
HO O
HCOHN OH
O O
OH
OH
HO OH OH
6-deoxy-L-talopyranose 3,6-dideoxy-L-xylo-hexose 2-acetamido-2,3,6-
(Colitose) trideoxy-3-formamido-D-mannose
COOH OH
OH
O
H3C O O
HO OH HO O
H AcHN
OH O HO OH
OH
OH
H COOH
H3C
4-[(R)-1-carboxyethyl]-D- 3-[(R)-1-carboxyethyl]-L- 4-acetamido-4,6-dideoxy-D-glucopyranose

glucopyranose rhamnopyranose
CH2OH CH3
COOH COOH
HO H
HO OH HO H
H AcHN O OH
O O OH
HO AcHN
HO OH H
OH OH
OH AcHN
6-deoxy-D-manno-heptopyranose N-acetyl-neuraminic acid 5,7-diacetamido-3,5,7,9-
tetradeoxy-L-glycero-L-
manno-nonulosonic acid
(Pseudaminic acid)
Scheme 1. Unusual glycosyl residues in Escherichia coli O-antigens.
O1A, O2 and possibly O149 antigens, where D-GlcNAc is 4- biological repeating unit has also been determined on
substituted by a b-L-Rhap residue, or in O83 and O136, medium-sized O-antigens with a degree of polymerization
where it is substituted by a b-D-Galp residue, i.e. the of 13 for E. coli O126 and 10 for E. coli O91 (Larsson
structural element is N-acetyl-lactosamine. These results are et al., 2004; Lycknert & Widmalm, 2004). The three-sub-
in good agreement with the few examples when the biologi- stituted D-GlcNAc residue was present in these three O-
cal repeating unit has been determined by NMR spectro- polysaccharides at the reducing end of the repeating unit.
scopy, e.g. in semi-rough type of LPS containing only one Genetic analysis of E. coli O26 and O172 has revealed
repeating unit as for E. coli O6 (Grozdanov et al., 2002). The that the second sugar is added to the D-GlcNAc-PP-

c 2006 Federation of European Microbiological Societies
Table 3. Topology of the O-antigen repeating units glucosyl residue, i.e. the backbone, or part of it, has the
Topology Abundance (%) following structure: ! X)-a-D-Glc-(1 ! 3)-a-L-FucNAc-
(1 ! 3)-b-D-GlcNAc(1 ! , where X represents different
2-residue backbone 5
3 linkage positions. Further genetic similarities may be pre-
sent, e.g. in O4:K52, ! 2)-a-L-Rha-(1 ! 6)-a-D-Glc-
1
(1 ! 3)-a-L-FucNAc-(1 ! 3)-b-D-GlcNAc(1 ! , and O26
1 (e.g. without the glucosyl residue), where the last sugar is an
3-residue backbone 27 a-linked rhamnosyl residue, which is possibly also the case
6 for O25. The latter strain carries an additional D-Glc residue
that forms a substituted branch-point residue. In analogy to
8
the hypothesis described above, close structural relation-
3 ships are observed between, for example, O6, O17, O44,
O58, O77, O78 and O88, having a Man-Man-GlcNAc
7 sequence at the reducing end. Although in E. coli the
numbering of serogroups is chronological, at least with
3
newly described strains, with the most recent ones covering
4-residue backbone 52 O174-O181 (Scheutz et al., 2004), subgroups are present in
22 some cases based on cross-reactivity, e.g. in O1, O18 and
27 most recently in O5, (Urbina et al., 2005) similar to the
Danish serotyping scheme for Streptococcus pneumoniae
2 capsular polysaccharides, which is based on cross-reactivity,
1
in contrast to the American system for which up to almost
100 different CPS serotypes have been described (Tomasz,
5-residue backbone 15
2000). In the future one may also type E. coli based on
11
genetic resemblance between the strains which then should
4 explain both structural and cross-reactivity relationships.
6-residue backbone 1 Furthermore, other structural similarities such as those of
1
blood-group determinants are present for the O86, O90,
O127 and O128 O-antigens, and these strains presumably
utilize the concept of molecular mimicry, thereby evading
undecaprenol carrier by a UDP-L-FucNAc transferase to the immune system of the human host (Moran et al., 1996).
form an a-(1 ! 3)-linkage (Guo et al., 2004; D’Souza In some of the E. coli strains the O-antigen structures
et al., 2002). Analysis of the O-antigen structures hitherto contain terminal glucosyl or N-acetylglycosamine residues,
determined indicates that in the serogroups O4, O25 and e.g. in O23A, O139 and O142, as side-chains. Whether these
O172 the third sugar to be added is an a-(1 ! 3)-linked residues are added by a phage-induced glycosyl transferase
Table 4. O-antigens synthesised by the ABC-transporter-dependent pathway

Serogroup Structure Ref.
O8 ! 2)-a-D-Man-(1 ! 2)-a-D-Man-(1 ! 3)-b-D-Man-(1 ! Jansson et al. (1985)
O9a ! 2)-a-D-Man-(1 ! 2)-a-D-Man-(1 ! 3)-a-D-Man-(1 ! 3)-a-D-Man-(1 ! Parolis et al. (1986)
O9 ! 2)-[a-D-Man-(1 ! 2)]2-a-D-Man-(1 ! 3)-a-D-Man-(1 ! 3)-a-D-Man-(1 ! Prehm et al. (1976)
O20ab ! 2)-b-D-Ribf-(1 ! 4)-a-D-Gal-(1 ! Vasil’ev & Zakharova (1976)
O20ac a-D-Gal-(1 ! 3) Vasil’ev & Zakharova (1982)
|
! 2)-b-D-Ribf-(1 ! 4)-a-D-Gal-(1 !
O52 ! 3)-b-D-Fucf-(1 ! 3)-b-D-6dmanHep2Ac-(1 ! Feng et al. (2004a)
O97z ! 3)-a-L-Rha-(1 ! 3)-b-L-Rha-(1 ! Perry & MacLean (1987)
| |
b-D-Xulf-(2 ! 2)b-D-Xulf-(2 ! 2)
O101 ! 6)-a-D-GlcNAc-(1 ! 4)-a-D-GalNAc-(1 ! Staaf et al. (1997)
Biosynthetic pathway proven experimentally.
w
b-D-6dmanHep2Ac is 2-O-acetyl-6-deoxy-b-D-manno-heptopyranosyl.
z
b-D-Xulf is b-D-threo-pentofuranosyl.

Table 5. O-antigens synthesised by the polymerase-dependent pathway with four or less residues in the backbone
O1A, O1A1 ! 3)-a-L-Rha-(1 ! 3)-a-L-Rha-(1 ! 3)-b-L-Rha-(1 ! 4)-b-D-GlcNAc-(1 ! Baumann et al. (1991)
| Jann et al. (1992b)
b-D-ManNAc-(1 ! 2)
O1B ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 2)-a-D-Gal-(1 ! 3)-b-D-GlcNAc-(1 ! Gupta et al. (1992)
|
b-D-ManNAc-(1 ! 2)
O1C ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 3)-a-D-Gal-(1 ! 3)-b-D-GlcNAc-(1 ! Gupta et al. (1992)
|

b-D-ManNAc-(1 ! 2)
O2 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 3)-b-L-Rha-(1 ! 4)-b-D-GlcNAc-(1 ! Jansson et al. (1987a)
Escherichia coli O-polysaccharide antigens
|
a-D-Fuc3NAc-(1 ! 2)
O3 b-L-RhaNAc(1 ! 4) a-D-Glc-(1 ! 4) Medina et al. (1994)
| |
! 3)-b-D-GlcNAc-(1 ! 3)-a-D-Gal-(1 ! 3)-b-D-GlcNAc-(1 !
O4:K52 ! 2)-a-L-Rha-(1 ! 6)-a-D-Glc-(1 ! 3)-a-L-FucNAc-(1 ! 3)-b-D-GlcNAc(1 ! Jann et al. (1993)
O4:K6 a-D-Glc-(1 ! 3) Jann et al. (1993)
|
! 2)-a-L-Rha-(1 ! 6)-a-D-Glc-(1 ! 3)-a-L-FucNAc-(1 ! 3)-b-D-GlcNAc(1 !
O5ab ! 4)-b-D-Qui3NAc-(1 ! 3)-b-D-Ribf-(1 ! 4)-b-D-Gal-(1 ! 3)-a-D-GalNAc(1 ! MacLean & Perry (1997)
O5ac ! 2)-b-D-Qui3NAc-(1 ! 3)-b-D-Ribf-(1 ! 4)-b-D-Gal-(1 ! 3)-a-D-GalNAc(1 ! Urbina et al. (2005)
(strain 180/C3)
O6:K2; K13; K15 ! 4)-a-D-GalNAc-(1 ! 3)-b-D-Man-(1 ! 4)-b-D-Man-(1 ! 3)-a-D-GlcNAc-(1 ! Jansson et al. (1984)
|
b-D-Glc-(1 ! 2)
O6:K54 ! 4)-a-D-GalNAc-(1 ! 3)-b-D-Man-(1 ! 4)-b-D-Man-(1 ! 3)-a-D-GlcNAc-(1 ! Jann et al. (1994c)
|
b-D-GlcNAc-(1 ! 2)
c

O7 a-L-Rha-(1 ! 3) L’vov et al. (1984)
|
! 3)-b-D-Qui4NAc-(1 ! 2)-a-D-Man-(1 ! 4)-b-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 !
O10 ! 3)-a-L-Rha-(1 ! 3)-a-L-Rha-(1 ! 3)-a-D-Gal-(1 ! 3)-b-D-GlcNAc-(1 ! Kenne et al. (1986)
|
a-D-Fuc4NAcyl-(1 ! 2)
Acyl = acetyl (60%) or (R)-3-hydroxybutyryl (40%)
O16 ! 2)-b-D-Galf-(1 ! 6)-a-D-Glc-(1 ! 3)-a-L-Rha2Ac-(1 ! 3)-a-D-GlcNAc-(1 ! Jann et al. (1994b)
O17 a-D-Glc-(1 ! 6) Masoud & Perry (1996)
|
! 6)-a-D-Man-(1 ! 2)-a-D-Man-(1 ! 2)-b-D-Man-(1 ! 3)-a-D-GlcNAc(1 !
O18A, O18ac ! 2)-a-L-Rha-(1 ! 6)-a-D-Glc-(1 ! 4)-a-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 ! Jansson et al. (1989)
|
b-D-GlcNAc-(1 ! 3)
391
2006 Federation of European Microbiological Societies

Table 5. Continued.
c

392

O18A1 a-D-Glc-(1 ! 6) Jann et al. (1992a)
|
! 2)-a-L-Rha-(1 ! 6)-a-D-Glc-(1 ! 4)-a-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 !
|
b-D-GlcNAc-(1 ! 3)
O18B ! 3)-a-L-Rha-(1 ! 6)-a-D-Glc-(1 ! 4)-a-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 ! Jann et al. (1992a)
|
b-D-Glc-(1 ! 3)
O18B1 a-D-Glc-(1 ! 4) Jann et al. (1992a)
|
! 3)-a-L-Rha-(1 ! 6)-a-D-Glc-(1 ! 4)-a-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 !
|

b-D-Glc-(1 ! 3)
O21 b-D-Gal-(1 ! 4) Staaf et al. (1999a)
|
! 3)-b-D-Gal-(1 ! 4)-b-D-Glc-(1 ! 3)-b-D-GalNAc-(1 !
|
b-D-GlcNAc-(1 ! 2)
O23A a-D-Glc-(1 ! 6) Bartelt et al. (1993)
|
! 6)-a-D-Glc-(1 ! 4)-b-D-Gal-(1 ! 3)-a-D-GalNAc-(1 ! 3)-b-D-GlcNAc-(1 !
|
b-D-GlcNAc(1 ! 3)
O24 ! 7)-a-Neu5Ac-(2 ! 3)-b-D-Glc-(1 ! 3)-b-D-GalNAc-(1 ! Torgov et al. (1995)
|
a-D-Glc-(1 ! 2)
O25 b-D-Glc-(1 ! 6) Kenne et al. (1983a)
|
! 4)-a-D-Glc-(1 ! 3)-a-L-FucNAc-(1 ! 3)-b-D-GlcNAc-(1 !
|
a-L-Rha-(1 ! 3)
O26 ! 3)-a-L-Rha-(1 ! 4)-a-L-FucNAc-(1 ! 3)-b-D-GlcNAc-(1 ! Manca et al. (1996)
O28 ! 2)-(R)-Gro-1-P ! 4)-b-D-GlcNAc-(1 ! 3)-b-D-Galf2Ac-(1 ! 3)-a-D-GlcNAc-(1 ! Rundlöf et al. (1996)
O44 a-D-Glc-(1 ! 4) Staaf et al. (1995)
|
! 6)-a-D-Man-(1 ! 2)-a-D-Man-(1 ! 2)-b-D-Man-(1 ! 3)-a-D-GlcNAc(1 !
O45 ! 2)-b-D-Glc-(1 ! 3)-a-L-6dTal2Ac-(1 ! 3)-a-D-FucNAc-(1 ! Jann et al. (1995)
O45rel ! 2)-b-D-Glc-(1 ! 3)-a-L-6dTal2Ac-(1 ! 3)-b-D-GlcNAc-(1 ! Jann et al. (1995)
O55 ! 6)-b-D-GlcNAc-(1 ! 3)-a-D-Gal-(1 ! 3)-b-D-GalNAc-(1 ! Lindberg et al. (1981)
|
a-Col-(1 ! 2)-b-D-Gal-(1 ! 3)
O56 ! 7)-a-Neu5Ac-(2 ! 3)-b-D-Glc-(1 ! 3)-b-D-GlcNAc-(1 ! Gamian et al. (1994)
|
R. Stenutz et al.

a-D-Gal-(1 ! 2)
O58 3-O-[(R)-1-carboxyethyl]-a-L-Rha -(1 ! 3) Dmitriev et al. (1977)
|
! 4)-a-D-Man-(1 ! 4)-a-D-Man2Ac-(1 ! 3)-b-D-GlcNAc-(1 !
O64 b-D-Gal-(1 ! 6) Perry et al. (1993)
|
! 3)-a-D-ManNAc-(1 ! 3)-b-D-GlcA-(1 ! 3)-b-D-Gal-(1 ! 3)-b-D-GlcNAc(1 !
O69 ! 2)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 2)-a-D-Gal-(1 ! 3)-b-D-GlcNAc-(1 ! Erbing et al. (1977)
O73 a-D-Glc-(1 ! 3) Weintraub et al. (1993)
(Strain 73-1) |

! 4)-a-D-Man-(1 ! 2)-a-D-Man-(1 ! 2)-b-D-Man-(1 ! 3)-a-D-GalNAc(1 !
O75 b-D-Man-(1 ! 4) Erbing et al. (1978)
|
! 3)-a-D-Gal-(1 ! 4)-a-L-Rha-(1 ! 3)-b-D-GlcNAc-(1 !
O77 ! 6)-a-D-Man-(1 ! 2)-a-D-Man-(1 ! 2)-b-D-Man-(1 ! 3)-a-D-GlcNAc(1 ! Yildirim et al. (2001)
O78 ! 4)-b-D-GlcNAc-(1 ! 4)-b-D-Man-(1 ! 4)-a-D-Man-(1 ! 3)-b-D-GlcNAc-(1 ! Jansson et al. (1987b)
O86 a-D-Gal-(1 ! 3) Andersson et al. (1989)
|
! 4)-a-L-Fuc-(1 ! 2)-b-D-Gal-(1 ! 3)-a-D-GalNAc-(1 ! 3)-b-D-GalNAc-(1 !
O88 a-L-6dTal-(1 ! 3) Torgov et al. (1996)
|
! 4)-a-D-Man-(1 ! 3)-a-D-Man-(1 ! 3)-b-D-GlcNAc-(1 !
O90 ! 4)-a-L-Fuc2/3Ac-(1 ! 2)-b-D-Gal-(1 ! 3)-a-D-GalNAc-(1 ! 3)-b-D-GalNAc-(1 ! Ratnayake et al. (1994b)
O98 ! 3)-a-L-QuiNAc-(1 ! 4)-a-D-GalNAcA-(1 ! 3)-a-L-QuiNAc-(1 ! 3)-b-D-GlcNAc-(1 ! Marsden et al. (1994)
O104 ! 4)-a-D-Gal-(1 ! 4)-a-Neu5,7,9Ac3-(2 ! 3)-b-D-Gal-(1 ! 3)-b-D-GalNAc-(1 ! Gamian et al. (1992)
O111 a-Col-(1 ! 6) Eklund et al. (1978)
|
! 4)-a-D-Glc-(1 ! 4)-a-D-Gal-(1 ! 3)-b-D-GlcNAc-(1 !
|
a-Col-(1 ! 3)
c

O113 ! 4)-a-D-GalNAc-(1 ! 4)-a-D-GalA-(1 ! 3)-a-D-Gal-(1 ! 3)-b-D-GlcNAc-(1 ! Parolis & Parolis (1995)
|
b-D-Gal-(1 ! 3)
O114 ! 4)-b-D-Qui3N(N-acetyl-L-seryl)-(1 ! 3)-b-D-Ribf-(1 ! 4)-b-D-Gal-(1 ! 3)-a-D-GlcNAc(1 ! Dmitriev et al. (1983)
O119 b-D-RhaNAc3NFo-(1 ! 3) Anderson et al. (1992)
|
! 2)-b-D-Man-(1 ! 3)-a-D-Gal-(1 ! 4)-a-L-Rha-(1 ! 3)-a-D-GlcNAc-(1 !
O121 ! 3)-b-D-Qui4N(N-acetyl-glycyl)-(1 ! 4)-a-D-GalNAc3AcA6N-(1 ! 4)-a-D-GalNAcA-(1 ! 3)-a-D-GlcNAc-(1 ! Parolis et al. (1997)
O124 4-O-[(R)-1-carboxyethyl]-b-D-Glc-(1 ! 6)-a-D-Glc(1 ! 4) Dmitriev et al. (1976)
|
! 3)-a-D-Gal-(1 ! 6)-b-D-Galf-(1 ! 3)-b-D-GalNAc-(1 !
O125 a-D-Glc-(1 ! 3) Kjellberg et al. (1996)
|
! 4)-b-D-GalNAc-(1 ! 2)-a-D-Man-(1 ! 3)-a-L-Fuc-(1 ! 3)-a-D-GalNAc-(1 !
|
393

b-D-Gal-(1 ! 3)
Table 5. Continued.
c

394
O126 ! 2)-b-D-Man-(1 ! 3)-b-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 ! 3)-b-D-GlcNAc-(1 ! Larsson et al. (2004)

|
a-L-Fuc-(1 ! 2)
O127 ! 2)-a-L-Fuc-(1 ! 2)-b-D-Gal-(1 ! 3)-a-D-GalNAc-(1 ! 3)-a-D-GalNAc-(1 ! Widmalm & Leontein, (1993)
O128 a-L-Fuc-(1 ! 2) Sengupta et al. (1995)
|
! 6)-b-D-Gal-(1 ! 3)-b-D-GalNAc-(1 ! 4)-a-D-Gal-(1 ! 3)-b-D-GalNAc-(1 !
O136 ! 4)-b-Pse5Ac7Ac-(2 ! 4)-b-D-Gal-(1 ! 4)-b-D-GlcNAc-(1 ! Staaf et al. (1999c)
b-Pse5Ac7Ac = 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-b-L-manno-nonulosonic acid
O138 ! 2)-a-L-Rha-(1 ! 3)-a-L-Rha-(1 ! 4)-a-D-GalNAcA-(1 ! 3)-b-D-GlcNAc-(1 ! Linnerborg et al. (1997a)
O141 a-L-Rha-(1 ! 3) Färnbäck et al. (1998)
|
! 4)-a-D-Man-(1 ! 3)-a-D-Man6Ac-(1 ! 3)-b-D-GlcNAc-(1 !

|
b-D-GlcA-(1 ! 2)
O142 ! 2)-a-L-Rha-(1 ! 6)-a-D-GalNAc-(1 ! 4)-a-D-GalNAc-(1 ! 3)-a-D-GalNAc-(1 ! Landersjö et al. (1997)
|
b-D-GlcNAc-(1 ! 3)
O143 ! 2)-b-D-GalA6R3,4Ac-(1 ! 3)-a-D-GalNAc-(1 ! 4)-b-D-GlcA-(1 ! 3)-b-D-GlcNAc-(1 ! Landersjö et al. (1996)
R = 1,3-dihydroxy-2-propylamino
O147 ! 2)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 4)-b-D-GalA-(1 ! 3)-b-D-GalNAc-(1 ! Hygge Blackeman et al. (1998)
O149 ! 3)-b-D-GlcNAc-(S)-4,6Py-(1 ! 3)-b-L-Rha-(1 ! 4)-b-D-GlcNAc-(1 ! Adeyeye et al. (1988)
(S)-4,6Py = 4,6-O-[(S)-1-carboxyethylidene]-
O152 b-L-Rha-(1 ! 4) Olsson et al. (2005)
|
! 3)-a-D-GlcNAc-(1-P ! 6)-a-D-Glc-(1 ! 2)-b-D-Glc-(1 ! 3)-b-D-GlcNAc-(1 !
O157 ! 2)-a-D-Rha4NAc-(1 ! 3)-a-L-Fuc-(1 ! 4)-b-D-Glc-(1 ! 3)-a-D-GalNAc-(1 ! Perry et al. (1986)
O158 a-D-Glc-(1 ! 6) Nishiuchi et al. (2000)
| Nishiuchi et al. (2002)
! 4)-a-D-Glc-(1 ! 3)-a-D-GalNAc-(1 ! 3)-b-D-GalNAc-(1 ! Datta et al. (1999)
|
a-L-Rha-(1 ! 3)
O159 a-L-Fuc-(1 ! 4) Linnerborg et al. (1999c)
|
! 3)-b-D-GlcNAc-(1 ! 4)-a-D-GalA-(1 ! 3)-a-L-Fuc-(1 ! 3)-b-D-GlcNAc-(1 !
O164 b-D-Glc-(1 ! 6)-a-D-Glc(1 ! 4) Linnerborg et al. (1999a)
|
! 3)-b-D-Gal-(1 ! 6)-b-D-Galf-(1 ! 3)-b-D-GalNAc-(1 !
O173 a-L-Fuc-(1 ! 4) Linnerborg et al. (1999b)
|
! 3)-a-D-Glc-(1-P ! 6)-a-D-Glc-(1 ! 2)-b-D-Glc-(1 ! 3)-b-D-GlcNAc-(1 !
62D1 a-D-Gal(1 ! 6) Staaf et al. (1999b)
|
R. Stenutz et al.

! 2)-b-D-Qui3NAc-(1 ! 3)-a-L-Rha-(1 ! 3)-b-D-Gal-(1 ! 3)-a-D-FucNAc-(1 !
Suggested as Erwinia herbicola
Table 6. O-antigens synthesized by the polymerase-dependent pathway with five or six residues in the backbone

O22 ! 6)-a-D-Glc-(1 ! 4)-b-D-GlcA-(1 ! 4)-b-D-GalNAc3Ac-(1 ! 3)-a-D-Gal-(1 ! 3)-b-D-GalNAc-(1 ! Bartelt et al. (1994)
O35 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 3)-b-D-GlcNAc-(1 ! Rundlöf et al. (1998)

|
a-D-GalNAcA6N-(1 ! 2)
O65 ! 2)-b-D-Qui3NAc-(1 ! 4)-a-D-GalA6N-(1 ! 4)-a-D-GalNAc-(1 ! 4)-b-D-GalA-(1 ! 3)-a-D-GlcNAc-(1 ! Perry & MacLean (1999)
O66 ! 2)-b-D-Man-(1 ! 3)-a-D-GlcNAc-(1 ! 2)-b-D-Glc3Ac-(1 ! 3)-a-L-6dTal-(1 ! 3)-a-D-GlcNAc(1 ! Jann et al. (1995)
O83 ! 6)-a-D-Glc-(1 ! 4)-b-D-GlcA-(1 ! 6)-b-D-Gal-(1 ! 4)-b-D-Gal-(1 ! 4)-b-D-GlcNAc-(1 ! Jann et al. (1994a)
O91 ! 4)-a-D-Qui3NAcyl-(1 ! 4)-b-D-Gal-(1 ! 4)-b-D-GlcNAc-(1 ! 4)-b-D-GlcA6NGly-(1 ! 3)-b-D-GlcNAc-(1 ! Kjellberg et al. (1999)
Acyl = (R)-3-hydroxybutyryl
O105 b-D-Ribf-(1 ! 3) Tao et al. (2004)
|
! 4)-a-D-GlcA2Ac3Ac-(1 ! 2)-a-L-Rha4Ac-(1 ! 3)-b-L-Rha-(1 ! 4)-b-L-Rha-(1 ! 3)-b-D-GlcNAc6Ac-(1 !
O116 ! 2)-b-D-Qui4NAc-(1 ! 6)-a-D-GlcNAc-(1 ! 4)-a-D-GalNAc-(1 ! 4)-a-D-GalA-(1 ! 3)-b-D-GlcNAc-(1 ! Leslie et al. (1999)
O117 ! 4)-b-D-GalNAc-(1 ! 3)-a-L-Rha-(1 ! 4)-a-D-Glc-(1 ! 4)-b-D-Gal-(1 ! 3)-a-D-GalNAc-(1 ! Leslie et al. (2000)
O139 b-D-Glc-(1 ! 3) Marie et al. (1998)
|
! 3)-a-L-Rha-(1 ! 4)-a-D-GalA-(1 ! 2)-a-L-Rha-(1 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 3)-a-D-GlcNAc-(1 !
O153 ! 2)-b-D-Ribf-(1 ! 4)-b-D-Gal-(1 ! 4)-a-D-GlcNAc-(1 ! 4)-b-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 ! Ratnayake et al. (1994a)
O167 a-D-Galf-(1 ! 4) Linnerborg et al. (1997b)
c

|
! 2)-b-D-GalA6N(L)Ala-(1 ! 3)-a-D-GlcNAc-(1 ! 2)-b-D-Galf-(1 ! 5)-b-D-Galf-(1 ! 3)-b-D-GlcNAc-(1 !
O172 ! 3)-a-L-FucNAc-(1 ! 4)-a-D-Glc6Ac-(1-P ! 4)-a-D-Glc-(1 ! 3)-a-L-FucNAc-(1 ! 3)-a-D-GlcNAc-(1 ! Landersjö et al. (2001)
395

machinery or by another mechanism is of great interest for The E. coli database implemented facilitates a more rapid
future genetic studies as the positioning of the side-chain identification of strains that are difficult to type or suggests
onto the structure differs and sometimes leads to a doubly similarities to previously determined O-antigens in the case
branched residue, e.g. in O141. In many cases the repeating of novel isolates. Analysis of the O-antigen structures
unit is formed and structurally determined by the polymer- revealed that 3-linked N-acetylglucosamine or N-acetylga-
ization process, e.g. in O55 and O164, often occurring at the lactosamine residues should be present at the reducing end
penultimate sugar residue of the linear undecaprenol-PP- of the biological repeating unit, in accordance with NMR
oligosaccharide leading to a single sugar residue in the side- spectroscopy and genetic data. In a limited number of cases,
chain, e.g. in O35, O113, O152, O159 and O167. a 4-linked N-acetylglucosamine residue is instead observed.
The O-antigen of Shigella boydii type 13 was recently both The topology with four sugar residues in the backbone of
structurally and genetically characterized (Feng et al., the O-antigen is present in half of the hitherto determined
2004b). Although this strain is more distantly related to E. structures. Future structural studies should be combined
coli and other Shigella species, its O-antigen shows a quite with genetic analysis of the O-antigen cluster to facilitate
close structural resemblance to that of E. coli O172. The insight into structural patterns and biosynthetic pathways.
linear pentasaccharide of S. boydii type 13 has the following As part of this effort, amino acid sequences of flippases and
chemical structure: ! 3)-a-L-QuipNAc-(1 ! 4)-a-D-Glcp- polymerases are being added as elements of the entries in the
(1 ! P-4)-a-D-GlcpNAc-(1 ! 3)-a-L-QuipNAc-(1 ! 3)-a- database.
D-GlcpNAc-(1 ! , in which the 4-linked GlcNAc residue is
6-O-acetylated to 15%. Based on biosynthetic considera-
Note
tions where an N-acetylglycosamine residue should be present
at the reducing end, two possibilities were suggested for the This review covers structures reported up to early 2005. In
biological repeating unit, i.e. the one above or the frame- addition, recently reported E. coli O-antigen structures are
shifted one with the 4-linked GlcNAc residue at the reducing those of serogroups O178 (Ali et al., 2005) and O145 (Feng
end. From the structural results presented in this article we et al., 2005). Noteworthy is also the fact that phenotypically
propose that the biological repeating unit of the O-antigen rough E. coli K12 have been genetically complemented to
from S. boydii type 13 has a 3-linked GlcNAc residue at the produce its O-antigen, an O16 variant with cross-reactivity
reducing end as presented in the above structure. In both the to O17 (Liu & Reeves, 1994; Stevenson et al., 1994).
E. coli O172 and the S. boydii type 13 O-antigens the glucosyl-
1-phosphoryl residue is the penultimate one (as presented) in
the assembled linear undecaprenol-PP-oligosaccharide. The Acknowledgements
O-antigens of E. coli O152 and O173 have branched struc-
This work was supported by grants from the Swedish
tures with one sugar residue as the side-chain and, most
Research Council and from the Swedish Agency for Research
notably, the branching sugar is a glycosyl-1-phosphoryl
Cooperation with Developing Countries SIDA/SAREC.
residue being the penultimate one, suggesting similar biosyn-
thetic pathways. Future detailed investigations will clarify the
whole assembly of these E. coli O-antigen units and that of S.
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Escherichia Coli: The Structures of O-Polysaccharide Antigens

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Escherichia Coli: The Structures of O-Polysaccharide Antigens

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The structures of Escherichia coli O-polysaccharide antigens

Roland Stenutz1, Andrej Weintraub2 & Göran Widmalm1

Correspondence: Andrej Weintraub, Abstract

plasmid DNA encoding enterotoxins or invasion factors

FEMS Microbiol Rev 30 (2006) 382–403

Sepsis/meningitis 2002). Each O antigen defines a serogroup. E. coli of specific

FEMS Microbiol Rev 30 (2006) 382–403

infections with S. dysenteriae serotype 1. Closely related Structural determination of O-antigens

FEMS Microbiol Rev 30 (2006) 382–403

D-ribofuranose D-fucofuranose D-threo-pentulofuranose

4-[(R)-1-carboxyethyl]-D- 3-[(R)-1-carboxyethyl]-L- 4-acetamido-4,6-dideoxy-D-glucopyranose

Scheme 1. Unusual glycosyl residues in Escherichia coli O-antigens.

FEMS Microbiol Rev 30 (2006) 382–403

Table 4. O-antigens synthesised by the ABC-transporter-dependent pathway

FEMS Microbiol Rev 30 (2006) 382–403

2006 Federation of European Microbiological Societies

Serogroup Structure Ref.

Published by Blackwell Publishing Ltd. All rights reserved

FEMS Microbiol Rev 30 (2006) 382–403

FEMS Microbiol Rev 30 (2006) 382–403

2006 Federation of European Microbiological Societies

O126 ! 2)-b-D-Man-(1 ! 3)-b-D-Gal-(1 ! 3)-a-D-GlcNAc-(1 ! 3)-b-D-GlcNAc-(1 ! Larsson et al. (2004)

Published by Blackwell Publishing Ltd. All rights reserved

FEMS Microbiol Rev 30 (2006) 382–403

FEMS Microbiol Rev 30 (2006) 382–403

O35 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 3)-a-L-Rha-(1 ! 2)-a-L-Rha-(1 ! 3)-b-D-GlcNAc-(1 ! Rundlöf et al. (1998)

2006 Federation of European Microbiological Societies

FEMS Microbiol Rev 30 (2006) 382–403

FEMS Microbiol Rev 30 (2006) 382–403

FEMS Microbiol Rev 30 (2006) 382–403

FEMS Microbiol Rev 30 (2006) 382–403

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