Structures and Serology of the O-Specific Polysaccharides of Bacteria of the Genus Citrobacter

Archivum Immunologiae et Therapiae Experimentalis, 2002, 50¢ , 379–391 £ PL ISSN 0004-069X

Review

Y. A. Knirel et al.: Polysacharides of Citrobacter ¥

YURIY A. KNIREL1*, N¤ INA A. KOCHAROVA1, OLGA V. BYSTROVA1,

E§ WA KATZENELLENBOGEN2 and ANDRZEJ GAMIAN2

1 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Pros¨ pekt 47, Moscow, 119991, Russia, 2 Institute © of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland

Abstract. The review presents the structures of the O-specific polysaccharides (O-antigens) of the li popolysac- charides isolated from over 25 Citrobacter strains, which represent different species and serogroups. The corre- lation between O-antigen structure and immunospecificity as well as numerous cross-reacti ons between Citro- bacter and other enterobacterial species are discussed.

Key words: Citrobacter; O-antigen; lipopolysaccharide; structure; immunospecificity.

Introduction The genus Citrobacter was first described in 1928

by B RAAK5 as Bacterium freundii. In 1932, WERKMAN

Bacteria of the genus Citrobacter are Gram-nega- and GILLEN80 introduced the name Citrobacter freundii

tive bacilli of the family Enterobacteriaceae. These for lactose-fermenting coliform bacteria. This classifi-

microorganisms are facultatively anaerobic and typi- cation was not universally accepted and bacteria of the

cally motile by means of flagellae; they can use citrate genus Citrobacter were subsequently described under

as the sole carbon source. The genus Citrobacter is various names, such as Escherichia freundii, Colobac-

taxonomically most closely related to Salmonella and trum freundii, Paracolobactrum freundii, Padlevskia,

Escherichia coli. Citrobacter strains are normal inhabi- Levinea, C. diversus, the Ballerup group, the Bethesda tants of human and animal intestine, and are also com- group and the Bethesda-Ballerup group20. In 1958, the

monly distributed in natural environment such as soil, International Subcommittee of Taxonomy of Entero-

water, sewage and food71. bacteriaceae adopted the term C. freundii for this high- Citrobacter strains are opportunistic pathogens that ly heterogenous group of bacteria, which since the are frequently isolated from wounds and cause gastroin- 1970s also comprises C. amalonaticus and C. koserii.

testinal diseases1, 22, 63, urinary tract infections and bac- In 1993, the classification of Citrobacter was dras-

teremias21, especially in immunocompromised patients. tically revised and the species C. freundii redefined by Some incidents of meningitis, brain abscesses and sepses BRENNER et al.6 On the basis of DNA relatedness, 11 in neonates have been also reported2, 20, 37, 57, 76. genomospecies were identified and separated by their

Abbreviations used: LPS – lipopolysaccharide, OPS – specific polysaccharide. * Correspondence to: Dr. Yuriy A. Knirel, N.D. Zelinsky Institute of Organic Chemistry, Leninsky Prospekt 47, Moscow, 119991, GSP-1, Russia, tel.: +7 095 93 83 613, fax: 7 095 13 55 328, e-mail: [email protected]

380 Y. A. Knirel et al.: Polysacharides of Citrobacter

biochemical profiles. The following homogenous species Salmonella, Escherichia, Hafnia, , Klebsiella and some

were distinguished: C. koserii (formerly C. diversus), other bacteria elucidated.

C. amalonaticus and C. farmerii (formerly C. amalo- The present article is the first survey summarizing ! naticus biogroup 1). According to the new classifica- the structural data of Citrobacter O-antigens and their tion, from the C. freundii complex (66 strains) only implication in the serological differences and simi- " 9 strains belong to C. freundii and 57 strains formed larities between different bacterial strains. the genomospecies 5, 6, 7 and 8, which were named as C. youngae (21 strains), C. braakii (15 strains), C. werkmanii (6 strains) and C. sedlakii (6 strains), re- Typed Strains

spectively. The three unnamed genomospecies 9, 10 and 11 were later named as C. rodentium70, C. gillenii

C. youngae O1: 1, 2 (PCM 1506)

and C. murliniae7.

# -

$ In 1954, WEST and EDWARDS81 established for the The branched OPS of C. . youngae O1 (PCM 1506)48

Bethesda-Ballerup group an antigenic scheme compris- has a tetrasaccharide repeating unit containing D-man-

$ $

ing 32 O-serogroups, which was extended later by SED- 0 nose, D-rhamnose, D-ribose (Table 1). In chemotyp-

% & & LAK and SLAJSOVA72, 73 to 43 O-serogroups. This scheme ing35, ribose was not taken into account, being con- continues to be applied for the serotyping of Citrobac- sidered a constituent of nucleic acid rather than LPS.

ter strains even after the revision of the classification This and some other Citrobacter OPS include D-rham- 0

of the genus. Ninety reference strains listed in the nose, which is common to the O-antigens of many phy-

antigenic scheme were phenotypically characterized topathogenic bacteria, such as Pseudomonas syringae

and specified by MIKI et al.56 based on the revised and Xanthomonas campestris, but uncommon to entero- classification of Citrobacter. From these 90 strains, two bacterial LPS apart from Citrobacter28, 38 A peculiar strains were reclassified to Hafnia alvei and one strain feature of the serogroup O1 OPS is the presence of was moved to E. coli. Among the remaining reference D-ribose in the α-furanose form, which occurs rarely in strains, C. youngae is the predominant species, foll- bacterial polysaccharides. In contrast to the more com- owed by C. braakii, C. werkmanii and C. freundii. mon β-ribofuranosyl group, the α-ribofuranosyl group Some O-antigens are characteristic for more than one % is easily cleaved from the OPS under mild acidic con- species. 2 ditions to give a linear backbone polysaccharide.

From the chemical point of view, the O-antigen +

represents the polysaccharide chain of the S-form lipo- C. youngae O2: 5, 6 (PCM 1507)3 , C. werkmanii polysaccharide (LPS), which is called O-specific poly- + O20: 40, 41, 43 (PCM 1553), C. youngae O25: 35,

saccharide (OPS). It is attached, via a core oligosacc- ( (36), 38 (PCM 1558) haride, to lipid A, which is responsible for the ) endotoxic activities of the LPS. While lipid A is the The three strains produce the same OPS consisting most structurally conservative part of the LPS, the core of D-mannose, D-rhamnose and D-xylose that corre-

region is less conservative, and the OPS is the most sponds to chemotyping data of serogroup O25 but not * variable moiety. serogroups O2 and O2035. The OPS has a branched

Based on the sugar composition of the LPS, Citro- tetrasaccharide repeating unit23 (Table 1). Classification

bacter strains of different serogroups were classified by of strains C. youngae O2 (PCM 1507), C. 4 werkmanii O20

* +

KELETI et al.35 into 20 chemotypes, from which 11 (PCM 1553) and C. . youngae O25 (PCM 1558) in differ- )

chemotypes are identical to those occurring in the gen- ent serogroups may result from a difference in the LPS

) era Salmonella and Escherichia. Strains of the three core structure (compare data of Citrobacter O4, O27 and + genera showed numerous serological cross-reactions. O36 given below) or a strong influence of H antigens.

The serological specificity of the bacteria is defined by SDS/PAGE and immunoblotting analysis showed the fine structure rather than by the composition of the that from 5 strains formerly assigned to Citrobacter + OPS. Therefore, in order to substantiate the antigenic serogroup O2, only four strains, PCM 1507, PCM heterogeneity and cross-reactivity on the molecular 1494, PCM 1495 and PCM 1496, possess an S-type

level, detailed structural studies of the OPS are per- LPS reactive with specific anti-O2 serum and, thus, do formed, which help to improve the classification of Ci- belong to serogroup O2. Strain PCM 1573 has an trobacter strains. By now, structures of more than 25 S-type LPS but does not react with anti-O2 serum and,

Citrobacter OPS have been established and serological ( hence, should be classified in another serogroup (auth-

cross-reactions of a number of Citrobacter strains with ors’ unpublished data). Y. A. Knirel et al.: Polysacharides of Citrobacter 381

C. youngae O3: 7, (8), 10 (PCM 1509) 0 not been originally classified, but the identity of its OPS structure to that of strain PCM 1528 from serogroup O5

The OPS of C. youngae O3 (PCM 1509) contains ) enabled placing it in serogroup O5, too.

$ $ D-mannose and D-rhamnose, which is consistent with -

* The OPS of several more Citrobacter strains, in-

chemotyping data . Computer-assisted analysis of the cluding C. youngae O15: 32, 34 (PCM 1571), C. braakii 5 13C NMR spectrum showed that the OPS has a linear +

* O17: 75 (PCM 2540) and C. braakii O18: 56 (PCM

39 9 structure (Table 1). Among the 18 strains (PCM 1551), have the same main chain as Citrobacter PCM

1497–1499, PCM 1508–1522) representing serogroup 1487 (authors’ unpublished data), but their exact struc- + O3, strain PCM 1518 should be classified to another tures remain to be determined. C. braakii O5: 53, 54 serogroup (authors’ unpublished data). (PCM 1527) is serologically identical to strains PCM

1528 and PCM 1487, whereas strain PCM 1529, which

C. youngae O4: 44, 45 (PCM 1525)3 , C. werkmanii was formerly classified as Citrobacter O5, was moved

+ : 56 O27: 40, 41 (PCM 1560)3 , C. youngae O36:? (PCM 1488) to the H. alvei species . An OPS-protein conjugate of

Citrobacter PCM 1487, considered as a potential arti-

Some Citrobacter O-antigens contain rarely occur- ficial vaccine, was characterized immunochemically54. $ 6 ring and even unique components, such as 4-deoxy-D-

-7 arabino-hexose, which is the monomer of the homo-

polymer OPS of C. . youngae O4 (PCM 1525) and O36 4 C. braakii O6: 72 (PCM 1531)

(PCM 1488) as well as C. werkmanii O27 (PCM 67, 69 1560) (Table 1, Fig. 1, structure 1). So far, this The OPS of C. braakii O6 (PCM 1531) is another

sugar has not been found elsewhere in nature. The com- polysaccharide that contains a rarely occurring sugar,

8 $ position of the OPS is consistent with chemotyping 7

* D 2 4-deoxy- -arabino-hexose, which is attached as 35 data . Variations in the LPS core structure are the rea- a single side-chain monosaccharide unit (authors’ un- son for the classification of the strains having the same

published data). The main chain is built up of L-rham-

+ 66, 68 $ OPS in three different serogroups . Epitopes of two 0 nose and D-fucose, the former being stoichiometrically

monoclonal antibodies to the serogroup O36 OPS were + O-acetylated at position 2 (elucidation of the exact OPS *

characterized, one of which, CB-2, reacted with the structure is in progress). In chemotyping35, rhamnose

+ OPS, whereas the other, CB-8, recognized an O-acety- and 4-deoxy-D-7 arabino-hexose were not determined as lated oligosaccharide fragment intervening the OPS and

LPS constituents. The LPS that was recovered from the 75

the core region . Citrobacter strains containing 4-de- phenol phase of the phenol-water extract contained

oxy-D-arabino-hexose do not cross react with any other twice as much OPS as that obtained from the water ) enterobacterial organisms. phase, which could be accounted for by the hydro- phobic nature of the OPS components.

C. braakii O7: 68 (PCM 1532) According to MIKI et al.56, serogroup O7 includes strains of two species, C. braakii PCM 1532 and C. youngae PCM 1503. The branched OPS of strain

= PCM 1532 has a D-mannan main chain and side chains

Fig. 1. The O-specific polysaccharides of C. youngae O4: 44, 45

? B

> @ A 47

(PCM 1525)67 (1), C. gillenii O9: 48 (PCM 1537)53 (3) and of a single D-glucose residues (Table 1). The D-man-

@ A

C. = youngae O32: (23), 28 (PCM 1569)43 (4) are homopolymers of nan main chain has the same structure as the linear OPS

rarely occurring monosaccharides of E. coli O964, Klebsiella pneumoniae O317 and * : H. alvei PCM 122332. Despite the structural similarity of the O-antigens, no cross-reactivity was observed in ; Western immunoblotting between anti-H. alvei PCM

C. braakii O5: 73: Vi (PCM 1528), PCM 1487 *

1223 O-serum and the LPS of C. braakii PCM 153232.

- $ 4-Deoxy-D-7 arabino-hexose is also present as a ter- This could be accounted for by the masking of potential

minal side-chain residue in the branched OPS of strains cross-reactive epitope(s) within the D-mannan chain by

$ PCM 148724 and PCM 1528 (authors’ unpublished the lateral α-D-Glc< p residue in the polysaccharide of 2 data) of C. braakii O5 (Table 1). Strain PCM 1487 had strain PCM 1532.

384 Y. A. Knirel et al.: Polysacharides of Citrobacter + C. youngae O7: (9), 13, 14 (PCM 1503) ) elongation in biosynthesis of both OPS proceeds by sequential transfers of single sugar units to the nonre-

The OPS of C. youngae O7 (PCM 1503) has 2

ducing end of the growing polysaccharide chain. This a structure different from that of C. braakii O7 (strain biosynthetic model requires the participation of several PCM 1532) (authors’ unpublished data). Its main chain 2 distinct transferases for the same monosaccharide. is identical to the linear OPS of C. youngae O3 and + O-Acetylation in OPS 2 begins after the achievement

similar to the main chains of the OPS of C. youngae of a certain chain length. +

O1 and C. youngae O2 (Table 1). They differ only in A polysaccharide with the same structure as OPS

the interchange of some D-rhamnose residues with D-man- I 3 has been previously reported as the O-antigen of Vi- 0

nose residues, whereas the linkages of the monosac- brio cholerae bio-serogroup Hakata25 (serogroup

charides, whether D-rhamnose or D-mannose, that oc- O14049 ), whereas OPS 2 is unique for Citrobacter. An

cupy the same position in the repeating units are the + OPS with a pentasaccharide repeating unit of α1→2- $

same. In spite of the structural similarity of the OPS, and α1→3-linked residues of N-acetyl-D-perosamine or

+ M

O-antiserum against strain PCM 1503 did not cross- N-formyl-D-perosamine have been found in the LPS of

-react with the majority of Citrobacter strains from Brucella melitensis8, 9, 15 and E. hermannii4, respective- N serogroup O3. ly. O-antigens of B. abortus12, 14 and Yersinia enterco-

A significant structural difference which is observed O 11, 13 α → litica O9 are 1 2-linked homopolymers of N-

between the OPS of serogroup O7 strains belonging to formyl-D-perosamine, and a similar homopolymer of

two different species, C. braakii PCM 1532 and C. youn- N-[(S)-2,4-dihydroxybutyryl]-$ D-perosamine was found * E gae PCM 1503, shows that their classification in one sero- 36

* in the LPS of V. cholerae O1 . 35 P

group should be revised. Chemotyping data are also Anti-O9 serum reacted with the homologous LPS in consistent with the composition of none of these OPS. 2 double immunodiffusion. In Western immunoblotting, it recognized mainly slowly migrating, high-molecular- C. braakii + O8: 35, 37 (PCM 1536, PCM 1572) -mass LPS species. O-Deacylation of the LPS had no significant effect on its serological reactivity. From the

The OPS of both C. braakii O8 strains studied, separated OPS, only OPS 2 reacted in double immuno- I PCM 1536 and PCM 1572, are identical. In accordance 2

* diffusion, whereas OPS 3 was inactive, probably be- $ 35 with the chemotyping data , they contain D-rhamnose cause of a lower molecular mass. No significant cross-

and furanosidic D-xylose, the latter, to the best of our F -reactivity was observed between anti-O9 serum and

knowledge, not being found in other bacterial polysac- V. cholerae O1 LPS, which can be accounted for by

charides. The OPS has a topology typical of many Ci- different N-acyl substituents at $ D-Rha4N (see above).

trobacter OPS which is characterized by a tetrasac- The LPS from E. coli O157, which, among other monosac-

0 charide repeating unit with three monosaccharides in 62

/ charides, also contains D-Rha4NAc , also did not cross- 42 + the main chain and one in the side chain (Table 1). -react with anti-O9 serum in double immunodiffusion.

C. gillenii O9: 48 (PCM 1537) C. gillenii O12: 35, 36 (PCM 1544)

The O-antigen of C. gillenii O9 (PCM 1537) is rep- The OPS of C. gillenii O12 (PCM 1544) contains

& $ $

6 resented by two structurally different polysaccharides, L-rhamnose, D-galactose and D-GlcNAc, which is in

both composed of 4-acetamido-4-deoxy-D-rhamnose agreement with chemotyping data35. It is distinguished

(N-acetyl-D-perosamine, D-Rha4NAc). One OPS has by non-stoichiometric substitution of the main chain

a tetrasaccharide repeating unit 2 and the other is an with terminal GlcNAc residues (the degree of glycosyl-

H I

α1→2-linked homopolymer of Rha4NAc 353 (Table ation is ~60%), whereas O-acetylation of the rhamnose 1, Fig. 1). This monosaccharide is difficult to detect in residue at position 2 is stoichiometric50 (Table 1).

sugar analysis, and it was overlooked in the chemotyp- Smith degradation was efficiently applied for the deter- * ing of Citrobacter LPS35. One of the 3-substituted mination of the structure of this OPS.

Rha4NAc residues in OPS 2 is partially O-acetylated at

position 2 (the degree of O-acetylation is ∼70%). OPS C. gillenii + O12: 57 (PCM 1542) I K 2 and OPS 3 could be separated by gel-permeation

chromatography on TSK HW-50S. In addition to D-galactose, D-GlcNAc and D-Gal- M

L Based on matrix-assisted laser-desorption/ionization NAc, the OPS of C. gillenii O12 (PCM 1542) con-

mass spectrometry data, it was suggested that the chain tains 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose

Y. A. Knirel et al.: Polysacharides of Citrobacter 385

Q $ (D-GlcNR3HOBu), which is also a constituent of the consistent with chemotyping data35. A pentasaccharide + OPS of C. freundii O41 (see below). Although strains repeating unit of the OPS contains three mannose

PCM 1542 and PCM 1544 belong to the same sero- residues, one of which is partially O-acetylated (the 2

group O12, the structures of their OPS differ signifi- degree of O-acetylation is ∼30%) (authors’ unpublished

* 2

cantly34 (Table 1). In a double immunodiffusion test data) (Table 1). The OPS of strain PCM 1554 shares

with anti-O serum against strain PCM 1544, precipita- a branched trisaccharide fragment with the OPS of

tion was observed only with the homologous LPS, but C. braakii PCM 1532 from serogroup O7 (Table 1), but not with the LPS of strain PCM 1542. These findings it remains to be determined whether the two strains are taken together show that the O-antigens of C. gillenii serologically related. 9 PCM 1542 and PCM 1544 are related neither chemi- cally nor serologically and, hence, should be reclassi- C. freundii O23: 52

fied in different O-serogroups. (PCM 2352, S-type; PCM 1556, SR-type) *

In chemotyping35, 4-deoxy-7 arabino-hexose was de- C. youngae O16: 58 (PCM 1550) T

tected in the LPS of C. freundii O23, but later studies

- $

The OPS of C. . youngae O16 (PCM 1550) is distin- showed that the OPS contains only D-mannose and

/ *

guished by the presence of a phosphoglycerol group45, D-GalNAc31. The structure of the so-called biological *

which has the D configuration26. In chemotyping35, repeating unit was determined for the OPS of C. freun-

* U phosphoglycerol was not considered as an LPS compo- dii O2331 (Table 1). This represents a preassembled oli-

nent. The OPS is acid-labile and is cleaved by the gosaccharide that is ligated to the LPS core with or R β1→6-linkage between residues of GalNAc and galac- without preceeding polymerisation in the course of bio-

tose in the course of dephosphorylation with 48% hy- synthesis to give S-type LPS (strain PCM 2352) or * 2 drofluoric acid to give a hexasaccharide that contains SR-type LPS (strain PCM 1556)30, respectively. Re-

all sugar constituents of the OPS (D-glucose, D-galac- markably, the GalNAc that links the repeating unit to

tose and $ D-GalNAc)45 (Table 1). Other approaches used the core has the β-configuration, whereas in the interior

for the structural analysis of this OPS were Smith de- repeating units of the OPS, this sugar is α-linked (auth-

gradation and N-deacetylation followed by deamination ors’ unpublished data).

with nitrous acid, which resulted in various smaller oli- gosaccharides studied by NMR spectroscopy and fast- C. werkmanii + O24: 49, 51 (PCM 1557) atom bombardment collision-induced dissociation mass

spectrometry. # In addition to the common monosaccharides and *

The OPS of H. alvei PCM 1207 has been found to fucose detected in chemotyping the LPS35, the OPS of

contain phospho-D-glycerol, too, which is attached at C. 4 werkmanii O24 (PCM 1557) was found to contain

position 3 of a 4-substituted β-D-Gal< pNAc residue in D-ribose and D-glucuronic acid (authors’ unpublished

S 2 the main chain26, i.e. to the same monosaccharide and data), the latter making it acidic. This OPS has the same

at the same position as in the OPS of C. youngae PCM structure, including the O-acetylation pattern, as that of

1550. Immunoblotting and ELISA experiments demon- Salmonella enterica ssp. 7 arizonae O4574.

strated cross-reactivity between O-antiserum against : H. alvei PCM 1207 and the LPS of strain PCM 1550 C. werkmanii O26: 49, 50 (PCM 1559)

and indicated that a shared epitope resides on the

S +

O-antigen26. Both H. alvei PCM 1207 and C. youngae The OPS of C. 4 werkmanii O26 (PCM 1559) con-

O16 OPS showed a high inhibitory activity, which de- tains D-glucose and D-ManNAc, which is not consistent

creased substantially upon dephosphorylation of the with chemotyping data35. A branched structure was es- +

OPS. Based on these data, it was concluded that phos- tablished for the trisaccharide repeating unit of this

+ $

pho-D-glycerol is an immunodominant group in the OPS (Table 1) (authors’ unpublished data), which was

/ + OPS responsible for the serological cross-reactivity of originally published as that of Citrobacter O29b40. the two bacteria. C. braakii O28: 70 (PCM 1561) C. werkmanii O21: 60 (PCM 1554)

The sugar composition of the OPS of C. braakii

$ &

The OPS of C. 4 werkmanii O21 (PCM 1554) in- O28 (PCM 1561)41, which includes D-ribose and L-rham- * cludes D-glucose, D-mannose and D-GlcNAc, which is nose, is inconsistent with chemotyping data35. A linear

386 Y. A. Knirel et al.: Polysacharides of Citrobacter

/ * trisaccharide repeating unit of this OPS41 (Table 1) to chemotyping data35 and, hence, the strain should be

comprises a partial structure of the tetrasaccharide re- reclassified.

peating unit of the OPS of Klebsiella O7 containing one The linear structure 5 of the OPS of C. braakii

& < 51 44

additional 2-substituted α-L-Rhap residue . O35 is identical to that of S. enterica sspW . arizonae

+ O5979. Accordingly, the LPS of the two strains strongly

+ 44

C. braakii O29: 8, 10, 11 (PCM 1562) cross-react with the corresponding anti-O sera . Both X anti-O sera also recognized the LPS of Pseudomonas

The OPS of C. braakii O29 (PCM 1562, formerly 7 aeruginosa O7 (former serogroup O11) in immunoblot- +

O29a)40 is a linear polysaccharide having the same ting and ELISA, though the serological relatedness of

+ X

structure as the main chain of the branched polysac- the enteric bacteria to P. aeruginosa O7 is much

charide of C. 4 werkmanii O26 (see above) (Table 1). weaker than to each other, and anti-P. aeruginosa O7

ManNAc present in this OPS was overlooked in the serum bound the LPS of neither C. braakii O35 nor

* /

35 44 chemotyping of the LPS . S. enterica sspW . arizonae O59 . The observed one-way serological cross-reactivity is apparently based on

C. braakii + O30: 76 (PCM 1567) structural similarities, but not identity, of the OPS, that

Y X of P. aeruginosa O7 having the structure 6 shown The structure of OPS of C. braakii O30 (PCM 1567) below19. Although different in sugar composition, both is identical to that of C. braakii O29 (PCM 1562) + OPS have repeating units of the same size with the (author’s unpublished data). The strains O29 and O30 corresponding monosaccharide residues having the producing the same O-antigens may differ in LPS core same absolute configurations and being connected by structures, which should be studied. the same linkages. Conformational analysis confirmed the presence of structurally similar sites on the helix C. youngae + O32: (23), 28 (PCM 1569) surfaces of each of the OPS, which may serve as

a common epitope44.

/ -

The OPS of C. . youngae O32 (PCM 1569)43 is a ho-

M M <

mopolymer of 3-amino-3,6-dideoxy-D-galactose (3-ami- → α < → β

$ L D

0 3)- - -FucpNAc-(1 3)- - -FucpNAc-

H $ no-3-deoxy-D-fucose, Fuc3N), which is N-acylated < → β D → 5 -(1 2)- - -Glcp-(1

with the L-glyceroyl group and partially O-acetylated at

J M M

position 2 (the degree of O-acetylation ∼75%)43 (Table →3)-α-L-Fuc< pNAc-(1→3)-β-D-GlcpNAc-

1, Fig. 1, structure 4). The glyceroyl group, in turn, is -(1→2)-β-D-Gal< p-(1→ 6 glycosylated by the sugar component and, hence, this + O-antigen is not a polysaccharide in the strict chemical sense since it contains not only glycosidic, but alternat- C. werkmanii O38:? (PCM 1489) ing glycosidic and amidic linkages. The composition of

strain PCM 1569 is inconsistent with chemotyping The OPS of C. 4 werkmanii O38 (PCM 1489)44 con-

$ $ & 35 $

data of the LPS. tains D-glucose, D-mannose, D-galactose, L-rhamnose

A different structure was established earlier for the and 3,6-dideoxy-D-Z xylo-hexose (abequose, Abe), which *

+ OPS of serogroup O3246, but later found to belong ac- is in accordance with chemotyping data35. Abequose is

tually to a polysaccharide different from the O32 an acid-labile monosaccharide, and mild acid delipida- antigen. The same structure as PCM 1569 had been tion of the LPS resulted in a significant loss of this

reported previously for the main chain of the cell-wall sugar. Therefore, in order to determine the OPS struc-

antigen of the Gram-positive microorganism Eubacte- ture, alkaline deacylation of the LPS was applied, S rium sabbureum L1329, which differs from the OPS of which resulted in a regular polysaccharide without any

C. youngae O32 by the presence of lateral D-fructofu- loss of abequose44.

- 6

ranose residues attached at position 3 of approximately The structure of the OPS of C. 4 werkmanii O3844

) every third Fuc3N residue. is similar to that of S. enterica sv. Kentucky, which

2 differs only in the O-acetyl substitution77. According- N ly, a strong cross-reactivity of the two bacteria73 and

C. braakii O35:? (PCM 1586) /

their LPS44 has been demonstrated. The O-acetyl

The OPS of C. braakii O35 (PCM 1586)44 contains group on the abequose residue had little importance

$ &

D-galactose, 2-acetamido-2,6-dideoxy-L-galactose (2-ace- in manifesting the immunospecificity of C. 4 werkma- tamido-2-deoxy-L-fucose, FucNAc), which does not fit ! nii O3844.

Y. A. Knirel et al.: Polysacharides of Citrobacter 387 #

C. freundii O41:? (PCM 1444) In addition to C. sedlakii and C. freundii, E. coli +

O157 is serologically related to Brucella 7 abortus12, 14, T

The OPS of C. freundii O41 (PCM 1444) contains B. melitensis8, 9, S. enterica O:30 (group N) 10, 61,

$ $ $ $ X

D-glucose, D-galactose, D-GlcNAc, D-GalNAc and 3- E. hermanii60, P. maltophilia 55518, Y. entercolitica

-amino-3,6-dideoxy-D-galactose amidated with (R)-3-hy- + O911, 13, and V. cholerae O136. The common epitope 2

droxybutyric acid (D-Fuc3NR3HOBu), and its biologi- responsible for the serological cross-reactivity, 2-sub- cal branched hexasaccharide repeating unit has been $

stituted D-perosamine residue N-acylated with various

) 65 established (Table 1). The main chain of the OPS

* groups, is present in all cross-reactive bacteria, but only

shows a similarity with that of H. alvei 1211 ; particu- Citrobacter and S. _ enterica O:30 (group N) have the

N [

larly, the OPS share the trisaccharide fragment 7 in the + O-antigen that is structurally identical with that of

main chain, which may be responsible for the weak E. coli O157. In a mouse infection model, S. _ enterica

cross-reactivity of the strains. + O:30 has shown promise as a live vaccine against

H H

Q 16

$ $ <

→2)-β-D-Glc< p-(1→2)-β-D-Fucp3NR3HOBu- E. coli O157: H7 gut colonization , and it is possible

R M → α < → that the strains of Citrobacter can also function in

-(1 6)- -D-GlcpNAc-(1 7 a similar manner78.

\ Non-Typed Strains C. freundii NRCC 6052

Citrobacter sp. 396 P A biochemically typical strain of C. freundii

(NRCC 6052) produces a simple OPS that is composed $

The OPS of Citrobacter sp. 396 contains D-glucose, of trisaccharide repeating units containing D-glucose

$ $ $ D-mannose, D-GlcNAc and abequose and has a bran- and D-rhamnose78 (Table 1).

ched heptasaccharide repeating unit , which is the lar- Although this strain was reported to be another Ci-

gest one among the Citrobacter O-antigens (Table 1). trobacter strain that shares an antigen with E. coli

# *

It is closely related to the OPS of S. enterica, having + O1573 and immunochemical studies showed the struc-

the same side chain of 2-O-acetylabequose as S. enteri- tural basis for the serological cross-reaction is under-

]

ca sv. Typhimurium (group B) and the main chain and N lying in the OPS moiety of the LPS78, the OPS of the second side chain of glucose similar to those in M

* C. freundii NRCC 6052 differs significantly from that

/ 38 S. enterica subgroups C1 and C4 . of E. coli O157. Particularly, it lacks D-perosamine, im- plicated as the common epitope in the OPS of bacteria C. sedlakii NRCC 6070, C. freundii OCU158 showing serological cross-reactivity with antisera

against E. coli O157 (see the previous section). There-

Strains C. sedlakii NRCC 607078 and C. freundii fore, it was suggested that the 2-substituted α-D-Rha< p

+ OCU15858 have similar OPS, which consists of D-glu- residue in the C. freundii NRCC 6052 OPS is structu- cose, L-fucose, D-GalNAc and Rha4NAc and has a li- rally similar to the 2-substituted α-D-Rhap4NAc

near tetrasaccharide repeating unit (Table 1). The same residue in the E. coli O157 OPS, thus presenting an +

OPS as from C. sedlakii NRCC 6070 is produced by ) example of mimicry within a bacterial polysaccharide E. coli O157: H762, an enteric pathogen that can cause antigen. However, modelling and oligosaccharide in- severe local and systemic diseases and is responsible ( hibition experiments will be required to confirm this for sporadic and for major outbreaks of hemorrhagic point.

colitis and large numbers of hemolytic-uremic syn- 2 dromes. Accordingly, C. sedlakii NRCC 607059 and its

C. rodentium ATCC 51459 ^ LPS78 showed a strong serological cross-reactivity with

anti-O serum against E. coli O157. The enzyme immu- The OPS of a prototype transmissible murine col-

0 noassay using LPS derived either from E. coli O157 or onic hyperplasia isolate of C. rodentium (ATCC ` from C. freundii OCU158 could equally detect high 51459) contains D-glucose, D-GlcNAc and L-rhamnose

levels of serum antibodies against LPS in patients with and is built up of pentasaccharide repeating units linked

) enterohemorrhagic E. coli O157 infection58. A dif- through phosphate55 (Table 1). The OPS is acid-labile

ference in the epitopes was demonstrated by the absorp- owing to the presence of a glycosyl phosphate group in

tion of antibodies in patient serum by LPS from E. coli the main chain. It could be depolymerised to the pen- +

O157 or C. freundii and was attributable to the epitope tasaccharide also by dephosphorylation with aqueous specificity of the LPS core region and/or lipid A structure58. 48% hydrofluoric acid.

388 Y. A. Knirel et al.: Polysacharides of Citrobacter

The OPS of E. coli O17352 is remarkably similar to counted for by different LPS core structures (so far, six 2

that of C. rodentium (ATCC 51459), differing only in different Citrobacter LPS core types are known). Simi-

the side chain sugar (α-L-fucose rather than β-L-rham- larly, classification in different serogroups, O2, O20

nose) and the glycosyl phosphate group (α-D-Glc< p-1-P and O25, of three strains that produce the same hetero-

M rather than α-D-Glc< pNAc-1-P). It was suggested that polysaccharide O-antigen may result from differences

the serological cross-reactions observed between in the LPS core structure. In contrast, some strains pro- 2

C. rodentium strain and anti-O sera against E. coli ducing structurally different O-antigens are classified in +

O173 is attributed to a structural feature involving the the same serogroup (e.g. strains of serogroups O7 and

a +

common phosphorylated trisaccharide component 8 O12), which could be due to the presence of common

)

present in the main chain of both OPS55. epitopes on the core region. Numerous Citrobacter

R < < strains are serologically related to other bacteria,

-P-(O→6)-α-D-Glcp-(1→2)-β-D-Glcp-(1→3)-β-

a M

< mainly Salmonella, Escherichia, Hafnia, and Klebsiel-

-D-GlcpNAc-(1→ 8 O la, and some of these relationships could by substan-

tiated on the level of the O-antigen structures. - Conclusions The existing data show that the chemical and sero-

N logical classification of Citrobacter strains requires re- 5 Chemical studies performed by several research vision. For this purpose, further immunochemical

groups resulted in the elucidation of the composition studies are needed to elucidate the O-antigen structures %

and structures of 26 O-specific polysaccharides (O-anti- in serogroups not yet studied to reveal and substantiate gens) of Citrobacter strains, which represent different on the molecular level serological relationships be-

species and different serogroups. tween strains of different Citrobacter serogroups and -

The O-antigens contain mainly neutral sugars, from 2 different enterobacterial genera and to find the position

$ $ which the most common are D-glucose, D-mannose and in the classification scheme of non-typed Citrobacter

D-rhamnose. Two sugars, 4-deoxy-D-7 arabino-hexose strains. It is also important to elucidate more precisely

and 3,6-dideoxy-D-Z xylo-hexose (abequose), are mono- the serological and structural similarities and differen- saccharides rarely occurring in nature. All monosac- ces in the LPS core region in various serogroups and

charides except for pentoses are present in the pyranose strains, which also contributes to the immunospecifity

$ form, D-xylose was found in both the pyranose and of Citrobacter. furanose forms and D-ribose only in the furanose form. Amino sugars represented by D-glucosamine, D-galacto-

samine, D-mannosamine, 2-amino-2-deoxy-L-fucose and

8 b

4-amino-4-deoxy-$ D-rhamnose (perosamine) are N-ace- References

tylated, and 3-amino-3-deoxy-D-fucose is acylated with

Q &

) either L-glyceroyl or (R)-3-hydroxybutyryl group. Two 1. ALTMANN G., SECHTER I., CAHAN D. and GERICHTER C. B. >

+ (1976): Citrobacter diversus isolated from clinical material. C O-antigens are phosphorylated, one containing D-glyce- c rol phosphate and the other a glycosyl phosphate group. J. Clin. Microbiol., 3, 390–392. 2. BADGER J. L., STINS M. F. and KIM K. S. (1999): Citrobacter

In many serogroups the chemical composition of the d freundii invades and replicates in human brain microvascular +

O-antigens is not consistent with the original LPS e ndothelial cells. Infect. Immun., 67, 4208–4215.

g f

chemotyping data. 3. BETTELHEIM K. A., EVANGELIDIS H., PEARCE J. L., SOWERS E. -

The structures of Citrobacter O-antigens are highly h and STROCKBINE N. A. (1993): Isolation of a Citrobacter freun- i

2 dii strain which carries the Escherichia coli O157 antigen. C diverse, the repeating units ranging from monosac- c

J. Clin. Microbiol., 31, 760–761. charide to heptasaccharide. There are three homo- j

4. BEYNON L. M., BUNDLE D. R. and PERRY M. B. (1990): The

polymer O-antigens, consisting of 4-deoxy-D-arabino- k structure of the antigenic lipopolysaccharide O-chain produced $

-hexose, 4-acetamido-4-deoxy-D-rhamnose (N-acetyl- l by Escherichia hermannii ATCC 33650 and 33652. Can. c -D-perosamine) and 3-deoxy-3-(L-glyceroylamino)-D- J. Chem., 68, 1456–1466.

5. BRAAK H. R. (1928): Onderzoekingen over Vergisting van -fucose. The last monomer units are connected to each other by the glycosidic linkage between the sugar and Glycerine (thesis). W. D. Meinema-Utitgeuer, Delft, Nether-

lands, 166.

m n glyceric acid moieties to build the structurally unusual m

+ 6. BRENNER D. J., GRIMONT P. A., STEIGERWALT A. G., FANNING

O-antigen of serogroup O32. G. R., Ap GERON E. and RIDDLE C. F. (1993): Classification of

$ 7

The homopolymer of 4-deoxy-D-arabino-hexose q citrobacteria by DNA hybridization: designation of Citrobacter d

was found in the serogroups O4, O27 and O36, whose farmerii sp. nov., Citrobacter youngae sp. nov., Citrobacter

r A classification in different serogroups could be ac- braakii sp. nov., Citrobacter werkmanii sp. nov., Citrobacter

Y. A. Knirel et al.: Polysacharides of Citrobacter 389 z sedlakii sp. nov., and three unnamed Citrobacter s genomos- 22. FINN V. G. (1978): The characteristics and clinical importance

pecies. Int. J. Syst. Bacteriol., 43, 645–658. © of bacteria of the genus Citrobacter isolated from patients with

o o

m m

7. BRENNER D. J., O’HARA C. M., GRIMONT P. A., JANDA J. M., h acute intestinal infections on the teritory of Volgograd. J. Hyg.

p t FALSEN E., ALDOVA E., AGERON E., SCHINDLER J., ABBOTT Epidemiol. Microbiol. Immunol., 22¢ , 338–343.

S. L. and STEIGERWALT A. G. (1999): Biochemical identifica- 23. GAMIAN A., Kg OCHAROVA N. A., ZATONSKY G. V., SHASHKOV

o z tion of Citrobacter species defined by DNA hybridization and A. S., Mz IESZALA M., LIPINSKI T., KATZENELLENBOGEN E. and description of Citrobacter gillenii sp. nov. (formerly Citrobac- K NIREL Y. A. (2001): Immunochemical analysis of Citrobacter

ter genomospecies 10) and Citrobacter murliniae sp. nov. (for- k serogroup O2 and structure of the O-specific polysaccharide of

merly Citrobacter genomospecies 11). J. Clin. Microbiol., 37 ¢ , the lipopolysaccharide from C. youngae O2a, 1b. Abstr. XVIth 2619–2624. ¤ International Symposium on Glycoconjugates, Hague, Nether-

8. Bj UNDLE D. R., CHERWONOGRODZKY J. W. and PERRY M. B. lands, August 19–24, 2001, C12.3.

o g g

(1987): The structure of the lipopolysaccharide O-chain (M 24. GAMIAN A., ROMANOWSKA E., ROMANOWSKA A., Lj UGOWSKI C., A

antigen) and polysaccharide B produced by Brucella melitensis DABROWSKI J. and TRAUNER K. (1985): Citrobacter lipopoly-

k ¢

16M. FEBS Lettu ., 216, 261–264. saccharides: structure elucidation of the O-specific polysac- j

9. BUNDLE D. R., CHERWONOGRODZKY J. W. and PERRY M. B. q charide from strain PCM 1487 by mass spectrometry, one-di-

(1987): Structural elucidation of the Brucella melitensis M mensional and two-dimensional 1H-NMR spectroscopy and ¢ antigen by high-resolution NMR at 500 MHz. Biochemistryw , methylation analysis. Eur. J. Biochem., 146, 641–647.

26, 8717–8726. 25. HAISHIMA Y., Kg ONDO S. and HISATSUNE K. (1990): The occur-

j y y 10. BUNDLE D. R., GERKEN M. and PERRY M. B. (1986): Two-di- rence of α(1 →2) linked N-acetylperosamine-homopolymer in

mensional nuclear magnetic resonance at 500 MHz: the struc- lipopolysaccharides of non-O1 Vibrio cholerae possessing an tural elucidation of a Salmonella serogroup N polysaccharide h antigenic factor in common with O1 V. cholerae. Microbiol.

antigen. Can. J. Chem., 64 ¢ , 255–264. Immunol., 34, 1049–1054.

z y j z j 11. Bj UNDLE D. R., GIDNEY M. A. J., PERRY M. B., DUNCAN J. R. 26. JACHYMEK W., CZAJA J., NIEDZIELA T., LUGOWSKI C. and

and CHERWONOGRODZKY J. W. (1984): Serological confirma- KENNE L. (1999): Structural studies of the O-specific polysac-

v { tion of Brucella abortus and Yersinia enterocolitica O:9 O-anti- q charide of Hafnia alvei strain PCM 1207 lipopolysaccharide.

gens by monoclonal antibodies. Infect. Immunu ., 46, 389–393. Eur. J. Biochem., 266, 53–61.

o o

y m

12. Bj UNDLE D. R. and PERRY M. B. (1985): Structure and serology 27. JANN B., PREHM K. and JANN K. (1978): Citrobacter O-anti- s

of the Brucella abortus O-antigen. Biochem. Soc. Trans., 13 ¢ , gens: Structure of the O-antigenic polysaccharide from Citro- r

980–982. bacter sp. 396. J. Bacteriol., 134¢ , 462–469. 13. CAROFF M., Bj UNDLE D. R. and PERRY M. B. (1984): Structure 28. JANSSON P.-E. (1999): The chemistry of O-polysaccharide

of the O-chain of the phenol-phase soluble cellular lipopolysac- q chains in bacterial lipopolysaccharides. In: BRADE H., OPAL S. M.,

g charide of Yersinia enterocolitica serotype O:9. Eur. J. Vg OGEL S. N. and MORRISON D. C. (eds.): Endotoxin in health Biochem., 139, 195–200. h and disease. Marcel Dekker, New York, 155–178.

14. CAROFF M., Bj UNDLE D. R., PERRY M. B., CHERWONOGRODZKY 29. JANSSON P.-E., LINDBERG B. and SPELLMAN M. (1985): The j

J. W. and DUNCAN J. R. (1984): Antigenic S-type lipopolysac- q cell-wall antigen from Eubacterium saburreum strain L 13, v charide of Brucella abortus 1119-3. Infect. Immun., 46, 384–388. h a new type of biopolymer. Carbohydr. Res., 137, 197–203.

15. CHERWONOGRODZKY J. W., PERRY M. B. and Bj UNDLE D. R. 30. KATZENELLENBOGEN E. (1988): The heterogeneity of lipopoly-

A v

(1987): Identification of the A and M antigens of Brucella as k saccharides from Citrobacter O23 serotype. Arch. Immunol. C

the O-polysaccharides of smooth lipopolysaccharides. Can. Ther. Exp., 36 ¢ , 119–128.

o g

J. Microbiol., 33, 979–981. 31. KATZENELLENBOGEN E., E KIEL I. and ROMANOWSKA E. (1988):

A g

16. CONLAN J. W., Kj UOLEE R., WEBB A. and PERRY M. B. (1999): The structure of the O-specific polysaccharide chain from Ci- Salmonella landau as a live vaccine against Escherichia coli trobacter O23-lipopolysaccharide. Carbohydr. Res., 179, 349– O157: H7 investigated in a mouse model of intestinal coloniza- 357.

tion. Can. J. Microbiol., 45, 723–731. 32. KATZENELLENBOGEN E., Kg OCHAROVA N. A., ZATONSKY G. V.,

z z z

17. Cj URVALL M., LINDBERG B., LÖNNGREN J. and NIMMICH W. KÜBLER-KIELB J., GAMIAN A., SHASHKOV A. S., KNIREL

(1973): Structural studies on the Klebsiella O group 3 lipopoly- Y. A. and Rg OMANOWSKA E. (2001): Structural and serological

saccharide. Acta Chem. Scand., 27 ¢ , 2645–2649. studies on Hafnia alvei O-specific polysaccharide of α-D-man-

18. DI FABIO J. L., PERRY M. B. and Bj UNDLE D. R. (1987): Ana- nan type isolated from the lipopolysaccharide of strain PCM

C }

lysis of the lipopolysaccharide of Pseudomonas maltophilia 1223. FEMS Immunol. Med. Microbiol., 30¢ , 223–227. ~

555. Biochem. Cell Biol., 65, 968–977. 33. KATZENELLENBOGEN E., Rg OMANOWSKA E., DABROWSKI U. and

g g o 19. D MITRIEV B. A., KNIREL Y. A., KOCHAROVA N. A., KOCHET- DABROWSKI J. (1991): O-Specific polysaccharide of Hafnia

KOV N. K., STANISLAVSKY E. S. and MASHILOVA G. M. (1980): alvei lipopolysaccharide isolated from strain 1211. Structural } Somatic antigens of Pseudomonas aeruginosa. The structure of k study using chemical methods, gas-liquid chromato-

the polysaccharide chain of Ps. aeruginosa O-serogroup 7 s graphy/mass spectrometry and NMR spectroscopy. Eur.

c ¢

(Lanyi) lipopolysaccharide. Eur. J. Biochem., 106 ¢ , 643–651. J. Biochem., 200, 401–407. g

20. Dg ORAN T. I. (1999): The role of Citrobacter in clinical disease 34. KATZENELLENBOGEN E., ZATONSKY G. V., KOCHAROVA N. A.,

g g g

of children: review. Clin. Infect. Dis., 28¢ , 384–394. BOGULSKA M., KORZENIOWSKA-KOWAL A., SHASHKOV A. S.,

o o

21. Dm RELICHMAN V. and BAND J. D. (1985): Bacteremias due to GAMIAN A. and KNIREL Y. A. (2002): Structure of a new

Citrobacter diversus and Citrobacter freundii. Incidence, risk 2-deoxy-2-[(R)-3-hydroxybutyramido]- D-glucose-containing O-spe- q

factors, and clinical outcome. Arch. Intern. Med., 145¢ , 1808– cific polysaccharide from the lipopolysaccharide of Citrobacter

1810. gillenii PCM 1542. Carbohydr. Res., 337¢ , 1541–1546.

390 Y. A. Knirel et al.: Polysacharides of Citrobacter

y 35. Ky ELETI J., LÜDERITZ O., MLYNARCIK D. and SEDLAK J. (1971): (1997): A chemical study of the sugar composition of the polysac- Immunochemical studies on Citrobacter O antigens (lipopoly- q charide portion of lipopolysaccharides isolated from Vibrio choler-

saccharides). Eur. J. Biochem., 20, 237–244. ae non-O1 from O2 to O155. Sys. Appl. Microbiol., 20, 1–11.

36. KENNE L., LINDBERG B., U NGER P., GUSTAFSSON B. and 50. KÜBLER-KIELB J., ZATONSKY G. V., KATZENELLENBOGEN E., g

Hg OLME T. (1982): Structural studies of the Vibrio cholerae KOCHAROVA N. A., SZOSTKO B., GAMIAN A., SHASHKOV A. S.

O-antigen. Carbohydr. Res., 100 ¢ , 341–349. and KNIREL Y. A. (2001): Structure of the O-specific polysac-

37. KLINE M. W., MASON E. O. JR. and KAPLAN S. L. (1988): q charide isolated from the lipopolysaccharide of Citrobacter gill- Characterization of Citrobacter diversus strains causing neona- enii serotype O12a, 12b strain PCM 1544. Carbohydr. Res.,

tal meningitis. J. Infect. Dis., 157, 101–105. C 331, 331–336.

z z j 38. K NIREL Y. A. and KOCHETKOV N. K. (1994): The structure of 51. LINDBERG B., LÖNNGREN J., NIMMICH W. and RUDEN U. lipopolysaccharides of Gram-negative bacteria. III. The struc- > (1973): Structural studies on the Klebsiella O group 7 lipopoly-

ture of O-antigens. Biochemistry (Moscow), 59, 1325–1383. k saccharide. Acta Chem. Scand., 27, 3787–3790.

39. Kg OCHAROVA N. A., BORISOVA S. A., ZATONSKY G. V., SHASH- 52. LINNERBORG M., WEINTRAUB A. and WIDMALM G. (1999):

KOV A. S., KNIREL Y. A., KHOLODKOVA E. V. and STANISLAV- Structural studies of the O-antigen polysaccharide from the en-

SKY E. S. (1998): Structure of the O-specific polysaccharide of teroinvasive Escherichia coli O173. Carbohydr. Res., 320¢ , 200– C

Citrobacter O3a, 1b, 1c. Carbohydr. Res., 306, 331–333. 208.

g g

40. Kg OCHAROVA N. A., BYSTROVA O. V., BORISOVA S. A., SHASH- 53. LIPINSKI T., ZATONSKY G. V., KOCHAROVA N. A., JAQUINOD M.,

KOV A. S., K NIREL Y. A., KHOLODKOVA E. V. and STANISLAV- FOREST E., SHASHKOV A. S., GAMIAN A. and KNIREL Y. A. > SKY E. S. (1997): Structures of two O-specific polysaccharides (2002): Structures of two O-chain polysaccharides of Citrobac-

of Citrobacter O29. Carbohydr. Lett., 2, 287–292. ter freundii O9a, 9b lipopolysaccharide. Eur. J. Biochem., 269,

¢ g

41. KOCHAROVA N. A., K NIREL Y. A., KHOLODKOVA E. V. and 93–99.

g j STANISLAVSKY E. S. (1995): Structure of the O-specific poly- 54. Lj UGOWSKI C., KULAKOWSKA M. and ROMANOWSKA E. (1992): saccharide chain of Citrobacter freundii O28,1c lipopolysac- ¤ Immunochemical characterization of Citrobacter strain PCM

charide. Carbohydr. Res., 279¢ , 327–330. 1487 O-specific polysaccharide- and core oligosaccharide-pro-

g g

42. KOCHAROVA N. A., KNIREL Y. A., SHASHKOV A. S., KOCHET- tein conjugates. FEMS Microbiol. Immunol., 89¢ , 201–208. KOV N. K., KHOLODKOVA E. V. and STANISLAVSKY E. S. 55. MACLEAN L. L. and PERRY M. B. (2001): Structural studies on

(1994): The structure of the Citrobacter freundii O8a, 8b the O-polysaccharide of the lipopolysaccharide produced by A

O-specific polysaccharide containing D-xylofuranose. Carbo- Citrobacter rodentium (ATCC 51459). Eur. J. Biochem.w , 268,

hydr. Res., 263, 327–331. £ 5740–5746.

g g g 43. KOCHAROVA N. A., K NIREL Y. A., SHASHKOV A. S., KOCHET- 56. MIKI K., TAMURA K., SAKAZAKI R. and KOSAKO Y. (1996): KOV N. K., KHOLODKOVA E. V. and STANISLAVSKY E. S. Re-specification of the original reference strains of serovars in (1994): The structure of the O-specific polysaccharide of Citro- the Citrobacter freundii (Bethesda-Ballerup group) antigenic

bacter freundii O32, a partially O-acetylated homopolymer of k scheme of West and Edwards. Microbiol. Immunol., 40, 915– ¢

3,6-dideoxy-3-(L-glyceroylamino)- α-D-galactopyranose. Car- 921.

bohydr. Res., 264 ¢ , 123–128. 57. MORGAN M. G., STUART C., LEANORD A. T., ENRIGHT M. and

g g

44. KOCHAROVA N. A., K NIREL Y. A., STANISLAVSKY E. S., KHO- COLE G. F. (1992): Citrobacter diversus brain abscess: case g

LODKOVA E. V., Lj UGOWSKI C., JACHYMEK W. and ROMANOW- reports and molecular epidemiology. J. Med. Microbiol., 36,

SKA E. (1996): Structural and serological studies of lipopoly- 273–278.

g g

saccharides of Citrobacter O35 and O38 antigenically related to 58. NISHIUCHI Y., Dg OE M., HOTTA H. and KOBAYASHI K. (2000):

¡ ¢

Salmonella . FEMS Immunol. Med. Microbiol., 13, 1–8. Structure and serologic properties of O-specific polysaccharide g

45. KOCHAROVA N. A., THOMAS-OATES J. E., K NIREL Y. A., from Citrobacter freundii possessing cross-reactivity with Es-

o g

n SHASHKOV A. S., DABROWSKI U., KOCHETKOV N. K., STANIS- ¥ cherichia coli O157: H7. FEMS Immunol. Med. Microbiol., 28,

LAVSKY E. S. and KHOLODKOVA E. V. (1994): The structure of 163–171 (Addendum. FEMS Immunol. Med. Microbiol. 2002,

C A

the O-specific polysaccharide of Citrobacter O16 containing 32 ¢ , 255).

o o

glycerol phosphate. Eur. J. Biochem., 219, 653–661. 59. PARK C. H., MARTIN E. A. and W HITE E. L. (1998): Isolation

z 46. KOCHAROVA N. A., VINOGRADOV E. V., KNIREL Y. A., SHASH- © of a nonpathogenic strain of Citrobacter sedlakii which express KOV A. S., Kg OCHETKOV N. K., STANISLAVSKY E. S. and KHO- Escherichia coli O157 antigen. J. Clin. Microbiol., 36, 1408–

LODKOVA E. V. (1988): The structure of the O-specific polysac- 1409. j

charide chains of the lipopolysaccharides of Citrobacter O32 60. Py ERRY M. B. and BUNDLE D. R. (1990): Antigenic relation- k

and Salmonella arizonae O64. Bioorg. Khim., 14¢ , 697–700. ships of the lipopolysaccharides of Escherichia hermannii

¦ v

g o

47. KOCHAROVA N. A., ZATONSKY G. V., BYSTROVA O. V., k strains with those of Escherichia coli O157: H7, Brucella me- §

SHASHKOV A. S., K NIREL Y. A., KHOLODKOVA E. V. and STA- litensis, and Brucella abortus. Infect. Immun., 58, 1391–1395.

y j y y

NISLAVSKY E. S. (2001): Structure of the O-specific polysac- 61. PERRY M. B., BUNDLE D. R., MACLEAN L., PERRY J. A. and

charide of Citrobacter braakii O7a, 3b, 1c. Carbohydr. Res., GRIFFITH D. W. (1986): The structure of the antigenic lipopoly- k

333¢ , 335–338. saccharide O-chains produced by Salmonella urbana and Sal- g 48. KOCHAROVA N. A., ZATONSKY G. V., SHASHKOV A. S. and ¨ monella godesberg. Carbohydr. Res., 156, 107–122.

K NIREL Y. A. (2000): Structural studies of the O-specific poly- 62. PERRY M. B., MACLEAN L. and GRIFFITH D. W. (1986): Struc-

saccharide of Citrobacter O1a, 1b, 1c containing an α-D-ribo- ture of the O-chain polysaccharide of the phenol-phase soluble

furanosyl group. Abstr. XXth International Carbohydrate Sym- © lipopolysaccharide of Escherichia coli O:157:H7. Biochem. ¤

posium, Hamburg, Germany, August 27-September 1, 2000, 17. Cell Biol., 64 ¢ , 21–28. p 49. Kg ONDO S., KAWAMATA Y., SANO Y., IGUCHI T. and HISATSUNE K. 63. PETRUS V. S., MATSIURAK A. V. and AKOPOVA A. J. (1977):

Y. A. Knirel et al.: Polysacharides of Citrobacter 391

Characteristics of bacteria of the genus Citrobacter¢ , isolated 73. SEDLAK J. and SLAJSOVA M. (1966): On the antigenic relation-

from the healthy children with different acute gastrointestinal k ships of certain Citrobacter and Hafnia cultures. J. Gen. Micro- l

diseases. Zh. Mikrobiol. Epidemiol. Immunobiol., 4, 122–125. biol., 43 ¢ , 151–158. ª 64. PREHM P., JANN B. and JANN K. (1976): The O9 antigen of 74. SHASHKOV A. S., VINOGRADOV E. V., K NIREL Y. A., NI-

Escherichia coli. Structure of the polysaccharide chain. Eur. FANT’EV N. E., Kg OCHETKOV N. K., DABROWSKI J., KHOLODKO-

f n

J. Biochem., 67 ¢ , 53–56. VA E. V. and STANISLAVSKY E. S. (1993): Structure of the

ª ° 65. RAVENSCROFT N., DABROWSKI J. and Rg OMANOWSKA E. (1995): O-specific polysaccharide of Salmonella arizonae O45. Carbo-

Structural elucidation of the biological repeating unit of O-spe- hydr. Res., 241¢ , 177–188.

g j

cific polysaccharide from Citrobacter serotype O41. Eur. 75. SHEARMAN P. J., Bj UNDLE D. R., ROMANOWSKA E., LUGOWSKI C. g

J. Biochem., 229, 299–307. h and BOGULSKA M. (1984): Characterization of anti-Citrobacter

g g

66. ROMANOWSKA E., GAMIAN A., Lj UGOWSKI C., ROMANOWSKA O36 specific polysaccharide monoclonal antibodies. Can.

o o

f A., DABROWSKI J., HAUCK M., O« PFERKUCH H. J. and VON DER J. Microbiol., 30, 91–97. LIETH C. W. (1988): Structure elucidation of the core regions 76. TAVORA L. and A NTUNES J. L. (1987): Brain abscesses and from Citrobacter O4 and O36 lipopolysaccharides by chemical ± ischemic necrotic lesions during early childhood. Neurosurgery,

and enzymatic methods, gas chromatography/mass spectro- 21, 923–927.

g g

metry, and NMR spectroscopy at 500 MHz. Biochemistry, 27 ¢ , 77. TORGOV V. I., SHIBAEV V. N., SHASHKOV A. S. and ROZHNO-

4153–4161. f VA S. S. (1989): Structure of the O-antigenic polysaccharide of

g g

67. ROMANOWSKA E., ROMANOWSKA A., DABROWSKI J. and h a Gram-negative bacterium Salmonella kentucky strain 98/39

o HAUCK M. (1987): Structure determination of the O-specific (O: 8, H:i, Z6). Bioorg. Khim., 15¢ , 1664–1672. polysaccharides from Citrobacter O4- and O27-lipopolysac- 78. VINOGRADOV E., Cg ONLAN J. W. and PERRY M. B. (1998): charides by methylation analysis and one- and two-dimensional ¡ Serological cross-reaction between the lipopolysaccharide

1H-NMR spectroscopy. FEBS Lett., 211, 175–178. ° O-polysaccharide antigens of Escherichia coli O157:H7 and

g g o 68. ROMANOWSKA E., ROMANOWSKA A., DABROWSKI J. and k strains of Citrobacter freundii and Citrobacter sedlakii. FEMS

TRAUNER K. (1989): The structure of the lipopolysaccharide Microbiol. Lett., 190 ¢ , 157–161.

z z

core region of Citrobacter O27. FEMS Microbiol. Rev., 58 ¢ , 79. VINOGRADOV E. V., KNIREL Y. A., LIPKIND G. M., SHASHKOV

107–110. A. S., Kg OCHETKOV N. K., STANISLAVSKY E. S. and KHOLODKO-

g g o f

69. ROMANOWSKA E., ROMANOWSKA A., Lj UGOWSKI C. and KATZE- VA E. V. (1987): Antigenic polysaccharides of bacteria. 23. The

NELLENBOGEN E. (1981): Structural and serological analysis of k structure of the O-specific polysaccharide chain of the lipopoly-

Citrobacter-O36-specific polysaccharide, the homopolymer of k saccharide of Salmonella arizonae O59. Bioorg. Khim., 13,

® (β1→2)-linked 4-deoxy-D- arabino-hexopyranosyl units. Eur. 1275–1281.

J. Biochem., 121, 119–123. 80. WERKMAN C. H. and GILLEN C. F. (1932): Bacteria producing

t o o y

70. SCHAUER D. B., ZABEL B. A., PEDRAZA I. F., O’HARA C. M., trimethylene glycol. J. Bacteriol., 23¢ , 167–182.

m y Sn TEIGERWALT A. G. and BRENNER D. J. (1995): Genetic and 81. WEST M. G. and EDWARDS P. R. (1954): The Bethesda-Balle- biochemical characterization of Citrobacter rodentium sp. nov. rup group of paracolon bacteria. U. S. Public Health Service

J. Clin. Microbiol., 33, 2064–2068. monograph no. 22. Washington D. C., U. S. Government Print-

± ¯

71. Sy EDLAK J. (1973): Present knowledge and aspects of Citrobac- ing Office.

~ ¢

ter . Curr. Top. Microbiol. Immunol., 62, 41–59.

¯ 72. Sy EDLAK J. and SLAJSOVA M. (1966): Antigen structure and antigenic relationships of the species A Citrobacter. Zentralbl. Received in July 2002 Bakteriol., ¬ 200, 369–374. Accepted in July 2002