Báo cáo khoa học: Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups ppt

Based on the O-polysaccharide structure and serological data, we propose classifying Proteus genomospecies 4 into a new, separate Proteus serogroup, O56.. A weak cross-reactivity of Prot

Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups

Krystyna Zych1, Andrei V Perepelov2, Małgorzata Siwinˇska1, Yuriy A Knirel2 and

Zygmunt Sidorczyk1

1 Department of General Microbiology, Institute of Microbiology and Immunology, University of Ło´dz´, Poland

2 N.D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation

Gram-negative bacteria of the genus Proteus are

com-mon in human and animal intestines but under

favour-able conditions they cause infections of wounds, burns,

skin, eyes, ears, nose and throat, as well as intestinal

and urinary tract infections The last illness can lead to

severe complications, such as acute or chronic

pyelone-phritis and formation of bladder and kidney stones In

the genus Proteus there are four clinically important

named species: P mirabilis, P vulgaris, P penneri and

P hauseri as well as three unnamed Proteus

genomo-species 4, 5 and 6 [1]

The outer membrane lipopolysaccharide (LPS) is a somatic antigen and is considered to be one of the most important potential virulence factors of Proteus [2] The serological O-specificity of Gram-negative bacteria is defined by the structure of the polysaccha-ride chain of LPS (O-antigen) Based on the somatic antigens, two Proteus species: P mirabilis and P vul-garis, have been classified into 49 O-serogroups [3] This classification, however, does not include two groups of P mirabilis and P vulgaris representatives (totally 20 strains), which were described later [4,5] or

Keywords

Proteus; O-antigen; O-serogroup; serological

classification;

3-acetamido-3,6-dideoxy-D -glucose

Correspondence

Z Sidorczyk, Department of General

Microbiology, Institute of Microbiology and

Immunology, University of Ło´dz´, Banacha

12 ⁄ 16, 90–237 Lo´dz, Poland

Tel: +48 42 6354467

Fax: +48 42 6655818

E-mail: zsidor@biol.uni.lodz.pl

(Received 16 May 2005, revised 30 August

2005, accepted 5 September 2005)

doi:10.1111/j.1742-4658.2005.04958.x

An acidic branched O-polysaccharide was isolated by mild acid degrada-tion of the lipopolysaccharide (LPS) of Proteus genomospecies 4 and stud-ied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected

1H,13C HSQC experiments The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established, which is unique among Proteus polysaccharide structures:

where Qui3NAc stands for 3-acetamido-3,6-dideoxyglucose Based on the O-polysaccharide structure and serological data, we propose classifying Proteus genomospecies 4 into a new, separate Proteus serogroup, O56 A weak cross-reactivity of Proteus genomospecies 4 antiserum with LPS of Providencia stuartiiO18 and Proteus vulgaris OX2 was observed and is dis-cussed in view of a similarity of the O-polysaccharide structures Structural and serological investigations showed that Proteus genomospecies 5 and 6 should be classified into the existing Proteus serogroups O8 and O69, respectively

Abbreviations

EIA, enzyme immunosorbent assay; GalA, galacturonic acid; LPS, lipopolysaccharide; PIH, passive immunohemolysis test; Qui3NAc, 3-acetamido-3,6-dideoxyglucose.

two other recognized medically important species,

P penneri and P hauseri, nor Proteus genomospecies

4, 5 and 6 [6–9] Previously, as a result of the chemical

and serological studies of LPS, a number of new

Pro-teus serogroups were proposed for P penneri strains

[9,10] Here we report on the structure of the O-specific

polysaccharide from LPS of Proteus genomospecies 4

and propose to classify this strain into a new Proteus

serogroup O56 We found also that Proteus

genomo-species 5 and 6 should be classified into the existing

Proteusserogroups O8 and O69, respectively

Results and Discussion

Structural studies

The O-polysaccharide was obtained by mild acid

degra-dation of the LPS, isolated from dried cells by the

phenol⁄ water procedure, and separated from lower

molecular mass substances, including a core

oligosac-charide, by Sephadex G-50 gel-permeation

chromato-graphy Sugar analyses by GLC of the alditol acetates

derived after full acid hydrolysis of the polysaccharide

revealed Glc, GlcN, GalN and

3-amino-3,6-dideoxyglu-cose (Qui3N) in the ratio 1 : 0.7 : 1 : 0.6 In addition,

GLC analysis of the acetylated methyl glycosides

showed the presence of GalA GLC analyses of the

acetylated (S)-2-(+)-butyl glycosides demonstrated the

dconfiguration of Glc, GlcN, GalN, and GalA The d

configuration of Qui3N was established by the analysis

of the 13C-NMR chemical shifts of the polysaccharide

using the known regularities in glycosylation effects [11]

The 13C NMR spectrum of the polysaccharide (Fig 1) contained signals for five anomeric carbons at

d 97.6, 98.1, 102.5, 104.0 and 104.3, three OCH2-C groups (C6 of Glc, GalN, and GlcN) at d 61.7, 62.3, and 69.9 [data of a distortionless enhancement of polari-zation transfer (DEPT) experiment], one CH3-C group (C6 of Qui3N) at d 19.8, one COOH group (C6 of GalA) at d 174.3, three nitrogen-bearing carbons (C2 of GlcN and GalN and C3 of Qui3N) at d 49.0–58.0, 17 oxygen-bearing ring carbons in the region d 68.8–81.8, and three N-acetyl groups at d 23.4–23.8 (CH3) and 175.1–176.0 (CO) Accordingly, the1H NMR spectrum

of the polysaccharide contained signals of equal inten-sity for five anomeric protons at d 4.52, 4.53, 4.74, 5.21 and 5.24 as well as signals for one CH3-C group (H6 of Qui3N) at d 1.36, three N-acetyl groups at d 1.98–2.05, and other signals at d 3.27–4.36 These data indicate that the polysaccharide has a regular structure with a pentasaccharide repeating unit

Methylation analysis of the polysaccharide, including GLC of partially methylated alditol acetates, resulted in the identification of the derivatives of terminal Glc, 3-substituted GalNAc, 6-substituted GlcNAc and 4-substituted Qui3NAc in the ratio 0.9 : 1 : 1 : 0.9 When the methylated polysaccharide was carboxyl-reduced prior to hydrolysis, 3-O-methylgalactose was identified in addition to the sugars mentioned above, which was evidently derived from 2,4-disubstituted GalA Therefore, the O-polysaccharide has a branched pentasaccharide repeating unit containing terminal

d-Glc, 3-substituted d-GalNAc, 6-substituted d-GlcNAc, 4-substituted d-Qui3NAc, and 2,4-disubstituted d-GalA

Fig 1 125-MHz 13 C-NMR spectrum of the O-polysaccharide of Proteus genomospecies 4 Arabic numerals refer to carbon positions in sugar residues.

The 1H- and 13C-NMR spectra of the

O-polysaccha-ride were assigned using 2D COSY, TOCSY, ROESY,

H-detected 1H,13C HSQC and HMBC experiments

(Table 1) The spin systems for GalA and Qui3NAc were

identified by correlations of H1 with H2-H5 and H2-H6,

respectively, in the TOCSY spectrum and with H3,H5 in

the ROESY spectrum Correlations of H1 with H2-H6

in the TOCSY spectrum demonstrated Glc and GlcNAc

GalNAc showed correlations for H1 with H2-H4 in the

TOCSY spectrum and H4 with H5,H6 in the ROESY

spectrum The carbonyl signal of GalA was distinguished

by an H5⁄ C6 correlation in the HMBC spectrum

J1,2 coupling constant values of  3 Hz indicated

that GalNAc and Glc are a-linked, whereas J1,2-values

of 7–8 Hz showed that Qui3NAc, GlcNAc, and GalA

are b-linked The pyranose form of all monosaccharide

residues originated from the absence from the

13C-NMR spectrum of any signals for nonanomeric

ring carbons at a field lower than d 82 [12]

A relatively low-field position of the signals for C6

of GlcNAc (d 69.9), C3 of GalNAc (d 77.5), C4 of

Qui3NAc (d 77.5) as well as C2 and C4 of GalA

(d 77.2 and 81.8, respectively), as compared with their

position in the corresponding nonsubstituted

monosac-charides [13,14], demonstrated the modes of

glycosyla-tion of the constituent sugar residues

A ROESY experiment (Fig 2) revealed interresidue

cross-peaks between the anomeric protons and

glyco-sidically linked protons, which were assigned to

Qui3NAc H1⁄ GlcNAc H6a and H6b (d 4.52 ⁄ 3.90 and 4.17); GlcNAc H1⁄ GalA H4 (d 4.53 ⁄ 3.80); GalA H1⁄ GalNAc H3 (d 4.74⁄ 4.25); GalNAc H1⁄ Qui3NAc H4 (d 5.24 ⁄ 3.52); and Glc H1 ⁄ GalA H2 (d 5.21⁄ 3.54) correlations In accordance with the ROESY data, the HMBC experiment showed the following inter-residue cross-peaks between the atoms separated by three bonds: Qui3NAc H1⁄ GlcNAc C6 (d 4.52⁄ 69.9); GlcNAc H1 ⁄ GalA C4 (d 4.53 ⁄ 81.8); GalA H1⁄ GalNAc C3 (d 4.74 ⁄ 77.5); GalNAc C1 ⁄ Qui3-NAc H4 (d 97.6⁄ 3.52) and Glc C1 ⁄ GalA H2 (d 98.1⁄ 3.54) These data were in agreement also with the glycosylation pattern determined from methylation and

13C-NMR chemical shift data (see above) and defined the monosaccharide sequence in the repeating unit Therefore, it was concluded that the O-polysaccharide

of Proteus genomospecies 4 has the structure:

The O-polysaccharide has a unique structure among Proteus polysaccharides and is distinguished by the presence of 3-acetamido-3,6-dideoxy-d-glucose While this is a rarely occurring component, it was found in several other bacterial polysaccharides [15], including O-polysaccharides of Proteus [16]

Studies of the O-polysaccharides of Proteus genomo-species 5 and 6 and the comparison of the correspond-ing 13C NMR spectra indicated that they are identical

to those of P vulgaris O8 [17] and P penneri 25 (O69) [18], respectively

Table 1 1 H- and 13 C-NMR data of the polysaccharide of Proteus genomospecies 4 (d, p.p.m) The chemical shifts for the N-acetyl groups are dH1.98, 2.04 and 2.05; dC23.4, 23.6, 23.8 (all Me), 175.1, 175.3 and 176.0 (all CO).

Atom number

13

fi2,4)-b- D -GalpA-(1fi 1

13

In particular, the 13C NMR spectrum of Proteus

genomospecies 5 contained signals for four anomeric

carbons at d 99.3, 99.8, 102.4 and 105.1 (d 99.6,

100.0, 102.5 and 105.3 in P vulgaris O8), two OCH2-C

groups at d 62.2 and 63.3 (d 62.4 and 63.4 in P

vul-garisO8), two nitrogen-bearing carbons at d 50.4 and

55.4 (d 50.6 and 55.5 in P vulgaris O8), one CH3-C

group at d 16.6 (d 16.7 in P vulgaris O8), one COOH

group at d (d 173.5 in P vulgaris O8), two N-acetyl

groups at d 23.5 and 23.8 (CH3; 23.7 and 23.9 in

P vulgaris O8) and 176.1 and 176.2 (CO; 176.2 and

176.3 in P vulgaris O8) as well as 12 other

oxygen-bearing ring carbons in the region d 69.6–84.0 (d 69.7–

84.2 in P vulgaris O8)

Similarly, the13C NMR spectrum of the

O-deacetyl-ated polysaccharide of Proteus genomospecies 6

con-tained signals for four anomeric carbons at d 102.2,

102.3, 102.9 and 104.1 (d 101.9, 102.0, 102.7 and 104.0

in P penneri 25), two OCH2-C groups at d 61.8 and

68.7 (d 61.9 and 68.8 in P penneri 25), two

nitrogen-bearing carbons at d 55.4 and 56.1 (d 55.3 and 56.0

in P penneri 25), two COOH groups at d 174.6 and

175.7 (d 174.9 and 176.0 in P penneri 25), one

N-ace-tyl group at d 23.6 (CH3; 23.7 in P penneri 25) and 175.7 (CO; 175.7 in P penneri 25), 14 other oxygen-bearing ring carbons in the region d 69.6–84.0 (d 69.7–84.2 in P penneri 25) as well as signals for alanine at d 172.5, 50.8 and 17.7 (d 172.5, 50.8 and 17.6 in P penneri 25)

Serological studies Serological investigations were performed using LPS-specific rabbit polyclonal antisera against strains of Proteus genomospecies 4, 5 and 6 They were tested with LPS of a number of Proteus strains with known O-polysaccharide structure in passive immunohemoly-sis (PIH) In addition, LPS of Providencia stuartii O18 was investigated in order to confirm the serological relatedness between this bacterium and Proteus genomospecies 4 reported earlier [19]

From 92 tested Proteus lipopolysaccharides, inclu-ding those of 27 strains of P vulgaris, 39 strains of

P mirabilis, 24 strains of P penneri, one strain of

P myxofaciens, one strain of P hauseri and Providen-cia stuartii O18, only LPS of P vulgaris OX2 and

Fig 2 Part of a 2D ROESY spectrum of the

polysaccharide of Proteus genomospecies

4 The corresponding parts of the 1 H-NMR

spectrum are displayed along the axes

Ara-bic numerals refer to proton positions in

sugar residues.

Providencia stuartii O18 cross-reacted with Proteus

genomospecies 4 antiserum As opposed to the

reac-tion of Proteus genomospecies 4 antiserum with the

homologous LPS (1 : 51 200), the cross-reactions were

rather weak (1 : 1600 and 1 : 6400, respectively) Their

specificity was confirmed using enzyme immunosorbent

assay (EIA) (Fig 3A) (1 : 256 000, 1 : 8000 and

1 : 32 000, respectively) and inhibition of the reaction

in both PIH and EIA Again, the homologous LPS

was a strong inhibitor (1–4 ng) whereas the inhibiting

activity of the cross-reactive antigens was significantly

weaker (1250–5000 ng)

The antigens studied were tested in PIH with

Proteus genomospecies 4 antiserum, which was

absorbed with the respective LPS The reactivity of

antiserum against Proteus genomospecies 4 with all

tested antigens was completely abolished when it was

absorbed with the homologous LPS Absorption with

LPS of either Providencia stuartii O18 or P vulgaris

OX2 removed antibodies to the corresponding cross-reactive LPS but influenced the reactivity with the homologous LPS insignificantly Hence, cross-reactive

is only a minor population of antibodies

In western blot (SDS⁄ PAGE separation of the investi-gated LPSs also presented on Fig 4A), Proteus genomospecies 4 antiserum (Fig 4 B) strongly reacted with slow migrating bands of the homologous LPS and that of Providencia stuartii O18 These and absorption data suggest that the antiserum contains both major O-polysaccharide-specific antibodies, which recognize

an epitope on the homologous O-antigen, and a minor population of antibodies, which react with a common epitope on the O-antigen of Proteus genomospecies 4 and Providencia stuartii O18 In addition, Proteus ge-nomospecies 4 antiserum recognized fast migrating LPS bands of the two bacteria as well as those of P vulgaris OX2; these correspond to the LPS core with no O-poly-saccharide attached Therefore, there are also LPS core-specific cross-reactive antibodies, which recognize different epitopes on the Providencia stuartii O18 and

P vulgaris OX2 LPS, as followed from combined western blot and absorption data

Serological relatedness of Proteus genomospecies 4 and Providencia stuartii O18 demonstrated in this work is in agreement with the cross-reactivity of LPS

of the Proteus strain with anti-Providencia stuartii O18 serum [19] Comparison of the O-polysaccharide structures of Proteus genomospecies 4 and Providencia stuartii O18 showed that they share a b-d-Quip3NAc-(1fi 6)-d-GlcNAc disaccharide fragment (a), which, most likely, occupies the nonreducing end of the polysaccharide chain (Fig 5) Moreover, the O-polysaccharides possess also a common interior a-d-GalpNAc-(1fi 4)-b-d-Quip3NAc-(1 fi 6)-d-GlcNAc trisaccharides fragment (b), and both common features seem to be responsible for the cross-reactivity of these strains Proteus vulgaris OX2 has no similarities with Proteus genomospecies 4 in O-antigen structure [20], which is consistent with the western blot data showing the absence of any common epitope on the O-polysac-charides (Fig 4B)

Based on the results of serological studies and the uniqueness of the O-polysaccharide structure we pro-pose classifying Proteus genomospecies 4 into a new Proteusserogroup, O56, in which at present this strain

is the single representative

In PIH and EIA LPS from P vulgaris O8 cross-reacted with Proteus genomospecies 5 antiserum (Fig 3B) and that from P penneri O69 with Proteus genomospecies 6 antiserum (Fig 3C), the cross-reac-tions being almost identical to the reaccross-reac-tions of the homologous LPS (1 : 51 200) Inhibition of the

reac-Fig 3 EIA binding curves of Proteus genomospecies 4 (A), 5 (B)

and 6 (C) antisera to the investigated LPS.

tions in both PIH and EIA was also very strong and similar (1–2 ng), indicating a high level of serological relatedness between the two pairs of bacteria The reactivity of both Proteus genomospecies 5 and 6 anti-sera was completely abolished by absorption with the respective homologous and cross-reactive antigens In western blot, Proteus genomospecies 5 and 6 antisera (Fig 4C,D) reacted similarly with both homologous and heterologous P vulgaris O8 and P penneri O69 LPS, respectively The reactions were strong with slow migrating LPS bands and weaker with fast migrating bands, which correspond to the LPS core with and without the O-polysaccharide, respectively

The serological data obtained and the identity of the O-polysaccharide structures showed that Proteus genomospecies 5 and 6 strains should be classified into the existing Proteus serogroups O8 and O69 [17,18,21], respectively

Fig 4 SDS ⁄ PAGE (A) and western blot of Proteus genomospecies 4 (B), 5 (C) and 6 (D) antisera with LPS from investigated strains Abbre-viations: P.s., Providencia stuartii; P.g., Proteus genomospecies; P.p., P penneri; P.v., P vulgaris The antisera were diluted in blotting buffer (1 : 300); 3–5 ng LPS were applied per lane Concentration of acrylamide in the gel was 12%.

Fig 5 Structures of the O-polysaccharides of cross-reactive

bac-teria Proteus genomospecies 4 (this work), Providencia stuartii O18

[19] and P vulgaris OX2 [20].

Experimental procedures

Bacterial strains and growth

Proteus genomospecies 4 (ATCC 51469), 5 (ATCC

51470), 6 (ATCC 51471), P hauseri (ATCC 700826) and 24

P penneri strains were kindly provided by C M O’Hara

and D J Brenner (Centres for Disease Control and

Preven-tion, Atlanta, GA, USA) Thirty-nine strains of P mirabilis

and 27 P vulgaris strains were from the Czech National

Collection of Type Cultures (CNCTC, National Institute of

Public Health, Prague, Czech Republic) Proteus

myxofac-iensstrain (CCUG 18769) was kindly provided by E

Fal-sen [Cultures Collection, University of Goeteborg (CCUG),

Goeteborg, Sweden] and Providencia stuartii O18 was from

the Hungarian National Collection of Medical Bacteria

(National Institute of Hygiene, Budapest, Hungary) Dry

bacterial mass was obtained from aerated culture as

des-cribed previously [22]

Isolation and degradation of lipopolysaccharide

Lipopolysaccharides were obtained by extraction of

bacter-ial mass with a hot phenol⁄ water mixture [23] and purified

by treatment with aqueous 50% (v⁄ v) CCl3CO2H at 4
C

followed by dialysis of the supernatant Alkali-treated LPS

were prepared by saponification of LPS with 0.25 m NaOH

(56
C, 2 h) followed by precipitation with ethanol

Acid degradation of Proteus genomospecies 4 LPS

(226 mg) was performed with aqueous 2% HOAc at 100
C

until lipid A precipitation The precipitate was removed by

centrifugation (13 000 g, 20 min), and the supernatant was

fractionated on a column (56· 2.6 cm) of Sephadex G-50

(Pharmacia, Fairfield, CT, USA) in 0.05 m pyridinium

acet-ate buffer, pH 4.5, monitored using a Knauer differential

refractometer (Berlin, Germany) A high-molecular mass

polysaccharide was obtained in a yield of 10% of the LPS

weight All experiments followed 36⁄ 609 EEC guidelines

Rabbit antiserum and serological assays

Polyclonal LPS-specific antisera were obtained in a

stand-ard procedures by immunization of New Zealand white

rabbits (two for one bacterial strain) with heat-inactivated

(100
C, 2,5 h) bacteria of Proteus genomospecies 4, 5 and

6, as described previously [24] Passive immunohemolysis,

absorption, EIA and inhibition experiments as well as

SDS⁄ PAGE and western blot were carried out as described

[24] Lipopolysaccharide (EIA) or alkali-treated LPS (PIH)

were used as antigens

Sugar analyses

The polysaccharide was hydrolysed with 2 m CF3CO2H

(120
C, 2 h), monosaccharides were reduced with 0.25 m

NaBH4 in 1 m ammonia (20
C, 1 h), acetylated with a

1 : 1 (v⁄ v) mixture of pyridine and acetic anhydride (120
C, 30 min) and analysed by GLC, using a Hewlett-Packard 5890 Series II instrument, equipped with a

DB-225 capillary column (30 m· 0.25 mm) and a temperature gradient 170–180
C at 1
CÆmin)1 and then 180–230
C

at 7
CÆmin)1 Methanolysis of the polysaccharide (1 mg) was performed with 1 m HCl in MeOH (85
C, 16 h) and was followed by acetylation and GLC analysis as des-cribed above The absolute configuration of the mono-saccharides was determined by GLC of the acetylated (S)-(+)-2-butyl glycosides according to the published method [25]

Methylation analysis Methylation of the polysaccharide was performed with

CH3I in dimethylsulfoxide in the presence of sodium meth-ylsulfinylmethanide [26] A portion of the methylated poly-saccharide was reduced with LiBH4 in 70% (v⁄ v) aqueous 2-propanol (20
C, 16 h) Partially methylated monosaccha-rides were derived by hydrolysis under the same conditions

as in sugar analysis, reduced with NaBH4or NaBD4, acet-ylated and analysed by GLC-MS using a Trace GC 2000 instrument and a temperature gradient 150–250
C at

10
CÆmin)1

NMR spectroscopy NMR spectra were recorded on a Bruker DRX-500 spec-trometer (Karlsruhe, Germany) for a solution in D2O at

40
C using internal acetone (dH 2.225 p.p.m., dC

31.45 p.p.m.) as reference Prior to the measurements, sam-ples were deuterium-exchanged by freeze-drying twice from

D2O Bruker software xwinnmr 2.1 was used to acquire and process the NMR data A mixing time of 200 and

300 ms was used in two-dimensional TOCSY and ROESY experiments, respectively The other parameters used for 2D experiments were essentially the same as described previously [27]

Acknowledgements

The work was supported by the following grants: Uni-versity of Lodz (grant 801), 02-04-48767 of the Russian Foundation for Basic Research, RFMK-226.2003.03 and RF State Contract RI–112⁄ 001 ⁄ 272

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