Structure and serological studies of the O-polysaccharide of<i>Proteus penneri</i>75

The tribe Proteeae consists of three genera: Proteus, Providencia and Morganella. These bacteria are usually found in soil, water and sewage, and also are part of normal faecal microflora. They are important human opportunistic pathogens that cause a variety of community-acquired and nosocomial diseases, including urinary tract infections, septicemia and wound infections [1,2]. The genus Proteus includes five named species, including P. mirabilis, P. vulgaris, P. myxofaciens, P. penneri and P. hauseri, and three unnamed genomospecies 4, 5 and 6 [3,4]. Proteus penneri, formerly known as Proteus vulgaris biogroup 1 or indol-negative P. vulgaris, was described as a new species within the genus Proteus in 1982 [5]. These bacteria are isolated mainly from urine of patients with urinary tract abnormalities [5,6] and from abdominal wounds, particularly, after bowel resections [6]. Strains of P. penneri also caused urosepsis [7], subcutaneous [8] and nosocomial infections in a Spanish hospital [9]. Recently, Proteus infections have received special attention owing to the emergence of resistance of some species to a variety of antibiotics, in particular, to β-lactams [10]. The serospecificity of Gram-negative bacteria is defined by the structure of the O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS). Based on the O-antigens, strains of two species, P. vulgaris and P. mirabilis, have been classified first into 49 O-serogroups [11,12] and later into 12 additional O-serogroups [13,14]. Further 15 O-serogroups have been proposed for the strains of P. penneri[15–17]. In order to create the molecular basis for the classification of Proteus strains, structures of the O-polysaccharides of serologically distinguishable strains of P. mirabilis, P. vulgaris, P. penneri and P. myxofaciens have been elucidated [14,15,18–20]. Many of them have been found to contain uncommon non-sugar components, such as residues of lactic, pyruvic, hydroxy and amino acids, phosphate-linked polyalcohols and amino alcohols. Now we report on a new structure of a ribitol-5-phosphate containing O-polysaccharide from the LPS of Proteus penneri 75, which is similar to that of the O-polysaccharide of P. penneri 103 reported earlier [21]. Serological investigations showed a serological relatedness of these strains to each other and to P. penneri strains 48, 90 and 128 classified into Proteus serogroup O73 [22]. The results obtained allowed a subdivision of the Proteus O73 serogroup into two subgroups and a suggestion about epitopes that are responsible for the cross-reactivity. P. penneri 75 (3PC(8)K/VIII/95) was isolated from urine of women with bacteriuria in a hospital in Łódź, Poland. The other strains were kindly provided: P. penneri 103 (No CL 191/90 = 21B) by Dr. Barry Holmes (National Collection of Type Cultures, Central Public Health Laboratory, London, UK); P. penneri 128 (2175) by Dr. Miguel Valvano (Department of Microbiology and Immunology, Ontario, Canada); P. penneri 90 (17518R) by Dr. Bernard Senior (Department of Medical Microbiology, Dundee, UK); P. penneri 48 (CCUG 33352) by Dr. Enevold Falsen (Culture Collection University of Goeteborg, Department of Clinical Bacteriology, Goeteborg, Sweden). P. mirabilis O20 (strain 38/57) came from the Czech National Collection of Type Cultures (CNCTC, Institute of Microbiology and Genetics, Prague, Czech Republic). Dry bacterial cells were obtained from the aerated cultures as described previously [23]. LPSs were obtained by extraction of dried bacterial cells with a hot phenol–water mixture [24] and purified by treatment with cold aqueous 50% CCl3CO2H, followed by dialysis of the supernatant [25]. Degradation of the LPSs of P. penneri 75 and 103 was performed with 0.1 M sodium acetate buffer pH 4.5 at 100 °C for 2 h. The O-specific polysaccharides were isolated by gel-permeation chromatography on a column (2.6 × 56 cm) of Sephadex G-50 in 0.05 M pyridinum acetate buffer pH 4.5 monitored using a Knauer differential refractometer (Germany). Alkali-treated LPSs were prepared by saponification of the LPSs with 0.25 M NaOH (56 °C, 2 h) followed by precipitation with ethanol. The polysaccharide from P. penneri 75 (0.5 mg) was hydrolysed with 2 M CF3CO2H (120 °C, 2 h), monosaccharides were reduced with 0.25 M NaBH4 in aqueous 1 M ammonia (25 °C, 1 h), acetylated with a 1:1 (v/v) mixture of pyridine and acetanhydride (120 °C, 0.5 h) and analysed by GLC. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (+)-2-butyl glycosides [26,27]. GLC was performed using a Hewlett-Packard 5890 Series II instrument equipped with an HP-1 fused silica column (0.20 mm × 25 m) and a temperature program of 170 to 180 °C at 1 °C min−1 followed by a program of 180 to 230 °C at 7 °C min−1. The absolute configuration of ribitol was determined by oxidation with 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO) followed by acid hydrolysis and GLC analysis as described [28]. Methylation of the polysaccharide from P. penneri 75 (2 mg) was performed with CH3I in dimethylsulfoxide in the presence of sodium methylsulfinylmethanide [29]. Partially methylated monosaccharides were derived by hydrolysis under the same conditions as in sugar analysis, converted into the alditol acetates and analysed by GLC-MS on a TermoQuest Finnigan mass spectrometer model Trace GC 2000 equipped with an EC-1 column (0.32 mm × 30 m), using a temperature gradient of 150 (2 min) to 250 °C at 10 °C min−1. Prior to the measurements, samples were freeze-dried from D2O. 1H, 13C and 31P NMR spectra were recorded with a Bruker DRX-500 spectrometer in D2O at 20 °C using internal acetone (δH 2.225, δC 31.45) or external aqueous 85% H3PO4 (δP 0) as reference. 1D and 2D experiments were performed using standard pulse sequences and data obtained were processed using Bruker software XWINNMR 2.6. Polyclonal P. penneri 75 and 103 O-antisera were obtained by immunization of rabbits with heat-inactivated bacteria according to a published procedure [30]. SDS–PAGE, electrotransfer of LPSs from the gel to nitrocellulose sheets, immunostaining and absorption experiments were carried out as described [31]. LPS was used as antigen in enzyme immunosobrent assay (EIA) [32]. Passive immunohemolysis (PI) was performed with increasing amounts (2–200 µg) of alkali-treated LPS [25]. LPS was isolated from dried bacterial cells of P. penneri 75 by the phenol–water extraction [24] and degraded under mild acidic conditions to give a high-molecular-mass polysaccharide. Sugar analysis of the polysaccharide revealed Glc, Gal and GalN in the ratios ~ 2:1:1 as well as ribitol (Rib-ol). Determination of the absolute configurations by GLC of the acetylated (S)-(+)-2-butyl glycosides derived from sugars showed that all monosaccharides have the D configuration. The absolute configuration of ribitol was determined by GLC of the acetylated octyl ester of ribonic acid derived by TEMPO oxidation [28]. Methylation analysis of the polysaccharide resulted in the identification of 2,3,4,6-tetra-O-methylhexose (from terminal α-Glc), 2,4,6-tri-O-methylhexose (from 3-substituted Gal), 2,6-di-O-methylhexose (from 3,4-disubstituted β-Glc) and 2-deoxy-4,6-di-O-methyl-2-(N-methyl)acetamidohexose (from 3-substituted GalNAc). Therefore, the polysaccharide is branched. The 13C NMR spectrum of the polysaccharide (Fig. 1) contained signals for four anomeric carbons at dd 101.0–105.7, one carbon linked to nitrogen at dd 52.5, one N-acetyl group at dd 23.6 (CH3) and 176.5 (CO), five C-C H2O groups at dd 61.4–66.8 (C6 of three sugar residues, C1 and C5 of ribitol) and 17 other carbons linked to oxygen at dd 69.2–84.3. Accordingly, the 1H NMR spectrum contained signals for four anomeric protons at dd 4.52–5.23, one N-acetyl group at dd 2.07 and other signals in the region dd 3.47–4.17. The 31P NMR spectrum contained signals for one phosphate group at dd 0.7. Therefore, the polysaccharide has a repeating unit containing two residues of Glc and one residue each of Gal, GalNAc and ribitol as well as phosphate. Part of a 13C NMR spectrum of the O-specific polysaccharide of P. penneri 75. The 1H and 13C NMR spectra of the polysaccharide were assigned using 2D COSY, TOCSY and 1H, 13C HMQC experiments (Table 1). Based on typical JH,H coupling constant values, the three sugar spin systems were assigned. The Glc residue was identified by large J3,4 and J4,5 coupling constant values of 9–10 Hz, as compared with the values of ≤3 Hz for the sugars with the galacto configuration (Gal and GalNAc). The spin system of Rib-ol was identified based on 1H,13C correlations for five remaining oxygen-linked carbons in the 1H,13C HMQC spectrum. As judged by relatively small J1,2 values of 3 Hz determined from the 1H NMR spectrum, one of the Glc residues is α-linked and the other three sugar residues are bb-linked (J1,2 values of ~8–9 Hz). This conclusion was confirmed by a ROESY experiment, which revealed typical intraresidue H-1,H-3 and H-1,H-5 correlations for three beta pyranosides and a H-1,H-2 correlation for the α-Glc residue. 1H NMR and 13C NMR data of the O-specific polysaccharide from P. penneri 75 (δ, ppm) Chemical shift for NAc is δ 2.07 (proton spectra) and δ 23.6 (Me) and 176.5 (CO) (carbon spectra). H-1a; H-1b at δ 4.85. H-5a; H-5b at δ 4.20. 1H NMR and 13C NMR data of the O-specific polysaccharide from P. penneri 75 (δ, ppm) Chemical shift for NAc is δ 2.07 (proton spectra) and δ 23.6 (Me) and 176.5 (CO) (carbon spectra). H-1a; H-1b at δ 4.85. H-5a; H-5b at δ 4.20. Low-field displacements of the signals for C-3 and C-4 of β-Glc (+7.5 and +3.0 ppm), C-3 of Gal (+9.6 ppm), C-3 of GalNAc (+9.1 ppm), and C-4 of Rib-ol (+6.6 ppm), as compared with their positions in the corresponding non-substituted compounds [33], demonstrated the modes of substitution of the monosaccharides and ribitol in the repeating unit, which were in agreement with methylation analysis data (see above). Phosphorylation also caused a positive but smaller effect on the C5 signal of Rib-ol (+3.0 ppm). Sequence analysis of the O-polysaccharide was performed using a ROESY experiment, which showed the following interresidue correlations: α-Glc H-1/β-Glc H-3, β-Glc H-1/Gal H-3, Gal H-1/GalNAc H-3 and GalNAc H-1/Rib-ol H-4 at dd 5.23/3.80, 4.71/3.78, 4.52/3.88 and 4.74/4.14, respectively. The glycosylation pattern and the monosaccharide sequence were confirmed also by a 1H,13C HMBC experiment, which showed the following correlations: α-Glc H-1/β-Glc C-3, β-Glc H-1/Gal C-3, Gal H-1/GalNAc C-3 and GalNAc H-1/Ribitol, C4 at dd 5.23/84.3, 4.71/83.4, 4.52/81.2 and 4.74/80.1, respectively. A 1H,31P HMQC experiment showed a correlation of the phosphorus signal at dd 0.7 with the signals for H5a,H5b of Rib-ol at dd 3.99 and 4.20 as well as with the H4 signal of β-Glc at dd 4.15. Therefore, the ribitol and β-Glc residues are connected via a phosphate group. On the basis of the data obtained, it was concluded that the O-polysaccharide of P. penneri 75 has the structure shown in Fig. 3. This structure is unique among the known structures of Proteus O-antigens. On the other hand, P. penneri 75 shares the same O-polysaccharide main chain with P. penneri 103 [21], whereas the lateral substituents of the glucose residue (either another glucose or phosphoethanolamine) are different (Fig. 3). Structures of the O-specific polysaccharides of P. penneri 75, 103 and P. mirabilis O20 [34]. Etn-P stands for phosphoethanolamine. Shown are putative epitopes on the O-polysaccharides. LPS from 92 strains representing all Proteus O-serogroups were tested in PI and EIA with polyclonal rabbit O-antiserum against P. penneri 75. The cross-reactivity was observed for the LPSs of P. penneri 48, 90, 103 and 128 and P. mirabilis O20 (38/57). In further studies an additional O-antiserum, viz., that against P. penneri 103, was employed. The reactivity of the O-antisera with the LPSs of P. penneri 48, 75, 90, 103 and 128 and P. mirabilis O20 is shown in Table 2. In PI and EIA, P. penneri 75 O-antiserum reacted equally strongly with the homologous and P. penneri 128 LPS. A slightly weaker reaction was observed with the other P. penneri LPSs studied and a markedly weaker reaction with the LPS of P. mirabilis O20. Accordingly, the homologous LPS and the LPS of P. penneri 128 were the strongest inhibitors of the reaction in both PI and EIA, whereas the LPS of P. mirabilis O20 showed almost no inhibitory activity. P. penneri 103 O-antiserum exhibited strong reactivity with P. penneri 48, 90 and 103 LPS, and these antigens were the strongest inhibitors, whereas no cross-reaction was observed for the P. mirabilis O20 LPS. Reactivity of O-antisera against P. penneri 75 and 103 with the Proteus lipopolysaccharides LPS and alkali-treated LPS were used as antigen in EIA and PI, respectively. The data for the homologous LPS are shown in bold type. Reactivity of O-antisera against P. penneri 75 and 103 with the Proteus lipopolysaccharides LPS and alkali-treated LPS were used as antigen in EIA and PI, respectively. The data for the homologous LPS are shown in bold type. In Western blot, both P. penneri 75 and 103 O-antisera clearly recognized both high-molecular-mass O-polysaccharide-containing LPS species and low-molecular-mass LPS species restricted to the core-lipid A moiety from all P. penneri strains studied (Fig. 2(a) and (b)). P. penneri 75 O-antiserum (Fig. 2(a)), but not P. penneri 103 O-antiserum (Fig. 2(b)), bound also to high-molecular-mass LPS species from P. mirabilis O20. Western blot of Proteus LPS with O-antisera against P. penneri 75 (a) and P. penneri 103 (b). Absorption of P. penneri 75 O-antiserum with each of P. penneri 75 and 128 LPS completely abolished their reactivity with all tested antigens (Table 3). Absorption with each LPS of P. penneri 48, 90 and 128 removed antibodies recognizing these particular LPSs but left antibodies reacting with P. penneri 75 and 128 LPS as well as with the P. mirabilis O20 LPS. The last antigen affected binding of P. penneri 75 O-antiserum with none of the tested P. penneri LPSs but with the LPS of P. mirabilis O20. The reactivity of P. penneri 103 O-antiserum with all tested antigens in PI was completely abolished after absorption with the LPSs of P. penneri 48, 90 and 103. Absorption with the P. penneri 75 and 128 LPSs removed all antibodies against these LPSs and significantly decreased the reactivity with the LPSs of P. penneri 48, 90 and 103. These data showed close serological relatedness between P. penneri 75 and 128, on the one hand, and P. penneri 48, 90 and 103, on the other hand, as well as the serological homogeneity of strains belonging to each of the two groups. PI of the alkali-treated Proteus LPS with absorbed O-antisera against P. penneri 75 and 103 Sheep red blood cells were used as a control. Pp, Proteus penneri; Pm, Proteus mirabilis. PI of the alkali-treated Proteus LPS with absorbed O-antisera against P. penneri 75 and 103 Sheep red blood cells were used as a control. Pp, Proteus penneri; Pm, Proteus mirabilis. Comparison of the P. penneri O-polysaccharide structures shown in Fig. 3 suggested that the major epitope (epitope a) responsible for the cross-reactivity of P. penneri 75 and 103 O-antisera with the P. penneri LPSs studied is associated with the common main chain. The distinct parts of the O-polysaccharide structures, viz., a lateral glucose residue in P. penneri 75 and 128 (epitope b) and phosphoethanolamine in P. penneri 48, 90 and 103 (epitope c), seem to play a role of the particular, minor epitopes. Evidently, the existence of the major common epitope was the reason for complete or almost complete absorption of both P. penneri 75 and 103 O-antisera by each of the P. penneri LPSs (Table 3). The remaining antibodies in P. penneri 75 O-antiserum after absorption with the LPSs of P. penneri 48, 90 and 103 recognised the minor epitope b on the O-polysaccharides of P. penneri 75 and 128. Vice versa, the remaining antibodies in the cross-absorbed P. penneri 103 O-antiserum bound to the minor epitope c. Therefore, the structural and serological data enabled classification of P. penneri strains 48, 75, 90, 103 and 128 into one Proteus serogroup, which was named as serogroup O73 and which is characterised by the major group epitope 73a. According to the existence of different minor epitopes, the O73 serogroup is sudivided into two subgroups: 73a,73b for P. penneri 75 and 128 and 73a,73c for P. penneri 48, 90 and 103. The cross-reactivity of the P. mirabilis O20 LPS with P. penneri 75 O-antiserum with no reactivity with P. penneri 103 O-antiserum suggested that the shared epitope is associated with the lateral glucose residue (epitope b in the P. penneri 75 O-polysaccharide) since there is no other common structural element in the two O-polysaccharides that could be responsible for the serological relatedness. This work was supported by grants No. 2 PO5A 085 26 of the Sciences Research Committee (KBN, Poland), 02-04–48767 of the Russian Foundation for Basic Research and MK-226.2003.03 of the Russian Federation for support of young scientists.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.