Structure of the O-polysaccharide of<i>Proteus penneri</i>28 and<i>Proteus vulgaris</i>O31 and classification of<i>P. penneri</i>26 and 28 in<i>Proteus</i>serogroup O31

Bacteria of the genus Proteus from the family Enterobacteriaceae are a common cause of urinary tract infections, which can lead to severe complications such as formation of bladder and kidney stones as well as acute or chronic pyelonephritis [1,2]. Recently, it has been suggested that Proteus mirabilis plays an ethiopathogenic role in rheumatoid arthritis [3]. The genus Proteus includes five species: P. vulgaris, P. mirabilis, P. penneri, P. myxofaciens and P. hauseri[4]. Genetic studies showed the existence of four additional genomospecies 3, 4, 5 and 6 [5] (genomospecies 3 was renamed as P. hauseri). Based on the immunospecificity of the lipopolysaccharides (LPS), two species, P. mirabilis and P. vulgaris, have been classified into 60 O-serogroups [6,7], and recently new O-serogroups proposed for P. penneri strains [8–10]. Aiming at the creation of the molecular basis for classification of Proteus strains, structures of the O-polysaccharide chains of the LPS of a number of serologically distinguishable strains of P. vulgaris, P. mirabilis and P. penneri have been elucidated [10–13]. Many from them have been found to contain non-sugar components that are uncommon for O-polysaccharide, such as residues of lactic, pyruvic, hydroxy and amino acids, phosphate groups and phosphate-linked polyalcohols and amino alcohols. Now we report on the structure of a new lactic acid-containing O-polysaccharide, which was isolated from the LPS of P. penneri 28 and P. vulgaris O31 and which is similar to the O-polysaccharide of P. penneri 26 whose structure has been determined earlier [14]. The corresponding LPS were characterised serologically using polyclonal rabbit O-antisera, and it was found reasonable to combine the three strains in Proteus serogroup O31. P. penneri strains 28 (STI 339, isolated from urine of a patient with bacteriuria in Toronto, Canada), 16 (0465–75), 20 (0766–80), 26 (STI 021–2) and 31 (TGH 629) were kindly provided by Prof. D. J. Brenner (Centre for Diseases Control and Prevention, Atlanta, GA, USA). P. penneri 62 was from the collection of the Department of General Microbiology (Institute of Microbiology and Immunology, University of Lodz, Lodz, Poland) and P. vulgaris O31 (PrK 55/57) from the Czech National Collection of Type Cultures (CNCTC, Institute of Epidemiology and Microbiology, Prague, Czech Republic). The bacteria were grown on nutrient broth (Warsaw Laboratory of Sera and Vaccines, Poland) supplemented with 1% glucose. Bacterial cells were separated by centrifugation, washed with distilled water and lyophilised. Crude LPS were isolated in yields 3.8 and 6.7% from dry bacterial cells of P. penneri 28 and P. vulgaris O31, respectively, by extraction with hot aqueous phenol [15] and purified by treatment with aqueous 50% CCl3CO2H at 4°C [16]. Degradation of the LPS was performed with aqueous 1% HOAc at 100°C for 2 h and followed by fractionation by gel-permeation chromatography on Sephadex G-50 (S) (Amersham Biosciences, Sweden) using 0.05 M pyridinium acetate buffer as eluent (10 ml HOAc and 4 ml pyridine in 1 l water); monitoring was performed using a differential refractometer (Knauer, Germany). Polyclonal O-antisera were obtained by immunisation of rabbits with heat-inactivated bacteria of P. penneri 26 and 28 and P. vulgaris O31 according to the published procedure [17]. SDS–PAGE (with use of 9.5% acrylamide), immunoblotting, absorption experiments, enzyme immunosorbent assay (EIA) using LPS and passive immunohemolysis test (PI) using alkali-treated LPS as antigen as well as inhibition experiments were performed as described in detail previously [18]. The polysaccharide was hydrolysed with 2 M CF3CO2H (120°C, 3 h). Amino sugars were identified using a Biotronik LC-2000 amino acid analyser equipped with a column (22×0.4 cm) of Ostion LG AN B cation-exchange resin in 0.2 M sodium citrate buffer pH 3.25 at 65°C. Muramic acid from purchased N-acetylmuramic acid and isomuramic acid from the O-polysaccharide of P. penneri 19 [19] were used as reference compounds; the retention times of muramic acid, isomuramic acid, GlcN, and 2-acetamido-2,6-dideoxyglucose (QuiN) were 21, 29, 59 and 75 min, respectively. The absolute configurations of GlcN and QuiN were determined by GLC of the acetylated (+)-2-octyl glycosides [20,21]. 1H and 13C NMR spectra were recorded on a Bruker DRX-500 spectrometer in D2O at 60°C using internal acetone (δH 2.225, δc 31.45) as reference. Standard Bruker software was used to acquire and process the data. A mixing time of 150 and 300 ms was used in 2D TOCSY and ROESY experiments, respectively. The O-polysaccharide was prepared by mild acid degradation of the LPS of P. penneri 28 followed by gel-permeation chromatography on Sephadex G-50. Analyses using an amino acid analyser after hydrolysis of the polysaccharide revealed GlcN and QuiN in the ratio ∼1:1 as well as another amino component, which has the same retention time as isomuramic acid and different from muramic acid. The identity of the amino sugars was confirmed and their absolute configurations determined by GLC of the acetylated (+)-2-octyl glycosides, which showed that GlcN has the d-configuration and QuiN the l-configuration. The structure of isomuramic acid, including the position of the lactic acid residue (Lac) and the absolute configuration of the sugar residue, were confirmed by NMR spectroscopic studies of the polysaccharide (see below). The 13C NMR spectrum of the polysaccharide contained signals for three anomeric carbons at δ 98.0–98.8, one unsubstituted and one substituted OC H2-C group at δ 61.9 and 66.2, respectively (C6 of GlcN), two C H3-C groups at δ 17.8 (C6 of QuiN) and δ 19.8 (C3 of Lac), three nitrogen-bearing carbons at δ 54.0–54.9 (C2 of GlcN and QuiN), 11 oxygen-bearing carbons in the region δ 69.0–82.4, a COOH group of lactic acid at δ 179.9 and three N-acetyl groups (CH3 at δ 23.3, 23.4, and 23.6; CO at δ 174.5, 174.8, and 175.2). The 1H NMR spectrum of the polysaccharide contained, inter alia, signals for three anomeric protons at δ 4.92–5.07, two CH3-C groups at δ 1.34 and 1.43 (H6 of QuiN and H3 of Lac, both doublets, J 6.0 and 7.8 Hz, respectively) and three N-acetyl groups at δ 2.03–2.16 (all singlets). Therefore, the polysaccharide has a trisaccharide repeating unit containing one residues each of d-GlcNAc, l-QuiNAc (QuiN (N-acetylquinovosamine)) and N-acetylisomuramic acid. The 1H NMR spectrum of the polysaccharide was assigned using a 2D COSY experiment, which showed correlations between all protons within each sugar system, and confirmed by a 2D TOCSY experiment (Table 1). The spin system of QuiNAc was distinguished from those of GlcNAc (designated as GlcNAcI and GlcNAcII) by a H5/H6 correlation at δ 4.26/1.34 in the COSY spectrum. An H2/H3 cross-peak at δ 4.29/1.43 was observed for the lactic acid residue in both 2D NMR spectra. With the 1H NMR spectrum assigned, the 13Ñ NMR spectrum of the polysaccharide was assigned using a 1H,13Ñ HMQC experiment (Table 1). H and 13C NMR data of the O-polysaccharide of P. penneri 28 (δ, p.p.m.) Additional chemical shifts for NAc are δH 2.03, 2.08 and 2.16; δC 23.3, 23.4, 23.6 (all Me), 174.5, 174.8 and 175.1 (all CO). S-Lac: (S)-1-carboxyethyl [a residue of (S)-lactic acid]. The appearance of the signals for the anomeric protons as broadened singlets indicated small J1,2 coupling constants and thus the α-configuration of all three sugar residues. In accordance with this conclusion, a 2D ROESY experiment showed an H1/H2 correlation for each sugar residue typical of 1,2-cis orientation of these protons but no correlation between H1 and H3,H5, which would be observed for β-linked monosaccharides. The HMBC spectrum showed a correlation between Lac H2 and GlcNAcII C3 at δ 4.29/82.4, thus demonstrating the attachment of the lactic acid residue at position 3 of GlcNAcII. Accordingly, the signal for C3 of GlcNAcII is shifted downfield to δ 82.4 due to alkylation. Low-field displacements of the signals for C3 of QuiNAc and GlcNAcI and C6 of GlcNAcII to δ 77.5, 76.9 and 66.2, respectively, i.e. by 4–6 p.p.m. as compared with their positions in the spectra of the corresponding non-substituted monosaccharides [22,23], revealed the glycosylation pattern of the monosaccharide residues and showed that the polysaccharide is linear. The ROESY experiment showed cross-peaks between GlcNAcII H1 and QuiNAc H3 at δ 5.07/3.81 and between QuiNAc H1 and GlcNAcI H3 at δ 5.06/3.93. Each of the signals for GlcNAcI H1 at δ 4.92 and Lac H2 at δ 4.29 gave a cross-peak with a signal at δ 3.66, which was interpreted as GlcNAcI H1/GlcNAcII H6a and Lac H2/GlcNAcII H3 correlations. These data were in agreement with the positions of substitution of the monosaccharides and defined the monosaccharide sequence in the repeating unit. A relatively large negative effect (−2.2 p.p.m.) on C4 of l-QuiNAc caused by its glycosylation by α-GlcNAcII indicated different absolute configurations of the linked sugar residues, i.e. the d-configuration of GlcNAcII (compare the corresponding glycosylation effects of −2.0 and +0.7 p.p.m. in α1→3-linked disaccharides of 2-acetamido-2-deoxy-l- and d-galacturonic acid with d-QuiNAc, respectively [23]). This finding confirmed finally 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-d-glucose (N-acetylisomuramic acid). On the basis of these data, it was concluded that the O-polysaccharide of P. penneri 28 has the structure shown in Fig. 1. The 13C NMR spectrum of the polysaccharide of P. vulgaris O31 was found to be identical to that of P. penneri 28. Particularly, it showed signals for three anomeric carbons, three nitrogen-bearing carbons, two C H3-C group, one unsubstituted and one substituted OC H2-C group, 11 other oxygen-bearing carbons, one COOH group and three N-acetyl groups, all signals having the same chemical shifts as the corresponding signals in the spectrum of the polysaccharide of P. penneri 28. Hence, both polysaccharides have the same structure of the repeating unit. The structure of the O-polysaccharide of P. penneri 28 resembles also that of P. penneri 26 studied earlier, which has the same carbohydrate backbone and differs only in the absence of the lactic acid residue (Fig. 1) [14]. Structures of the O-polysaccharides of P. penneri 28 and P. vulgaris O31 and related O-polysaccharides [24,25]. Various Proteus LPS were tested in PI, EIA and immunoblotting with polyclonal rabbit O-antisera against strains that have the identical (P. penneri 28 and P. vulgaris PrK 55/57 from serogroup O31) or structurally similar (P. penneri 26) O-polysaccharides (Fig. 1). A cross-reactivity was observed for the LPS of P. penneri 16, 20, 26, 28, 31 and 62 and P. vulgaris O31 (Table 2). Reactivity of O-antisera against P. penneri 26 and 28 and P. vulgaris O31 with the Proteus LPS. LPS and alkali-treated LPS were used as antigen in EIA and PI, respectively The data of the homologous LPS are shown in bold type. In PI and EIA, P. penneri 28 O-antiserum reacted strongly with the homologous LPS and weaker with all heterologous LPS studied (Table 2). As expected, P. vulgaris O31 O-antiserum showed a strong reactivity with the LPS of P. vulgaris O31and P. penneri 28 as well as with the P. penneri 62 LPS, the cross-reactivity being markedly stronger than that of P. penneri 28 O-antiserum. No reaction was observed between P. vulgaris O31 O-antiserum and the LPS from P. penneri 16, 20 and 31, and both O-antisera showed only a weak cross-reactivity with the P. penneri 26 LPS. P. penneri 26 O-antiserum bound strongly to the homologous LPS and the LPS from P. penneri 28 and P. vulgaris O31, much weaker to the P. penneri 62 LPS and did not bind to the LPS of P. penneri 16, 20 and 31. Results of inhibition experiments with the corresponding LPS in both assays (Table 2) were in agreement with these data. In Western blot after SDS–PAGE (Fig. 2A), P. penneri 28 O-antiserum recognised high-molecular-mass species corresponding to the O-polysaccharide-containing LPS molecules from all strains studied, binding to the P. penneri 26 LPS being weaker than to the other LPS. It reacted also with low-molecular-mass species of the core-lipid A moiety of the LPS from all investigated strains except for P. penneri 26 and 62. P. vulgaris O31 O-antiserum (Fig. 2B) strongly bound to high-molecular-mass species of the LPS from P. vulgaris O31, P. penneri 28 and 62 LPS and much weaker to those from P. penneri 26. No cross-reactivity was observed between P. vulgaris O31 O-antiserum and the core-lipid A moiety of any LPS studied. P. penneri 26 O-antiserum (Fig. 2C) strongly reacted with high- and low-molecular-mass species of the LPS from P. penneri 26, P. penneri 28 and P. vulgaris O31 and weaker with high-molecular-mass species of the P. penneri 62 LPS. As opposite to P. penneri 28 O-antiserum, P. vulgaris O31 and P. penneri 26 O-antisera showed no cross-reactivity with the LPS from P. penneri 16, 20 and 31. Western blot of Proteus LPS with O-antisera against P. penneri 28 (A), P. vulgaris O31 (B) and P. penneri 26 (C). Pp 16, 20, 26, 28, 31 and 62 refer to P. penneri LPS and Pv O31 refers to P. vulgaris LPS. The epitope specificity of the cross-reactive LPS was studied by absorption of the three O-antisera tested with the respective LPS and repeated examination in PI (Table 3). Absorption of P. penneri 28 O-antiserum with the P. vulgaris O31 LPS decreased the reactivity with the homologous LPS, abolished the reactivity with the LPS of P. penneri 26 and 62 and had no influence on the binding to the other cross-reactive LPS. Western blot with absorbed sera showed that the P. penneri 31 LPS removed from P. penneri 28 O-antiserum cross-reactive antibodies against the core-lipid A region and did not affect antibodies that recognise an epitope on the O-polysaccharides of P. penneri 28, 62 and P. vulgaris O31 (data not shown). These data together suggest that (i) P. penneri 28, 62 and P. vulgaris O31 share an epitope on the O-polysaccharides, (ii) core-specific antibodies are abundant in P. penneri 28 O-antiserum and (iii) the LPS core region in P. penneri 28 is similar to that in P. penneri 16, 20 and 31 and different from the core region in P. vulgaris O31 and P. penneri 62 LPS. These conclusions were confirmed by complete removal of antibodies from P. penneri 28 O-antiserum by absorption with both P. penneri 31 and P. vulgaris O31 LPS, which eliminated antibodies specific to the core and to the O-polysaccharide, respectively. PI of the alkali-trated Proteus LPS with absorbed O-antisera against P. penneri 28 and P. vulgaris O31 Sheep red blood cells were used as control. Similar results were obtained with P. vulgaris O31 O-antiserum absorbed with the LPS of P. vulgaris O31 and P. penneri 28 (Table 3). Absorption with the P. penneri 62 LPS significantly decreased the reactivity with both P. vulgaris O31 and P. penneri 28 LPS, whereas absorption with the P. penneri 26 LPS did not influence the cross-reactivity of P. vulgaris O31 O-antiserum. Absorption of P. penneri 26 O-antiserum with the LPS of P. penneri 28 and P. vulgaris O31 completely removed antibodies that cross-react with these LPS and with the LPS of P. penneri 62 and significantly decreased the reactivity with the homologous antigen. From the structures of the O-polysaccharides shown in Fig. 1, it can be inferred that the epitope that is responsible for the cross-reactivity of P. penneri 28 and P. vulgaris O31 O-antisera with the LPS of P. penneri 62 is associated with N-acetylisomuramic acid, which is the only sugar that is shared by the O-polysaccharides of the three strains. The cross-reactivity of P. penneri 28 and P. vulgaris O31 O-antisera with the P. penneri 26 LPS is evidently due to a common oligosaccharide fragment (most likely, a α-l-Quip NAc-(1→3)-α-d-Glcp NAc disaccharide fragment) within the O-polysaccharide backbone, which is identical in the LPS of the three strains. That the cross-reactive epitopes in P. penneri 26 and 62 are different is confirmed by the finding that the P. penneri 62 LPS had no influence on the reaction of the P. penneri 26 LPS and, vice versa, the P. penneri 26 LPS did not effect the reaction of the P. penneri 62 LPS with P. penneri 28 and P. vulgaris O31 O-antisera. The nature of a minor common epitope on the O-polysaccharides that is responsible for the reaction of P. penneri 26 O-antiserum with the P. penneri 62 LPS remains unknown. The cross-reactivity of P. penneri 28 O-antiserum with the LPS of P. penneri 16, 20 and 31 is evidently accounted for by sharing an epitope on the LPS core, whose structure in these strains remains to be determined. On the basis of the data obtained, we suggest to classify three of the strains studied, P. vulgaris O31, P. penneri 28 and P. penneri 26, in two subgroups within one Proteus serogroup, O31: subgroup O(31a), 31b for P. vulgaris PrK 55/57 (former P. vulgaris O31 [6]) and P. penneri 28 and subgroup O31a for P. penneri 26 (formerly classified to a separate Proteus serogroup, O70 [8]). In these antigenic formulae, factor O31a is associated with the O-polysaccharide backbone common for all three strains and factor O31b with N-acetylisomuramic acid present in the O-polysaccharide of strains from subgroup O(31a), 31b only. Designation O(31a) indicates that the epitope is present only passively (a silent epitope), i.e. it is well recognised by O31a-specific antibodies against P. penneri 26 from subgroup O31a, but strains from subgroup O(31a), 31b that carry this epitope do not induce O31a-specific antibodies themselves. The LPS of P. penneri 62 also possess epitope O31b that is linked to N-acetylisomuramic acid but it would not be reasonable classifying it to Proteus serogroup O31 since (i) it has been soundly classified in Proteus serogroup O64 together with several other serologically related strains, which have the O-antigens that are structurally more close to that of P. penneri 62 than strains of Proteus serogroup O31, and (ii) the LPS of P. penneri 62 showed a lower level of cross-reactivity with P. penneri 26 O-antiserum than the LPS of P. penneri 28 and P. vulgaris O31, as it is most clearly seen from the inhibition data (Table 2, columns 4 and 5). enzyme immunosorbent assay lipopolysaccharide passive immunohemolysis 2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine) This work was supported by grants 02-04-48767 from the Russian Foundation for Basic Research, YS 2001–2/1 from INTAS, 6 P 04A 074 20 and 3 P 05A 073 22 from the Sciences Research Committee (KBN, Poland).

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