These rickettsiae were purified by Ficoll density gradient

Transcription

1 INFECrION AND IMMUNrrY, Oct. 1993, p /93/ $02.00/0 Copyright 1993, American Society for Microbiology Vol. 61, No. 10 Chemical Properties of Lipopolysaccharides from Spotted Fever Group Rickettsiae and Their Common Antigenicity with Lipopolysaccharides from Proteus Species KEN-ICHI AMANO,1* MIKI FUJITA,1 AND TSUNEHISA SUTO2 Central Research Laboratory, Akita University School of Medicine, Hondo, 1 and Akita Prefectural Institute ofpublic Health, 2 Akita 010, Japan Received 26 February 1993/Returned for modification 13 April 1993/Accepted 16 July 1993 The lipopolysaccharides (LPS) isolated from spotted fever group (SFG) rickettsia strains Thai tick typhus TT-118 and Katayama were characterized by chemical analyses, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. These LPS did not contain heptose, but they contained 3-deoxy-D-manno-octulosonic acid (KDO), glucosamine, quinovosamine, phosphate, ribose, an unknown neutral sugar, and palmitic acid. Resolution of the apparent molecular masses of these LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with silver showed ladder-like bands. In an ELISA, convalescent-phase sera from 10 patients with Japanese spotted fever reacted with LPS from the Katayama strain, and 90%o (9 of 10) of these sera also reacted with LPS isolated from Proteus vulgaris OX2. Immunoblotting revealed that the sera reacted with the high-molecular-mass bands of LPS from SFG rickettsiae, in addition to those of OX2 LPS. In an ELISA, immunoglobulin M antibodies from these sera reacted with the O-polysaccharide and lipid A portions of LPS from P. vulgaris OX2. The epitopes common to LPS of SFG rickettsiae and P. vulgaris OX2 may be in the O-polysaccharide and lipid A portions. Spotted fever group (SFG) rickettsiosis in Japan was first observed in 1984 by Mahara et al. (19). Uchida et al. (25) isolated the rickettsia causing the disease that Mahara (18) had named Japanese spotted fever. The Weil-Felix test is used as a presumptive-diagnosis test for SFG rickettsiosis. This test employs Proteus vulgaris OX2 antigen, which gives characteristic agglutination patterns with the sera of persons infected with SFG rickettsiae. The common antigens between SFG rickettsiae and P. vulgaris OX2 were recently suggested to be the lipopolysaccharides (LPS) of SFG rickettsiae and P. vulgaris OX2 which act as the common antigens (1). In this study, we isolated LPS from SFG rickettsia strains Katayama and TT-118 and compared them by chemical and immunochemical methods with LPS obtained from P. vulgaris OX2. The reactivities of paired sera from persons with Japanese spotted fever with LPS from SFG rickettsiae and P. vulgaris OX2 were compared. Fragments of the LPS from strain OX2 were used to define the fragments that carry antigenicities common to LPS from rickettsiae and P. vulgaris OX2. MATERIALS AND METHODS Microorganisms and extraction of LPS. SFG rickettsia strains Thai tick typhus TT-118 and Rickettsia sibirca 246 were obtained from D. J. Kelly of the U.S. Army Medical Research Unit, Kuala Lumpor, Malaysia. Japanese spotted fever rickettsia strains Katayama and Abe were isolated from the blood of patients with Japanese spotted fever as described previously (2, 4, 12). Propagation and purification of the rickettsiae were also described previously (2, 12). Briefly, the rickettsiae were propagated in green monkey kidney cells which were established and passaged several times at the National Institute of Health, Tokyo, Japan. * Corresponding author These rickettsiae were purified by Ficoll density gradient centrifugation. The growth conditions used for Proteus strains were reported by Mizushiri et al. (20). LPS were extracted from whole cells (WC) of SFG rickettsiae and Proteus strains by the hot phenol-water method described by Mizushiri et al. (20). The phenol-water extracts were treated with DNase I (Sigma Chemical Co., St. Louis, Mo.) and RNase A (Sigma Chemical Co.) at 37 C overnight and then treated with proteinase K (Sigma Chemical Co.) at 37 C for 1 day as described by Amano and Williams (6). After dialysis for 4 days at room temperature against three changes of distilled water, the samples were centrifuged at 100,000 x g for 2 h (Proteus strains) or 15 h (SFG rickettsia strains). The precipitates (designated LPS) were washed twice and lyophilized. Preparation of fragments from P. vulgaris OX2 LPS. The LPS fragments were isolated from P. vulgaris OX2 by hydrolysis with acetic acid, followed by ultracentrifugation and Sephadex G-50 column chromatography as described by Mizushiri et al. (20). Analytical methods. Neutral sugar was determined by the phenol-sulfuric acid method (11) with glucose as the reference standard. Heptose was assayed by the cystein-sulfuric acid method (21) with D-glucoheptose (Sigma Chemical Co.) as the reference standard. KDO was assayed by the thiobarbituric acid method (14). Total phosphorus was assayed by the method of Lowry et al. (16). Uronic acid was assayed by the method of Blumenkrantz and Asboe-Hansen (10) with glucuronic acid as the reference standard. Protein was determined by the method of Lowry et al. (17). Amino acids and amino compounds were determined with an amino acid analyzer after hydrolysis in 4 N HCl at 100 C for 8 h as described previously (5). The composition of neutral sugar was analyzed as described previously (20). Briefly, samples (0.2 mg) were hydrolyzed in 4 N HCl at 100 C for 2 h. After reduction with NaBH4, samples were applied to a column of Dowex 50 and

2 VOL. 61, 1993 COMMON ANTIGENICITY BETWEEN SFGR AND PROTEUS LPS 4351 TABLE 1. Chemical compositionsa of LPS from P. vulgaris and spotted fever group rickettsiae Strain Heptose Neutral KDO Uronic Phosphate G pcn GacN- GalN QuiN Ethanolamine Protein sugar acid phosphate (%) Katayama 5 (0.0) 1,034 (4.2) 78 (0.3) 0 (0) 2,003 (8.1) 246 (1) 34 (0.1) 0 (0) 108 (0.4) 21 (0.1) 15.8 TT (0) 1,046 (6.8) 69 (0.5) 0 (0) 2,011 (13) 153 (1) 26 (0.2) 0 (0) 71 (0.5) 21 (0.1) 7.5 OX2 255 (0.4) 1,339 (2.2) 121 (0.2) 390 (0.6) 454 (0.8) 603 (1) 56 (0.1) 21 (0.0) 195 (0.3) 46 (0.1) 1.8 OX (0.6) 1,267 (2.8) 140 (0.3) 379 (0.8) 571 (1.2) 459 (1) 86 (0.2) 47 (0.1) 61 (0.1) 56 (0.1) 0.4 a Expressed as nanomoles per milligram, unless otherwise indicated. The values in parentheses are molar ratios relative to GlcN. treated with acetic anhydride and pyridine at 100 C for 1 h. After drying, acetate derivatives were analyzed in a Hitachi G-300 gas chromatograph on an OV-1701 bonded capillary glass column (0.25 mm by 25 m; GL Science Inc., Tokyo, Japan) with a temperature increase of 1 C/min from 240 to 270 C. Fatty acids were analyzed as methyl esters in the gas chromatograph on a PEG-20M bonded capillary glass column (0.25 mm by 25 m; GL Science Inc.) at 220 C. Sera from patients with Japanese spotted fever or scmb typhus. Paired sera from 10 patients with Japanese spotted fever were obtained between 1984 and 1991 on the island of Shikoku, Japan, as described previously (1, 4). These patients were diagnosed clinically and immunologically by F. Mahara, Tokushima, Japan (19). Sera from patients with scrub typhus were obtained from Akita prefecture, Japan. These sera were stocked in a -40 C deep freezer before use in this study. Weil-Felix test. The Weil-Felix test was performed as described previously (3). A suspension of formalin-killed WC of P. vulgaris (0.8 mg/ml of physiological saline) was used as the antigen in this test. ELISA. Enzyme-linked immunosorbent assay (ELISA) was performed as described previously (3). MicroELISA plates (type H; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) were coated with 50,ul of antigens per well (10,ug/ml of 0.1 M carbonate buffer, ph 9.6). Antibodies were separately assayed with horseradish peroxidase-conjugated goat antihuman immunoglobulin G (IgG) and anti-human IgM antibodies (DAKO Immunoglobulins, a/s, Copenhagen, Denmark). Adsorption of sera with LPS. Adsorption studies were performed with either WC or LPS fractions of SFG rickettsiae and P. vulgaris OX2 as described previously (3). Sera of patients infected with Japanese spotted fever rickettsiae were diluted 50-fold in dilution buffer and mixed with an equal volume of the inhibiting materials. The mixture was incubated for 90 min at 37 C, and then the remaining antibody titers were determined by ELISA as described above. SDS-PAGE and immunoblotting. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (15), with a 12.5% polyacrylamide gel concentration. Immunoblotting was accomplished by a modification (3) of the techniques of Towbin et al. (23). The electrophoretically separated components were stained by the silver staining method described by Hitchcock and Brown (13) or were electrophoretically transferred from the gel onto nitrocellulose sheets (Bio-Rad Laboratories, Richmond, Calif.). The secondary antibodies used for immunoblotting were the same kinds of antibodies as those used for the ELISA. RESULTS Isolation and chemical compositions of LPS fractions from SFG rickettsiae and P. vulgaris OX2. Ultracentrifugation of the hot phenol-water extracts of strain Katayama and TT- 118 WC yielded 1.3 and 0.9% LPS, respectively. The yields of LPS from strains OX2 and OX19 were 6.1 and 2.7%, respectively. The chemical compositions of the LPS of the microorganisms are given in Table 1. The LPS of strains Katayama and TT-118 contained a neutral sugar, phosphate, glucosamine (GlcN), and quinovosamine (QuiN) as major components, and heptose was rarely found. GlcN-phosphate and ethanolamine were also contained at low concentrations in these two LPS. Strain OX2 LPS contained heptose, a neutral sugar, KDO, uronic acid, phosphate, GlcN, and QuiN as major components. Strain OX19 LPS contained heptose, a neutral sugar, KDO, uronic acid, phosphate, and GlcN as major components. Although the neutral sugars of the LPS from strains Katayama and TT-118 contained ribose and unknown sugar as major neutral sugars and rhamnose, galactose (Gal), and glucose (Glc) as minor components, heptose was not detected (Table 2). Glc, D-glycero-D-mannoheptose, D-glycero-L-mannoheptose, and unknown sugar 1 were the major neutral sugars in LPS from strains OX2 and OX19. On the basis of the chemical analyses described above and the previous report (20), we speculate that the O-polysaccharide portion of strain OX2 LPS was composed of Glc, GlcN, and QuiN and that of strain OX19 LPS was composed of Glc, GlcN, GalN, and QuiN. However, we cannot speculate on the composition of the O-polysaccharide portions of two rickettsial LPS because of the small yields of these LPS. TABLE 2. Analysis of alditol acetate derivatives from LPS of P. vulgaris and spotted fever group rickettsiae Relative Alditol acetate (%) of LPS from retention Carbohydrate strain: timea Katayama 1T-118 OX2 OX Unknown sugar Rhamnose b Ribose Unknown sugar Galactose Glucose DD-heptosec LD-heptosec a The retention time of hexaacetylglucitol (relative retention time = 1) was 10.9 min under the conditions described in Materials and Methods. b _, Undetectable. C DD-heptose and LD-heptose are D-glycero-D-mannoheptose and L-glyCero-D-mannoheptose, respectively (20).

3 4352 AMANO ET AL. TABLE 3. Titersa of antibodies to LPS from Japanese spotted fever rickettsiae and P. vulgaris strains in sera from patients with Japanese spotted fever or scrub typhus Weil-Felix Antibody titer Days after test against ELISA against LPS of from strain: no. of strain: Patieb onset antigen fever Katayama OX2 OX19 OX2 OX19 IgG IgM IgG IgM IgG IgM < ,600 6,400 3,200 3, < ,400 6,400 1,600 6,400 < < < <100 < ,600 12,800 3,200 6, <20 < < ,600 3,200 1,600 1,600 < < ,400 1,600 3,200 3, <20 <20 < ,600 <100 <100 < ,200 1,600 6, < , < , ,400 25,600 3,200 3,200 < <20 < < < , , < , ,200 3,200 6,400 6,400 < <20 <20 1, , , , < < < , < <20 <20 6,400 <100 3,200 < <20 <20 6, ,200 < <20 < < <20 < <20 < < <20 < a Measured by the Weil-Felix test and ELISA b Patients 1 to 10 were infected with Japanese spotted fever; patients 11 to 14 had scrub typhus. The LPS from strains Katayama and TT-118 contained mainly palmitic acid, but 3-hydroxy fatty acids, which are characteristic components of enterobacterial LPS, were not detected (data not shown). Strain OX2 and OX19 LPS contained myristic acid, palmitic acid, and j3-hydroxymyristic acid as major fatty acids (20). On the basis of the data given above, the two LPS from the SFG rickettsiae are composed of GlcN, QuiN, KDO, Glc, ribose, some unknown sugar, phosphate, and palmitic acid. These LPS did not contain heptose or,3-hydroxy fatty acid. Immunoreactivity of LPS from SFG rickettsiae and P. vulgaris OX2. Paired sera from 10 human patients with A i I &L., --j W:4, i, Silver stain B..s!.F lre.;, r.a;. FT.l t:'fe!%ft. X FtWFT r INFECT. IMMUN ;e....,,.jx's;' - - S,. :e 1 Lt) 9>@ sz r u Z:ga' 4s 1t}E 1. S. iliti s... J s _L '.s: B.v_ t +1-,... Immunoblotting FIG. 1. (A) Silver-stained SDS-PAGE gel of LPS from P. vulgaris OX2 and SFG rickettsiae. Ten micrograms of strain OX2 LPS and 20 p,g of SFG rickettsial LPS were loaded per lane. (B) Immunoblot of LPS from P. vulgaris OX2 and SFG rickettsiae with the IgM antibody of convalescent-phase sera of patient 3. Lanes: 1, strain OX2 LPS; 2, strain Katayama LPS; 3, strain TT-118 LPS. Japanese spotted fever were analyzed by the Weil-Felix test and ELISA (Table 3). With the exception of the sera from patients 6 and 10, the Weil-Felix test gave higher titers to the OX2 antigen than to the OX19 antigen. By ELISA, all of the convalescent-phase sera taken 11 or more days after onset of fever reacted with the strain Katayama LPS. A fourfold or greater increase in IgM antibody titers was seen in convalescent-phase sera compared with those found in acutephase sera of nine patients, except patient 10, against strain OX2 LPS. This increase was associated with the increased titers obtained by the Weil-Felix test against OX2 antigen. In the acute-phase sera, IgG antibodies of patients 3, 4, 5, and 10 already reacted weakly with LPS of both strains Katayama and OX2, and those of patients 6 and 7 reacted with strain OX2 LPS. It is possible that these patients had been infected with spotted fever rickettsiae and/or a Proteus strain previously. The titers found in these sera against the OX19 antigen by the Weil-Felix test and against strain OX19 LPS by ELISA were lower than those against the strain OX2 antigen and strain OX2 LPS, respectively. We also analyzed sera from patients with scrub typhus by the Weil-Felix test and ELISA (Table 3). The Weil-Felix test gave negative titers to strain OX2 and OX19 antigens. In ELISA of the convalescent-phase sera of patients with scrub typhus (no. 11 to 14), a fourfold or greater increase in IgG or IgM antibody titers was not seen, in contrast to those found in acute-phase sera against any LPS fractions. SDS-PAGE and immunoblotting of LPS from SFG rickettsiae and P. vulgaris OX2. When the polyacrylamide gel was stained with silver (Fig. 1A), strain OX2 LPS showed closely spaced ladder-structural bands in the gel lane. The LPS from strains Katayama and TT-118 showed several weakly stained bands in the middle of the gel lane. The LPS from strain Katayama gave two strong bands at the bottom of the gel, and that of strain TT-118 gave only one. Both LPS also showed a diffused smear throughout the gel lanes. By

4 VOL. 61, 1993 COMMON ANTIGENICITY BETWEEN SFGR AND PROTEUS LPS 4353 A *#*~~~~WA i4 Silver stain B Immunoblotting FIG. 2. (A) Silver-stained SDS-PAGE gel of PK-WC of P. vulgaris 0X2 and SFG rickettsiae. (B) Immunoblot of PK-WC of P. vulgaris 0X2 and SFG rickettsiae with the IgM antibody of convalescent-phase sera of patient 3. Lanes: 1, strain 0X2 WC; 2, strain T WC; 3, R. sibirica 246 WC; 4, strain Katayama WC; 5, strain Abe WC. immunoblotting (Fig. ib), the IgM antibody in the convalescent-phase of sera patient 3 reacted with the ladder-like bands of strain 0X2 LPS and with the broad bands of strain Katayama and TT-118 LPS in the gel lanes. In contrast, the bands which were stained strongly by silver among the lowest-molecular-mass bands were not immunoreactive. The IgG antibody in the same sera also reacted with the same bands from the three LPS that reacted with the igm antibody (data not shown). Immunoblotting with the sera of the other Japanese spotted fever patients gave results comparable to those obtained by ELISA (Table 3). The results described above suggested strongly that the sera from the patients with Japanese spotted fever reacted with highermolecular-mass bands of LPS but not with the lowermolecular-mass bands of strain 0X2 LPS. The proteinase K-treated WC (PK-WC) from SFG rickettsia strains TTh-118, sibiica 246, Katayama, and Abe and P. vulgaris 0X2 were run in SDS-PAGE and stained with silver (Fig. 2A) and also submitted to immunoblotting (Fig. 2B). By silver staining, PK-WC from strain 0X2 showed the same patten as strain 0X2 LPS (Fig. 1A). PK-WC from four strains of SFG rickettsiae showed the same ladder-like banding patters. By immunoblotting, the patters of PK-WC of strain OX2 and SFG rickettsiae were similar to the patterns obtained by silver staining. These results indicated the similarities among the LPS of SFG rickettsiae. However, because the patterns of SFG rickettsial LPS (Fig. 1) were different from those of PK-WC (Fig. 2), it is suggested that these purified LPS were degraded in the purification process or aggregated by themselves after purification. Antigenic portion of strain OX2 LPS cross-reacted with sera of patients with Japanese spotted fever. Table 4 shows the reactivity of the convalescent-phase sera of patients with Japanese spotted fever against LPS from strain OX2 and the fractions eluted from a Sephadex G-50 column of the acetic acid-treated LPS from strain OX2 (20). The IgG antibodies of patients 5 and 6 and the IgM antibodies of patients 2, 3, 5, 7, and 9 reacted with strain OX2 fragment 1 corresponded to the polysaccharide portion of strain OX2 LPS, while the IgG antibody of patient 6 reacted only with strain OX2 fragment TABLE 4. Antibody titers of sera of patients with Japanese spotted fever to LPS fractions of Proteus strain OX2 measured by ELISA Antibody titer against: Days Patient after OX2 LPS fae l g 2 OX2 lipid A no.onset of ia fragment r X fever IgG IgM IgG IgM IgG IgM IgG IgM ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~fragment ,200 3, < <100 1, ,600 6, ,200 < < ,200 6, , , ,600 1,600 < <100 <100 < ,200 3,200 3, ,600 1, , , ,600 < ,200 3, < < , < <100 <100 <100 < ,400 6, ,600 < <100 1, , <100 < a Strain OX2 fragments 1 and 2 were obtained by 2% acetic acid hydrolysis (100 C, 90 min) of strain OX2 LPS, centrifugation, and gel filtration of the supernatant with Sephadex G-50 as described previously (20). Strain OX2 lipid A was obtained by 0.1 N HCl hydrolysis (100'C, 90 min) of strain OX2 LPS and precipitation with a centrifuge. 2 corresponded to the oligosaccharide portion. The IgG antibodies of two patients (no. 3 and 5) and the IgM antibodies of six patients (no. 1, 2, 4, 5, 8, and 9) reacted with the lipid A of strain OX2 LPS. We immunoblotted the fractions of strain OX2 LPS with the convalescent-phase sera of these patients (data not shown). The three fractions were scarcely stained with silver in the gels after SDS-PAGE. Both IgG and IgM antibodies of all of the patients did not react with three fractions (fractions 1 and 2 and lipid A), but strain OX2 LPS did react with these sera. TABLE 5. Inhibition of ELISA-reactive antibody to LPS from Japanese spotted fever rickettsiae and P. vulgaris OX2 by LPS fractions Antibody titer against: Mixture of antisera + Amt Strain Katayama Strain OX2 LPS or LPS fraction' (mg/ml) LPS LPS IgG IgM IgG IgM Patient serab alone 25,600 12,800 3,200 3,200 Patient sera + strain , ,600 <100 Katayama LPS Patient sera + strain , , Tr-118 LPS Patient sera + strain ,600 1,600 1, OX2 LPS Patient sera + strain ,600 6,400 3,200 3,200 OX2 fragment , , Patient sera + strain ,600 12,800 3,200 3,200 OX2 fragment ,600 3,200 1,600 1,600 Patient sera + strain ,600 12,800 3,200 3,200 OX2 lipid A ,800 3,200 1,600 1,600 a Antisera were incubated with the LPS fractions described in Table 4 at 37 C for 90 min before ELISA. b Patient 3 (19 days after onset of fever) sera were used.

5 4354 AMANO ET AL. We performed inhibition tests by ELISA (Table 5). Absorption of the sera of patient 3 (19 days after onset of fever) with LPS from strains Katayama and 1T-118 decreased the titers of IgM antibody against strain Katayama LPS or strain OX2 LPS 32-and 16-fold, respectively. Absorption with OX2 LPS decreased the IgM antibody titers against strain Katayama and OX2 LPS eight- and fourfold, respectively. The IgM titers of the same sera against strain Katayama and OX2 LPS, absorbed with 0.5 mg of strain OX2 fragment 1 per ml, decreased 16- and 8-fold, respectively. Absorption with only 0.15 mg of the same fragment per ml did not change the IgM antibody titers. Similarly, the decreases in the IgM antibody titers in the sera against strain Katayama and OX2 LPS, absorbed with 0.5 mg of OX2 fragment 2 or OX2 lipid A per ml, were only four- and twofold, respectively. IgG antibody titers were not decreased by absorption with any LPS fractions or the complete LPS. The results of the inhibition test indicated that the IgM antibodies in the convalescent-phase sera of patient 3 that reacted with strain OX2 LPS, especially with the polysaccharide portion, are the same antibodies that reacted with SFG rickettsial LPS. DISCUSSION The presence of LPS in SFG rickettsial SDS-PAGE patterns has been reported by Anacker et al. (9) for R. rickettsii and by Teysseire and Raoult (22) for R. conorii. These reports state that LPS from SFG rickettsiae showed a ladder-like banding pattern similar to those observed with enterobacterial LPS (13, 24). However, purification and characterization of SFG rickettsial LPS has not been reported. This is possibility due to the difficulty of growing SFG rickettsiae on a large scale. In a previous report (1), we showed that the reactivity of sera from patients with Japanese spotted fever against the strain OX2 antigen in the Weil-Felix test was probably due to IgM antibodies against LPS from strain OX2. In the present study, we harvested and purified SFG rickettsia strains 1T-118 and Katayama and prepared small amounts of highly purified LPS. Strain 1T-118 and Katayama LPS were chemically similar, containing KDO, GlcN, QuiN, phosphate, ribose, an unknown neutral sugar, and palmitic acid as major components. No heptose or,-hydroxy fatty acids were detected. This chemical composition is somewhat different from that of enterobacterial LPS. The structural difference is mainly in the core oligosaccharide and lipid A. Similarly, Amano et al. (7, 8) reported that the structure of Coxiella bumetii LPS differed from that of enterobacterial LPS in its core oligosaccharide and lipid A. C. burnetii LPS is deficient in KDO and,-hydroxy fatty acid. Evaluation of the structural and antigenic microheterogeneity of LPS by SDS-PAGE and silver staining and immunoblotting with the sera of patients with Japanese spotted fever showed that the fastest-migrating band of these LPS did not react with the sera but stained strongly with silver. We speculate that these fast-migrating LPS bands are the molecules containing the core oligosaccharide and lipid A portions without the O-polysaccharide and that the sera react only with the O-polysaccharide-containing LPS. The results of ELISA with the fragments from strain OX2 indicated that the epitopes of strain OX2 LPS that reacted with these sera are in both the O-polysaccharide and lipid A. On the basis of the results of immunological studies, the reactivity of these sera with strain OX2 LPS may need all portions of the LPS, including the O-polysaccharide, core oligosaccharide, and lipid A. The O-polysaccharide of the rickettsial LPS could include structures antigenically similar to the O-polysaccharide portion of strain OX2 LPS, which contains Glc, GlcN, and QuiN (20). Recently, Vinogradov et al. (26) reported that the structure of the O-polysaccharide of strain OX19 LPS consists of Glc, GlcNAc, and QuiNAc. Because this sugar composition was similar to that of the O-polysaccharide of strain OX2 LPS (20), we suggest that the strain OX19 LPS isolated by Vinogradov et al. (26) may react with the sera of persons infected with Japanese spotted fever rickettsiae. In the preceding study (1), we reported that the sera of patients who had IgG antibodies against LPS from a P. vulgaris OX2-like strain reacted a little with LPS from SFG rickettsiae and their IgM antibodies did not react with strain OX2 LPS by ELISA and immunoblotting when the patients were infected with Japanese spotted fever rickettsiae. Furthermore, on the basis of the preceding report (1) and this study, we conclude that the LPS from strain OX2 is responsible for the Weil-Felix test cross-reaction produced by IgM antibodies in the sera of patients with Japanese spotted fever, regardless of the IgG antibodies titers, and that the reaction is due to common antigenic epitopes between O-polysaccharides of the LPS of SFG rickettsiae and the LPS of strain OX2. ACKNOWLEDGMENTS We are grateful to Y. Nakamura and R. Ito for excellent technical assistance and to F. Mahara for providing sera from patients with Japanese spotted fever. We also thank J. J. Munoz, Rocky Mountain Laboratories, NIAID, for correcting the manuscript. This work was supported partly by a grant from the Ohyama Health Foundation. REFERENCES 1. Amano, K., H. Hatakeyama, M. Okuta, T. Suto, and F. Mahara Serological studies of antigenic similarity between Japanese spotted fever rickettsiae and Weil-Felix test antigens. J. Clin. Microbiol. 30: Amano, K., H. 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