Identification of Lectin-Binding Proteins in Chlamydia Species

Transcription

1 INFECTION AND IMMUNITY, Feb. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 58, No. 2 Identification of Lectin-Binding Proteins in Chlamydia Species ALBERTINA F. SWANSON AND CHO-CHOU KUO* Department of Pathobiology, University of Washington, Seattle, Washington Received 15 September 1989/Accepted 14 November 1989 Lectin-binding proteins of chlamydiae were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. All three Chlamydia species tested expressed two proteins when wholeelementary-body lysates were reacted with the biotinylated lectin Dolichos biflorus agglutinin. The protein with a molecular mass of 18 kilodaltons (kda) responded strongly compared with a higher-molecular-mass protein that varied from 27 to 32 kda with each chlamydia strain tested. Among six lectins tested, including concanavalin A, D. biflorus agglutinin, Ulex europaeus agglutinin, soybean agglutinin, peanut agglutinin, and wheat germ agglutinin, the latter was the only lectin that did not recognize any chlamydial protein. For each lectin that reacted against the elementary body of serovar L2 of Chlamydia trachomatis, the same two peptides, an 18-kDa peptide and a 32-kDa peptide, were revealed. These two polypeptides adhered to HeLa cell surface components. Binding of a lectin to the L2 reticulate body resulted in a reduced response at the 18-kDa peptide. The 18- and 32-kDa peptides were purified from L2 serovar elementary bodies by affinity chromatography. The two proteins isolated from a concanavalin A-agarose column maintained their lectin-binding capacities and elicited hemagglutinating properties against mouse erythrocytes. Periodate oxidation abolished the abilities of the peptides to adhere to any of the lectins tested. These results suggest that these lectin-binding proteins are glycoproteins that may be an essential factor for attachment of chlamydial organisms to host cells. Chiamydia species are obligate intracellular bacteria with a unique life cycle (20). The infectious elementary body (EB) attaches to its host cell and then is endocytosed. Once inside, the EB undergoes changes to form a metabolically active reticulate body (RB). RBs multiply by binary fission and then mature into EBs. Attachment and internalization of chlamydia to host cells is poorly understood. However, adhesion of chlamydia to eucaryotic cells has been speculated to involve the following physicochemical properties: surface charge (15), hydrophobic interactions (23), and surface receptors such as lectins (1, 15, 18, 24). Lectins are nonimmunogenic proteins that bind to specific carbohydrate structures and have been used to investigate external and internal functions of cells (22). Recent studies have shown the effects of chlamydial organisms interacting with lectinbound eucaryotic cells. Wheat germ agglutinin (WGA) was found to inhibit attachment of Chiamydia trachomatis to HeLa (1) and McCoy (24) cells and that of C. psittaci to L cells (18). Carlson et al. (3) attempted to identify chlamydial glycoproteins by lectin-binding assays by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting (immunoblotting). The 40- kilodalton (kda) major outer membrane protein, the 60-kDa peptide, and many minor proteins bound to five of the lectins tested. In our investigation, we used immunochemical techniques and affinity chromatography to isolate lectin-binding proteins in chlamydial organisms which may be required in the mechanism of attachment to eucaryotic cells. MATERIALS AND METHODS Organisms. The chlamydial organisms used were C. trachomatis C/TW-3/OT, E/UW-5/Cx, and L2/434/Bu (16); C. psittaci mammalian strain meningopneumonitis (5) and avian strain 6BC (8); and C. pneumoniae TW-183 and AR-39 (9). The organisms (EBs) were grown in HeLa 229 cell cultures (16), harvested 3 days postinoculation, and purified by one * Corresponding author. 502 cycle of differential centrifugation, pelleting through a 30% Hypaque-76 cushion (Winthrop-Breon Laboratories, Sterling Drug Inc., New York, N.Y.), and banding by centrifugation in a 30 to 65% linear gradient of Hypaque-76. The final products usually contained 5 x 108 to 1 x 109 inclusionforming units per ml of organisms. RB preparations were prepared from C. trachomatis L2/434/Bu. Cultures were harvested at 19 h postinoculation, and organisms were purified as described above. Lectins. The lectins used were concanavalin A (ConA), specific to at-d-mannose and at-d-glucose; Dolichos biflorus agglutinin (DBA), specific to N-acetyl-D-galactosamine; WGA, specific to N-acetyl-D-glucosamine; Ulex europaeus agglutinin (UEAI), specific to cx-l-fucose; soybean agglutinin, specific to N-acetyl-D-galactosamine and D-galactose; and peanut agglutinin (PNA), specific to galactosyl(p-1,3) N-acetyl-D-galactosamine. The biotinyl derivatives of plant lectins were obtained from Vector Laboratories Inc. (Burlingame, Calif.). Antiserum. Rabbit anti-l2 and anti-hela cell immune sera were used for immunoblotting. Anti-L2 serum was prepared by immunization of New Zealand White rabbits with purified organisms grown in HeLa cells used in our earlier studies (2). Anti-HeLa serum was prepared by immunization of rabbits with whole HeLa cells (107/ml), an intramuscular injection of 2 ml of antigen with Freund incomplete adjuvant, followed by a series of intravenous injections of 0.1, 0.2, 0.3, 0.4, and 1.0 ml of antigen in phosphate-buffered saline over a 14-day period, and the rabbits were bled 7 days after the final intravenous injection as described by Kenny (12). SDS-PAGE and immunoblotting. SDS-PAGE and immunoblotting were conducted as described by Laemmli (17) and Towbin et al. (25), respectively. Briefly, whole organisms were solubilized and reduced by boiling in 2% SDS with 2-mercaptoethanol. Solubilized antigens (20,ug per 5-mmwide well) were separated on a 5% stacking gel and a 12.5% running gel. Following electrophoretic transfer of proteins to nitrocellulose papers, the paper slips were reacted with

2 VOL. 58, 1990 antiserum. Immune reactions were detected with peroxidase-conjugated anti-rabbit immunoglobulin (Organon Teknika, Malvern, Pa.), and the bands were visualized by using 4-chloro-1-naphthol as the substrate. The molecular mass markers used were phosphorylase b (94 kda), bovine serum albumin (67 kda), ovalbumin (43 kda), carbonic anhydrase (30 kda), soybean trypsin inhibitor (20.1 kda), and a-lactalbumin (14.4 kda). They were purchased from Pharmacia (Piscataway, N.J.). Protein bands were stained by Aurodye (Janssen Life Science Products, Piscataway, N.J.). Lectin-binding assay. The lectin-binding assay was performed as described by Gordon and Pena (7). Proteins separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose paper were first blocked against nonspecific protein binding with 3% (wt/vol) bovine serum albumin in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) basic solution (HBS) (137 mm NaCl, 2.7 mm KCl, 0.9 mm CaCl2, 0.5 mm MgCl2, 30 mm HEPES, ph 7.4) for 1 h at 37 C. After the nitrocellulose strips were rinsed in HBS, the proteins were reacted with an individual biotinylated lectin at a concentration of 10,ug/ml in HBS containing 3% bovine serum albumin for 1 h at room temperature. The protein strips were again rinsed with HBS, and then the preformed avidin-biotinyl peroxidase (Vectastain ABC Mouse IgG Kit; Vector Laboratories) in 12% bovine serum albumin was applied for 1 h at room temperature. The substrate used to detect the lectin-binding proteins was diaminobenzidine tetrahydrochloride. Hapten inhibition of lectin binding. For carbohydrate hapten inhibition tests, the lectin-binding assay as described above was used, but an additional step was included. The lectin was preincubated with a 0.25 M concentration of one of the following sugars; a-lactose, D-mannose, D-galactose, N-acetyl-D-galactosamine, or a.-l-fucose (Sigma Chemical Co., St. Louis, Mo.) for 1 h at room temperature before reaction with the protein strips. Affinity chromatography. Chromatography was performed by applying solubilized EBs to a lectin affinity column. A ConA lectin-agarose (Vector) column (1.0 by 3.5 cm) was equilibrated against 5 column volumes with buffer (10 mm Tris hydrochloride [ph 7.5], 0.15 M NaCl, 1 mm CaCl2, 1 mm MnCl2, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride). C. trachomatis EBs of serovar L2 (equivalent of 8 ml of purified organisms or about 10 mg of proteins) were treated with 4 ml of 2% SDS in 0.1 M phosphate-buffered saline (ph 7.4) for 1 h at 37 C and then centrifuged at 35,000 x g for 1 h at 30 C. The supernatant was dialyzed overnight at room temperature. The solubilized EBs were adsorbed onto the ConA lectin column for 30 min at room temperature. The column was washed with buffer (5 column volumes) until the optical density at 280 nm became 0, and the proteins that specifically bound to the lectin were eluted with 10 ml each of 0.1 M and 0.3 M methyl-a-d-mannopyranoside (Sigma) in the same buffer. To maximize recovery of protein from the original sample, this procedure was repeated twice by reloading the first 6 ml of the unbound sample wash onto the column and collecting the eluted material. One-milliliter samples were collected and subjected to SDS-PAGE. The fractions that contained the lectin-binding proteins were pooled, dialyzed, and concentrated by the Micro-ProDiCon (Bio-Molecular Dynamics, Beaverton, Oreg.), which usually yielded 100,ug of protein. Chemical and biological assays. (i) Periodate oxidation. Proteins fractionated from lectin affinity columns were LECTIN-BINDING PROTEINS OF CHLAMYDIA SPP. 503 FIG. 1. Detection of chlamydial lectin-binding proteins. Electrophoretically transferred proteins (20 jig per lane) of the L2 serovar of C. trachomatis were exposed to six different biotinylated lectins (10,ug/ml) and stained with 3,3'-diaminobenzidine. Lane A contained Aurodye-stained marker proteins (top to bottom: 94, 67, 43, 30, 20.1, and 14.4 kda). Lanes B through G contained the six lectins used in the experiment. Lanes: B, ConA; C, DBA; D, WGA; E, UEAI; F, soybean agglutinin; G, PNA. The arrowheads indicate 32- and 18-kDa proteins. treated with 0.05 M potassium metaperiodate (Sigma) in 50 mm sodium acetate buffer (ph 4.5) at 4 C for 24 h. (ii) Hemagglutination assay. The technique used for the hemagglutination assay was based on that of Watkins et al. (26). Freshly harvested mouse (BALB/c) erythrocytes (RBCs) were washed with Alsever solution, centrifuged, and suspended in MacIllvaine-saline solution. The concentration of the RBC solution was 1%. Fifty microliters of the 1% RBC suspension was delivered to each well of a round-bottom microtiter plate, and an equal volume of the protein fraction eluted from the lectin affinity column was added. Threefold dilutions of the protein samples or buffer were prepared and incubated at 4 C for 2 h. RESULTS Demonstration of lectin-binding proteins. The ability of chlamydial proteins to bind to lectins was examined. Chlamydial proteins of C. trachomatis serovar L2 EBs that were separated by SDS-PAGE from whole-cell lysates and electrotransferred onto nitrocellulose paper were reacted with six different lectins (Fig. 1). ConA, DBA, UEAI, soybean agglutinin, and PNA exposed the same two proteins with molecular masses of 32 and 18 kda. No binding was shown with WGA. To determine that the proteins bind specifically to the

3 504 SWANSON AND KUO INFECT. IMMUN. 43' c ID A B C D E F G 43k k- 20k k-.1 _w_r 1441 FIG. 2. Chlamydial proteins show binding specificity to lectins by hapten inhibition tests. The biotinylated lectin UEAI (10 p.g/ml) was preincubated with a carbohydrate (2.5 M) for 1 h at room temperature before exposure to nitrocellulose strips consisting of electrophoretically transferred proteins of the L2 serovar of C. trachomatis. The lectin bound to the proteins was detected by 3,3'-diaminobenzidine. Marker proteins stained by Aurodye were in lane A (k, kilodaltons). Lane B contained the positive control; there was no carbohydrate preincubation with UEAI. Lanes C to E contained the lectin UEAI preincubated with one of the haptens tested. Lanes: C, cx-l-fucose; D, D-mannose; E, D-galactose. Lane F contained the negative control, with which no lectin or sugar was used. lectins, hapten inhibitory tests were performed. These experiments showed that the carbohydrate specific to the lectin was able to inhibit the lectin from binding to the proteins. Figure 2 illustrates inhibition by ot-l-fucose of UEAI binding to the proteins of the L2 serovar of C. trachomatis. The unrelated carbohydrates D-mannose and D-galactose did not inhibit binding. Binding of lectins to several strains from the three Chlamydia species was tested (Fig. 3). Each strain detected two proteins, a low-molecular-mass protein at or about 18 kda and a higher-molecular-mass protein that varied from 27 to 32 kda, depending on the particular strain examined. For example, serovar C of C. trachomatis expressed the protein at 29 kda, while the high-molecular-mass protein for serovar E of C. trachomatis was 32 kda. The two C. pneumoniae strains expressed equivalent protein bands with molecular masses of 32 and 18 kda. Each strain of C. trachomatis disclosed a different higher-molecular-mass protein adhering to the lectin, but all within close range of 30 kda. The greatest contrast of staining between the two proteins was revealed on the representative strains of C. psittaci in which the 30-kDa band was faint. The 18-kDa band was the FIG. 3. Identification of lectin-binding proteins of different chlamydial strains. Twenty micrograms of chlamydial proteins was separated by SDS-PAGE and transblotted to nitrocellulose paper. HeLa cell lysates were run in parallel as a control. Proteins were probed with 10,ug of the lectin DBA per ml and then stained with 3,3'-diaminobenzidine. Lanes: A, HeLa cells; B, serovar C of C. trachomatis; C, serovar E of C. trachomatis; D, C. pneumoniae AR-39; E, C. pneumoniae TW-183; F, meningopneumonitis strain of C. psittaci; G, C. psittaci 6BC. The numbers on the left indicate the migration of the molecular mass markers (k, kilodaltons). predominant protein by the biotinyl-avidin-peroxidase method. The binding assay with HeLa cell proteins as a control did not express any reaction in the 32-kDa range. However, there was a band near but not exactly in the 18-kDa area. Immunoblotting with rabbit anti-hela cell serum did not recognize the 32- and 18-kDa lectin-binding proteins of chlamydiae, although the antiserum reacted with many HeLa cell proteins tested in parallel. We compared the binding of lectins between equivalent protein amounts of EBs and RBs of the L2 serovar of C. trachomatis. Only the 18-kDa peptide of the RB elicited a reduced response by the biotinylated lectins. There was no reaction at 32 kda (data not shown). Isolation of lectin-binding proteins. SDS-solubilized whole EBs of serovar L2 C. trachomatis were applied to a ConA lectin-agarose column. Proteins of 18 and 32 kda were recovered when an inhibitory sugar eluted the lectin-binding proteins from the column. SDS-PAGE of the concentrated fraction revealed the two peptides by staining with Aurodye (Fig. 4). Both proteins retained the ability to bind to the lectin DBA as shown by the biotin-avidin-peroxidase technique. Adhesion of HeLa cell components to lectin-binding proteins of Chlamydia spp. Since the proteins of C. trachomatis that adhere to the surface of eucaryotic cells have molecular weights comparable to those of the lectin-binding proteins we have isolated (10, 27), we tested whether the lectinbinding proteins were eucaryotic-cell-binding proteins. L2 proteins run on SDS-PAGE and electrotransferred onto nitrocellulose paper were incubated with Triton X-100-solubilized HeLa cells. The 32- and 18-kDa chlamydial peptides were shown to bind the HeLa cell proteins (Fig. 5). In addition, a protein corresponding to the molecular mass of the major outer membrane protein (40 kda) reacted with the eucaryotic cell components. Rabbit anti-hela cell serum did

4 VOL. 58, k 18k t.?o-:1.* r1 FIG. 4. Isolation of lectin-binding proteins by affinity chromatography. The proteins of the L2 serovar of C. trachomatis were fractionated by a ConA-agarose column. The bound proteins were eluted with methyl-a-d-mannopyranoside. The concentrated eluate was subjected to SDS-PAGE and transferred onto nitrocellulose paper. Reaction with the lectin DBA revealed the 18- and 32-kDa proteins shown. not react with chlamydial proteins that were not exposed to Triton X-100-solubilized HeLa cells (data not shown). Chemical and biological properties. (i) Periodate oxidation. The ability to bind complex carbohydrates is one of the criteria of a lectin. To help determine whether the proteins that bound to the lectins were actually glycoproteins, we conducted periodate oxidation experiments. Treatment of 40k- s 32k- _ 18k- _ FIG. 5. Immunoblot showing that chlamydial lectin-binding proteins also bind HeLa cell components. C. trachomatis serovar L2 organisms were run on SDS-PAGE and electroblotted to nitrocellulose. The separated chlamydial proteins were reacted with Triton X-100-solubilized HeLa cells and then probed with rabbit anti-hela cell serum. The reaction was revealed with peroxidase and 4- chloro-1-naphthol stain. Three chlamydial proteins with molecular masses of 40, 32, and 18 kda (k) bound HeLa cell components. LECTIN-BINDING PROTEINS OF CHLAMYDIA SPP. 505 L2 proteins fractionated from the lectin affinity column with periodate abolished the binding abilities of lectins. When the sample was incubated with buffer only, the biotinyl lectin reacted with the 32- and 18-kDa peptides. (ii) Hemagglutination. We tested the abilities of fractionated lectin-binding proteins to hemagglutinate mouse RBCs. The results showed that the 18- and 32-kDa peptides caused hemagglutination of BALB/c mouse RBCs at a minimum concentration of 18,ug. Contamination with chlanydial lipopolysaccharide, which has been shown also to cause hemagglutination (26), was ruled out by testing affinitypurified preparations in immunoblots using monoclonal antibody CF-2, which is specific to chlamydial lipopolysaccharide (14). DISCUSSION We identified and isolated two lectin-binding proteins of Chlamydia spp. that correspond in molecular weight to eucaryotic cell surface-binding proteins reported in the literature (10, 27). We further demonstrated the ability of the lectin-binding protein to adhere to HeLa cell components and cause agglutination of mouse RBCs. Preliminary data indicate that the chemical structures of these compounds contain carbohydrate as well as protein. Using the same lectin-binding assay, Carlson et al. (3) investigated the binding of electrophoretically separated proteins of three C. trachomatis and two C. psittaci strains (all strains were different from ours) to the same lectins. They found chlamydial proteins that reacted to ConA, DBA, PNA, UEAI, and soybean agglutinin. There was no binding to WGA and Ricinus communis agglutinin I; the latter was not tested in our studies. This finding was similar to ours. However, the results obtained with stained lectin-bound peptides were different. They showed that the predominant binding proteins were the 40-kDa major outer membrane protein and the 60-kDa protein, in addition to several minor reacting proteins which varied among the lectins. Contrary to these findings, for each separate strain, we consistently detected only two proteins, an 18-kDa protein and a highermolecular-mass protein that ranged from 27 to 32 kda, that bound to the lectins. Furthermore, we were able to purify these two proteins from solubilized whole chlamydial organisms by lectin affinity chromatography. The discrepancy cannot be explained by strain differences, because our studies showed consistent reactivities between different strains, or by the difference in the reagents used, because the biotinylated lectins were obtained from the same source. The correlation between the lectin-binding proteins detected in this study and the proteins which bind eucaryotic cell surface components reported by earlier investigations (10, 27) is supported by several facts. (i) The molecular weights are identical. Hackstadt (10) determined the HeLa cell-binding proteins among eight C. trachomatis serovars to be an 18-kDa peptide and a higher-molecular-mass peptide that varied from 23 to 32 kda. The two C. trachomatis strains tested by Wenman and Meuser (27) showed these two proteins to be 18 and 31 kda. Our study showed that the lectin-binding proteins of three strains of C. trachomatis have a molecular mass identical to that of the 18-kDa peptide, while the higher-molecular-mass proteins ranged from 27 to 32 kda. In addition, Hackstadt showed only a single HeLa cell-binding protein of 18 kda for the C. psittaci strains. In our results, the 18-kDa peptide also was displayed

5 506 SWANSON AND KUO as the dominant band in comparison with the higher-molecular-mass peptide. (ii) Using rabbit anti-hela cell serum for immunoblotting, we demonstrated that both the 18- and 32-kDa proteins bound HeLa cell components. (iii) Both HeLa cell-binding proteins reported by Hackstadt (10) and Wenman and Meuser (27) and our lectin-binding proteins are developmental cycle dependent in that they are prominently displayed in the EB stage and are reduced in amount or absent in the RB stage. The composition of the lectin-binding proteins of chlamydiae is still undefined. The preliminary data from the periodate oxidation experiment suggest that the structure that bound to the lectin(s) contains carbohydrate moieties; this is compatible with the glycoprotein nature of the lectin-binding proteins (19). The selected lectins covered a wide range of carbohydrate specificity. In our assays, the same two proteins adhered to a variety of sugar-specific lectins, which suggests a complex oligosaccharide structure. Similar results have previously been found when fetuin was tested with R. communis agglutinin, PNA, and Limulus polyphemus agglutinin (13), and the lectin probes of ConA, WGA, and lotus lectin revealed the same four proteins from the parotid salivary gland (21). Hemagglutination of mouse RBCs by chlamydiae was first reported by Hilleman et al. (11) in The hemagglutination activity was group reacting. We found that fractions from serovar L2 EBs of C. trachomatis isolated by lectin affinity chromatography exhibited mouse RBC-hemagglutinating activity. Chong et al. (4) have reported a hemagglutinin from the L2 serovar of C. trachomatis that has attributes similar to those of our lectin-binding compound in that it was nonimmunogenic and temperature independent and appeared in both life forms of chlamydiae. The components in chlamydia hemagglutinins have been determined to contain phospholipid lecithin and a nucleoprotein by Gogolak and Ross (6) and have been identified as lipopolysaccharide most recently by Watkins et al. (26). If lectin-binding proteins are determined to be glycoproteins, they will represent another class of chlamydia hemagglutinins. The role that these lectin-binding proteins play in the life cycle of chlamydiae has yet to be determined. Whether these components serve as sites of binding to mammalian cell surface receptors is an important question. These studies will further our understanding of the pathogenesis and prevention of chlamydial infections. ACKNOWLEDGMENT This study was supported by Public Health Service grants EY and Al from the National Institutes of Health. LITERATURE CITED 1. Bose, S. K., G. B. Smith, and R. G. 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