Antibody-Mediated Immobilization of Campylobacter fetus:

Transcription

1 INFECTION AND IMMUNITY, Apr. 1976, p Copyright X 1976 American Society for Microbiology Vol. 13, No. 4 Printed in U.S.A. Antibody-Mediated Immobilization of Campylobacter fetus: Inhibition by a Somatic Antigen E. C. McCOY,' H. A. WILTBERGER, AND A. J. WINTER* New York State College of Veterinary Medicine, Cornell University, Ithaca, New York Received for publication 25 November 1975 Immobilization tests were conducted on a wild-type strain of Campylobacter fetus subsp. intestinalis and on a mutant lacking an antiphagocytic cell surface component. Highly effective immobilization of the mutant, both as single cells and clumps of cells, was produced with an antiserum containing antibodies specific for the flagellar hook and filament and for the 0 antigen. Damage to flagellar hooks after reaction with this antiserum was observed only with cells of the mutant. Single-cell immobilization of the mutant was also produced with an antiserum specific for a heat-stable somatic antigen which was distinct from the 0 antigen and was exposed on the cell surface only of the mutant. Minimal immobilization of the wild strain was brought about by either of these antisera. It was shown also that 0 antibodies had no effect on the motility of either the wild strain or mutant. These findings indicate that antibody-mediated immobilization may be brought about by effects on the flagellar hook or cell body, as well as on the flagellar filament. Furthermore, the protection from immobilization afforded the bacterium by the antiphagocytic surface structure suggests a dual function for this virulence factor in the infected animal. Campylobacter fetus is a monotrichous, gram-negative bacterium that produces infertility and abortion in cattle and sheep. In cattle the disease is venereal, and prior studies in this laboratory have demonstrated that one effect of vaginal antibodies in vaccinated or convalescent animals was to immobilize the organism (2). Mucosal antibodies of the immunoglobulin A (IgA) class were particularly effective in this function, and it was postulated that immobilization might constitute an important protective quality of secretory IgA in bacterial infections (2). Recent findings with Vibrio cholerae also have prompted the hypothesis that motility constitutes an important virulence mechanism (8) and that immobilization of the organism is consequently a key factor in protective immunity (16). The means whereby antibodies immobilize monotrichous bacteria are incompletely understood. That immobilization occurs in ways other than cross-linkage of filaments (1, 4) is indicated by the fact that IgA antibodies can effect single-cell immobilization of C. fetus (2) and that V. cholerae is immobilized by somatic antibodies (16). Evidence that flagella move rigidly (1) through rotary movement of the flagellar hook (13) and that motility requires functional integrity of the cell envelope (9, 14, 15) is I Present address: Department of Microbiology, New York Medical College, Valhalla, N.Y consistent with the possibility that antibodies directed at components other than flagellar filaments may produce immobilization. The purpose of the present experiments was to gain further insight into the mechanisms of antibody-mediated immobilization of C. fetus. A previous observation (11) that the glycoprotein having an antiphagocytic function (10) might cover sites critical for immobilization of the bacterium was examined in this study. MATERIALS AND METHODS Bacterial strains and growth requirements. C. fetus subsp. intestinalis strains 23D and 23B were used through this study. The isolation and characterization of these strains and the medium and conditions used in their cultivation have been described previously (11). For antigen preparation, cultures were grown in flasks; for immobilization studies, overlay bottles were used. Strain 23D contains an antiphagocytic surface component, antigen [a], whereas the mutant strain 23B lacks antigen [a] (10). Preparation of antigens. Methods used for the preparation of flagellar antigens, 0 antigen, and ultrasonic extracts of C. fetus have been described previously (10, 11). The 60 C extract was produced by placing a suspension of whole 23B cells in saline (1 g [wet weight]/3 ml of saline) into a 60 C water bath for 20 min. The supernatant was separated by centrifugation and stored at 4 C with phenol (2% final concentration) as a preservative. 1266

2 VOL. 13, 1976 Preparation of antisera. Antisera were produced in male New Zealand White rabbits. Rabbit 339 was immunized with an aqueous suspension of flagella. Quantities of 200 gg were injected on three occasions, 3 days apart (11). Rabbit 370 was immunized with an immunoprecipitate prepared by immunoelectrophoresis of the 60 C saline extract with antiserum directed against an extract of strain 23D cell envelopes produced with Triton X-100-ethylenediaminetetraacetic acid (EDTA) (12). The precipitate was washed and emulsified in complete Freund adjuvant and injected by the route and schedule employed previously (5). Rabbits were exsanguinated by cardiac puncture within a week after the last dose of antigen. An 0 antiserum (313) had been produced previously with an immunoprecipitate from a trichloroacetic acid extract of strain 23D (10). 2-Mercaptoethanol reduction. The procedure used for reduction by 2-mercaptoethanol was that described by Grubb and Schwann (7). Isolation of flagella. The method used for the purification of flagella was that of DePamphilis and Adler (3), except that penicillin was employed for the production of spheroplasts. For spheroplast induction, flasks of brucella broth were inoculated with overlay cultures of strain 23D or 23B to an estimated optical density at 525 nm of 0.05, and incubated at 37 C in a shaker incubator at 175 rpm. Cultures were gassed continously with a mixture of 95% N2 and 5% 02. After 6 h, penicillin was added to give a final concentration of 500 gg/ml. The cultures were returned to the shaker and the cells were collected after an additional 18 h of incubation. Preparation of samples for electron microscopy. Methods used for the preparation of thin sections and for ferritin labeling have been described previously (10). Negative stains were prepared by floating carbon-stabilized, 200-mesh, Formvar-coated grids on a drop of the sample. Grids were drained on filter paper, floated on distilled water for 10 to 15 s, drained, and placed on a drop of antiserum for 5 min. The grids were washed for 1 min by holding them on the surface of distilled water in a petri plate. The water was agitated gently with a magnetic stirrer during the washing procedure. The grids were drained and transferred to a drop of 0.5% uranyl acetate (no ph adjustment). Sections and IMMOBILIZATION OF C. FETUS 1267 negative stains were viewed in a Phillips E.M.-300 electron microscope at 80 kv. Serological reactions. Agglutination, immunoelectrophoresis, immunodiffusion, and immobilization tests were performed as described previously (5, 11, 17). RESULTS Antigenic structure of C. fetus 23. Antigens of strain 23, which have been identified in prior and current studies, are indicated in Table 1. The lipopolysaccharide component of the endotoxin, comparable to that in other gram-negative genera, contains the 0 antigen (17). Antigens [a], [b], and [c] are superficial somatic antigens readily released from the cell upon brief treatment in acid buffer (10, 11). Antigen [a], which has an antiphagocytic effect and is probably a microcapsule, lies most superficially on the cell and is missing from the mutant strain 23B (10). Antigens [d] and [e] are located on the unsheathed flagellum, but their identity as specific flagellar structures has not been determined (11). Antigen [g] corresponds to the major envelope protein antigen (12). Antibodies specific for this antigen fail to react with whole cells of strain 23D, indicating either a deep location of this antigen in the outer membrane or a blocking effect of antigen [a] (12). Specificity of antiserum 339. C. fetus 23D flagella, separated on a CsCl gradient, were almost entirely free from globular bodies (11). Rabbit 339 was immunized intravenously with small dosages of this preparation to minimize immune response to 0 and [a] antigens, known to be present in the globular bodies (A. J. Winter, unpublished data). Nevertheless, immunodiffusion reactions with 60 C extracts revealed the presence of 0 antibodies in this serum (Table 1). No other precipitins were detectable in reactions with ultrasonic extracts, acid extracts, or solubilized flagella (Table 1). Despite the lack of precipitins for flagellar antigens [d] TABLE 1. Antiserum no. Specificity of rabbit antisera for C. fetus strain 23 antigens as determined by immunodiffusion reactions and by binding of antibodies onto whole cells or isolated flagella Somatic antigen Flagellar antigen [a]p [b] [c] [fl [g] O [d] [e] Hooke Filament' Letters indicate antigens defined by immunodiffusion reactions. Antigen [a] is an antiphagocytic component; antigen [g] is a major envelope protein; antigen 0 is the 0 antigen of the lipopolysaccharide. The identity of the remaining antigens is not established. Specificity of antibodies for these components defined solely by visualization of antibody binding.

3 1268 McCOY, WILTBERGER, AND WINTER and [e], the presence of antibodies specific for hook and filament were demonstrated by negative stains (Table 1). Serum 339 was bound to filament and hook portions of isolated flagella from both strains 23B and 23D (Fig. 1B and E). When intact, exponential-phase cells of the respective strains were reacted with serum 339, antibody attached to the filaments of both cell types but bound to the cell surface only in strain 23B (Fig. 2B and E). Deposition of antibody on the flagellar hook could not be distinguished with certainty in these preparations. However, after treatment with serum 339 many hooks of strain 23B cells appeared to be poorly stained, and bends and breaks were frequently observed. Whole cells of strains 23D and 23B were agglutinated by serum 339. In triplicate INFECT. IMMUN. experiments with cells from 12- and 18-h overlay cultures, average titers were 40 with strain 23D and 80 with strain 23B. Specificity of antiserum 370. In immunodiffusion reactions with 60 C extracts, ultrasonic extracts, acid extracts, and solubilized flagella, antiserum 370 produced a single line of identity with the 60 C and ultrasonic extracts. Immunoelectrophoresis of these preparations with antiserum 370 produced a precipitate with a cathodally migrating component, designated antigen [fi (Table 1, Fig. 3). Antigen [f] is distinct from the 0 antigen and from other antigens of C. fetus that have been characterized (10, 11), including the major envelope protein antigen [g] (12). In addition to its presence in 60 C saline extracts, antigen Lf] is found in K. A '5 NY FIG. 1. Negative san of C. fetus flagella. (A) Strain 23B, utetd (B) Strain 23B after treatment with antiserum 339. (C) Strain 23B after treatment with antiserum 370. (D) Strain 23D, untreated. (E) Strain 23D after treatment with antiserum 339. (F) Strain 23D after treatment with antiserum 370. Antiserum 339 is deposited on filaments and hooks of both strains. Stained with uranyl acetate. x137,000.

4 VOL. 13, 1976 IMMOBILIZATION OF C. FETUS 1269., FIG. 2. Negative stains of C. fetus cells. (A) Strain 23B, untreated. x43,000. (B) Strain 23B after treatment with antiserum 339. x43,000. (C) Strain 23B after treatment with antiserum 370. x69,000. (D) Strain 23D, untreated. x49,000. (E) Strain 23D after treatment with antiserum 339. Lack of antibody deposition on the cell surface is best shown on the lower cell (arrow). x43,000. (F) Strain 23D after treatment with antiserum 370. x43,000. Stained with uranyl acetate. extracts produced with Triton X-100-EDTA or 2% sodium dodecyl sulfate (12). It is heat stable as evidenced by the presence of [f] antibodies in the majority of antisera produced with boiled or steamed cells (Winter, unpublished data). The absence of flagellar antibodies in antiserum 370 is further evidenced by the failure to bind to flagellar filaments or hooks of either strain 23B or 23D (Table 1; Fig. 1C and F; Fig. 2C and F). Heavy deposition of antibodies occurred on the cell surface of strain 23B (Fig. 2C). A thin, stained zone of uniform thickness was noted on the cell border of strain 23D cells treated with antiserum 370 (Fig. 2F). Because it was unclear whether this represented antibody deposition, immunoferritin labeling was performed. Exponential-phase cells of strains 23D and 23B were FIG. 3. Immunoelectrophoresis of C. fetus strain 23 extracts with antiserum 370. Top well: 60 C water extract of strain 23B. Bottom well: ultrasonic extract of whole 23D cells. The precipitin arc is produced by antigen [fi. Anode is to the right. incubated for 30 min at 37 C with antiserum 370 before treatment with ferritin-conjugated goat anti-rabbit IgG antiserum (10). The majority of 23D cells were unlabeled, although minimal binding was occasionally observed on the cell body (Fig. 4A). The cell bodies of strain 23B were labeled abundantly (Fig. 4B and C), indi-

5 1270 McCOY, WILTBERGER, AND WINTER INFECT. IMMUN. II ' I;iJ'-' ;. * '- FIG. 4. Ferritin labeling of C. fetus strains 23D and 23B after reaction with antiserum 370. (A) Strain 23D. x119,000. (B) Strain 23B. x 71,000. (C) Strain 23B. x119,000. Abundant label is present on the cell surface of strain 23B. cating ready accessibility of antigen [fi at the cell surface in the absence of antigen [a]. However, antiserum 370 failed to agglutinate whole cells of either strain 23B or 23D. Specificity of antiserum 313. Agglutination and immunodiffusion reactions indicated the presence solely of 0 antibodies (Table 1). Immobilization tests. Immobilization data are presented in Table 2. Two types of immobilization were observed: single-cell immobilization and immobilization accompanying clump formation. When cells of strain 23B were examined immediately (30 s) after the addition of antiserum 339, the most pronounced effect was single-cell immobilization of approximately 50% of the bacteria. Upon longer incubation (3 and 30 min), increases in number and size of clumps were noted. Results with strain 23D were markedly different. Antiserum 339 produced no effect on motility during the first 3 min. A low proportion of cells was immobilized by 30 min, principally in large clumps (Table 2). The phase of growth did not influence immobilization of 23B cells. However, with strain 23D, clump formation appeared to be increased in cells from the late stationary phase. In two separate trials, treatment of antiserum 339 with 2-mercaptoethanol did not reduce its capacity to immobilize strain 23B cells, either singly or in clumps. Complete loss of translational motility was TABLE 2. Immobilization ofc. fetus strains 23D and 23B by antiseraa Immobilizationb Time of Strain Antiserum treatment Immobile Immobile (min) single clumps of cells cells 23D B D _ + 23B _ adata represent averages of at least four separate experiments. b Defined as loss of translational motility. Symbols for approximate percentages of immobilized cells in a field, single or clumped, are as follows: -, 1%; -, 1 to 10%; +, 10 to 25%; ++, 25 to 50%; + + +, 50 to 75%; , 75 to 100%. A minimum of five separate fields were examined in each preparation. minimal in cells of strain 23B that had been mixed with antiserum 370 for time intervals up to 3 min, and clump formation was absent. However, a remarkable deceleration in the rate of movement of individual cells was observed by 3 min when antiserum-treated cells were

6 VOL. 13, 1976 compared to controls. By 30 min the majority of bacteria were either immobilized as single cells (Table 1) or greatly slowed in their movement. Antiserum 370 had no effect on the motility of strain 23D except for an occasional clump formed by 30 min (Table 2). Monospecific 0 antiserum 313 failed to immobilize either strain 23B or 23D. DISCUSSION The only demonstrable differences between C. fetus strains 23D and 23B are those directly related to the presence of the antiphagocytic factor antigen [a] on the surface of 23D cells (10). Antigen [a] has been visualized as a discrete component on the cell surface and is responsible, at least in part, for the inagglutinability of cells in 0 antiserum (10). Since the presence of this component inhibits immobilization by antibodies, its selective advantage to the bacterium may be dual in nature: preventing immobilization as well as phagocytosis. The specificities of the immobilizing antibodies in this study have not been defined fully. Serum 339 contained antibodies reactive with the flagellar filament and hook and with the 0 antigen (Table 1). Filament antibodies were doubtlessly instrumental in immobilization with clumping, as observed previously (4). The low proportion of strain 23D cells immobilized in clumps is surprising, however, since antibodies reacted abundantly with filaments of both 23D and 23B. Specificity of serum 339 antibodies for filaments of both strains, together with the failure of monospecific 0 antiserum 313 to immobilize either strain, suggests the possibility that hook antibodies were principally instrumental in the single-cell immobilization of strain 23B. Damage produced to 23B hooks after treatment with antiserum 339 supports this possibility; the antigen [a] layer, if overlying critical sites on the hook, could account for the resistance of strain 23D to this antiserum. Similarly, antiserum 370 contained antibodies to a somatic antigen, If], exposed on the surface of 23B but not 23D cells by reason of antigen [a] on the latter cell surface. Immobilization of C. fetus by antiserum 370 provides another instance in which somatic antibodies inhibit motility of monotrichous bacteria (16). The marked slowing effect of serum 370 on cell movement resembles observations in Escherichia coli treated with colicin El, which was attributed to the inhibition of energy transfer reactions (6). The lack of complete correlation of agglutination results with immunoferritin or direct antibody labeling data probably stems from the IMMOBILIZATION OF C. FETUS 1271 more ready detection of primary antigen-antibody interactions than of secondary phenomena. Thus, the presence of antigen [a] on 23D cells prevents their agglutination by 0 antiserum in a 24-h test (10). Although the deposition of 0 antibodies onto the cell surface was inhibited during a 5-min treatment period (cf. Fig. 2B and E), 23D cells are well stained after 30 min of incubation with fluorescein-conjugated 0 antibodies (Winter, unpublished data). Similarly, [f] antibodies failed to agglutinate 23B cells despite abundant interactions at the cell surface as evidenced directly (Fig. 2C) or in immunoferritin labeling reactions (Fig. 4B and C). The reason for these distinctions is not clear, but steric factors no doubt play some role. Comparison of immobilization data of C. fetus reported by Corbeil et al. (2) with those obtained in the present study must be restricted, since the former investigation dealt with antibodies of undefined specificities induced in cattle with a venereal strain. Corbeil et al. (2) found that IgA antibodies immobilized single cells whereas IgG antibodies immobilized with clumping, but whether this difference stemmed from variations in specificities or in functional attributes between IgA and IgG antibodies was not resolved. The lack of effect of 2-mercaptoethanol on immobilization in this study provides limited evidence that in some circumstances IgG as well as IgA (or IgM) antibodies can effect single-cell immobilization of monotrichous organisms. Immobilization detected by Corbeil et al. (2) occurred despite the presence on the bacterial cells of the antiphagocytic antigen, whereas in the present study effective immobilization occurred only in the absence of the [a] antigen. These findings, taken together, are consistent with the suggestion that bacterial immobilization mediated by antibody may occur through any of several mechanisms and that wild-type strains have evolved protective adaptations toward some of these. ACKNOWLEDGMENTS We thank Joyce Reyna for preparation of the manuscript. Electron microscopy was performed in the Richard King Mellon Laboratory of Electron Microscopy at the James A. Baker Institute for Animal Health. This investigation was supported by Public Health Service Research Grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Berg, H. C., and R. A. Anderson Bacteria swim by rotating their flagellar filaments. Nature (London) 245: Corbeil, L. B., G. D. Schurig, J. R. Duncan, R. R. Corbeil, and A. J. Winter Immunoglobulin classes and biological function of Campylobacter (Vi-

7 1272 McCOY, WILTBERGER, AND WINTER brio) fetus antibodies in serum and cervicovaginal mucus. Infect. Immun. 10: DePamphilis, M. L., and J. Adler Purification of intact flagella from Escherichia coli and Bacillus subtilis. J. Bacteriol. 105: DiPierro, J. M., and R. N. Doetsch Enzymatic reversibility of flagellar immobilization. Can. J. Microbiol. 14: Duncan, J. R., B. N. Wilkie, F. Hiestand, and A. J. Winter The serum and secretory immunoglobulins of cattle: characterization and quantitation. J. Immunol. 128: Fields, K. L., and S. E. Luria Effects of colicins El and K on cellular metabolism. J. Bacteriol. 97: Grubb, R., and B. Schwann Destruction of some agglutinins but not of others by two sulfhydryl compounds. Acta Pathol. Microbiol. Scand. 43: Guentzel, M. N., and L. J. Berry Motility as a virulence factor for Vibrio cholerae. Infect. Immun. 11: Lederberg, J Bacterial protoplasts induced by penicillin. Proc. Natl. Acad. Sci. U.S.A. 42: McCoy, E. C., D. Doyle, K. Burda, L. B. Corbeil, and A. J. Winter Superficial antigens of Campylobacter (Vibrio) fetus: characterization of an antiphag- INFECT. IMMUN. ocytic surface component. Infect. Immun. 11: McCoy, E. C., D. Doyle, H. Wiltberger, K. Burda, and A. J. Winter Flagellar ultrastructure and flagella-associated antigens of Campylobacter fetus. J. Bacteriol. 122: McCoy, E. C., H. A. Wiltberger, and A. J. Winter Major outer membrane protein of Campylobacter fetus: physical and immunological characterization. Infect. Immun. 13: Silverman, M., and M. Simon Flagellar rotation and the mechanism of bacterial motility. Nature (London) 249: Vaituzis, Z., and R. N. Doetsch Relationship between cell wall, cytoplasmic membrane, and bacterial motility. J. Bacteriol. 100: Weibull, C The isolation of protoplasts from Bacillus megaterium by controlled treatment with lysozyme. J. Bacteriol. 66: Williams, H. R., Jr., W. F. Verwey, G. D. Schrank, and E. K. Hurry An in vitro antigen-antibody reaction in relation to a hypothesis of intestinal immunity to cholera, p In Proc. 9th Joint Cholera Res. Conf. U.S. Department of State, Publ. no Winter, A. J An antigenic analysis of Vibrio fetus. III. Chemical, biologic and antigenic properties of the endotoxin. Am. J. Vet. Res. 27: