Vol. 13, No. 6
INFECTION AND IMMUNITY, June 1976, p. 1663-1670 Copyright © 1976 American Society for Microbiology
Printed in U.SA.
Complement-Mediated Killing of Acholeplasma laidlawii by Antibodies to Various Membrane Components ISOLDE DORNER, HELMUT BRUNNER,* HANS-GERD SCHIEFER, AND HANSJOBST WELLENSIEK Institut fur Medizinische Mikrobiologie, Justus Liebig-Universitfft, D-63 Giessen, Federal
Republic of Germany Received for publication 2 January 1976
Mycoplasmas are useful models for biochemical studies of the mechanism of complement-mediated killing by antibodies to various membrane components. The purpose of this study was to determine the membrane antigens involved in immune killing of Acholeplasma laidlawii. Antibodies to A. laidlawii membrane total lipids, glycolipids, and phospholipids could be induced in rabbits after injection of reaggregates of the purified lipids with Mycoplasma hominis protein as the carrier. Killing ofA. laidlawii occurred after the combined action of antibodies and complement. Antisera to A. laidlawii membrane lipids were less effective than anti-membrane protein antisera in killing the organisms. Of the antisera to lipid components of A. laidlawii membranes, antiserum to phospholipids showed a more pronounced killing effect than antiserum to glycolipids. The antibodies to A. laidlawii in the rabbit antisera belong predominantly to the immunoglobulin G class of immunoglobulins. Double-diffusion tests in agar indicated that two immunologically reactive proteins are located on the membrane surface.
It has been shown by several authors during the past 8 years that immune killing of some mycoplasma species is a complement-dependent process (2, 6, 10, 14, 20). Morphological alterations of the mycoplasma membrane after immune killing are similar to those described for other biological membranes (5). Mycoplasmas lack a rigid cell wall. In addition, their membrane components can easily be labeled by incorporation of radioactive precursors. Mycoplasmas are therefore useful models for biochemical studies of the mechanism of complement-mediated killing by antibodies to various membrane components. Several aspects of the effect of antibody and complement on biological membranes could not be resolved using erythrocytes or lipid model membranes. For example it has been suggested but not clearly demonstrated that antibody and complement act on the lipid components of biological membranes (13). We therefore felt that it might be worthwhile to further investigate the effect of antibodies and complement on mycoplasma membranes. The membrane antigens involved in the complement-mediated immune killing vary among different mycoplasma species. Acholeplasma laidlawii was chosen for these experiments because this organism can be cultivated in relatively large quantities and pure membranes
can easily be obtained after osmotic shock. The purpose of this study was to determine the membrane antigens involved in immune killing of A. laidlawii. (This work is part of a doctoral thesis presented by I. Dorner to the Fachbereich Veterinarmedizin der Justus Liebig-Universitat Giessen.) MATERIALS AND METHODS Organisms and culture conditions. A. laidlawii, oral strain, and Mycoplasma hominis, strain DC 63, originally obtained from R. M. Chanock, National Institutes of Health, Bethesda, Md., were used. A. laidlawii had been subcultured several times on artificial medium. M. hominis was used in the seventh passage on growth medium. The medium for A. laidlawii consisted of 350 ml of PPLO broth (Difco), supplemented with 5 ml of PPLO serum fraction (Difco Laboratories Inc, Detroit, Mich.), 50 ml of 1% yeast extract (Oxoid Nahrboden und Chemie GmbH, Wesel), 10 ml of 50% glucose, 10 ml of 0.1% phenol red, 12.5 ml of 2% thallium acetate, and 1,000 U of penicillin G per ml (7). The pH was adjusted to 7.8 with 1 N sodium hydroxide. The growth medium for M. hominis contained 10 ml of 50% argininehydrochloride instead of glucose. The pH was adjusted to 6.9 with 1 N hydrochloric acid. The organisms were grown in 10- to 20-liter batches. They were harvested after incubation at 37 C for 36 h, when they were still in their logarithmic growth phase. The suspension was distributed 1663
1664
DORNER ET AL.
in small quantities in glass ampoules, sealed, shell frozen in a dry ice-alcohol bath, and stored at - 70 C. Once thawed they were not refrozen. Isolation of cell membranes. Cell membranes were isolated after lysis of the organisms by osmotic shock (18) or by digitonin (23). For osmotic lysis (18), the cell suspension of A. laidlawii was centrifuged at 36,000 x g for 30 min (Sorvall RC 2-B centrifuge). The sedimented cells from 10 liters of growth medium were washed five times in saline, resuspended in 250 ml of deionized water, and incubated at 37 C for 15 min to lyse the cells. After incubation, the mycoplasma suspension was centrifuged at 8,000 x g for 5 min to remove clumps of unbroken cells. The supernatant fluid was then centrifuged at 36,000 x g for 30 min to collect the membranes. The osmotically more resistant M. hominis was lysed by digitonin treatment (23). For digitonin lysis, the washed cell suspension (20 to 25 mg of cell protein) was added to 200 ml of 0.25 M NaCl containing 25 ,ag of digitonin per ml, preheated to 37 C. After 15 min of further incubation at 37 C, membranes were collected by centrifugation at 36,000 x g for 60 min. Membranes were washed 10 times alternately with deionized water and dilute ,3-buffer (19) [0.15 M NaCl, 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.01 M 2-mercaptoethanol in deionized water, adjusted to pH 7.4 with 1 N HCl and diluted 1:20 in deionized water]. The amount of protein in the membrane suspensions was determined according to Lowry et al. (15). Separation of membrane proteins from membrane lipids. The method of Fleischer and Fleischer (9) was used to separate membrane proteins from membrane lipids. The washed membranes were suspended in deionized water (approximately 10 mg of protein/ml) and added to nine times the volume of acetone. One drop of 28% NH4OH per 50 ml of the suspension was added. The mixture was kept at room temperature for 30 min and then centrifuged at 25,000 x g for 30 min. The sediment was resuspended in deionized water, again extracted with acetone-NH4OH, and centrifuged. This second sediment contained the lipid-depleted materials henceforth referred to as membrane protein. The two supernatant fluids contained the lipids. Separation of membrane lipids. The aqueous acetone extract of A. laidlawii membrane lipids was evaporated at 30 C. The crude lipids were separated into neutral lipids, glycolipids, and phospholipids by column chromatography on silicic acid (Kieselgel 60 extra pure, 70- to 230-mesh ASTM, Merck AG, Darmstadt) (26). The silica gel was prewashed with chloroform-methanol (2:1, vol/vol), and activated at 110 to 120 C for 24 h. Fifteen grams of the activated silica gel was suspended in chloroform, and the suspension was poured into a glass column with 1-cm inner diameter. The crude lipids were dissolved in 1 to 2 ml of chloroform and applied to the column. Neutral lipids were eluted from the column with 150 ml of chloroform, glycolipids were eluted with 150 ml of acetone, and phospholipids were obtained after elution with 150 ml of chloroform-methanol (2:1, vol/vol) (21, 26). The fractions collected were
INFECT. IMMUN. dried by evaporation of the solvent under nitrogen and weighed. They were then redissolved in chloroform-methanol (2:1) and stored at -20 C until used. Thin-layer chromatography. Samples (100 gg) of the different lipid fractions were chromatographed on thin-layer plates, which were coated with a 0.25mm-thick layer of silica gel. The plates were prewashed with the developing solvent chloroformmethanol-water (70:25:5) before activation. The separated lipid spots were detected by iodine vapor, phospholipid spots were visualized with the molybdate, and glycolipids were visualized with the anaphthol and the diphenylamine spray reagents (12,
28).
Reaggregation of membrane proteins and lipids. Lipid-depleted membranes of M. hominis (50 mg of total protein) were suspended in 4 ml of deionized water and solubilized by the addition of 1 ml of 0.1 M sodium dodecyl sulfate (SDS). The neutral, glyco-, and phospholipid fractions of A. laidlawii were dis-
solved in 0.5 ml of methanol. Two milliliters of deionized water and 0.5 ml of 0.1 M SDS were added. The solutions were combined (10 mg of each lipid component per 20 mg of membrane protein) and dialyzed at 4 C for 4 days against 3 liters of dilute /3buffer (19, 24) containing 20 mM Mg24. The reaggregated material formed in the dialysis bag was collected by centrifugation at 36,000 x g for 90 min and resuspended in saline. The successful reaggregation to membrane-like structures was visualized by electron microscopy (8). Immunization of rabbits. Young rabbits, weighing 2 to 3 kg, were immunized with antigen containing 3 to 6 mg of total protein per injection, according to the following schedule. In the first injection the rabbits received intradermally a mixture of equal volumes of the antigen and complete Freund adjuvant. After 3 weeks the animals were immunized with equal volumes of the antigen and incomplete Freund adjuvant intracutaneously. Again 3 weeks later the antigen was given intraperitoneally without adjuvant. One week after the last injection, the animals were bled and the serum was separated. The antisera were heat inactivated (56 C, 30 min), divided into small portions, and stored at -20 C. Partial purification of immunoglobulin G and immunoglobulin M. Fractions of antisera to A. laidlawii cells containing predominantly immunoglobulin G or immunoglobulin M were prepared by gel filtration on Sephadex G-200. Antiserum (10 ml) was applied to a column (90 by 5 cm, Sephadex G-200) and eluted with 0.1 M tris(hydroxymethyl)aminomethane-buffered saline, pH 8.0. Fractions of 6 ml were collected, combined corresponding to the peaks, and concentrated 10-fold by pressure dialysis. Complement. Whole guinea pig serum (GPS) served as the source of complement. The animals were sacrificed, and their blood was collected by cardiac puncture. The serum was separated after clotting overnight at 4 C, distributed in small quantities, and stored at -85 C. A single pool of GPS was used for all experiments. Naturally occurring antibodies to A. laidlawii in the GPS were removed by absorption. The GPS was incubated with a suspen-
VOL. 13, 1976
IMMUNE KILLING OF A. LAIDLAWII
sion of A. laidlawii organisms at 4 C for 1 h. The mixture was centrifuged at 36,000 x g for 20 min, and the supernatant was filtered through a 450-nm pore size membrane filter (Millipore Corp.). The filtrate was stored in small quantities at -85 C and used as the source of complement. Complement fixation test. The complement fixation (CF) test was performed using microtiter equipment (30). It included overnight incubation at 4 C of serial twofold dilutions of the antisera with the antigen suspension and 2 units of complement. A washed and concentrated suspension ofA. laidlawii organisms (approximately 10 mg of protein per ml) in veronal buffer was used as the antigen. All antisera were tested for complement-fixing antibodies to this antigen. Additionally, the antisera to the lipids were tested for antibody titers against the homologous antigen. The lipid antigen was prepared by mixing egg lecithin (0.1 mg of lipid per 2 mg of lecithin, L-a-phosphatidyl choline from egg yolk type III E; Sigma Chemical Co., St. Louis, Mo.) in chloroform-methanol (2:1, vol/vol). The solvent was evaporated to dryness under nitrogen. After the lipids were redissolved in 0.1 ml of ethanol, 0.9 ml of Mayer buffer (preheated to 60 C) was added and mixed well. Metabolism inhibition test. The metabolism inhibition test was performed as described by TaylorRobinson et al. (29). The organism suspension contained 10W colony-forming units (CFU)/ml. The time of incubation was 36 to 48 h at 37 C. Agglutination of whole cells. A drop of a concentrated suspension of A. laidlawii organisms (approximately 10 mg of protein per ml) was mixed at room temperature with an equal amount of antiserum on a microscope slide. A visible agglutination appeared within a few seconds when the reaction was positive. Double-diffusion test in agar. The test was performed according to Ouchterlony (17). Purified agar
acid (TES)-buffered saline, pH 7.1, containing 0.15 mM Ca2, 0.5 mM Mg2+ and 0.1% gelatin. Then 0.1 ml of the filtered organism suspension (containing 0.5 x 107 to 1.5 x 107 CFU/ml) was added. The mixture was incubated for 60 min at 4 C. Then 0.15 ml of undiluted absorbed GPS was added. After further incubation for 120 min at 37 C, 0.1-ml amounts of the mixtures were removed and diluted 1:100 in icecold TES-buffered saline containing no Ca 2 and Mg2+ in order to stop the reaction. Controls with test serum alone (at the highest concentration employed in the test), GPS alone, and buffer alone were included. After stopping the reaction, two further 10fold dilutions were prepared in PPLO broth without additives (pH 7.1) and 0.1-ml quantities of each dilution were inoculated in triplicate onto agar medium. The plates were incubated at 37 C for 2 days, and the number of colonies was counted by using a dissecting microscope at a magnification of x 20. Based on the average number of viable organisms in the controls, the extent of killing by test serum in the presence of complement was determined. The highest dilution of the test serum which produced a 90% decrease in viability was calculated and considered to be the titer.
4% (wt/vol) (Behringwerke AG, Marburg) was mixed with 0.15 M phosphate-buffered saline, pH 7.1, and diluted 1:4 in deionized water supplemented with merthiolate 1:10,000. The mixture was heated to 100 C. Microscope slides were covered with melted agar. Six peripheral holes and one central hole of 2-mm diameter were cut into the agar and filled with 5 pIA of antiserum or antigen solution in veronal-buffered saline containing 1% SDS. The lipid antigens were prepared as described for the CF test. Each antigen was tested against different antisera in twofold dilutions. Veronal-buffered saline containing 1% SDS served as the control. The slides were kept at room temperature in a moist chamber. Optimal precipitation lines were detected after 48 h
of incubation. Mycoplasmacidal effect of antibody and complement. A. laidlawii was filtered through a prewashed 450-nm pore size membrane filter (Millipore Corp.) to obtain a homogeneous suspension of predominantly single organisms. The test was performed using microtiter equipment (3, 4). To determine the reduction in viable organisms, fourfold dilutions of the test sera were performed in 0.05 ml of 0.01 M N-
tris(hydroxymethyl)methyl-2-aminoethanesulfonic
1665
RESULTS Immunogenicity of membrane protein and membrane lipids of A. laidlawii. Antibodies to whole cells, membranes, and various membrane components of A. laidlawii were induced in rabbits. Antibody titers were determined with four serological procedures (Table 1). As can be seen, antibodies to A. laidlawii cells, purified A. laidlawii membranes, and A. laidlawii membrane protein could be demonstrated by the metabolism inhibition test, the CF reaction, agglutination, and the mycoplasmacidal test. In the CF test, the results of which are listed in Table 1, whole A. laidlawii cells were used as the antigen. The data in Table 1 also show that an antiserum against M. hominis protein apparently did not contain antibodies to A. laidlawii. This indicates that no detectable immunological relationship exists between the proteins of the two organisms. This was an important observation because M. hominis protein was used as the carrier for induction of antibodies to A. laidlawii lipids (Table 1). Antibodies reacting with living A. laidlawii cells could be demonstrated in metabolism inhibition and the mycoplasmacidal test in rabbit sera after immunization with reaggregates of A. laidlawii total lipids, glycolipids, and phospholipids, and M. hominis membrane protein as the carrier. Antibody titers to A. laidlawii membrane neutral lipids could not be detected. Antibody titers of A. laidlawii membrane protein were higher than the titers to the lipids. The less sensitive CF and agglutination reaction did not reveal antibody titers to the lipids
1666
DORNER ET AL.
INFECT. IMMUN.
when whole A. laidlawii cells were used as the antigen, whereas antibodies to the membrane proteins could be demonstrated under these experimental conditions. The low anti-lipid antibody titers in CF when whole organisms were used as the antigen may in part be explained by the high anticomplementary activity of whole A. laidlawii cells. The antigen had therefore to be used in low concentrations of 0.10 mg of total protein per ml. However, when homologous antigens were used in the CF test, antibodies to A. laidlawii total lipids, glycolipids, and phospholipids could easily be demonstrated in the rabbit sera by CF (Table 2). Since M. hominis membrane protein was used as the carrier for induction of antibodies to A. laidlawii membrane lipids, antibodies to the carrier M. hominis protein, could also be demonstrated in antisera to A. laidlawii lipids. In antisera to M. hominis, protein antibodies to A. laidlawii membrane protein could not be detected. This indicates that
membrane proteins of A. laidlawii and M. hominis do not have detectable antigenic determinants in common. It can therefore be excluded that antibodies to M. hominis protein, present in antisera to lipid components of A. laidlawii membranes, interfere in the CF test, when whole organisms are used as the antigen. Both M. hominis membrane protein and A. laidlawii membrane protein are potent antigens as indicated by the high antibody titers in the homologous system. Killing of A. laidlawii by antibody and complement. A significant decrease in the viable counts ofA. laidlawii occurred only when both specific antibodies and fresh GPS were present in the reaction mixture (Fig. 1). The GPS was absorbed at 0 C with A. laidlawii cells before use in this assay to remove naturally occurring antibodies to A. laidlawii antigens in GPS. A loss in viability was not observed by antibodies alone without the addition of GPS. Heating of the GPS at 56 C for 30 min also abolished the
TABLE 1. Immunogenicity of A. laidlawii membrane fractions Reciprocal of antibody titer to A. laidlawii as measured by: Material used for immunization MI (geometric CF (geometric Agglutination MC mean) mean) mean) A. laidlawii cells 512 16 ++ 600 A. laidlawii membranes 12 ++ 2,048 600 A. laidlawii membrane protein 380 2 + 140 M. hominis membrane protein
INFECTION AND IMMUNITY, June 1976, p. 1663-1670 Copyright © 1976 American Society for Microbiology
Printed in U.SA.
Complement-Mediated Killing of Acholeplasma laidlawii by Antibodies to Various Membrane Components ISOLDE DORNER, HELMUT BRUNNER,* HANS-GERD SCHIEFER, AND HANSJOBST WELLENSIEK Institut fur Medizinische Mikrobiologie, Justus Liebig-Universitfft, D-63 Giessen, Federal
Republic of Germany Received for publication 2 January 1976
Mycoplasmas are useful models for biochemical studies of the mechanism of complement-mediated killing by antibodies to various membrane components. The purpose of this study was to determine the membrane antigens involved in immune killing of Acholeplasma laidlawii. Antibodies to A. laidlawii membrane total lipids, glycolipids, and phospholipids could be induced in rabbits after injection of reaggregates of the purified lipids with Mycoplasma hominis protein as the carrier. Killing ofA. laidlawii occurred after the combined action of antibodies and complement. Antisera to A. laidlawii membrane lipids were less effective than anti-membrane protein antisera in killing the organisms. Of the antisera to lipid components of A. laidlawii membranes, antiserum to phospholipids showed a more pronounced killing effect than antiserum to glycolipids. The antibodies to A. laidlawii in the rabbit antisera belong predominantly to the immunoglobulin G class of immunoglobulins. Double-diffusion tests in agar indicated that two immunologically reactive proteins are located on the membrane surface.
It has been shown by several authors during the past 8 years that immune killing of some mycoplasma species is a complement-dependent process (2, 6, 10, 14, 20). Morphological alterations of the mycoplasma membrane after immune killing are similar to those described for other biological membranes (5). Mycoplasmas lack a rigid cell wall. In addition, their membrane components can easily be labeled by incorporation of radioactive precursors. Mycoplasmas are therefore useful models for biochemical studies of the mechanism of complement-mediated killing by antibodies to various membrane components. Several aspects of the effect of antibody and complement on biological membranes could not be resolved using erythrocytes or lipid model membranes. For example it has been suggested but not clearly demonstrated that antibody and complement act on the lipid components of biological membranes (13). We therefore felt that it might be worthwhile to further investigate the effect of antibodies and complement on mycoplasma membranes. The membrane antigens involved in the complement-mediated immune killing vary among different mycoplasma species. Acholeplasma laidlawii was chosen for these experiments because this organism can be cultivated in relatively large quantities and pure membranes
can easily be obtained after osmotic shock. The purpose of this study was to determine the membrane antigens involved in immune killing of A. laidlawii. (This work is part of a doctoral thesis presented by I. Dorner to the Fachbereich Veterinarmedizin der Justus Liebig-Universitat Giessen.) MATERIALS AND METHODS Organisms and culture conditions. A. laidlawii, oral strain, and Mycoplasma hominis, strain DC 63, originally obtained from R. M. Chanock, National Institutes of Health, Bethesda, Md., were used. A. laidlawii had been subcultured several times on artificial medium. M. hominis was used in the seventh passage on growth medium. The medium for A. laidlawii consisted of 350 ml of PPLO broth (Difco), supplemented with 5 ml of PPLO serum fraction (Difco Laboratories Inc, Detroit, Mich.), 50 ml of 1% yeast extract (Oxoid Nahrboden und Chemie GmbH, Wesel), 10 ml of 50% glucose, 10 ml of 0.1% phenol red, 12.5 ml of 2% thallium acetate, and 1,000 U of penicillin G per ml (7). The pH was adjusted to 7.8 with 1 N sodium hydroxide. The growth medium for M. hominis contained 10 ml of 50% argininehydrochloride instead of glucose. The pH was adjusted to 6.9 with 1 N hydrochloric acid. The organisms were grown in 10- to 20-liter batches. They were harvested after incubation at 37 C for 36 h, when they were still in their logarithmic growth phase. The suspension was distributed 1663
1664
DORNER ET AL.
in small quantities in glass ampoules, sealed, shell frozen in a dry ice-alcohol bath, and stored at - 70 C. Once thawed they were not refrozen. Isolation of cell membranes. Cell membranes were isolated after lysis of the organisms by osmotic shock (18) or by digitonin (23). For osmotic lysis (18), the cell suspension of A. laidlawii was centrifuged at 36,000 x g for 30 min (Sorvall RC 2-B centrifuge). The sedimented cells from 10 liters of growth medium were washed five times in saline, resuspended in 250 ml of deionized water, and incubated at 37 C for 15 min to lyse the cells. After incubation, the mycoplasma suspension was centrifuged at 8,000 x g for 5 min to remove clumps of unbroken cells. The supernatant fluid was then centrifuged at 36,000 x g for 30 min to collect the membranes. The osmotically more resistant M. hominis was lysed by digitonin treatment (23). For digitonin lysis, the washed cell suspension (20 to 25 mg of cell protein) was added to 200 ml of 0.25 M NaCl containing 25 ,ag of digitonin per ml, preheated to 37 C. After 15 min of further incubation at 37 C, membranes were collected by centrifugation at 36,000 x g for 60 min. Membranes were washed 10 times alternately with deionized water and dilute ,3-buffer (19) [0.15 M NaCl, 0.05 M tris(hydroxymethyl)aminomethane (Tris), 0.01 M 2-mercaptoethanol in deionized water, adjusted to pH 7.4 with 1 N HCl and diluted 1:20 in deionized water]. The amount of protein in the membrane suspensions was determined according to Lowry et al. (15). Separation of membrane proteins from membrane lipids. The method of Fleischer and Fleischer (9) was used to separate membrane proteins from membrane lipids. The washed membranes were suspended in deionized water (approximately 10 mg of protein/ml) and added to nine times the volume of acetone. One drop of 28% NH4OH per 50 ml of the suspension was added. The mixture was kept at room temperature for 30 min and then centrifuged at 25,000 x g for 30 min. The sediment was resuspended in deionized water, again extracted with acetone-NH4OH, and centrifuged. This second sediment contained the lipid-depleted materials henceforth referred to as membrane protein. The two supernatant fluids contained the lipids. Separation of membrane lipids. The aqueous acetone extract of A. laidlawii membrane lipids was evaporated at 30 C. The crude lipids were separated into neutral lipids, glycolipids, and phospholipids by column chromatography on silicic acid (Kieselgel 60 extra pure, 70- to 230-mesh ASTM, Merck AG, Darmstadt) (26). The silica gel was prewashed with chloroform-methanol (2:1, vol/vol), and activated at 110 to 120 C for 24 h. Fifteen grams of the activated silica gel was suspended in chloroform, and the suspension was poured into a glass column with 1-cm inner diameter. The crude lipids were dissolved in 1 to 2 ml of chloroform and applied to the column. Neutral lipids were eluted from the column with 150 ml of chloroform, glycolipids were eluted with 150 ml of acetone, and phospholipids were obtained after elution with 150 ml of chloroform-methanol (2:1, vol/vol) (21, 26). The fractions collected were
INFECT. IMMUN. dried by evaporation of the solvent under nitrogen and weighed. They were then redissolved in chloroform-methanol (2:1) and stored at -20 C until used. Thin-layer chromatography. Samples (100 gg) of the different lipid fractions were chromatographed on thin-layer plates, which were coated with a 0.25mm-thick layer of silica gel. The plates were prewashed with the developing solvent chloroformmethanol-water (70:25:5) before activation. The separated lipid spots were detected by iodine vapor, phospholipid spots were visualized with the molybdate, and glycolipids were visualized with the anaphthol and the diphenylamine spray reagents (12,
28).
Reaggregation of membrane proteins and lipids. Lipid-depleted membranes of M. hominis (50 mg of total protein) were suspended in 4 ml of deionized water and solubilized by the addition of 1 ml of 0.1 M sodium dodecyl sulfate (SDS). The neutral, glyco-, and phospholipid fractions of A. laidlawii were dis-
solved in 0.5 ml of methanol. Two milliliters of deionized water and 0.5 ml of 0.1 M SDS were added. The solutions were combined (10 mg of each lipid component per 20 mg of membrane protein) and dialyzed at 4 C for 4 days against 3 liters of dilute /3buffer (19, 24) containing 20 mM Mg24. The reaggregated material formed in the dialysis bag was collected by centrifugation at 36,000 x g for 90 min and resuspended in saline. The successful reaggregation to membrane-like structures was visualized by electron microscopy (8). Immunization of rabbits. Young rabbits, weighing 2 to 3 kg, were immunized with antigen containing 3 to 6 mg of total protein per injection, according to the following schedule. In the first injection the rabbits received intradermally a mixture of equal volumes of the antigen and complete Freund adjuvant. After 3 weeks the animals were immunized with equal volumes of the antigen and incomplete Freund adjuvant intracutaneously. Again 3 weeks later the antigen was given intraperitoneally without adjuvant. One week after the last injection, the animals were bled and the serum was separated. The antisera were heat inactivated (56 C, 30 min), divided into small portions, and stored at -20 C. Partial purification of immunoglobulin G and immunoglobulin M. Fractions of antisera to A. laidlawii cells containing predominantly immunoglobulin G or immunoglobulin M were prepared by gel filtration on Sephadex G-200. Antiserum (10 ml) was applied to a column (90 by 5 cm, Sephadex G-200) and eluted with 0.1 M tris(hydroxymethyl)aminomethane-buffered saline, pH 8.0. Fractions of 6 ml were collected, combined corresponding to the peaks, and concentrated 10-fold by pressure dialysis. Complement. Whole guinea pig serum (GPS) served as the source of complement. The animals were sacrificed, and their blood was collected by cardiac puncture. The serum was separated after clotting overnight at 4 C, distributed in small quantities, and stored at -85 C. A single pool of GPS was used for all experiments. Naturally occurring antibodies to A. laidlawii in the GPS were removed by absorption. The GPS was incubated with a suspen-
VOL. 13, 1976
IMMUNE KILLING OF A. LAIDLAWII
sion of A. laidlawii organisms at 4 C for 1 h. The mixture was centrifuged at 36,000 x g for 20 min, and the supernatant was filtered through a 450-nm pore size membrane filter (Millipore Corp.). The filtrate was stored in small quantities at -85 C and used as the source of complement. Complement fixation test. The complement fixation (CF) test was performed using microtiter equipment (30). It included overnight incubation at 4 C of serial twofold dilutions of the antisera with the antigen suspension and 2 units of complement. A washed and concentrated suspension ofA. laidlawii organisms (approximately 10 mg of protein per ml) in veronal buffer was used as the antigen. All antisera were tested for complement-fixing antibodies to this antigen. Additionally, the antisera to the lipids were tested for antibody titers against the homologous antigen. The lipid antigen was prepared by mixing egg lecithin (0.1 mg of lipid per 2 mg of lecithin, L-a-phosphatidyl choline from egg yolk type III E; Sigma Chemical Co., St. Louis, Mo.) in chloroform-methanol (2:1, vol/vol). The solvent was evaporated to dryness under nitrogen. After the lipids were redissolved in 0.1 ml of ethanol, 0.9 ml of Mayer buffer (preheated to 60 C) was added and mixed well. Metabolism inhibition test. The metabolism inhibition test was performed as described by TaylorRobinson et al. (29). The organism suspension contained 10W colony-forming units (CFU)/ml. The time of incubation was 36 to 48 h at 37 C. Agglutination of whole cells. A drop of a concentrated suspension of A. laidlawii organisms (approximately 10 mg of protein per ml) was mixed at room temperature with an equal amount of antiserum on a microscope slide. A visible agglutination appeared within a few seconds when the reaction was positive. Double-diffusion test in agar. The test was performed according to Ouchterlony (17). Purified agar
acid (TES)-buffered saline, pH 7.1, containing 0.15 mM Ca2, 0.5 mM Mg2+ and 0.1% gelatin. Then 0.1 ml of the filtered organism suspension (containing 0.5 x 107 to 1.5 x 107 CFU/ml) was added. The mixture was incubated for 60 min at 4 C. Then 0.15 ml of undiluted absorbed GPS was added. After further incubation for 120 min at 37 C, 0.1-ml amounts of the mixtures were removed and diluted 1:100 in icecold TES-buffered saline containing no Ca 2 and Mg2+ in order to stop the reaction. Controls with test serum alone (at the highest concentration employed in the test), GPS alone, and buffer alone were included. After stopping the reaction, two further 10fold dilutions were prepared in PPLO broth without additives (pH 7.1) and 0.1-ml quantities of each dilution were inoculated in triplicate onto agar medium. The plates were incubated at 37 C for 2 days, and the number of colonies was counted by using a dissecting microscope at a magnification of x 20. Based on the average number of viable organisms in the controls, the extent of killing by test serum in the presence of complement was determined. The highest dilution of the test serum which produced a 90% decrease in viability was calculated and considered to be the titer.
4% (wt/vol) (Behringwerke AG, Marburg) was mixed with 0.15 M phosphate-buffered saline, pH 7.1, and diluted 1:4 in deionized water supplemented with merthiolate 1:10,000. The mixture was heated to 100 C. Microscope slides were covered with melted agar. Six peripheral holes and one central hole of 2-mm diameter were cut into the agar and filled with 5 pIA of antiserum or antigen solution in veronal-buffered saline containing 1% SDS. The lipid antigens were prepared as described for the CF test. Each antigen was tested against different antisera in twofold dilutions. Veronal-buffered saline containing 1% SDS served as the control. The slides were kept at room temperature in a moist chamber. Optimal precipitation lines were detected after 48 h
of incubation. Mycoplasmacidal effect of antibody and complement. A. laidlawii was filtered through a prewashed 450-nm pore size membrane filter (Millipore Corp.) to obtain a homogeneous suspension of predominantly single organisms. The test was performed using microtiter equipment (3, 4). To determine the reduction in viable organisms, fourfold dilutions of the test sera were performed in 0.05 ml of 0.01 M N-
tris(hydroxymethyl)methyl-2-aminoethanesulfonic
1665
RESULTS Immunogenicity of membrane protein and membrane lipids of A. laidlawii. Antibodies to whole cells, membranes, and various membrane components of A. laidlawii were induced in rabbits. Antibody titers were determined with four serological procedures (Table 1). As can be seen, antibodies to A. laidlawii cells, purified A. laidlawii membranes, and A. laidlawii membrane protein could be demonstrated by the metabolism inhibition test, the CF reaction, agglutination, and the mycoplasmacidal test. In the CF test, the results of which are listed in Table 1, whole A. laidlawii cells were used as the antigen. The data in Table 1 also show that an antiserum against M. hominis protein apparently did not contain antibodies to A. laidlawii. This indicates that no detectable immunological relationship exists between the proteins of the two organisms. This was an important observation because M. hominis protein was used as the carrier for induction of antibodies to A. laidlawii lipids (Table 1). Antibodies reacting with living A. laidlawii cells could be demonstrated in metabolism inhibition and the mycoplasmacidal test in rabbit sera after immunization with reaggregates of A. laidlawii total lipids, glycolipids, and phospholipids, and M. hominis membrane protein as the carrier. Antibody titers to A. laidlawii membrane neutral lipids could not be detected. Antibody titers of A. laidlawii membrane protein were higher than the titers to the lipids. The less sensitive CF and agglutination reaction did not reveal antibody titers to the lipids
1666
DORNER ET AL.
INFECT. IMMUN.
when whole A. laidlawii cells were used as the antigen, whereas antibodies to the membrane proteins could be demonstrated under these experimental conditions. The low anti-lipid antibody titers in CF when whole organisms were used as the antigen may in part be explained by the high anticomplementary activity of whole A. laidlawii cells. The antigen had therefore to be used in low concentrations of 0.10 mg of total protein per ml. However, when homologous antigens were used in the CF test, antibodies to A. laidlawii total lipids, glycolipids, and phospholipids could easily be demonstrated in the rabbit sera by CF (Table 2). Since M. hominis membrane protein was used as the carrier for induction of antibodies to A. laidlawii membrane lipids, antibodies to the carrier M. hominis protein, could also be demonstrated in antisera to A. laidlawii lipids. In antisera to M. hominis, protein antibodies to A. laidlawii membrane protein could not be detected. This indicates that
membrane proteins of A. laidlawii and M. hominis do not have detectable antigenic determinants in common. It can therefore be excluded that antibodies to M. hominis protein, present in antisera to lipid components of A. laidlawii membranes, interfere in the CF test, when whole organisms are used as the antigen. Both M. hominis membrane protein and A. laidlawii membrane protein are potent antigens as indicated by the high antibody titers in the homologous system. Killing of A. laidlawii by antibody and complement. A significant decrease in the viable counts ofA. laidlawii occurred only when both specific antibodies and fresh GPS were present in the reaction mixture (Fig. 1). The GPS was absorbed at 0 C with A. laidlawii cells before use in this assay to remove naturally occurring antibodies to A. laidlawii antigens in GPS. A loss in viability was not observed by antibodies alone without the addition of GPS. Heating of the GPS at 56 C for 30 min also abolished the
TABLE 1. Immunogenicity of A. laidlawii membrane fractions Reciprocal of antibody titer to A. laidlawii as measured by: Material used for immunization MI (geometric CF (geometric Agglutination MC mean) mean) mean) A. laidlawii cells 512 16 ++ 600 A. laidlawii membranes 12 ++ 2,048 600 A. laidlawii membrane protein 380 2 + 140 M. hominis membrane protein
