Vol. 24, No. 1
INFECTION AND IMMUNITY, Apr. 1979, P. 39-46 0019-9567/79/04-0039/08$02.00/0
Immunogenicity of Bacteroides Isolated from Mice: Relationship Between Immunogenicity and Cell Wall Antigens HISAKO YASUI,* NOBUYOSHI YASUTAKE, AND MAKOTO OHWAKI Yakult Institute for Microbiological Research, Kunitachi, Tokyo 186, Japan Received for publication 5 January 1979
Three different strains of Bacteroides were isolated from feces and cecal contents of mice. The immunogenicity of the strains was determined by measuring the serum agglutinin titers after intraperitoneal antigen injection. There were marked differences in quantity and quality of produced antibodies among the three strains. One strain (2-2) induced low antibody titers in both the primary and secondary responses, and a significant 2-mercaptoethanol (2-ME)-resistant antibody production occurred. Another strain (Y) induced low antibody titers in the primary response and high titers in the secondary response, but 2-MEresistant antibody production did not occur. The third strain (2-4) induced very high antibody titers in both the responses, and a large amount of 2-ME-resistant antibody production occurred. Further, heat-ethanol-treated strain Y induced only immunoglobulin M antibody, but periodate-treated strain Y induced no antibody. Heat-ethanol- or periodate-treated strain 2-4 induced immunoglobulin M or G antibody, respectively. These observations suggest that the surface antigens of the two strains are distinctly different: the antigen of strain Y would be mostly O-antigen, whereas those of strain 2-4 would be O-antigen and protein moieties.
It has been suggested that Bacteroides strains are present at very high population levels in mice ceca (19) and that one species of Bacteroides is poorly immunogenic in mice (4, 9). However, it has recently been reported that a number of different species of Bacteroides are found in the cecal contents of conventional mice (25). It has been reported that one principal factor in the immunogenicity, especially the determination of the antibody class, is the nature of the cell wall constituents (3, 10, 20). In the present studies, we compared the immunogenicity among three different strains of Bacteroides isolated from the feces or cecal contents of mice and investigated certain features of the cell wall constituents, based on agglutination and immunofluorescence tests. MATERIALS AND METHODS Isolation and maintenance of Bacteroides. As soon as ICR and C3H/He mice were killed, samples of feces and cecal contents were removed into the glove box, diluted serially with diluent (22) in several 10-fold steps, and plated in appropriate dilution on modified NBGT medium (21). The composition of the modified NBGT medium was as follows: heart infusion agar, 1,000 ml; brilliant green, 0.001 g; sodium taurocholate, 1 g; L-cysteine-hydrochloride 0.3 g; neomycin, 0.2 g; and horse blood, 50 ml (pH 7.2). The atmosphere in the chamber used in the experiment was a mixture of 7% H2 and 5% C02 in N2. Seven colonies were trans-
ferred, grown in prereduced modified VL medium (1) supplemented with 0.5% glucose, harvested by centrifugation, resuspended in prereduced dispersion medium (skim milk, 1%; sodium glutamate, 2%; resazurin, 0.0001%; and L-cysteine-hydrochloride, 0.05%) (22), and stored at -70°C. The composition of the modified VL medium (per liter of medium) was as follows: salt solution I [KH2PO4, 0.3%; NaCl, 0.6%; (NH4)2SO4, 0.3%; CaCl2, 0.03%; MgSO4, 0.03%], 75 ml; salt solution II (K2HPO4, 0.3%), 75 ml; Trypticase, 10 g; yeast extract, 5 g; beef extract, 2 g; 0.1% resazurin, 1 ml; 8% Na2CO3, 50 ml; 3% L-cysteine-hydrochloride, 10 ml; and 0.1% hemin, 7 ml. The isolates were classified as members of the genus Bacteroicles since they were gram-negative rods, did not grow aerobically, and produced mainly acetic acid and succinic acid and did not produce butyric acid. In addition, each of the seven strains was examined for its ability to ferment various carbohydrates and to hydrolyze starch and esculin (12). The production of indole and coagulation of milk were also tested. The seven isolates were divided into three groups, and the three strains 2-2, Y, and 2-4, representing each group, were used for the experiments. Strain Y was isolated from feces of ICR mice and strains 2-2 and 2-4 were isolated from cecal contents of C3H/He mice. The mice were obtained from the Central Institute for Experimental Animals, Tokyo, Japan, and bred in our laboratories. Preparation of antigens. Three strains were cultivated in prereduced modified VL medium and washed three times with phosphate-buffered saline (PBS). The composition of PBS (per liter) was as follows: NaCl, 8 g; KC1, 0.2 g; Na2HPO4, 1.15 g; 39
40
YASUI, YASUTAKE, AND OHWAKI
KH2PO4, 0.2 g (pH 7.2). Antigens of the three strains were prepared by treatment with 0.3% Formalin at 370C overnight. The somatic antigens of strains Y and 2-4 were prepared by the heat-ethanol method (8). Cells of the strains oxidized with periodate were prepared by the modified Hofstad method (13). The method was as follows. The mixture of 2% bacterial suspension in PBS (pH 7.2) and equal volumes of 0.05 M sodium meta-periodate were incubated in the dark at 40C for 5 days and dialyzed against PBS (pH 7.2). Immunization schedule. Mice were injected intraperitoneally with 1 x 109 Formalin-, heat-ethanol-, or periodate-treated organisms. Male C57BL/6 and C3H/He mice, 6 weeks of age, were used for the immunization. The former mice were obtained from the National Institute of Genetics, Mishima, Japan. The primary response was determined 7 days after the primary injection and the secondary response was determined by agglutination tests at various times after the secondary injection. The results of the agglutinin tests in each point were observed on four to seven mice. Agglutination test. Immune sera with or without 2-mercaptoethanol (2-ME) treatment (13) were diluted twofold by using U-well plastic trays (14), and equal volumes of 5 x 108 Formalin-treated cell suspensions per ml were added. After incubation at 370C for 1 h and at 40C overnight, the patterns of agglutination were read. Treatment of immune sera with 2-ME was performed by adding equal volumes of a 0.2 M solution of 2-ME in PBS (pH 7.2) and keeping the solution at room temperature for 1 h. Ultracentrifugation of serum antibody. The immune serum was separated into immunoglobulin G (IgG) and IgM in a 10 to 30% sucrose density gradient [in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer at pH 7.5] after 18 h of centrifugation in an SW rotor at 36,000 rpm. The agglutination titers of each fraction were measured and the class of the antibody was confirmed by the Ouchterlony (23) and 2-ME treatment methods (13). Preparation and conjugation of antibodies in indirect fluorescent antibody test. As the first antibody, antisera to Formalin-, heat-ethanol-, or periodate-treated cells of strains Y and 2-4 were prepared from mice after the secondary injection of respective treated cells. As the second antibody, rabbit antimouse IgM (RAMIM) and rabbit anti-mouse IgG (RAMIG) were prepared. RAMIM was harvested from a rabbit after three subcutaneous injections with a mixture of mouse IgM antibody prepared with Sephadex G-200 (17) and complete Freund adjuvant and purified by absorption with a 1/20 volume of suckling mouse serum. RAMIG was harvested from a rabbit after several subcutaneous injections with a mixture of mouse IgG antibody prepared by diethylaminoethyl-cellulose (17) and complete Freund adjuvant. The specificities of RAMIM and RAMIG were tested by immunoelectrophoresis. Fluorescent antibody conjugates were prepared by the procedure of Goldstein et al. (11). RAMIM and RAMIG were precipitated with 33% saturated ammonium sulfate and conjugated with fluorescein isothiocyanate (FITC). The fluorescein-to-protein ratio of RAMIM or RAMIG was 1.4 or 1.3, respectively.
INFECT. IMMUN.
Indirect fluorescent antibody test. The smears of Formalin-treated strain Y or 2-4 and diluted antiserum to Formalin-, heat-ethanol-, or periodatetreated cells of strain Y or 2-4 were allowed to react for 30 min at 370C in the moist chamber. The smears were washed three times with PBS (pH 7.2) and allowed to react with FITC-conjugated RAMIM or RAMIG for 30 min at 37°C in the moist chamber. The stained smears were washed three times with PBS (pH 7.2) and examined with an Olympus fluorescent microscope. These were controlled by conducting parallel tests in which normal mouse sera or antisera absorbed by the respective Formalin-treated cells were used as the first antibody. These controls were not stained.
RESULTS Characteristics of three Bacteroides isolates. The characteristics of three Bacteroides isolates (strains 2-2, Y, and 2-4) from feces and cecal contents of ICR and C3H/He mice are summarized in Table 1. The three strains appeared to represent different species since they differed in their ability to ferment certain carbohydrates, to hydrolyze esculin and starch, to coagulate milk, to produce indole, and to liquefy gelatin. According to the Virginia Polytechnic Institute Anaerobe Laboratory Manual (15), strain 2-2 was closely akin to Bacteroides distasonis, except that milk was not coagulated. Strain Y was similar to B. fragilis but did not ferment maltose and did not hydrolyze esculin. Strain 2-4 resembled B. thetaiotaomicron, except that galactose and maltose were not fermented and gelatin was liquefied. Antibody response to three Bacteroides isolates. The primary and secondary antibody responses in C57BL/6 and C3H/He mice after intraperitoneal injections of strains 2-2, Y, and 2-4 treated with Formalin were studied on the basis of agglutination tests (Fig. 1-3). Only a few serum agglutinin titers could be detected in both the primary and secondary responses to Formalin-treated cells of strain 2-2, but a small amount of 2-ME-resistant antibody production occurred (Fig. 1A and B). On the other hand, in response to Formalin-treated cells of strain Y, a few serum agglutinin titers could be detected after the primary injection and a large amount of serum agglutinin titers could be detected after the secondary injection, but they were totally 2ME-sensitive antibodies (Fig. 2A and B). In contrast, a large amount of 2-ME-resistant agglutinin titers could be observed in both the primary and secondary responses to Formalintreated cells of strain 2-4 (Fig. 3A and B). Although the primary response to strain 2-4 seems to be anamnestic, the presence of preexisting antibodies to this strain was almost never detected.
IMMUNOGENICITY OF BACTEROIDES FROM MICE
VOL. 24, 1979
TABLE 1. Characteristics of Bacteroides isolates Strain
Substrate or test Y
2-2
2-4
Gram stain
-
-
Galactose Amygdalin Arabinose Cellobiose Esculin Fructose Glucose Glycogen Inositol Lactose Maltose Mannitol Mannose Raffinose Rhamnose Ribose Salicin Sorbose Starch Sucrose Trehalose Pyruvate
Wa
A
w
w
w w
-
A
A
A A
A A
A A
A
A
-
w
A A
A A
-
w
A A
A A
w
-
-
41
These were found to be the IgM component by the Ouchterlony method (Fig. 4A). The patterns of immune sera after the secondary injection with similarly treated strain 2-4 displayed 2-MEsensitive peaks toward the bottom of the tube and resistant ones above the mid-portion of the tube. These were considered to be IgM and IgG components on the basis of the Ouchterlony method (Fig. 4B). Differences in the nature of the cell surface antigens of strains Y and 2-4. To clarify the differences in the nature of the cell surface
w w
w A
w A A A
-
w
A
A
Esculin hydrolysis Starch hydrolysis
_b
+
+
_
+
Milk
ce
_
C
Indole production
_d
_
+
Gas from glucose NDe -f Gelatin + ND Catalase KNO3 reduction + +9 Growth in 20% bile a A, Fermented (
INFECTION AND IMMUNITY, Apr. 1979, P. 39-46 0019-9567/79/04-0039/08$02.00/0
Immunogenicity of Bacteroides Isolated from Mice: Relationship Between Immunogenicity and Cell Wall Antigens HISAKO YASUI,* NOBUYOSHI YASUTAKE, AND MAKOTO OHWAKI Yakult Institute for Microbiological Research, Kunitachi, Tokyo 186, Japan Received for publication 5 January 1979
Three different strains of Bacteroides were isolated from feces and cecal contents of mice. The immunogenicity of the strains was determined by measuring the serum agglutinin titers after intraperitoneal antigen injection. There were marked differences in quantity and quality of produced antibodies among the three strains. One strain (2-2) induced low antibody titers in both the primary and secondary responses, and a significant 2-mercaptoethanol (2-ME)-resistant antibody production occurred. Another strain (Y) induced low antibody titers in the primary response and high titers in the secondary response, but 2-MEresistant antibody production did not occur. The third strain (2-4) induced very high antibody titers in both the responses, and a large amount of 2-ME-resistant antibody production occurred. Further, heat-ethanol-treated strain Y induced only immunoglobulin M antibody, but periodate-treated strain Y induced no antibody. Heat-ethanol- or periodate-treated strain 2-4 induced immunoglobulin M or G antibody, respectively. These observations suggest that the surface antigens of the two strains are distinctly different: the antigen of strain Y would be mostly O-antigen, whereas those of strain 2-4 would be O-antigen and protein moieties.
It has been suggested that Bacteroides strains are present at very high population levels in mice ceca (19) and that one species of Bacteroides is poorly immunogenic in mice (4, 9). However, it has recently been reported that a number of different species of Bacteroides are found in the cecal contents of conventional mice (25). It has been reported that one principal factor in the immunogenicity, especially the determination of the antibody class, is the nature of the cell wall constituents (3, 10, 20). In the present studies, we compared the immunogenicity among three different strains of Bacteroides isolated from the feces or cecal contents of mice and investigated certain features of the cell wall constituents, based on agglutination and immunofluorescence tests. MATERIALS AND METHODS Isolation and maintenance of Bacteroides. As soon as ICR and C3H/He mice were killed, samples of feces and cecal contents were removed into the glove box, diluted serially with diluent (22) in several 10-fold steps, and plated in appropriate dilution on modified NBGT medium (21). The composition of the modified NBGT medium was as follows: heart infusion agar, 1,000 ml; brilliant green, 0.001 g; sodium taurocholate, 1 g; L-cysteine-hydrochloride 0.3 g; neomycin, 0.2 g; and horse blood, 50 ml (pH 7.2). The atmosphere in the chamber used in the experiment was a mixture of 7% H2 and 5% C02 in N2. Seven colonies were trans-
ferred, grown in prereduced modified VL medium (1) supplemented with 0.5% glucose, harvested by centrifugation, resuspended in prereduced dispersion medium (skim milk, 1%; sodium glutamate, 2%; resazurin, 0.0001%; and L-cysteine-hydrochloride, 0.05%) (22), and stored at -70°C. The composition of the modified VL medium (per liter of medium) was as follows: salt solution I [KH2PO4, 0.3%; NaCl, 0.6%; (NH4)2SO4, 0.3%; CaCl2, 0.03%; MgSO4, 0.03%], 75 ml; salt solution II (K2HPO4, 0.3%), 75 ml; Trypticase, 10 g; yeast extract, 5 g; beef extract, 2 g; 0.1% resazurin, 1 ml; 8% Na2CO3, 50 ml; 3% L-cysteine-hydrochloride, 10 ml; and 0.1% hemin, 7 ml. The isolates were classified as members of the genus Bacteroicles since they were gram-negative rods, did not grow aerobically, and produced mainly acetic acid and succinic acid and did not produce butyric acid. In addition, each of the seven strains was examined for its ability to ferment various carbohydrates and to hydrolyze starch and esculin (12). The production of indole and coagulation of milk were also tested. The seven isolates were divided into three groups, and the three strains 2-2, Y, and 2-4, representing each group, were used for the experiments. Strain Y was isolated from feces of ICR mice and strains 2-2 and 2-4 were isolated from cecal contents of C3H/He mice. The mice were obtained from the Central Institute for Experimental Animals, Tokyo, Japan, and bred in our laboratories. Preparation of antigens. Three strains were cultivated in prereduced modified VL medium and washed three times with phosphate-buffered saline (PBS). The composition of PBS (per liter) was as follows: NaCl, 8 g; KC1, 0.2 g; Na2HPO4, 1.15 g; 39
40
YASUI, YASUTAKE, AND OHWAKI
KH2PO4, 0.2 g (pH 7.2). Antigens of the three strains were prepared by treatment with 0.3% Formalin at 370C overnight. The somatic antigens of strains Y and 2-4 were prepared by the heat-ethanol method (8). Cells of the strains oxidized with periodate were prepared by the modified Hofstad method (13). The method was as follows. The mixture of 2% bacterial suspension in PBS (pH 7.2) and equal volumes of 0.05 M sodium meta-periodate were incubated in the dark at 40C for 5 days and dialyzed against PBS (pH 7.2). Immunization schedule. Mice were injected intraperitoneally with 1 x 109 Formalin-, heat-ethanol-, or periodate-treated organisms. Male C57BL/6 and C3H/He mice, 6 weeks of age, were used for the immunization. The former mice were obtained from the National Institute of Genetics, Mishima, Japan. The primary response was determined 7 days after the primary injection and the secondary response was determined by agglutination tests at various times after the secondary injection. The results of the agglutinin tests in each point were observed on four to seven mice. Agglutination test. Immune sera with or without 2-mercaptoethanol (2-ME) treatment (13) were diluted twofold by using U-well plastic trays (14), and equal volumes of 5 x 108 Formalin-treated cell suspensions per ml were added. After incubation at 370C for 1 h and at 40C overnight, the patterns of agglutination were read. Treatment of immune sera with 2-ME was performed by adding equal volumes of a 0.2 M solution of 2-ME in PBS (pH 7.2) and keeping the solution at room temperature for 1 h. Ultracentrifugation of serum antibody. The immune serum was separated into immunoglobulin G (IgG) and IgM in a 10 to 30% sucrose density gradient [in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer at pH 7.5] after 18 h of centrifugation in an SW rotor at 36,000 rpm. The agglutination titers of each fraction were measured and the class of the antibody was confirmed by the Ouchterlony (23) and 2-ME treatment methods (13). Preparation and conjugation of antibodies in indirect fluorescent antibody test. As the first antibody, antisera to Formalin-, heat-ethanol-, or periodate-treated cells of strains Y and 2-4 were prepared from mice after the secondary injection of respective treated cells. As the second antibody, rabbit antimouse IgM (RAMIM) and rabbit anti-mouse IgG (RAMIG) were prepared. RAMIM was harvested from a rabbit after three subcutaneous injections with a mixture of mouse IgM antibody prepared with Sephadex G-200 (17) and complete Freund adjuvant and purified by absorption with a 1/20 volume of suckling mouse serum. RAMIG was harvested from a rabbit after several subcutaneous injections with a mixture of mouse IgG antibody prepared by diethylaminoethyl-cellulose (17) and complete Freund adjuvant. The specificities of RAMIM and RAMIG were tested by immunoelectrophoresis. Fluorescent antibody conjugates were prepared by the procedure of Goldstein et al. (11). RAMIM and RAMIG were precipitated with 33% saturated ammonium sulfate and conjugated with fluorescein isothiocyanate (FITC). The fluorescein-to-protein ratio of RAMIM or RAMIG was 1.4 or 1.3, respectively.
INFECT. IMMUN.
Indirect fluorescent antibody test. The smears of Formalin-treated strain Y or 2-4 and diluted antiserum to Formalin-, heat-ethanol-, or periodatetreated cells of strain Y or 2-4 were allowed to react for 30 min at 370C in the moist chamber. The smears were washed three times with PBS (pH 7.2) and allowed to react with FITC-conjugated RAMIM or RAMIG for 30 min at 37°C in the moist chamber. The stained smears were washed three times with PBS (pH 7.2) and examined with an Olympus fluorescent microscope. These were controlled by conducting parallel tests in which normal mouse sera or antisera absorbed by the respective Formalin-treated cells were used as the first antibody. These controls were not stained.
RESULTS Characteristics of three Bacteroides isolates. The characteristics of three Bacteroides isolates (strains 2-2, Y, and 2-4) from feces and cecal contents of ICR and C3H/He mice are summarized in Table 1. The three strains appeared to represent different species since they differed in their ability to ferment certain carbohydrates, to hydrolyze esculin and starch, to coagulate milk, to produce indole, and to liquefy gelatin. According to the Virginia Polytechnic Institute Anaerobe Laboratory Manual (15), strain 2-2 was closely akin to Bacteroides distasonis, except that milk was not coagulated. Strain Y was similar to B. fragilis but did not ferment maltose and did not hydrolyze esculin. Strain 2-4 resembled B. thetaiotaomicron, except that galactose and maltose were not fermented and gelatin was liquefied. Antibody response to three Bacteroides isolates. The primary and secondary antibody responses in C57BL/6 and C3H/He mice after intraperitoneal injections of strains 2-2, Y, and 2-4 treated with Formalin were studied on the basis of agglutination tests (Fig. 1-3). Only a few serum agglutinin titers could be detected in both the primary and secondary responses to Formalin-treated cells of strain 2-2, but a small amount of 2-ME-resistant antibody production occurred (Fig. 1A and B). On the other hand, in response to Formalin-treated cells of strain Y, a few serum agglutinin titers could be detected after the primary injection and a large amount of serum agglutinin titers could be detected after the secondary injection, but they were totally 2ME-sensitive antibodies (Fig. 2A and B). In contrast, a large amount of 2-ME-resistant agglutinin titers could be observed in both the primary and secondary responses to Formalintreated cells of strain 2-4 (Fig. 3A and B). Although the primary response to strain 2-4 seems to be anamnestic, the presence of preexisting antibodies to this strain was almost never detected.
IMMUNOGENICITY OF BACTEROIDES FROM MICE
VOL. 24, 1979
TABLE 1. Characteristics of Bacteroides isolates Strain
Substrate or test Y
2-2
2-4
Gram stain
-
-
Galactose Amygdalin Arabinose Cellobiose Esculin Fructose Glucose Glycogen Inositol Lactose Maltose Mannitol Mannose Raffinose Rhamnose Ribose Salicin Sorbose Starch Sucrose Trehalose Pyruvate
Wa
A
w
w
w w
-
A
A
A A
A A
A A
A
A
-
w
A A
A A
-
w
A A
A A
w
-
-
41
These were found to be the IgM component by the Ouchterlony method (Fig. 4A). The patterns of immune sera after the secondary injection with similarly treated strain 2-4 displayed 2-MEsensitive peaks toward the bottom of the tube and resistant ones above the mid-portion of the tube. These were considered to be IgM and IgG components on the basis of the Ouchterlony method (Fig. 4B). Differences in the nature of the cell surface antigens of strains Y and 2-4. To clarify the differences in the nature of the cell surface
w w
w A
w A A A
-
w
A
A
Esculin hydrolysis Starch hydrolysis
_b
+
+
_
+
Milk
ce
_
C
Indole production
_d
_
+
Gas from glucose NDe -f Gelatin + ND Catalase KNO3 reduction + +9 Growth in 20% bile a A, Fermented (
