Vol. 25, No. 2
INFECTION AND IMMUNITY, Aug. 1979, p. 764-767 0019-9567/79/08-0764/04$02.00/0
Fimbrial Hemagglutinin in Stationary and Shake Cultures of Bordetella pertussis HIDEO ARAI AND J. J. MUNOZ* National Institute ofAllergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840 Received for publication 13 April 1979
Bordetella pertussis produced hemagglutinin in stationary cultures; in cultures kept under constant shaking, hemagglutinin was found only during the first 48 h of incubation but not after 3 to 5 days. The type of medium had a pronounced effect on production of hemagglutinin. Strain differences in ability to produce hemagglutinin were also detected. For many years it has been accepted that the hemagglutinin (HA) described by Keogh et al. (2) was not an important substance in protecting mice from intracerebral infection with Bordetella pertussis (4). This belief has come about as a result of observations made by Masry (3), Thiele (9), and Pillemer (6). These workers reported that the amount of HA found in fractions from B. pertussis cells did not correlate with mouse-protective activity (6); HA could be removed by absorption with erythrocytes, leaving mouse-protective activity in the supernatant (9), and semipurified HA or its antibody did not protect mice from intracerebral infection (3). Sato et al. (7) reopened this problem when they reported that HA correlated with mouse-protective activity of fractions. Furthermore, they found (8) that a purified preparation of lymphocytosis-promoting factor contained two HAs. One of these, called fimbrial HA, was a nontoxic filamentous protein (2 by 40 nm) under the electron microscope that protected mice from infection with B. pertussis, and antibodies against it could easily be produced in rabbits. The other HA (lymphocyte-promoting factor HA) was associated with a toxic spherical protein (about 6 nm in diameter), that induced lymphocytosis and histamine hypersensitivity in mice but did not protect mice from infection at the doses tested (1; L. I. Iron and A. P. MacLennan, Proc. Int. Symp. Pertussis, in press). Under the cultural conditions used at the Rocky Mountain Laboratory (4), fimbrial HA could be detected only during the first 24 to 48 h of incubation (5), whereas Sato et al. (8), employing a different strain and cultural conditions, have always found high titers (1/160 or higher) of HA in culture supernatants obtained from 5-day-old cultures. The present work was done to resolve this difference, and it was found that, when B.
pertussis was incubated under stationary conditions and in the medium of Sato and Arai (7), high titers of HA were produced, whereas in cultures kept under constant shaking no HA was found after 3 days of incubation. Differences were also obtained between the types of media used. Two strains of B. pertussis were used: the agglutinogen type 1, 3, and 6 strain 3779BL2S4 which has been used in the studies at the Rocky Mountain Laboratory (5), and the agglutinogen type 1, 2, and 4 strain Tohama I used by Sato and Arai and Sato et al. (7, 8) in their studies. With only minor modifications, the liquid medium employed by Sato et al. (8), referred to here as SAZ medium, and the medium previously used by Munoz et al. (5), referred to as RML medium, were employed. The composition of these media is listed in Table 1. These media were individually studied under two sets of experimental conditions. In one set, 200 ml of SAZ or RML media was used per 1-liter Blake bottle and seeded with a 24-h-old Bordet-Gengou agar culture to give a final cell concentration of 9 x 107 cells per ml of medium. The bottles (broad side on shaker) were then incubated at 350C under constant shaking (120 cycles per min, with a displacement of 1.2 cm/cycle). In another set of conditions, 100 ml of media was placed in 1liter Blake bottles, and after seeding (9 x 107 cells per ml of medium) with 2 ml of the same suspension used above in 100 ml of medium, the bottles were incubated (broad side on shelves) at 350C as stationary cultures. At various intervals thereafter, two bottles of each medium and strain were removed, and the cells were sedimented by centrifugation and suspended in 1/15 M sodium phosphate-buffered saline to their original volume. Both cells and culture supernatants were assayed for HA activity by using
764
VOL. 25, 1979
NOTES
765
HA, and to lower titers than in the SAZ medium. Under stationary incubation, the cell growth in Amt (g/liter of meboth media was inferior to that obtained with dium) Substance shaking conditions, but contrary to shaking culRML SAZ tures, the SAZ medium supported heavier growth than the RML medium (Fig. 2, bottom 14 Casamino Acids (technical 10 grade; Difco) frame). The HA titers in cells from stationary 1 Soluble starch (indicator 1.5 cultures were similar to those in culture supergrade; Difco) natants and for the purpose of simplicity are not Niacin 0.02 0.03 given in Fig. 2. 0.01 Reduced glutathione 0.01 The broken lines given in Fig. 1 and 2 indicate MgCl2.6H20 0.1 0.4 the results of HA tests on stationary cultures 0.005 CaCl2 0.01 that had been transferred to incubation under 0.001 FeSO4. 7H20 0.01 constant shaking. In all cases, the HA activity 0.0005 CuS04 *5H20 0.00075 Trizma base 6 disappeared within 24 to 48 h after transferring 0.607 Yeast extract (Difco) 3 to incubation under shaking conditions. When a 0.5 KH2PO4 solution of fimbrial HA was placed at 37°C and DL-Glutamic acid 0.2 shaken overnight, complete destruction of its Cysteine hydrochloride 0.03 hemagglutinating activity took place. It was important to know whether cultures a Sterilization was by autoclaving at 15 lb (ca. 6.8 kg) for 30 min. The reduced glutathione and the FeSO4- kept under constant shaking had inactive HA TABLE 1. Composition of RML and SAZ media'
7H20 were individually dissolved in water, sterilized by filtration, and then added aseptically to the sterilized SAZ medium. In the case of the RML medium, these substances were added before autoclaving. When water was added to a total volume of 1 liter and the pH was adjusted, the pH's of RML and SAZ were 7.5 and 7.1, respectively.
washed chicken erythrocytes (0.5% suspension). Microtiter plates with round-bottom wells were used, and 0.05-ml microtiter diluters were used to make the serial dilutions of cells and culture supernatants. After adding 0.05 ml of erythrocyte suspension to each well and mixing, we allowed the plates to stand at room temperature for 2 to 4 h before recording the results. The results showed that, when B. pertussis strain 3779BL2S4 was grown under constant shaking in RML medium, HA could not be detected either in cells or in culture supernatants (Fig. 1). Strain Tohama I grown under the same conditions produced very low titers of HA in cells and culture supernatant only during the first 24 h of incubation. Both strains grown in SAZ medium and incubated under constant shaking produced very low titers of HA in cells and culture supernatants during the 24-h period of incubation (Fig. 1). The cell growth of the Tohama I strain in RML medium incubated under constant shaking was excellent, and that of 3779BL2S4 was very good (Fig. 1, bottom frame). In SAZ medium, the growth of both strains under this condition of incubation was not as profuse. The results with stationary cultures were strikingly different. Good titers of HA were obtained in cells and culture supernatants of either strain grown in SAZ medium (Fig. 2). In RML medium, however, only the Tohama I strain produced
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INFECTION AND IMMUNITY, Aug. 1979, p. 764-767 0019-9567/79/08-0764/04$02.00/0
Fimbrial Hemagglutinin in Stationary and Shake Cultures of Bordetella pertussis HIDEO ARAI AND J. J. MUNOZ* National Institute ofAllergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840 Received for publication 13 April 1979
Bordetella pertussis produced hemagglutinin in stationary cultures; in cultures kept under constant shaking, hemagglutinin was found only during the first 48 h of incubation but not after 3 to 5 days. The type of medium had a pronounced effect on production of hemagglutinin. Strain differences in ability to produce hemagglutinin were also detected. For many years it has been accepted that the hemagglutinin (HA) described by Keogh et al. (2) was not an important substance in protecting mice from intracerebral infection with Bordetella pertussis (4). This belief has come about as a result of observations made by Masry (3), Thiele (9), and Pillemer (6). These workers reported that the amount of HA found in fractions from B. pertussis cells did not correlate with mouse-protective activity (6); HA could be removed by absorption with erythrocytes, leaving mouse-protective activity in the supernatant (9), and semipurified HA or its antibody did not protect mice from intracerebral infection (3). Sato et al. (7) reopened this problem when they reported that HA correlated with mouse-protective activity of fractions. Furthermore, they found (8) that a purified preparation of lymphocytosis-promoting factor contained two HAs. One of these, called fimbrial HA, was a nontoxic filamentous protein (2 by 40 nm) under the electron microscope that protected mice from infection with B. pertussis, and antibodies against it could easily be produced in rabbits. The other HA (lymphocyte-promoting factor HA) was associated with a toxic spherical protein (about 6 nm in diameter), that induced lymphocytosis and histamine hypersensitivity in mice but did not protect mice from infection at the doses tested (1; L. I. Iron and A. P. MacLennan, Proc. Int. Symp. Pertussis, in press). Under the cultural conditions used at the Rocky Mountain Laboratory (4), fimbrial HA could be detected only during the first 24 to 48 h of incubation (5), whereas Sato et al. (8), employing a different strain and cultural conditions, have always found high titers (1/160 or higher) of HA in culture supernatants obtained from 5-day-old cultures. The present work was done to resolve this difference, and it was found that, when B.
pertussis was incubated under stationary conditions and in the medium of Sato and Arai (7), high titers of HA were produced, whereas in cultures kept under constant shaking no HA was found after 3 days of incubation. Differences were also obtained between the types of media used. Two strains of B. pertussis were used: the agglutinogen type 1, 3, and 6 strain 3779BL2S4 which has been used in the studies at the Rocky Mountain Laboratory (5), and the agglutinogen type 1, 2, and 4 strain Tohama I used by Sato and Arai and Sato et al. (7, 8) in their studies. With only minor modifications, the liquid medium employed by Sato et al. (8), referred to here as SAZ medium, and the medium previously used by Munoz et al. (5), referred to as RML medium, were employed. The composition of these media is listed in Table 1. These media were individually studied under two sets of experimental conditions. In one set, 200 ml of SAZ or RML media was used per 1-liter Blake bottle and seeded with a 24-h-old Bordet-Gengou agar culture to give a final cell concentration of 9 x 107 cells per ml of medium. The bottles (broad side on shaker) were then incubated at 350C under constant shaking (120 cycles per min, with a displacement of 1.2 cm/cycle). In another set of conditions, 100 ml of media was placed in 1liter Blake bottles, and after seeding (9 x 107 cells per ml of medium) with 2 ml of the same suspension used above in 100 ml of medium, the bottles were incubated (broad side on shelves) at 350C as stationary cultures. At various intervals thereafter, two bottles of each medium and strain were removed, and the cells were sedimented by centrifugation and suspended in 1/15 M sodium phosphate-buffered saline to their original volume. Both cells and culture supernatants were assayed for HA activity by using
764
VOL. 25, 1979
NOTES
765
HA, and to lower titers than in the SAZ medium. Under stationary incubation, the cell growth in Amt (g/liter of meboth media was inferior to that obtained with dium) Substance shaking conditions, but contrary to shaking culRML SAZ tures, the SAZ medium supported heavier growth than the RML medium (Fig. 2, bottom 14 Casamino Acids (technical 10 grade; Difco) frame). The HA titers in cells from stationary 1 Soluble starch (indicator 1.5 cultures were similar to those in culture supergrade; Difco) natants and for the purpose of simplicity are not Niacin 0.02 0.03 given in Fig. 2. 0.01 Reduced glutathione 0.01 The broken lines given in Fig. 1 and 2 indicate MgCl2.6H20 0.1 0.4 the results of HA tests on stationary cultures 0.005 CaCl2 0.01 that had been transferred to incubation under 0.001 FeSO4. 7H20 0.01 constant shaking. In all cases, the HA activity 0.0005 CuS04 *5H20 0.00075 Trizma base 6 disappeared within 24 to 48 h after transferring 0.607 Yeast extract (Difco) 3 to incubation under shaking conditions. When a 0.5 KH2PO4 solution of fimbrial HA was placed at 37°C and DL-Glutamic acid 0.2 shaken overnight, complete destruction of its Cysteine hydrochloride 0.03 hemagglutinating activity took place. It was important to know whether cultures a Sterilization was by autoclaving at 15 lb (ca. 6.8 kg) for 30 min. The reduced glutathione and the FeSO4- kept under constant shaking had inactive HA TABLE 1. Composition of RML and SAZ media'
7H20 were individually dissolved in water, sterilized by filtration, and then added aseptically to the sterilized SAZ medium. In the case of the RML medium, these substances were added before autoclaving. When water was added to a total volume of 1 liter and the pH was adjusted, the pH's of RML and SAZ were 7.5 and 7.1, respectively.
washed chicken erythrocytes (0.5% suspension). Microtiter plates with round-bottom wells were used, and 0.05-ml microtiter diluters were used to make the serial dilutions of cells and culture supernatants. After adding 0.05 ml of erythrocyte suspension to each well and mixing, we allowed the plates to stand at room temperature for 2 to 4 h before recording the results. The results showed that, when B. pertussis strain 3779BL2S4 was grown under constant shaking in RML medium, HA could not be detected either in cells or in culture supernatants (Fig. 1). Strain Tohama I grown under the same conditions produced very low titers of HA in cells and culture supernatant only during the first 24 h of incubation. Both strains grown in SAZ medium and incubated under constant shaking produced very low titers of HA in cells and culture supernatants during the 24-h period of incubation (Fig. 1). The cell growth of the Tohama I strain in RML medium incubated under constant shaking was excellent, and that of 3779BL2S4 was very good (Fig. 1, bottom frame). In SAZ medium, the growth of both strains under this condition of incubation was not as profuse. The results with stationary cultures were strikingly different. Good titers of HA were obtained in cells and culture supernatants of either strain grown in SAZ medium (Fig. 2). In RML medium, however, only the Tohama I strain produced
SAZ Medium
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