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The effect of pH on the production of pertussis toxin by Bordetella pertussis
Journal of Biotechnology, 17 (1991) 189-193 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0168-1656/91/$03.50 ADONIS 016816569100059G
189
BIOTEC 00563
Short Communication
The effect of pH on the production of pertussis toxin by Bordetella pertussis Peter Licari, Larry Winberry and Randall Swartz Biotechnology Engineering Center, Tufts University, Medfora~ and Massachusetts Public Health Biologic Laboratories, Jamaica Plain, Massachusetts, U.S.A. (Received 14 February 1990; revision accepted 30 May 1990)
Summary The production of pertussis toxin by Bordetella pertussis was increased by controlling the pH at 7.0 through the addition of sulfuric acid. The more commonly used hydrochloric acid and Tris buffer were observed to be detrimental to toxin yields. Pertussin toxin; Bordetella pertussis; pH control
Introduction
The current whole cell vaccine for pertussis (whooping cough), although effective in providing immunity, has been associated with many adverse reactions. Thus, there is interest in the development of an acellular vaccine lacking possible reactogenic biomolecules, e.g., lipopolysaccharide endotoxins. However, this development will require substantial yields of pertussis toxin that have yet to be realized. The growth of Bordetella pertussis cells is unusual in that the cells are unable to utilize carbohydrates, pyruvate, lactate and intermediates of glycolysis (Jebb and Tomlinson, 1957; Rowatt, 1957; Parker, 1976). In place of these compounds the organisms use amino acids as their main carbon and energy source. Various studies have shown that glutamic acid is the predominant carbon and energy source for B. pertussis (Lane, 1970; Andorn et al., 1988). Correspondence to." R. Swartz, Biotechnology Engineering Center, 4 Colby Street, Tufts University, Medford, MA 02155, U.S.A.
190 In the utilization of glutamate, the a-amino group is removed and the carbon skeleton is converted to a-ketoglutarate, a metabolic intermediate. The oxidative deamination of glutamate, glutamate + NAD++ H20 = NH~- + NADH + H++ a-ketoglutarate is regulated by the enzyme glutamate dehydrogenase. Many other amino acids are deaminated in a similar fashion. If the enzyme for the direct breakdown of a particular amino acid is absent, degradation occurs through transamination followed by oxidative deamination. The deamination of amino acids by B. pertussis results in an increase in pH due to the reaction of ammonia to ammonium ions in the media. Pirt and Thackeray (1964) demonstrated that certain antigenic components of Yersinia pestis are produced only at pH below 6.9, the optimum being 5.9. It has also been shown that pH plays an important part in the cell wall composition of many Bacillus species (Ellwood and Tempest, 1972), hence one must consider the influence this rise in pH has on pertussis toxin production and secretion.
Materials and Methods
Bordetella pertussis strain SK101, an m o d ( - ) kanamycin resistant strain, was obtained from the Massachusetts Public Health Biologic Laboratories (Boston, MA). The medium was based on that proposed by Stainer and Scholte (1971) with modifications, namely the addition of cyclodextrin and a casein derivative. The growth medium was iron limited and consisted of 1.0 g 1-1 heptakis(2,6-Odimethyl)fl-cyclodextrin, 6.1 g Tris-(hydroxymethyl)aminomethane, 3.0 g Casamino Acids (Difco certified), 17.0 g monosodium glutamate, 2.5 g NaC1, 0.5 g KC1, 0.1 g MgC12 • 6H20, 0.02 g CaC12, 0.24 g L-proline, 0.004 g niacin, 0.001 g FeSO4 • 7H20, 0.04 g L-cysteine hydrochloride monohydrate, 0.15 g glutathione, 0.4 g ascorbic acid, and 0.06 g kanamycin sulfate per liter. The medium was filter sterilized. Cultivation was in a 2 1 BioFlo unit (New Brunswick Scientific). Temperature was controlled at 36°C. pH was controlled by the addition of 2 M H2SO4. Air flow rate was maintained at 0.8 SLPM by the use of a Tylan mass flow controller while the agitation was 500 rpm. Cell growth was monitored by the optical density at 530 nm with a Beckman Spectrophotometer (model DU-50). Samples were removed and centrifuged at 15,000 rpm for 10 rain. The supernatant was assayed for pertussis toxin. Dry cell weight was analyzed with the remaining biomass. Viable colonies were determined by plating samples on Bordet-Gengou blood agar plates (BG agar base, glycerine, defibrinated sheep blood). Colonies were counted after 3 days incubation at 37°C. Culture supernatant samples were assayed for pertussis toxin by a fetuin ELISA. The assay consists of sensitizing ELISA plates with fetuin, a bovine serum glycoprotein, to which toxin selectively binds. Mouse monoclonal antibodies, specific for the S1 subunit of the toxin are then allowed to bind to the pertussis toxin. This is
191
followed by the addition of alkaline phosphatase-labeled goat anti-mouse IgG. After sufficient color develops from the addition of p-nitrophenylphosphate, the reaction is stopped with 1.0 M NaOH and the OD405 recorded. The optical densities are converted to a toxin concentration by the use of a standard curve generated with purified pertussis toxin.
Results and Discussion To date, little information has been provided concerning pH changes in the cultivation of B. pertussis, pH is often allowed to increase as the organisms grow (Andorn et al., 1988), the only form of pH control being provided by the incorporation of "Iris buffer into the media. In a typical batch culture, pH increases from 7.6 to a final pH of 9.2. In this work, pH was maintained at 7.6 for batch cultures by the addition of hydrochloric acid. The value of 7.6 was selected based on values cited in literature (e.g. Stainer and Scholte, 1971; Andorn et al., 1988). The pH control resulted in no noticeable differences in cell density as measured by optical density or in the growth rates of the organism. However, at a controlled pH of 7.6 the number of CFU m l - t was 3.3 x 10 t° as compared to 2.0 X 101° in the absence of pH control. The growth rates were similar; however, differences resulted from a decline in numbers late in
-15
E 10
-
.~_
8"
k-
"1o
o.
o Q..
.
6.4
.
.
.
6.6
,
6.8
.
.
,
7.0
ell
7.2
.
.
0
7.4
7.6
Fig. 1. The effect of pH on cell density and toxin production in continuous culture at a dilution rate of 0.052 h-1. Steady state optical density (e) and toxin production (O) expressed in mg 1-1 are plotted against pH.
192 the bioprocess. Without p H control the expression level of toxin was 4 mg 1-1. With p H control (7.6) the concentration of toxin was 12 mg 1-1. Relatively large amounts of chloride ions accumulate in the culture from the use of HC1. Based on the successful use in other microbial processes, sulfuric acid was studied as an alternative. The use of sulfuric acid in place of hydrochloric acid resulted in increased toxin production. Considering the elemental balance of what the media provides and what the cells may require, the addition of sulfuric acid may also be beneficial in providing required sulfur to the culture. It should be noted that strains that do not possess the m o d ( - ) phenotype may down-regulate pertussis toxin production in the presence of excess sulfate. The influence of the high level of Tris (6.2 g 1-1) in the Stainer and Scholte media was investigated. In a batch culture with Tris reduced to 1.5 g 1-1, coupled with sulfuric acid addition for pH control at 7.6, there was a dramatic increase in toxin levels. Under these conditions the final concentration of pertussis toxin was 23 mg 1-1. The effects of various pH values were investigated by operating a chemostat at a dilution rate of 0.052 h -1 while altering the pH in increments. Steady states were obtained for pH values ranging from 7.6 to 6.5. The cell densities (OD530) and toxin levels as a function of p H for this experiment are presented in Fig. 1. The optimal pH for cell mass is 6.8. A dramatic decrease in cell density is seen for pH values greater and less than this value. Toxin production is strongly associated with the hydrogen ion concentration. The optimal pH set point for the production of the protein is 7.0. For p H values below 7.0 there is a sharp decrease in the amount of toxin produced. Between 7.0 and 7.4 there is a slight, but noticeable, decrease. Comparing the set point of 7.6 previously used, toxin production is approximately doubled at 7.0. In part, this is a result of the higher cell density at p H 7.0. The above data are for a chemostat operating at a dilution rate below /~max" A batch bioprocess with p H control at 7.0 verified the trends stated above. The final biomass was slightly greater than at 7.6 (OD530 of 8.4 versus 7.5). As expected the toxin production was significantly higher: 30 mg 1-1 as assayed by ELISA.
Conclusion In summary, pertussis toxin yields by the culture of B. pertussis are enhanced by controlling the p H at 7.0 by the addition of sulfuric acid and the omission of Tris buffer in the medium.
Acknowledgements This work was funded by the Massachusetts Research Institute, Boston, MA. Special thanks to Dr. George Siber, Dr. Larry Winberry, Ms. Leslie Wetterlow and Dr. Elizabeth Eubanks and the staff at the Massachusetts' Public Health Laboratories for their assistance.
193
References Andorn, N., Zhang, Y., Sekura, R. and Shiloach, J. (1988) Large scale cultivation of Bordetella pertussis in submerged culture for production of pertussis toxin. Appl. Microbiol. Biotechnol. 28, 356-360. Ellwood, D.C. and Tempest, D.W. (1972) Influence of culture pH on the content and composition of teichoic acids in the walls of Bacillus subtilis. J. Gen. Microbiol. 73, 395. Jebb, W.H. and Tomlinson, A.H. (1957) The minimal amino acid requirements of Haemophilus pertussis. J. Gen. Microbiol. 17, 59-63. Lane, A.G. (1970) Use of glutamic acid to supplement fluid medium for cultivation of Bordetella pertussis. Appl. Microbiol. 19, 512-520. Parker, C.D. (1976) Role of the genetics and physiology of Bordetella pertussis in the production of vaccine and the study of the host-parasite relationship in pertussis. Adv. Appl. Microbiol. 20, 27-42. Pirt, S.J. and Thackeray, E.J. (1964) Environmental influences on the growth of ERK mammalian cells in monolayer coverage. Exp. Cell Res. 33, 396. Rowatt, E. (1957) Some factors affecting the growth of Bordetella pertussis. J. Gen. Microbiol. 17, 279-296. Stainer, D.W. and Scholte, M.J. (1971) A simple chemically defined medium for the production of phase I Bordetella pertussis. J. Gen. Microbiol. 63, 211-220.
189
BIOTEC 00563
Short Communication
The effect of pH on the production of pertussis toxin by Bordetella pertussis Peter Licari, Larry Winberry and Randall Swartz Biotechnology Engineering Center, Tufts University, Medfora~ and Massachusetts Public Health Biologic Laboratories, Jamaica Plain, Massachusetts, U.S.A. (Received 14 February 1990; revision accepted 30 May 1990)
Summary The production of pertussis toxin by Bordetella pertussis was increased by controlling the pH at 7.0 through the addition of sulfuric acid. The more commonly used hydrochloric acid and Tris buffer were observed to be detrimental to toxin yields. Pertussin toxin; Bordetella pertussis; pH control
Introduction
The current whole cell vaccine for pertussis (whooping cough), although effective in providing immunity, has been associated with many adverse reactions. Thus, there is interest in the development of an acellular vaccine lacking possible reactogenic biomolecules, e.g., lipopolysaccharide endotoxins. However, this development will require substantial yields of pertussis toxin that have yet to be realized. The growth of Bordetella pertussis cells is unusual in that the cells are unable to utilize carbohydrates, pyruvate, lactate and intermediates of glycolysis (Jebb and Tomlinson, 1957; Rowatt, 1957; Parker, 1976). In place of these compounds the organisms use amino acids as their main carbon and energy source. Various studies have shown that glutamic acid is the predominant carbon and energy source for B. pertussis (Lane, 1970; Andorn et al., 1988). Correspondence to." R. Swartz, Biotechnology Engineering Center, 4 Colby Street, Tufts University, Medford, MA 02155, U.S.A.
190 In the utilization of glutamate, the a-amino group is removed and the carbon skeleton is converted to a-ketoglutarate, a metabolic intermediate. The oxidative deamination of glutamate, glutamate + NAD++ H20 = NH~- + NADH + H++ a-ketoglutarate is regulated by the enzyme glutamate dehydrogenase. Many other amino acids are deaminated in a similar fashion. If the enzyme for the direct breakdown of a particular amino acid is absent, degradation occurs through transamination followed by oxidative deamination. The deamination of amino acids by B. pertussis results in an increase in pH due to the reaction of ammonia to ammonium ions in the media. Pirt and Thackeray (1964) demonstrated that certain antigenic components of Yersinia pestis are produced only at pH below 6.9, the optimum being 5.9. It has also been shown that pH plays an important part in the cell wall composition of many Bacillus species (Ellwood and Tempest, 1972), hence one must consider the influence this rise in pH has on pertussis toxin production and secretion.
Materials and Methods
Bordetella pertussis strain SK101, an m o d ( - ) kanamycin resistant strain, was obtained from the Massachusetts Public Health Biologic Laboratories (Boston, MA). The medium was based on that proposed by Stainer and Scholte (1971) with modifications, namely the addition of cyclodextrin and a casein derivative. The growth medium was iron limited and consisted of 1.0 g 1-1 heptakis(2,6-Odimethyl)fl-cyclodextrin, 6.1 g Tris-(hydroxymethyl)aminomethane, 3.0 g Casamino Acids (Difco certified), 17.0 g monosodium glutamate, 2.5 g NaC1, 0.5 g KC1, 0.1 g MgC12 • 6H20, 0.02 g CaC12, 0.24 g L-proline, 0.004 g niacin, 0.001 g FeSO4 • 7H20, 0.04 g L-cysteine hydrochloride monohydrate, 0.15 g glutathione, 0.4 g ascorbic acid, and 0.06 g kanamycin sulfate per liter. The medium was filter sterilized. Cultivation was in a 2 1 BioFlo unit (New Brunswick Scientific). Temperature was controlled at 36°C. pH was controlled by the addition of 2 M H2SO4. Air flow rate was maintained at 0.8 SLPM by the use of a Tylan mass flow controller while the agitation was 500 rpm. Cell growth was monitored by the optical density at 530 nm with a Beckman Spectrophotometer (model DU-50). Samples were removed and centrifuged at 15,000 rpm for 10 rain. The supernatant was assayed for pertussis toxin. Dry cell weight was analyzed with the remaining biomass. Viable colonies were determined by plating samples on Bordet-Gengou blood agar plates (BG agar base, glycerine, defibrinated sheep blood). Colonies were counted after 3 days incubation at 37°C. Culture supernatant samples were assayed for pertussis toxin by a fetuin ELISA. The assay consists of sensitizing ELISA plates with fetuin, a bovine serum glycoprotein, to which toxin selectively binds. Mouse monoclonal antibodies, specific for the S1 subunit of the toxin are then allowed to bind to the pertussis toxin. This is
191
followed by the addition of alkaline phosphatase-labeled goat anti-mouse IgG. After sufficient color develops from the addition of p-nitrophenylphosphate, the reaction is stopped with 1.0 M NaOH and the OD405 recorded. The optical densities are converted to a toxin concentration by the use of a standard curve generated with purified pertussis toxin.
Results and Discussion To date, little information has been provided concerning pH changes in the cultivation of B. pertussis, pH is often allowed to increase as the organisms grow (Andorn et al., 1988), the only form of pH control being provided by the incorporation of "Iris buffer into the media. In a typical batch culture, pH increases from 7.6 to a final pH of 9.2. In this work, pH was maintained at 7.6 for batch cultures by the addition of hydrochloric acid. The value of 7.6 was selected based on values cited in literature (e.g. Stainer and Scholte, 1971; Andorn et al., 1988). The pH control resulted in no noticeable differences in cell density as measured by optical density or in the growth rates of the organism. However, at a controlled pH of 7.6 the number of CFU m l - t was 3.3 x 10 t° as compared to 2.0 X 101° in the absence of pH control. The growth rates were similar; however, differences resulted from a decline in numbers late in
-15
E 10
-
.~_
8"
k-
"1o
o.
o Q..
.
6.4
.
.
.
6.6
,
6.8
.
.
,
7.0
ell
7.2
.
.
0
7.4
7.6
Fig. 1. The effect of pH on cell density and toxin production in continuous culture at a dilution rate of 0.052 h-1. Steady state optical density (e) and toxin production (O) expressed in mg 1-1 are plotted against pH.
192 the bioprocess. Without p H control the expression level of toxin was 4 mg 1-1. With p H control (7.6) the concentration of toxin was 12 mg 1-1. Relatively large amounts of chloride ions accumulate in the culture from the use of HC1. Based on the successful use in other microbial processes, sulfuric acid was studied as an alternative. The use of sulfuric acid in place of hydrochloric acid resulted in increased toxin production. Considering the elemental balance of what the media provides and what the cells may require, the addition of sulfuric acid may also be beneficial in providing required sulfur to the culture. It should be noted that strains that do not possess the m o d ( - ) phenotype may down-regulate pertussis toxin production in the presence of excess sulfate. The influence of the high level of Tris (6.2 g 1-1) in the Stainer and Scholte media was investigated. In a batch culture with Tris reduced to 1.5 g 1-1, coupled with sulfuric acid addition for pH control at 7.6, there was a dramatic increase in toxin levels. Under these conditions the final concentration of pertussis toxin was 23 mg 1-1. The effects of various pH values were investigated by operating a chemostat at a dilution rate of 0.052 h -1 while altering the pH in increments. Steady states were obtained for pH values ranging from 7.6 to 6.5. The cell densities (OD530) and toxin levels as a function of p H for this experiment are presented in Fig. 1. The optimal pH for cell mass is 6.8. A dramatic decrease in cell density is seen for pH values greater and less than this value. Toxin production is strongly associated with the hydrogen ion concentration. The optimal pH set point for the production of the protein is 7.0. For p H values below 7.0 there is a sharp decrease in the amount of toxin produced. Between 7.0 and 7.4 there is a slight, but noticeable, decrease. Comparing the set point of 7.6 previously used, toxin production is approximately doubled at 7.0. In part, this is a result of the higher cell density at p H 7.0. The above data are for a chemostat operating at a dilution rate below /~max" A batch bioprocess with p H control at 7.0 verified the trends stated above. The final biomass was slightly greater than at 7.6 (OD530 of 8.4 versus 7.5). As expected the toxin production was significantly higher: 30 mg 1-1 as assayed by ELISA.
Conclusion In summary, pertussis toxin yields by the culture of B. pertussis are enhanced by controlling the p H at 7.0 by the addition of sulfuric acid and the omission of Tris buffer in the medium.
Acknowledgements This work was funded by the Massachusetts Research Institute, Boston, MA. Special thanks to Dr. George Siber, Dr. Larry Winberry, Ms. Leslie Wetterlow and Dr. Elizabeth Eubanks and the staff at the Massachusetts' Public Health Laboratories for their assistance.
193
References Andorn, N., Zhang, Y., Sekura, R. and Shiloach, J. (1988) Large scale cultivation of Bordetella pertussis in submerged culture for production of pertussis toxin. Appl. Microbiol. Biotechnol. 28, 356-360. Ellwood, D.C. and Tempest, D.W. (1972) Influence of culture pH on the content and composition of teichoic acids in the walls of Bacillus subtilis. J. Gen. Microbiol. 73, 395. Jebb, W.H. and Tomlinson, A.H. (1957) The minimal amino acid requirements of Haemophilus pertussis. J. Gen. Microbiol. 17, 59-63. Lane, A.G. (1970) Use of glutamic acid to supplement fluid medium for cultivation of Bordetella pertussis. Appl. Microbiol. 19, 512-520. Parker, C.D. (1976) Role of the genetics and physiology of Bordetella pertussis in the production of vaccine and the study of the host-parasite relationship in pertussis. Adv. Appl. Microbiol. 20, 27-42. Pirt, S.J. and Thackeray, E.J. (1964) Environmental influences on the growth of ERK mammalian cells in monolayer coverage. Exp. Cell Res. 33, 396. Rowatt, E. (1957) Some factors affecting the growth of Bordetella pertussis. J. Gen. Microbiol. 17, 279-296. Stainer, D.W. and Scholte, M.J. (1971) A simple chemically defined medium for the production of phase I Bordetella pertussis. J. Gen. Microbiol. 63, 211-220.