Appl Microbiol Biotechnol (1990) 33:389-394
Applied Microbiology Biotechnology © Springer-Verlag 1990
Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae Michele van de Walle, Raphael Fass*, and Joseph Shiloach Biotechnology Unit, LCDB, NIDDK, NIH, Building 6, Room B1-33, Bethesda, MD 20892, USA Received 19 September 1989/Accepted 6 February 1990
Summary. The B subunit (CTB) of cholera toxin (CT) can be used as a carrier protein for conjugate vaccines designed to elicit antipolysaccharide antibodies. A defined medium, AGM4, was designed to grow a highproducing mutant of Vibrio cholerae expressing only the B subunit of CT: V. cholerae 0395-NI. AGM4 contains four amino acids, asparagine, glutamic acid, arginine and serine, salts and a trace element solution. The carbon source is glucose. The fermentations performed in AGM4 indicated that CTB production paralleled the growth of the organism but that there was a maximal release of CTB during the stationary phase. There was a clear optimum of productivity at p H 8.0 and 30 ° C. The p H had an influence on CTB production and not only on its release. Analysis of the amino acids present in the medium showed a correlation between their consumption rates and CTB productivity.
Introduction Vibrio cholerae, a Gram-negative pathogenic bacterium, colonizes the upper intestine and produces an exotoxin, cholera toxin (CT), which is responsible for the diarrheal symptoms of the disease. CT is composed of two subunits: the A subunit (mol-wt. 27000) which has ADP ribosyl transferase activity, and the B subunit (CTB) which contains five identical polypeptides (mol. wt. 11000 each) and forms the mammalian cell receptor binding domain. The B subunit has high affinity for the oligosaccharide moiety of the ganglioside GM1 (Finkelstein and LoSpalluto 1969, 1970; Holmgren 1973). The A and B subunits are encoded by the etxA and e t x B genes regulated by the product o f the t o x R gene (Miller et al. 1987; Miller and Mekalanos 1984, 1985; Peterson and Mekalanos 1988). * Permanent address: Department of Biotechnology, IIBR, Ness
Ziona 70450, Israel Offprint requests to: J. Shiloach
The neutralizing activity of serum antibodies (antitoxin) is largely directed towards the B subunit. CT antitoxin also neutralizes the toxic activity of the structurally related and widely distributed cholera-like enterotoxin of Eseheriehia coli (de Aizpurua and RussellJones 1988; Finkelstein et al. 1987; Finkelstein 1973,. 1984). The B subunit of CT has been shown to be an immunogenic carrier protein for conjugate vaccines designed to elicit anti-polysaccharide antibodies (McKenzie and Halsey 1984; Schneerson et al. 1987; Taylor et al. 1988). The use of the B subunit, rather than the holotoxin in preparation of human vaccines, avoids the problem of holotoxin toxicity without loss of immunogenicity (Sanchez and Holmgren 1989). The procedure used to prepare the B subunit from CT includes denaturing conditions and may cause aggregation and loss of solubility of the B subunit. Accordingly, we have studied a high-producing mutant of V. eholerae expressing only the B subunit of CT to prepare CTB. In order to obtain large amounts of CTB we studied the optimization of its production in a defined medium using a computerized fermentation system. A defined medium could simplify and facilitate the recovery and purification of the toxin produced. The influence of the environmental parameters on the production kinetics of CTB was analyzed.
Materials and methods Microorganisms. Vibrio cholerae 0395-NI, which carries a deletion in the etxA gene and produces only the B subunit of the toxin
(Mekalanos et al. 1983; Taylor et al. 1987), was used. This bacterium was maintained in Luria-Bertani (LB) medium containing 15% glycerol at - 20° C. Media. Several synthetic media were tested (Table 1). The solutions of amino acids, salts, metals and sugars were prepared and autoclaved separately. Fermentations. Fermentations were performed at 30° C or as indi-
cated. To avoid the shear effect of agitation on the bacterial cells, two types of low shear fermentors were used: a 4-1 kluyver fermentor and a 3-1 air-lift fermentor (Wood and Thompson 1987). Further experiments were carried out in a 5-1 bench-top fermentor
390 Table 1. Influence of different media on cholera toxin subunit B (CTB) production Media LB a Nutrients (g. 1- 1) Tryptone Yeast extract Glucose Vitamins Thiamine HC1 Niacinamide Panthotenic acid Pyridoxine Amino acids Tryptophane Serine Glutamic acid Arginine Asparagine Salts Na2HPO4 NaC1 NaCHO3 KC1 K2HPO4 TRIS HC1
g/l DMM ~
AGc
Sagar a
6
2.5
0.5
3
2.5
2.5 2.5 2.5 2.5
3 4 4 3
0,2
1
2.5
2.5
2.5 1
10 5
0.5 6 2 0.4
8
5 5
2.5 8.71
8.71 0.01 M
Trace metals solution MgSO4, 5%; MnC12, 0.5%; FeCI3, 5%, 0.4% Nitrilotriacetic acid Flask CTB yield CTB 5-1 fermenter CTB yield CTB
AGM4
0.89 3.11
0.14 0.09
1.004 0.81
1 ml
1 ml
0 0
8 0.088
1.6 ml
10 2.53 4.40 36.67
LB: Luria Broth b D M M : developed by our laboratory c AG: Callahan and Richardson (1973) a Sagar: Sagar et al. (1979, 1981) Optical densities (OD) were measured at 600 nm after overnight incubation at 30 °C; CTB (mg 1-1) was measured using the GMI enzymelinked immunosorbent assay (Elisa); CTB yield was calculated in mg 1 - 1 0 D -~
a
(New Brunswick Scientific, Edison, N J, USA) equipped with either a single orifice sparger, a ring sparger or a sintered disk sparger. The fermentation system was connected to a computerized control system (Fass et al. 1989). This system regulated the pH and maintained the dissolved oxygen level above 30% saturation by adjusting the agitation a n d / o r the air-flow rate. The fermentations were performed using LB or defined media as indicated. The initial pH value was 6.5 or 8.0 as indicated. In pH-controlled fermentation the pH was regulated by the addition of 1 N N a O H or 40% H3PO4 solutions. Fermentors were inoculated with an overnight culture grown at 30 ° C, in 250-ml flasks containing 50 ml medium. During fermentation, samples were taken, centrifuged and their supernatants kept at - 20 ° C for analysis. At the end of fermentation the culture was centrifuged at 9000 rpm for 40 min and the supernatant was kept at 5°C for purification and analysis.
Purification. The supernatants of the cultures were microfiltrered and ultrafiltered using a tangential flow filtration system (Pellicon Cassette System, Millipore, Bedford, Ma, USA) with membranes of 0.22 Ix average pore size and 10000 mol. wt. cut-off, respectively. The concentrated extract was dialyzed against 10 m M NaPO4,
pH 7.4, and the B subunit was purified on a phosphocellulose column (Whatman P l l , lot no. 7611074, Whatman Biosystems, Springfield, USA) using 0.2 M NaPO4, pH 7.4, as eluent (Mekalanos 1988). The B-subunit-containing fractions were lyophilized and dialyzed against 0.2 M NaCI.
Analysis. The bacterial growth was monitored by measuring the optical density (OD) at 600 nm with a microsample spectrophotometer (Gilford 300-N, Gilford Instrument Labs, Oberlin, Ohio, USA). The CTB concentration was determined in the supernatants by a monosialoganglioside (GM1) specific enzyme-linked immunosorbent assay (ELISA; Holmgren 1973). The glucose was determined using a glucose and L-lactate analyzer (YSI Model 2000, YSI, Yellow Springs, Ohio, USA). The amino acid analysis was done by derivatizing the samples with phenylisothiocyanate (PITC) and separating the amino acids using reversed phase chromatography (MinoRPC column, Pharmacia, Uppsala, Sweden; Whitney and Chrustic 1986). The purified CTB was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% polyacrylamide gels in the presence of SDS, followed by staining with Coomassie Blue.
391 70 8 6 6
60A
,
rt~
~g
~
0
I I 20 0 2 4 6 8 10 121416 18 20 FERMENTATIONTIME(hrs)
Fig. 1. Growth of Vibrio cholerae 0395-NI and production of cholera toxin subunit B (CTB) in Luria-Bertani broth: ~ , optical density (OD); [~, CTB; @, pH; ©, dissolved oxygen
of increasing the CTB production, analyzing the influence of different constituents of the medium and simplifying the purification procedure. Based on AG medium (Callahan et al. 1971, Callahan and Richardson 1973) and on a medium developed by Sagar et al. (1979, 1981) and taking into account the composition of the Syncase medium (Finkelstein et al. 1966), and of TCY medium (Richardson et al. 1969) and of CAYE medium (Evans et al. 1973), several defined media were designed and tested. In shake flasks, the best results were achieved with AGM4 medium (Table 1). The addition of a combination of amino acids (serine, glutamic a d d asparagine and arginine), salts (sodium phosphate, sodium carbonate and potassium carbonate) and trace elements solution to the AG medium had a positive effect on the yield of the B subunit. Growth and production were well correlated; the pH of 8.0 at the start of fermentation increased up to 8.7 at the end of the growth phase, in parallel with an increase in CTB production.
Kinetics of CTB production in defined medium
Results Development of a defined medium for B subunit production Based on previous information on the optimization of CT production (Taylor et al. 1987), preliminary experiments were performed in a bench-top fermentor, using LB medium at an initial pH of 6.5 and a temperature of 30 ° C, with and without pH control. A good correlation between growth and CTB production was found (Fig. 1). In non-pH-controlled cultures the pH increased from 6.5 to 8.4 during the stationary phase. A slight increase in CTB production was observed at the onset of the stationary phase. Under these conditions the final concentration of CTB ranged between 0.69 and 1.5 mg1-1. A defined medium was developed with the purpose 400
During a representative fermentation performed in a bench-top fermentor with the defined medium AGM4 at pH 6.5 and 30 ° C, the concentration of the B subunit did not exceed 0.5 mg l - 1. In an attempt to increase the CTB concentration, we performed a fermentation under the same initial conditions of the shake flasks (pH 8.0, 30 ° C, AGM4 medium) (Fig. 2). In this fermentation the pH was maintained at 8.0 with 1 N NaOH and the dissolved oxygen was kept above 30% saturation by increasing the stirring speed. Oxygen-enriched air (Fass et al. 1989) was used to avoid a high stirring speed that could adversely affect the bacteria (Fig. 2a). After 8 h the OD reached its maximum and the CTB concentration was 36 mg 1-1. The maximum specific growth rate was 0.5 h -1 (Fig. 2b). The growth kinetics were similar at pH 6.5 and pH 8.0 and the maximum specific growth rates were identical, but the production kinetics were different: at pH 8.0, growth and production were corre-
40
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01 2 3 4 5 6 7 8 9 FERMENTATION TIME (hrs)
0
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89 0 1 234567 FERMENTATION TIME (hrs)
Fig. 2. Growth of V. cholerae 0395-NI in a 5-1 fermentor in AGM4 medium, a Fermentation parameters: ÷, stirring speed; B, dissolved O2 (DO2); Iq, CO2 production, b Growth and production: B, OD; *, specific growth rate; El, CTB yield; ÷, production
392 Table 2. Fermentations conducted in a top-bench fermentor at 30°C and at varying pH Parameters measured
pH
CTB (mg 1-~) Yield (rag 1- ~ OD- ~) Specific growth rate (h-~)
6.5
7.0
7.5
8.0
8.5
9.0
0.64 0.08 0.37
4.10 0.79 0.68
22.63 2.87 0.84
36;70 4.49 0.59
2.50 0.27 0.27
4.20 0.42 0.34
Table 3. Fermentations conducted in a top-bench fermentor at pH 8.0 at different temperatures Parameters measured
Temperature
CTB (mg I -~) Yield (rag 1-~ OD -~) Specific growth rate (h-a)
25°C
30°C
33.5°C
37°C
7.52 0.70 0.44
36.7 4.49 0.59
19.7 1.08 0.60
3.87 0.26 0.75
lated during the growth phase but productivity increased during the stationary phase (Fig. 2b), whereas at p H 6.5, the productivity was low for the duration of the fermentation. Using AGM4, at p H 8.0 and 30 ° C, the concentration of CTB obtained was ten times higher than that obtained under the initial conditions (pH 6.5, 30 ° C) in LB medium.
check whether the high p H enhanced the release o f the toxin from the cells, we increased the p H up to 8.0 at the end of the growth phase of a fermentation with p H control lower than 8.0. No sudden excess release of CTB was observed, indicating that a p H value o f 8.0 had an effect on CTB production but not on its release into the medium. The effect of the temperature on CTB production was also studied. Fermentations were performed at 25 ° , 30 °, 33.5 ° and 37 ° C, keeping the p H constant at 8.0. A CTB production optimum was observed at a temperature of 30 ° C. Even though the growth rates increased with temperature, the yields were optimal at 30 ° C (Table 3).
Utilization of glucose and amino acids durin# CTB production
Effects of environmental conditions on CTB production in defined medium Using the defined medium AGM4, fermentations at different p H values were performed at a constant temperature of 30 ° C. The optimal p H value for CTB production was 8.0 (Table 2). As mentioned previously, the fermentations performed at an initial p H o f 6.5 showed a constant increase up to p H 8.0 at the end of the growth phase. To
During fermentation the glucose and amino acid concentrations were analyzed. The concentrations of glucose, asparagine and glutamic acid are shown together with the O D and the concentration of CTB in Fig. 3. There was a correlation between the consumption o f amino acids and glucose and CTB production. Additional glucose was added after 3 h fermentation. After this addition, during the first phase of productivity and the exponential growth phase, it was consumed at a higher rate. The asparagine was also consumed at a higher rate during the growth phase. The glutamic acid
10
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4
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6
Fermentation Time (hrs)
7
8
9
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Fig. 3. Nutrient consumption by V. cholerae 0395-NI during fermentation in a 51 fermentor using AGM4 medium: - - ~ - - , glucose; -- A --, asparagine; - I - - , glutamic acid; . - - O . . . , OD; -- [] --, CTB. Addition of glucose (Gluc.) after 3 h, and asparagine (Asp.) after 4 h, are indicated by arrows
393
Fig. 4. Polyacrylamidegel electrophoresis of CTB from V. cholerae 0395-NI: lane A, marker; lane B, fermentationsupernatant; lane G, CTB, pool from phosphocellulose column; lane H, CTB, pool after lyophilization;lane L cholera toxin standard concentration decreased progressively during the whole fermentation. The concentrations of serine and arginine were also determined. They were consumed in a pattern similar to asparagine.
Purification o f CTB
A two-step purification process (see Materials and methods) was implemented to purify CTB from fermentations run in AGM4 at 30°C and pH 8. A pure CTB polypeptide of 11000 tool. wt. was obtained (Fig. 4) that precipitated with specific antibodies (ELISA, immunoblotting, results not shown). The CTB was purified with 80% recovery and 37-fold purification.
Discussion
The optimal conditions for cholera toxin production by V. cholerae mentioned in the literature were low temperature (250-30 °C), pH 6.5, high aeration, and rich medium. In well-aerated fermentors, using a new defined medium, AGM4, optimum productivity of the B subunit of CT was observed at pH 8.0 and 30 ° C. Under these conditions, high production of CTB was obtained, 36 mg 1-1. This concentration was ten times higher than the one obtained under the initial conditions (pH 6.5, 30 ° C,,LB medium) reported to be optimal for CT production. Independently of the pH, growth and production were associated during the growth phase, but productivity of the toxin was higher at the onset of the stationary phase. The last phenomenon has been observed in other strains of V. eholerae (Callahan et al. 1971, Callahan and Richardson 1973). The concentration of 36 mg 1-1 CTB can be compared to the CTB concentrations reported in the literature.
According to Sanchez and Holmgren (1989), strain 569B produces 1.28 mg1-1 CTB and strain JBK70 (pJS162), which possesses an overexpression system for CTB, placed under the control of the tacP promoter, produces 75 mg 1-1. Taylor et al. (1988) reported production of 10-15 mg 1-a by strain 0395-NI under conditions optimal for TcpA production. Strain 0395-NI was obtained by in vivo recombination of in vitro constructed deletions of the ctx operon into each of the two residents etx copies carried by this strain. The purification was based on the Mekalanos scheme, a two-step process: ultrafiltration and ion-exchange chromatography. A pure CTB polypeptide was obtained with 80% recovery. In the fermentations conducted in AGM4 medium, asparagine and, in lower amounts, serine and arginine were consumed during the growth phase in parallel to the first peak of productivity. Glutamic acid was consumed during the stationary phase and the second peak of productivity. As asparagine and arginine were consumed, aspartic acid and alanine appeared. Callahan and Richardson (1973) observed that the four amino acids used in AGM4, asparagine, serine, arginine and glutamic acid, stimulated production of the holotoxin and that serine and asparagine were more quickly exhausted than arginine and glutamic acid. Sagar et al. (1979, 1981) showed that the cholera toxin synthesis was related to serine metabolism. We observed that nutrient deprivation resulted in the formation of coccoid cells and in an increase in cell number of reduced volume, as reported previously (Baker et al. 1983). During fermentations conducted in two phases, growth at constant pH of 6.5 and the stationary phase at pH 8.0, the concentration of CTB did not increase and did not surpass 1.5 mg 1-1. Studies performed with the holotoxin in flasks (Callahan and Richardson 1973), have shown that release of the whole toxin is triggered by a rise in pH and concluded that cell growth should be performed at pH 6.5 and that the toxin should be released from the cell at alkaline pH. Their experiments demonstrated also the influence of pH on the release of the whole toxin and not on its production. The differences between our results and theirs could be due to the variety of structure and conformation of the holotoxin and the CTB. It can affect the mechanisms influencing their release. Hardy et al." (1988) demonstrated that the molecules excreted from the periplasm consist almost exclusively of fully assembled holotoxin rather than B pentamers. The production of CTB 0395-NI has been shown to be correlated with the expression of the protein TcpA constituting the pili and responsible for the agglutination of cells (Miller and Mekalanos 1988; Taylor et al. 1987). To avoid the effect of agitation on pili formation, which can effect the cells' agglutination and CTB production, we used low-shear fermentors. In the 5-1 top bench fermentor, we maintained a high air-flow rate (3 1. h - 1) and we used air enriched with oxygen. However, we obtained a high toxin level without cell agglutination. The cells agglutinated when grown in tubes or in the kluyver fermentor, but the toxin yields were lower
394 t h a n t h o s e o b t a i n e d in a c o n v e n t i o n a l s t i r r e d r e a c t o r . M o r e o v e r , w h e n t h e cells grew in a n air-lift f e r m e n t o r without agglutination no lower yields were observed. W e c o n c l u d e d t h a t t h e T c p A was e v e n t u a l l y e x p r e s s e d b u t t h a t a g i t a t i o n in t h e f e r m e n t o r s was p r e v e n t i n g t h e p i l l f r o m a g g l u t i n a t i n g t h e cells. T a y l o r et al. (1987) a n d M i l l e r a n d M e k a l a n o s (1988) s t u d i e d m u t a n t s o f V. cholerae p r o d u c i n g o n l y the B s u b u n i t , s u c h as 0 3 9 5 - N I . T h e o p t i m a l c o n d i t i o n s f o r e x p r e s s i o n o f t h e p i l l a r e l o w t e m p e r a t u r e (25 ° 30 ° C), i n i t i a l p H o f 6.5, m o d e r a t e a e r a t i o n , a salt c o n c e n t r a t i o n o f 60 m M , a n d a s p a r a g i n e , g l u t a m a t e , a r g i n i n e a n d s e r i n e c o n c e n t r a t i o n s o f 25 m M (close to t h e c o n c e n t r a t i o n s in A G M 4 ) . T h e c o m p o s i t i o n o f t h e i r m e d i u m w a s c l o s e in t e r m s o f a m i n o a c i d c o n c e n t r a t i o n s to A G M 4 b u t t h e y c o n c l u d e d f r o m t h e i r e x p e r i ments that media properties such osmolarity and the presence of amino acids are more important than temperature and pH for the expression of toxR regulated p r o t e i n s s u c h as C T B a n d o u t e r m e m b r a n e p r o t e i n s s u c h as T c p A , O m p U a n d O m p T . I n A G M 4 t h e r e was, however, an optimum of temperature and pH for CTB production. Acknowledgements. This work was supported by an NRC grant.
The authors would like to thank Dr. J. B. Robbins (LDM1, NICHD, NIH) for his help in conducting this research.
References Aizpurua HJ de, Russell-Jones GJ (1988) Oral vaccination. J Exp Med 167:440-451 Baker RM, Singleton FL, Hood MA (1983) Effects of nutrient deprivation on Vibrio cholerae. Appl Environ Microbiol 46:930940 Callahan LT, Richardson ST (1973) Biochemistry of Vibrio cholerae virulence. III. Nutritional requirements for toxin production and the effects of pH on toxin elaboration in chemically defined media. Infect Immun 7:567-572 Callahan LT, Ryder RC, Richardson ST (1971) Biochemistry of Vibrio cholerae virulence. II. Skin permeability factor/cholera enterotoxin production in a chemically defined medium. Infect Immun 4:611-618 Evans DG, Evans DJ Jr, Pierce NF (1973) Differences in the response of rabbit small intestine to the heat-labile and heatstable enterotoxins of Escherichia coll. Infect Immun 7:873-880 Fass R, Clem TR, Shiloach J (1989) Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coll. Appl Environ Microbiol 55:1305-1307 Finkelstein RA (1973) Cholera. CRC Crit Rev Microbiol 2:553623 Finkelstein RA (1984) Cholera. In: Germanier R (ed) Bacterial vaccines. Academic Press, New York, pp 107-129 Finkelstein RA, LoSpalluto JJ (1969) Pathogenesis of experimental cholera: preparation and isolation of choleragen and choleragenoid. J Exp Med 130:185-202 Finkelstein RA, LoSpalluto JJ (1970) Production of highly purified choleragen and choleragenoid. J Infect Dis 121:Suppl. $63-$72 Finkelstein RA, Atthasampunna P, Chulasamaya M, Charunme-
thee P (1966) Pathogenesis of experimental cholera: biologic activities of purified procholeragen A. J Immunol 96:440 Finkelstein RA, Burks MF, Zupan A, Dallas WS, Jacob CO, Ludwig DS (1987) Epitopes of the cholera family of enterotoxins. Rev Infect Dis 9:544-561 Hardy SJS, Holmgren J, Johansson S, Sanchez J, Hirt TR (1988) Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci 85:7109-7113 Holmgren J (1973) Comparison of the tissue receptors for Vibrio cholerae and Vibrio cholera enterotoxins by means of gangliosides and natural cholera toxoid. Infect Immun 8:851-859 McKenzie SJ, Halsey JF (1984) Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. J Immunol 133 : 1818-1824 Mekalanos JJ (1988) Production and purification of cholera toxin. Methods Enzymol 165 : 169-175 Mekalanos J J, Swartz D J, Pearson GDN, Harfoi'd N, Groyne N, DeWilde M (1983) Cholera toxin genes: nucleotide sequence, deletion analysis, and vaccine development. Nature 306:551557 Miller VL, Mekalanos JJ (1984) Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc Natl Acad Sci USA 81:3471-3475 Miller YL, Mekalanos JJ (1985) Genetic analysis of the cholera toxin positive regulatory gene toxR. J Bacteriol 163:580-585 Miller VL, Mekalanos JJ (1988) A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575-2583 Miller VL, Taylor RK, Mekalanos JJ (1987) Cholera toxin transcriptional activator ToxR is a transmembrane DNA binding protein. Cell 48: 271-279 Peterson KM, Mekalanos JJ (1988) Characterization of the Vibrio cholerae toxR region: identification of novel genes involved in intestinal colonization. Infect Immun 56:2822-2829 Richardson SH (1969) Factors influencing in vitro skin permeability factor production by Vibrio cholerae. J Bacteriol 100:2734 Sagar IK, Nagesha CN, Bhat JV (1979) Effect of metal ions on the production of vascular permeability factor by 569B strain of Vibrio cholerae. J Med Res 69:18-25 Sagar IK, Nagesha CN, Bhat JV (1981) The role of trace elements and phosphates in the synthesis of vascular permeability factor by Vibrio cholerae. J Med Microbiol 14:243-250 Sanchez J, Holmgren J (1989) Recombinant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development. Proc Natl Acad Sci USA 86:481485 Schneerson R, Robbins JB, Szu SC (1987) Vaccines composed of polysaccharides-protein conjugates. Current status, unanswered questions and prospects for the future. In: Bell R, Torrigiani G (eds) Towards better carbohydrates vaccines. World Health Organization. Wiley and Sons, NY, USA, pp 307-332 Taylor RK, Miller VL, Furlong DB, Mekalanos JJ (1987) Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci USA 84:2833-2837 Taylor RK, Shaw C, Peterson KM, Spears P, Mekalanos JJ (1988) Safe, live Vibrio cholerae vaccines? Vaccine 6:151-154 Whitney B, Chrustic T (1986) Quantitative amino acid analysis with FPLC. FPLC Biocommunique 3, 2:8-11. Pharmacia, Uppsala, Sweden Wood LA, Thompson PW (1987) Applications of the air lift fermenter. Appl Biochem Biotechnol 15:131-143
Applied Microbiology Biotechnology © Springer-Verlag 1990
Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae Michele van de Walle, Raphael Fass*, and Joseph Shiloach Biotechnology Unit, LCDB, NIDDK, NIH, Building 6, Room B1-33, Bethesda, MD 20892, USA Received 19 September 1989/Accepted 6 February 1990
Summary. The B subunit (CTB) of cholera toxin (CT) can be used as a carrier protein for conjugate vaccines designed to elicit antipolysaccharide antibodies. A defined medium, AGM4, was designed to grow a highproducing mutant of Vibrio cholerae expressing only the B subunit of CT: V. cholerae 0395-NI. AGM4 contains four amino acids, asparagine, glutamic acid, arginine and serine, salts and a trace element solution. The carbon source is glucose. The fermentations performed in AGM4 indicated that CTB production paralleled the growth of the organism but that there was a maximal release of CTB during the stationary phase. There was a clear optimum of productivity at p H 8.0 and 30 ° C. The p H had an influence on CTB production and not only on its release. Analysis of the amino acids present in the medium showed a correlation between their consumption rates and CTB productivity.
Introduction Vibrio cholerae, a Gram-negative pathogenic bacterium, colonizes the upper intestine and produces an exotoxin, cholera toxin (CT), which is responsible for the diarrheal symptoms of the disease. CT is composed of two subunits: the A subunit (mol-wt. 27000) which has ADP ribosyl transferase activity, and the B subunit (CTB) which contains five identical polypeptides (mol. wt. 11000 each) and forms the mammalian cell receptor binding domain. The B subunit has high affinity for the oligosaccharide moiety of the ganglioside GM1 (Finkelstein and LoSpalluto 1969, 1970; Holmgren 1973). The A and B subunits are encoded by the etxA and e t x B genes regulated by the product o f the t o x R gene (Miller et al. 1987; Miller and Mekalanos 1984, 1985; Peterson and Mekalanos 1988). * Permanent address: Department of Biotechnology, IIBR, Ness
Ziona 70450, Israel Offprint requests to: J. Shiloach
The neutralizing activity of serum antibodies (antitoxin) is largely directed towards the B subunit. CT antitoxin also neutralizes the toxic activity of the structurally related and widely distributed cholera-like enterotoxin of Eseheriehia coli (de Aizpurua and RussellJones 1988; Finkelstein et al. 1987; Finkelstein 1973,. 1984). The B subunit of CT has been shown to be an immunogenic carrier protein for conjugate vaccines designed to elicit anti-polysaccharide antibodies (McKenzie and Halsey 1984; Schneerson et al. 1987; Taylor et al. 1988). The use of the B subunit, rather than the holotoxin in preparation of human vaccines, avoids the problem of holotoxin toxicity without loss of immunogenicity (Sanchez and Holmgren 1989). The procedure used to prepare the B subunit from CT includes denaturing conditions and may cause aggregation and loss of solubility of the B subunit. Accordingly, we have studied a high-producing mutant of V. eholerae expressing only the B subunit of CT to prepare CTB. In order to obtain large amounts of CTB we studied the optimization of its production in a defined medium using a computerized fermentation system. A defined medium could simplify and facilitate the recovery and purification of the toxin produced. The influence of the environmental parameters on the production kinetics of CTB was analyzed.
Materials and methods Microorganisms. Vibrio cholerae 0395-NI, which carries a deletion in the etxA gene and produces only the B subunit of the toxin
(Mekalanos et al. 1983; Taylor et al. 1987), was used. This bacterium was maintained in Luria-Bertani (LB) medium containing 15% glycerol at - 20° C. Media. Several synthetic media were tested (Table 1). The solutions of amino acids, salts, metals and sugars were prepared and autoclaved separately. Fermentations. Fermentations were performed at 30° C or as indi-
cated. To avoid the shear effect of agitation on the bacterial cells, two types of low shear fermentors were used: a 4-1 kluyver fermentor and a 3-1 air-lift fermentor (Wood and Thompson 1987). Further experiments were carried out in a 5-1 bench-top fermentor
390 Table 1. Influence of different media on cholera toxin subunit B (CTB) production Media LB a Nutrients (g. 1- 1) Tryptone Yeast extract Glucose Vitamins Thiamine HC1 Niacinamide Panthotenic acid Pyridoxine Amino acids Tryptophane Serine Glutamic acid Arginine Asparagine Salts Na2HPO4 NaC1 NaCHO3 KC1 K2HPO4 TRIS HC1
g/l DMM ~
AGc
Sagar a
6
2.5
0.5
3
2.5
2.5 2.5 2.5 2.5
3 4 4 3
0,2
1
2.5
2.5
2.5 1
10 5
0.5 6 2 0.4
8
5 5
2.5 8.71
8.71 0.01 M
Trace metals solution MgSO4, 5%; MnC12, 0.5%; FeCI3, 5%, 0.4% Nitrilotriacetic acid Flask CTB yield CTB 5-1 fermenter CTB yield CTB
AGM4
0.89 3.11
0.14 0.09
1.004 0.81
1 ml
1 ml
0 0
8 0.088
1.6 ml
10 2.53 4.40 36.67
LB: Luria Broth b D M M : developed by our laboratory c AG: Callahan and Richardson (1973) a Sagar: Sagar et al. (1979, 1981) Optical densities (OD) were measured at 600 nm after overnight incubation at 30 °C; CTB (mg 1-1) was measured using the GMI enzymelinked immunosorbent assay (Elisa); CTB yield was calculated in mg 1 - 1 0 D -~
a
(New Brunswick Scientific, Edison, N J, USA) equipped with either a single orifice sparger, a ring sparger or a sintered disk sparger. The fermentation system was connected to a computerized control system (Fass et al. 1989). This system regulated the pH and maintained the dissolved oxygen level above 30% saturation by adjusting the agitation a n d / o r the air-flow rate. The fermentations were performed using LB or defined media as indicated. The initial pH value was 6.5 or 8.0 as indicated. In pH-controlled fermentation the pH was regulated by the addition of 1 N N a O H or 40% H3PO4 solutions. Fermentors were inoculated with an overnight culture grown at 30 ° C, in 250-ml flasks containing 50 ml medium. During fermentation, samples were taken, centrifuged and their supernatants kept at - 20 ° C for analysis. At the end of fermentation the culture was centrifuged at 9000 rpm for 40 min and the supernatant was kept at 5°C for purification and analysis.
Purification. The supernatants of the cultures were microfiltrered and ultrafiltered using a tangential flow filtration system (Pellicon Cassette System, Millipore, Bedford, Ma, USA) with membranes of 0.22 Ix average pore size and 10000 mol. wt. cut-off, respectively. The concentrated extract was dialyzed against 10 m M NaPO4,
pH 7.4, and the B subunit was purified on a phosphocellulose column (Whatman P l l , lot no. 7611074, Whatman Biosystems, Springfield, USA) using 0.2 M NaPO4, pH 7.4, as eluent (Mekalanos 1988). The B-subunit-containing fractions were lyophilized and dialyzed against 0.2 M NaCI.
Analysis. The bacterial growth was monitored by measuring the optical density (OD) at 600 nm with a microsample spectrophotometer (Gilford 300-N, Gilford Instrument Labs, Oberlin, Ohio, USA). The CTB concentration was determined in the supernatants by a monosialoganglioside (GM1) specific enzyme-linked immunosorbent assay (ELISA; Holmgren 1973). The glucose was determined using a glucose and L-lactate analyzer (YSI Model 2000, YSI, Yellow Springs, Ohio, USA). The amino acid analysis was done by derivatizing the samples with phenylisothiocyanate (PITC) and separating the amino acids using reversed phase chromatography (MinoRPC column, Pharmacia, Uppsala, Sweden; Whitney and Chrustic 1986). The purified CTB was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% polyacrylamide gels in the presence of SDS, followed by staining with Coomassie Blue.
391 70 8 6 6
60A
,
rt~
~g
~
0
I I 20 0 2 4 6 8 10 121416 18 20 FERMENTATIONTIME(hrs)
Fig. 1. Growth of Vibrio cholerae 0395-NI and production of cholera toxin subunit B (CTB) in Luria-Bertani broth: ~ , optical density (OD); [~, CTB; @, pH; ©, dissolved oxygen
of increasing the CTB production, analyzing the influence of different constituents of the medium and simplifying the purification procedure. Based on AG medium (Callahan et al. 1971, Callahan and Richardson 1973) and on a medium developed by Sagar et al. (1979, 1981) and taking into account the composition of the Syncase medium (Finkelstein et al. 1966), and of TCY medium (Richardson et al. 1969) and of CAYE medium (Evans et al. 1973), several defined media were designed and tested. In shake flasks, the best results were achieved with AGM4 medium (Table 1). The addition of a combination of amino acids (serine, glutamic a d d asparagine and arginine), salts (sodium phosphate, sodium carbonate and potassium carbonate) and trace elements solution to the AG medium had a positive effect on the yield of the B subunit. Growth and production were well correlated; the pH of 8.0 at the start of fermentation increased up to 8.7 at the end of the growth phase, in parallel with an increase in CTB production.
Kinetics of CTB production in defined medium
Results Development of a defined medium for B subunit production Based on previous information on the optimization of CT production (Taylor et al. 1987), preliminary experiments were performed in a bench-top fermentor, using LB medium at an initial pH of 6.5 and a temperature of 30 ° C, with and without pH control. A good correlation between growth and CTB production was found (Fig. 1). In non-pH-controlled cultures the pH increased from 6.5 to 8.4 during the stationary phase. A slight increase in CTB production was observed at the onset of the stationary phase. Under these conditions the final concentration of CTB ranged between 0.69 and 1.5 mg1-1. A defined medium was developed with the purpose 400
During a representative fermentation performed in a bench-top fermentor with the defined medium AGM4 at pH 6.5 and 30 ° C, the concentration of the B subunit did not exceed 0.5 mg l - 1. In an attempt to increase the CTB concentration, we performed a fermentation under the same initial conditions of the shake flasks (pH 8.0, 30 ° C, AGM4 medium) (Fig. 2). In this fermentation the pH was maintained at 8.0 with 1 N NaOH and the dissolved oxygen was kept above 30% saturation by increasing the stirring speed. Oxygen-enriched air (Fass et al. 1989) was used to avoid a high stirring speed that could adversely affect the bacteria (Fig. 2a). After 8 h the OD reached its maximum and the CTB concentration was 36 mg 1-1. The maximum specific growth rate was 0.5 h -1 (Fig. 2b). The growth kinetics were similar at pH 6.5 and pH 8.0 and the maximum specific growth rates were identical, but the production kinetics were different: at pH 8.0, growth and production were corre-
40
1.4
10 +
350
~
/
~
300 :~ E~
~
250
~ ~CL
200
~ ~.N~ ~ e ~ ~
150
~~
~11"2
/ / ~ 0 . 6
~
~
0A ~oo
0
6
0
0.80
,
50
8
3O
'
0o) 0
~'O~
~
E,~ 20 m I-O
.~ ~
~4 ~:~ ~
10
2
"X
O.R
01 2 3 4 5 6 7 8 9 FERMENTATION TIME (hrs)
0
0
89 0 1 234567 FERMENTATION TIME (hrs)
Fig. 2. Growth of V. cholerae 0395-NI in a 5-1 fermentor in AGM4 medium, a Fermentation parameters: ÷, stirring speed; B, dissolved O2 (DO2); Iq, CO2 production, b Growth and production: B, OD; *, specific growth rate; El, CTB yield; ÷, production
392 Table 2. Fermentations conducted in a top-bench fermentor at 30°C and at varying pH Parameters measured
pH
CTB (mg 1-~) Yield (rag 1- ~ OD- ~) Specific growth rate (h-~)
6.5
7.0
7.5
8.0
8.5
9.0
0.64 0.08 0.37
4.10 0.79 0.68
22.63 2.87 0.84
36;70 4.49 0.59
2.50 0.27 0.27
4.20 0.42 0.34
Table 3. Fermentations conducted in a top-bench fermentor at pH 8.0 at different temperatures Parameters measured
Temperature
CTB (mg I -~) Yield (rag 1-~ OD -~) Specific growth rate (h-a)
25°C
30°C
33.5°C
37°C
7.52 0.70 0.44
36.7 4.49 0.59
19.7 1.08 0.60
3.87 0.26 0.75
lated during the growth phase but productivity increased during the stationary phase (Fig. 2b), whereas at p H 6.5, the productivity was low for the duration of the fermentation. Using AGM4, at p H 8.0 and 30 ° C, the concentration of CTB obtained was ten times higher than that obtained under the initial conditions (pH 6.5, 30 ° C) in LB medium.
check whether the high p H enhanced the release o f the toxin from the cells, we increased the p H up to 8.0 at the end of the growth phase of a fermentation with p H control lower than 8.0. No sudden excess release of CTB was observed, indicating that a p H value o f 8.0 had an effect on CTB production but not on its release into the medium. The effect of the temperature on CTB production was also studied. Fermentations were performed at 25 ° , 30 °, 33.5 ° and 37 ° C, keeping the p H constant at 8.0. A CTB production optimum was observed at a temperature of 30 ° C. Even though the growth rates increased with temperature, the yields were optimal at 30 ° C (Table 3).
Utilization of glucose and amino acids durin# CTB production
Effects of environmental conditions on CTB production in defined medium Using the defined medium AGM4, fermentations at different p H values were performed at a constant temperature of 30 ° C. The optimal p H value for CTB production was 8.0 (Table 2). As mentioned previously, the fermentations performed at an initial p H o f 6.5 showed a constant increase up to p H 8.0 at the end of the growth phase. To
During fermentation the glucose and amino acid concentrations were analyzed. The concentrations of glucose, asparagine and glutamic acid are shown together with the O D and the concentration of CTB in Fig. 3. There was a correlation between the consumption o f amino acids and glucose and CTB production. Additional glucose was added after 3 h fermentation. After this addition, during the first phase of productivity and the exponential growth phase, it was consumed at a higher rate. The asparagine was also consumed at a higher rate during the growth phase. The glutamic acid
10
~E ~ -E:I v = ~ m ~"~
__
~
8
I
40
--
I ....t"'"'~ Gluc.
_ {3
Jf
~ o ° mr-El ~ ~_~ ~ '(9
.....I
30
~
...i: 2O
~ r~
~
~-
4 - ~---~-=--~=~_.~__=
10 "~ (.9 - -
2
0
.... • °""
0
1
2
----I~
3
4
5
6
Fermentation Time (hrs)
7
8
9
0
Fig. 3. Nutrient consumption by V. cholerae 0395-NI during fermentation in a 51 fermentor using AGM4 medium: - - ~ - - , glucose; -- A --, asparagine; - I - - , glutamic acid; . - - O . . . , OD; -- [] --, CTB. Addition of glucose (Gluc.) after 3 h, and asparagine (Asp.) after 4 h, are indicated by arrows
393
Fig. 4. Polyacrylamidegel electrophoresis of CTB from V. cholerae 0395-NI: lane A, marker; lane B, fermentationsupernatant; lane G, CTB, pool from phosphocellulose column; lane H, CTB, pool after lyophilization;lane L cholera toxin standard concentration decreased progressively during the whole fermentation. The concentrations of serine and arginine were also determined. They were consumed in a pattern similar to asparagine.
Purification o f CTB
A two-step purification process (see Materials and methods) was implemented to purify CTB from fermentations run in AGM4 at 30°C and pH 8. A pure CTB polypeptide of 11000 tool. wt. was obtained (Fig. 4) that precipitated with specific antibodies (ELISA, immunoblotting, results not shown). The CTB was purified with 80% recovery and 37-fold purification.
Discussion
The optimal conditions for cholera toxin production by V. cholerae mentioned in the literature were low temperature (250-30 °C), pH 6.5, high aeration, and rich medium. In well-aerated fermentors, using a new defined medium, AGM4, optimum productivity of the B subunit of CT was observed at pH 8.0 and 30 ° C. Under these conditions, high production of CTB was obtained, 36 mg 1-1. This concentration was ten times higher than the one obtained under the initial conditions (pH 6.5, 30 ° C,,LB medium) reported to be optimal for CT production. Independently of the pH, growth and production were associated during the growth phase, but productivity of the toxin was higher at the onset of the stationary phase. The last phenomenon has been observed in other strains of V. eholerae (Callahan et al. 1971, Callahan and Richardson 1973). The concentration of 36 mg 1-1 CTB can be compared to the CTB concentrations reported in the literature.
According to Sanchez and Holmgren (1989), strain 569B produces 1.28 mg1-1 CTB and strain JBK70 (pJS162), which possesses an overexpression system for CTB, placed under the control of the tacP promoter, produces 75 mg 1-1. Taylor et al. (1988) reported production of 10-15 mg 1-a by strain 0395-NI under conditions optimal for TcpA production. Strain 0395-NI was obtained by in vivo recombination of in vitro constructed deletions of the ctx operon into each of the two residents etx copies carried by this strain. The purification was based on the Mekalanos scheme, a two-step process: ultrafiltration and ion-exchange chromatography. A pure CTB polypeptide was obtained with 80% recovery. In the fermentations conducted in AGM4 medium, asparagine and, in lower amounts, serine and arginine were consumed during the growth phase in parallel to the first peak of productivity. Glutamic acid was consumed during the stationary phase and the second peak of productivity. As asparagine and arginine were consumed, aspartic acid and alanine appeared. Callahan and Richardson (1973) observed that the four amino acids used in AGM4, asparagine, serine, arginine and glutamic acid, stimulated production of the holotoxin and that serine and asparagine were more quickly exhausted than arginine and glutamic acid. Sagar et al. (1979, 1981) showed that the cholera toxin synthesis was related to serine metabolism. We observed that nutrient deprivation resulted in the formation of coccoid cells and in an increase in cell number of reduced volume, as reported previously (Baker et al. 1983). During fermentations conducted in two phases, growth at constant pH of 6.5 and the stationary phase at pH 8.0, the concentration of CTB did not increase and did not surpass 1.5 mg 1-1. Studies performed with the holotoxin in flasks (Callahan and Richardson 1973), have shown that release of the whole toxin is triggered by a rise in pH and concluded that cell growth should be performed at pH 6.5 and that the toxin should be released from the cell at alkaline pH. Their experiments demonstrated also the influence of pH on the release of the whole toxin and not on its production. The differences between our results and theirs could be due to the variety of structure and conformation of the holotoxin and the CTB. It can affect the mechanisms influencing their release. Hardy et al." (1988) demonstrated that the molecules excreted from the periplasm consist almost exclusively of fully assembled holotoxin rather than B pentamers. The production of CTB 0395-NI has been shown to be correlated with the expression of the protein TcpA constituting the pili and responsible for the agglutination of cells (Miller and Mekalanos 1988; Taylor et al. 1987). To avoid the effect of agitation on pili formation, which can effect the cells' agglutination and CTB production, we used low-shear fermentors. In the 5-1 top bench fermentor, we maintained a high air-flow rate (3 1. h - 1) and we used air enriched with oxygen. However, we obtained a high toxin level without cell agglutination. The cells agglutinated when grown in tubes or in the kluyver fermentor, but the toxin yields were lower
394 t h a n t h o s e o b t a i n e d in a c o n v e n t i o n a l s t i r r e d r e a c t o r . M o r e o v e r , w h e n t h e cells grew in a n air-lift f e r m e n t o r without agglutination no lower yields were observed. W e c o n c l u d e d t h a t t h e T c p A was e v e n t u a l l y e x p r e s s e d b u t t h a t a g i t a t i o n in t h e f e r m e n t o r s was p r e v e n t i n g t h e p i l l f r o m a g g l u t i n a t i n g t h e cells. T a y l o r et al. (1987) a n d M i l l e r a n d M e k a l a n o s (1988) s t u d i e d m u t a n t s o f V. cholerae p r o d u c i n g o n l y the B s u b u n i t , s u c h as 0 3 9 5 - N I . T h e o p t i m a l c o n d i t i o n s f o r e x p r e s s i o n o f t h e p i l l a r e l o w t e m p e r a t u r e (25 ° 30 ° C), i n i t i a l p H o f 6.5, m o d e r a t e a e r a t i o n , a salt c o n c e n t r a t i o n o f 60 m M , a n d a s p a r a g i n e , g l u t a m a t e , a r g i n i n e a n d s e r i n e c o n c e n t r a t i o n s o f 25 m M (close to t h e c o n c e n t r a t i o n s in A G M 4 ) . T h e c o m p o s i t i o n o f t h e i r m e d i u m w a s c l o s e in t e r m s o f a m i n o a c i d c o n c e n t r a t i o n s to A G M 4 b u t t h e y c o n c l u d e d f r o m t h e i r e x p e r i ments that media properties such osmolarity and the presence of amino acids are more important than temperature and pH for the expression of toxR regulated p r o t e i n s s u c h as C T B a n d o u t e r m e m b r a n e p r o t e i n s s u c h as T c p A , O m p U a n d O m p T . I n A G M 4 t h e r e was, however, an optimum of temperature and pH for CTB production. Acknowledgements. This work was supported by an NRC grant.
The authors would like to thank Dr. J. B. Robbins (LDM1, NICHD, NIH) for his help in conducting this research.
References Aizpurua HJ de, Russell-Jones GJ (1988) Oral vaccination. J Exp Med 167:440-451 Baker RM, Singleton FL, Hood MA (1983) Effects of nutrient deprivation on Vibrio cholerae. Appl Environ Microbiol 46:930940 Callahan LT, Richardson ST (1973) Biochemistry of Vibrio cholerae virulence. III. Nutritional requirements for toxin production and the effects of pH on toxin elaboration in chemically defined media. Infect Immun 7:567-572 Callahan LT, Ryder RC, Richardson ST (1971) Biochemistry of Vibrio cholerae virulence. II. Skin permeability factor/cholera enterotoxin production in a chemically defined medium. Infect Immun 4:611-618 Evans DG, Evans DJ Jr, Pierce NF (1973) Differences in the response of rabbit small intestine to the heat-labile and heatstable enterotoxins of Escherichia coll. Infect Immun 7:873-880 Fass R, Clem TR, Shiloach J (1989) Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coll. Appl Environ Microbiol 55:1305-1307 Finkelstein RA (1973) Cholera. CRC Crit Rev Microbiol 2:553623 Finkelstein RA (1984) Cholera. In: Germanier R (ed) Bacterial vaccines. Academic Press, New York, pp 107-129 Finkelstein RA, LoSpalluto JJ (1969) Pathogenesis of experimental cholera: preparation and isolation of choleragen and choleragenoid. J Exp Med 130:185-202 Finkelstein RA, LoSpalluto JJ (1970) Production of highly purified choleragen and choleragenoid. J Infect Dis 121:Suppl. $63-$72 Finkelstein RA, Atthasampunna P, Chulasamaya M, Charunme-
thee P (1966) Pathogenesis of experimental cholera: biologic activities of purified procholeragen A. J Immunol 96:440 Finkelstein RA, Burks MF, Zupan A, Dallas WS, Jacob CO, Ludwig DS (1987) Epitopes of the cholera family of enterotoxins. Rev Infect Dis 9:544-561 Hardy SJS, Holmgren J, Johansson S, Sanchez J, Hirt TR (1988) Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci 85:7109-7113 Holmgren J (1973) Comparison of the tissue receptors for Vibrio cholerae and Vibrio cholera enterotoxins by means of gangliosides and natural cholera toxoid. Infect Immun 8:851-859 McKenzie SJ, Halsey JF (1984) Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. J Immunol 133 : 1818-1824 Mekalanos JJ (1988) Production and purification of cholera toxin. Methods Enzymol 165 : 169-175 Mekalanos J J, Swartz D J, Pearson GDN, Harfoi'd N, Groyne N, DeWilde M (1983) Cholera toxin genes: nucleotide sequence, deletion analysis, and vaccine development. Nature 306:551557 Miller VL, Mekalanos JJ (1984) Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc Natl Acad Sci USA 81:3471-3475 Miller YL, Mekalanos JJ (1985) Genetic analysis of the cholera toxin positive regulatory gene toxR. J Bacteriol 163:580-585 Miller VL, Mekalanos JJ (1988) A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575-2583 Miller VL, Taylor RK, Mekalanos JJ (1987) Cholera toxin transcriptional activator ToxR is a transmembrane DNA binding protein. Cell 48: 271-279 Peterson KM, Mekalanos JJ (1988) Characterization of the Vibrio cholerae toxR region: identification of novel genes involved in intestinal colonization. Infect Immun 56:2822-2829 Richardson SH (1969) Factors influencing in vitro skin permeability factor production by Vibrio cholerae. J Bacteriol 100:2734 Sagar IK, Nagesha CN, Bhat JV (1979) Effect of metal ions on the production of vascular permeability factor by 569B strain of Vibrio cholerae. J Med Res 69:18-25 Sagar IK, Nagesha CN, Bhat JV (1981) The role of trace elements and phosphates in the synthesis of vascular permeability factor by Vibrio cholerae. J Med Microbiol 14:243-250 Sanchez J, Holmgren J (1989) Recombinant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development. Proc Natl Acad Sci USA 86:481485 Schneerson R, Robbins JB, Szu SC (1987) Vaccines composed of polysaccharides-protein conjugates. Current status, unanswered questions and prospects for the future. In: Bell R, Torrigiani G (eds) Towards better carbohydrates vaccines. World Health Organization. Wiley and Sons, NY, USA, pp 307-332 Taylor RK, Miller VL, Furlong DB, Mekalanos JJ (1987) Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci USA 84:2833-2837 Taylor RK, Shaw C, Peterson KM, Spears P, Mekalanos JJ (1988) Safe, live Vibrio cholerae vaccines? Vaccine 6:151-154 Whitney B, Chrustic T (1986) Quantitative amino acid analysis with FPLC. FPLC Biocommunique 3, 2:8-11. Pharmacia, Uppsala, Sweden Wood LA, Thompson PW (1987) Applications of the air lift fermenter. Appl Biochem Biotechnol 15:131-143
