STUDEES OF THE MODUCTEON AMI? TRYPTIE: AC‘NVM'IIDN OF THE TDXEN {3F CLQSTREWM BOTUL‘ENUM TYPE E Thesis for the Degree of M. S. MISHMN STATE UNIVERSITY MARJORIE MUELLER SCHA-EFFER 1957 'iy-LL-A A..-) I- —‘ , ~ er.“ 311:2»: 7“” r r R 4 RY ’ ‘; J J -' & --."; : 3‘21 State . itf V';'i’."‘. :-.:1-. 6.) ulV‘ LIL slty ABSTRACT STUDIES OF THE PRODUCTION AND TBYPTIC ACTIVATION OF THE TOXIN OF CLOSTRIDIUM BOTULINUM TYPE E by Marjorie Mueller Schaeffer This research was conducted to learn more about the toxin of Clostridium botulinum type E and its production. The optimum conditions for 9;. botulinum type E toxin activation in trypticase-peptone-sucrose-yeast extract (TPSY) medium were a pH level of 5.8, a trypsin concentration of 0.5 percent, trypsin activation at 1&5 C for 30 minutes, and a sodium chloride concentration of h.0 percent. Trypsin, protease (endo-and exo—proteolytic), proteinase (endo-proteolytic) and erepsin caused activation of the toxin while peptidase (exo-proteolytic) caused no activation. It was also observed that toxin titers increased as vegetative cell numbers increased upon incubation in TPSY at 30 C. However, after 2h hours of incubation in TPSY the toxin was unstable. Toxin production appeared to have no relationship to sporulation. Sucrose, glucose, fructose, maltose, and sorbitol as carbohydrate sources in TPSY afforded maximum growth and toxin production. Bibose and glycerol were less adequate energy sources. Acetate, used as a substitute for the carbohydrate source, allowed some growth and toxin production. No specific carbohydrate appeared to be required Marjorie Schaeffer for optimum toxin production or activation. A medium con- taining 5.0 percent trypticase, 5.0 percent sucrose, 1.0 per- cent yeast extract and 0.1 percent sodium thioglycolate pro- vided optimal conditions for growth and toxin production. Preliminary data was obtained to suggest that during trypsin activation of the toxin the rupture of the lysine carbonyl peptide bonds (possibly in addition to rupture of the arginine carbonyl peptide bonds) contributes in some degree to the acti- vation process. STUDIES OF THE PRODUCTION AND TBYPTIC ACTIVATION OF THE TOXIN OF CLOSTRIDIUM BOTULINUM TYPE E By Marjorie Mueller Schaeffer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1967 .ACKNOWLEDGMENT I am grateful to my husband, Steve, and my advisor, Dr. Lechowich, for their help in completing this project. 11 TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . 2 Activation of the Botulinal Toxin 2 Toxin Production 6 Nature of the Toxin 13 METHODS AND MATERIALS . . . . . . . . o . . . . . . . 15 Toxin Activation l6 Toxin Production and Stability as a Function of Time and Cell Development 18 Substrate Requirements for Optimum Toxin Production 20 Sucrose and Trypticase Concentrations 21 Masking of the Lysine 23 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 26 Conditions for Activation of the Toxin 26 Toxin Production versus Time and Cell Development 29 Substrate Requirements for Toxin Production 33 Trypticase and Sucrose Concentration versus Toxin Production and Growth 3h Toxin Activation after Attempted Masking of Lysine 36 SUMMARY AND CONCLUSIONS 0 O O O O O O O O O O O O O O 38 LITERATIIRE CITED 0 O O O O O O O O O O O C C O C O O 0 [+1 111 Table 1. 2. 10. 11. LIST OF TABLES Titers in MLD/ml of Clostridium botulinum type E toxin after activation at various temperatures and times . . . . . . . . . . . . Titers in MLD/ml of Clostridium botulinum type E toxin after activation using various trypsin concentrations . . . . . . . . . . . . Titers in MLD/ml of Clostridium botulinum type B dialyzed toxin after activation in media containing various NaCl concentrations .‘ Activation of strain 517 Clostridium botulinum type E toxin with various commercial preparations of trypsin and other enzymes . . Activation of strain VH Clostridium botulinum type E toxin with peptidase and trypsin . .-. Toxin production versus time and cell develop- ment for strain 517 Clostridium botulinum type E (Trial 1) o o o o o o o o o o 0 o o o o Toxin production versus time and cell develop- ment for strain 517 Clostridium botulinum type E (Trial 2 ) O O O O O I O O O O O O O O O Toxin production versus time and cell develop— ment for the Kalamazoo strain Clostridium botulinum type E . . . . . . . . . . . . . . . Toxin production versus time and cell develOp- ment for the VH strain Clostridium botulinum typeEoococoa-000000000009 Growth and toxin production of Clostridium botulinum type E with various carbohydrate sources or substitutes for carbohydrate sources in the media . . . . . . . . . . . . . Toxin production of Clostridium botulinum type B strain 26080 in media containing various sucrose concentrations . . . . . . . . iv Page 48 49 50 51 52 53 56 59 62 65 66 Table 12. 13. 14. Page Toxin production of Clostridium botulinum type E strain 26080 in media containing various trypticase concentrations . . . . . . . 67 Toxin production of Clostridium botulinum type E in tissue culture medium 109 . . . . . . 68 Activation and non-activation toxin titer data for strain Minneapolis Clostridium botulinum type E subjected to dinitrophenyla- tion in an attempt to mask the lysine in the toxj-n O O O 0 O O O O O O O O O O O O O O O O O 69 Figure 1. LIST OF FIGURES Page Titers of strains 517 and VB Clostridium botulinum type E toxin activated at various pH levels . . . . . . . . . . . . . . . “6 Toxin production versus time and cell - development for strain 517 Clostridium botulinum type E (Trial 1) . . . . . . . . . . 55 Toxin production versus time and cell development for strain 517 Clostridium botulinum type E (Trial 2) . . . . . . . . . . 58 Toxin production versus time and cell develOpment for the Kalamazoo strain Clostridium botulinum type E . . . . . . . . . 61 Toxin production versus time and cell development for the VB strain Clostridium botulinum type E . . . . . . . . . 64 vi LIST OF APPENDICES Appendix Page 1. Tables and figures 0 o o o o o o o o o o o o o o “’5 vii INTRODUCTION Although outbreaks of food poisoning due to the toxin of Clostridium botulinum are few in number the tremendous lethal capacity of the botulinal protein toxin is a curious property. The specific toxicity of 91. botulinum type E is less than that for types A and B, the other two types which most commonly affect man. Schantz (196h) reported that Cl. botulinum type B had a specific toxicity for mice of 0.6 X 1010 LD50 per gram of purified toxin and types A and B each had a specific toxicity of 3.8 x 1010 LD50 per gram of purified toxin. 9;. botulinum type E is also physiologically unique from types A and B in that it is considered primarily sac- charolytic instead of proteolytic as are types A and B. This research was conducted in an attempt to learn more about the toxin of 2;. botulinum type E and its produc- tion. Five areas were investigated: (1) optimum conditions for toxin activation, (2) toxin production as a function of time and cell development, (3) substrate requirements for toxin production, (u) toxin production and cell multiplication as a function of trypticase (N) and sucrose (CHO) concentration in the growth media, and (5) toxin activation after attempted masking of lysine in the toxin. REVIEW OF LITERATURE Activation of the Botulinal Toxin Five areas will be considered in reviewing the activa- tion of the toxin of Clostridium botulinum: (l) the mode of action of trypsin on the toxin of Cl. botulinum type E; (2) the effect of trypsin upon the toxin of g;. botulinum type E; (3) the optimum conditions for tryptic activation of type E toxin; (h) the effectiveness of enzymes other than trypsin which have been tested for enhancing toxicity of Cl. botulinum type E toxin; and (5) the similarities between the tryptic activation of type E toxin and the apparent toxin activation of types A and B during autolysis of the vegetative cells. The mode of action of trypsin on the toxin of Ql-.222' ulinum type B is assumed to be the same as on any other protein. Trypsin hydrolyzes links (not necessarily peptide links) in- volving the carboxyl groups of the basic amino acids lysine and arginine. Substitution of the side-chain amino (NHZ) group entirely prevents the action of trypsin, while substitution on theoeNHz group facilitates the hydrolysis. The side-chain must not be shorter than in lysine. An acidic side-chain on the neighboring amino acid residue tends to retard hydrolysis (Dixon and Webb, 1958). -2- -3- The effect of trypsin on the clostridial toxins has been studied by a number of investigators. Turner and Rodwell (19h3a,b) demonstrated that the epsilon toxin of Clostridium welchii type D could be activated by trypsin; and Ross, warren and Barnes (1949) demonstrated that 9;. welchii iota toxin could also be activated by trypsin. Sakaguchi and Tohyama (l955a,b) observed that Cl. botulinum type E cultures contam- inated with organisms of the genus Clostridium produced higher toxin titers than the pure culture. These observations, com- bined with the low titer data obtained for type E toxin when intraperitoneally injected into mice as compared to oral dosing, encouraged Duff, Wright, and Yarinsky (1956) to try activating the toxin of type E with trypsin. These workers demonstrated an increase in titers for the toxins of various strains of type E upon incubation with trypsin at 37 C for U5 minutes. This increase ranged from 12 to 47 fold (approximately 5800 LD5O/ml to 180,000 LD5O/ml for the VH strain). Dolman (196u), however, obtained slightly different results upon type E toxin activation with trypsin. He observed a titer increase of approximately #000 fold with the VH strain, from 2000 to 3000 minimum lethal doses (MLD) to 10,000,000 to 50,000,000 MLD, over a three day period of incubation with trypsin at 37 C. There also seems to be a difference of opinion between Duff and his coworkers and Dolman as to the optimum conditions for tryptic activation of type E toxin. Duff gt a1. (1956) established an optimum pH between 5.5 and 6.0 with pH levels below 5.0 and above 7.0 showing a marked decrease in activation. -4- A trypsin concentration of 1.0 percent and an incubation tem- perature of 37 C for any time in excess of 15 minutes and less than 120 minutes produced the greatest activation in his ex- periments. Dolman (1964), on the other hand, observed a maxi- mum activation of type E toxin in three days in one experiment and four hours in another experiment both conducted under simi- lar conditions: 1.0 percent trypsin, 37 C, pH 5.5, with un- purified toxin in beef infusion medium. In this same report he announced a marked decrease in titer of the unpurified toxin upon attempted tryptic activation at pH 7.5 for times of 30 minutes up to 20 hours. Gerwing, Dolman, and Ko (1965) reported slightly different activating conditions for the purified type B toxin. Again, they activated at 37 C with 1.0 percent trypsin; but this time they obtained a maximum activation of approximatebr 30 fold at pH 5.8 in about five hours followed by a gradual de- crease in toxicity. They also found that the purified toxin was activated approximately 20 fold at pH 7.5 in 15 minutes with a sharp decrease in titer after 15 minutes which they at- tributed either to toxin instability at that pH level or to progressive destruction of the toxic site by trypsin. Some enzymes other than trypsin have been tested for a capacity to activate the toxin of 91. botulinum type E. Dolman (196“) incubated type E toxin with pepsin at 37 C and pH levels of 5.5 and 2.5 (Optimum for pepsin activity) and ob- served no titer increase at pH 5.5 and a decrease at pH 2.5. Pepsin catalyzes the hydrolysis of peptide bonds involving the carbonyl groups of tryptophan, phenylalanine, tyrosine, -5- methionine and leucine (White, Handlen.and Smith, 1964). Be- sides pepsin, chymotrypsin and papain have been tested and found to be ineffective as activating agents (Lechowich, 1962). The major sites of action of chymotrypsin are tryptophan, phenylalanine and tyrosine. Chymotrypsin can also catalyze the hydrolysis of the peptide bonds of the carbonyl groups of leucine, methionine, asparagine and histidine. Papain has a wide specificity but does not act at acidic residues in a pro- tein. Its major sites of action are the bonds involving the carboxyl groups of arginine, lysine and glycine (White _3 El. 1964). Activation of types A and B 91. botulinum toxin, simi- lar to the tryptic activation of type E toxin, was stated to occur during autolysis of the vegetative cells of types A and B. Bonventre and Kempe (1959, 1960a, b) have investigated the toxin production of types A and B. They observed that types A and B synthesized enzymes capable of hydrolyzing protein, that no additional protein was formed after 24 hours of incubation by types A and B, and that the sum of the extracellular and intracellular toxin at the end of the logarithmic growth phase (24 hours) accounted for only 10 percent of the toxicity demon- strable when autolysis was complete (48-72 hours). They also reported that the titer of the toxin of young cultures incu- bated for only 12 hours could be increased by incubation with -either trypsin or pepsin. However, the toxin titer of an older culture which had proceeded through autolysis could not be en- hanced by incubation with trypsin or pepsin. From this -6- information they concluded that the toxin was synthesized dur- ing the logarithmic growth phase and that with types A and B Q1. botulinum a toxin activation process occurred naturally that involved proteolytic enzymes synthesized by the organisms and released upon autolysis. Toxin Production This discussion of toxin production by 91. botulinum will include: (1) the physiology of toxin production; and (2) the conditions required for growth, toxin production, and toxin stability. The physiology of toxin production has been studied more extensively in types A, B, C, and D than in E. Conse- quently, the physiology of toxin production of types A, B, C, and D will receive more attention in this review than that of type E. The work of Kindler, Mager, and Grossowicz (1956) and Bonventre and Kempe (1959, 19603, b) on types A and B indicated that the toxin was produced within the cell primarily during the logarithmic growth phase (18-24 hours of incubation) or shortly thereafter. However, most of this toxin was not re- leased until autolysis occurred (24—72 hours). Proteolytic enzymes were also released upon autolysis which activated the toxin to its maximum titer, which occurred after approximately 72 hours of incubation. Kindler gt_§l. (1956) also noted that toxin production occurred in the absence of cell multiplicaticn. They observed that aerobic conditions were not detrimental to toxin formation using resting cell suspensions (prepared by -7- centrifuging 18 hour cultures, washing the cells, and then resuspending them). . Toxin production of 91. botulinum type C was reported by Boroff (1955) to be quite similar to that of types A and B except that autolysis occurred over a longer period of time. Autolysis began after 24 hours of incubation in corn steep liquor and continued until the seventh to tenth day and only then was the maximum toxin titer obtained. It was demonstrated that proteolytic enzymes synthesized within the vegetative cell and released upon autolysis also caused toxin activation in type C 9;. botulinum. Boroff contrasted reports of types A and B toxin production to that of type C and hypothesized that toxin production occurred on the type C cell surface instead of internally for two reasons: (1) organisms from which the toxin has been extracted with 1N sodium chloride appeared to be all gram negative (the same as the lysed debris) but re- tained their shape as if only their cell walls were removed; and (2) washed cells intravenously injected into mice caused death as rapidly as the soluble toxin thus hinting that there was not time for cell lysis to occur. The physiology of toxin production of type D was de- scribed by Boroff, Raymaud, and Prevct (1952) to be very simi- lar to that of types A and B, with a low toxin titer detected during cell multiplication followed by autolysis and a marked toxin titer increase. Segner, Schmidt, and Boltz (1964) have studied toxin production of 91. botulinum type E in two laboratory culture -8- media, trypticase-peptone-glucose (TPG) and pea extract plus 2 percent peptone (PE2). They observed that a maximum toxin titer of 100,000 to 200,000 MLD/ml was achieved after three days of incubation in TPG or PE2 at 85 F. A peak population of 100 to 120 million cells was reached in TPG medium in 24 to 30 hours of incubation at 85 F. Therefore, they concluded that 500 to 1000 cells yielded one MLD toxin. The conditions required for growth, toxin production, and toxin stability for types A, B, and E have been studied, and will be reviewed here with primary emphasis being placed upon substrate requirements. Much of the work involving the relationship of substrate conditions to growth and toxin production of type A has been done by: Lewis and Hill (1947); Mager, Kindler, and Grossowicz (1954); Kindler gt a1, (1956); and Bonventre and Kempe (1959, 1960a, b). Lewis and Hill (1947) observed that growth in media containing 1.0 to 2.0 percent casein, 0.6 percent glucose, 0.05 percent thioglycollic acid, and either 0.5 percent yeast ex- tract or corn steep liquor produced the highest toxin titers when the cultures were incubated at 34 C for three days. In corn steep casein medium he noted that the rate of the toxin accumulation decreased as the concentration of glucose was in- creased, but in all instances reached a maximum level of 500,000 to 1,000,000 MLD/ml. The cultures containing 0.3 to 0.6 percent glucose reached their maxima in approximately 30 hours, while the cultures containing 1.0 percent glucose required nearly 60 hours. Mager _e_t a1. (1954) and Kindler gt a1. (1956) observed that all strains which they tested -9- showed good growth in media containing at least 2.5 percent casein hydrolysate, equivalent to about 0.18 percent nitrogen. They found that the casein hydrolysate could be replaced by a mixture of amino acids of which the following were essential: tryptophan, threonine, valine, leucine, isoleucine, methionine, arginine, phenylalanine, and tyrosine. The last three acids were required in unusually large amounts. To these amino acids they added glucose, vitamins (including biotin, thiamine, p-aminobenzoic acid, nicotinic acid, and pyridoxine), phos- phates, and magnesium to complete their defined medium for type A. Omission of the vitamins did not greatly affect toxin pro- duction in resting cells. However, elimination of glucose or one of the essential amino acids resulted in very low titers in suspensions of resting cells. Bonventre and Kempe (1960b) also noted that glucose in the medium was necessary during the first 24 hours of incubation to obtain maximum toxin production. However, removal of glucose after 24 hours did not result in a reduced toxin titer, thus indicating that toxin synthesis re- quires glucose and occurs during the first 24 hours of growth. Only glucose and its disaccharide maltose fully supported toxin synthesis. Of the other energy sources which Bonventre tested, only glycerol, pyruvate, and ribose partially supported toxin synthesis. The toxin produced in the presence of galactose, rhamnose, sorbitol, xylose, lactose, and inositol was no greater than in a control medium which contained no carbohydrate. Total growth (but not growth rate) was also less for the other energy sources than for glucose. However, this growth repression was -10- not sufficient to account for the differences in toxin produc- tion. It was also noted that autolysis did not go to comple- tion unless glucose or maltose was present in the medium. Sonic disintegration of the intact cells which remained after 72 hours in media containing the other carbohydrates did not result in increased toxicity of the supernatant, thus indicat— ing that toxin was not present intracellularly. Bonventre hypothesized that glucose was required, not solely as an energy source, but specifically for synthesis of the protein toxin because: (1) the three carbon compounds (members of the gly- colytic pathway) and ribose (able to contribute to the gly- colytic pathway if the pentose shunt is operative) were not effective substitutes for glucose in toxin production; and (2) the slight growth repression observed with energy sources other than glucose and maltose in the media could not account for the marked decrease in toxin production. Much of the recent work published on the substrate re- quirements for growth and toxin production of type E has come from Dolman's laboratories. Several media have been used by various researchers for the growth of g;. botulinum type E, including TPG,PE2 and Dolman's (1964) medium consisting of ground meat, 1.0 percent peptone, 0.5 percent NaCl, 0.08 percent NaZHPou, 0.5 percent glucose, and 0.2 percent sodium thiogly— collate (GPBI-meat). Dolman reported that growth resulted when the glucose in GPBI-meat medium was replaced by 1.0 percent of various carbohydrates and the cultures incubated at 37 C for 48 hours. He observed that large amounts of acid and gas were produced with glucose, fructose, maltose, sorbitol, and sucrose; -11. and slight acid and gas were produced with glycerol and adonitol. However, he noted no growth as measured by acid and gas with dextrin, salicin, inositol, and galactose.. No studies on toxin production with these energy sources was reported. Gunnison, Cummings, and Meyer (1936) noted slight fermentation differenoaa They reported that galactose and dextrin were actively fermented while little fermentation occurred with sucrose and maltose. This difference was possibly due to a strain difference. Dolman" (1964) also reported that the glucose requirement seemed to vary with incubation temperature. Several type E strains, grown at 23, 30 and 37 C, usually produced more toxin at the lower temperatures when little or no glucose was present; but generally at 37 C toxin titers were increased by the addition of more glucose (2.0 percent). Observations have also been made on the stability of botulinal toxin. Toxin production by types A and B 91.jggt- ulinum can occur in media with the initial pH between 5.3 and 8.5. However, the toxin is very unstable in an alkaline en- vironment (Bonventre, 1957; Lamanna and Glassman, 1947). Kindler gt a1. (1956) also observed the toxin to be less stable in their synthetic medium than in synthetic medium which had added protein. The type A toxin is also readily denatured by heat. Cartwright and Iauffer (1958) found that type A toxin in a solution at pH 6.9 could be heated at 40 C for one hour without loss of toxicity, but at 50 C or above rapid detoxifi- cation occurred within minutes. Schantz (1964) reported that distortion of the toxin molecule by spreading on a large surface causes complete loss of toxicity. -12- Schantz and Spero (1957) studied the reaction of the toxin with ketene and found that it was readily detoxified upon its reaction with the free amino groups of the protein. However, reaction of the ketene with the phenolic hydroxy and free sulfhydryl groups did not appear to affect the toxicity. Spero and Schantz (1957) found that deamination of the toxin with nitrous acid caused a rapid detoxification. Spero (1958) found, on the basis of a thermodynamical consideration, that the decrease in toxicity due to an increase in pH is associated with the ionization of a small number of €-amino groups of lysine. The toxin is readily denatured by heat, strong alkali, and many oxidizing agents. Schantz (1964), accumulated evidence which indicates that the toxicity is a result of a particular conformation (coiling and folding) of the molecule, in which at least the free amino groups are involved. Segner gt a1. (1964) have studied the stability of 91. botulinum type E toxin in two laboratory media. They observed that type E toxin was less stable in TPG medium than in PE2 medium. The toxin remained stable for approximately 21 days in TPG and PE2 at 85 F. However, the titer decreased more sharply after 52 days in TPG than in PE2. When TPG medium culture filtrate of BB strain containing 100,000 MLD of acti- vated toxin was diluted 1:10 with 0.067 molar phosphate buffer at a pH level of 6.0, the toxin remained stable for 7 days at 85 F. However, the titer determined on day 16 decreased from that measured on day 7 and decreased steadily after that until a titer of only 50 MLD of activated toxin was determined on day 84. -13- Nature 9: the Toxin Research conducted on the toxin of 9;. botulinum is extensive. However, only a brief review of the literature on type E botulinal toxin and its mode of action will be attempted. The most recent published work on the characterization of type E toxin has been done by Gerwing, Dolman, and Ko (1965). They have determined a molecular weight of approximately 18,600 for the non-activated toxin. This value is much smaller than that of approximately 900,000 for types A, B, C, and D (Schantz, 1964). However, some progress is being made in an attempt to find smaller toxic subunits for types A and B (Gerwing, Dolman, and Bains, 1965; Van Alstyne, Gerwing, and Tremaine, 1966). Gerwing, Dolman, and Ko (1965), also determined the amino acid composition of type E toxin which seems to be quite similar to that determined for the toxin of type A by Buehler, Schantz, and lamanna (1947). In both cases it was determined that no prosthetic groups were present on the toxin unless the pros— thetic groups were also made of amino acids. Gerwing, Dolman, and Ko (1965) also learned that the N-terminal residue of the tryptic activated toxic peptide was arginine. The N—terminal residue of the non-activated toxin was glycine. They arrived at the following amino acid composition for the activated and non-activated toxin of type E: -14- Amino Acid Toxin? Activated Toxina Cysteic acid Aspartic acid 1 Threonine Serine Glutamic acid 1 Proline Glycine l Alanine valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine HH wwwmtoommmmHmvmu H H mwnmtmmttmmwmvmm Toxicity 6 (MLD/mg N) 7.5x10 2.8x108 8amino acid residues The mode of action of botulinal toxin can be briefly summarized. It is characterized by its effects on the periph- eral nervous system. The central nervous system appears to function normally even after injection of toxin into the medulla or sciatic nerves. Intoxication results in inactivation of the cholinergic synapses while adrenergic fibers are not affected. Botulinum toxin blocks cholinergic junctions through irrevers- ible inactivation of the presynaptic release mechanism of acetylcholine (Brooks, 1964). Death usually results from re- spiratory paralysis (Burgen, Dickens, and Zatman, 1949; Davies, Morgan, Wright, and Wright, 1953). METHODS AND MATERIALS This research was carried out in five parts. The purpose of each of the parts was as follows: (1) determination of optimum conditions for toxin activation; (2) observation of toxin production and stability as a function of time and cell development; (3) determination of substrate requirements for toxin production; (4) determination of optimum sucrose and trypticase concentrations in the medium for toxin production; (5) determination of more information on the process of tryptic activation of the toxin, by masking the lysine prior to activa- tion. The growth medium used regularly throughout the re- search contained 5.0 percent trypticase, 0.5 percent peptone, 1.0 percent yeast extract, 0.2 percent sucrose, and 0.1 percent sodium thioglycolate, withthe pH level adjusted to 7.2 and was designated TPSY medium. The stock cultures which were used and their sources are listed below. Cultures Sources Kalamazoo (KalJ, 26080, Mr. R. W. Johnston, 517 Food and Drug Administration Detroit, Michigan Vancouver Herring (VH), Dr. C. F. Schmidt, Minneapolis (Minn.) Continental Can Co., Inc. Chicago, Illinois -15- -16- Toxin Activation Toxin activation eXperiments were performed on the toxins of the 517 and VH strains, which were in suspension in the TPSY growth medium. The 517 and VH strains had been in- cubated at 30 C for 30 hours. The cells and spores were re- moved by centrifugation, and the supernatant containing the toxin was reduced to a pH level of 5.0 and stored at 2 C. Under these storage conditions the toxin titer remained stable for at least three months. Conditions which were altered during toxin activation were the pH level, temperature and time, trypsin concentration, sodium chloride concentration, and the use of different acti- vating enzymes. The pH was adjusted in the toxin suspension by adding NaOH or HCl. The pH of the trypsin was adjusted by suspending it in phosphate buffer solutions which were prepared at the desired pH levels according to Sorensen's table of phosphate buffer mixtures so that the pH of the toxin suspension and the pH of the trypsin suspension matched when they were combined in equal parts. The pH levels tested ranged from 5.0 to 7.0. The toxin-trypsin suspension was incubated in a 37 C water bath for one hour using a final trypsin concentration of 0.5 percent. The times and temperatures of incubation were adjusted within ranges of 30 C to 50 C and from 10 minutes to 8 hours, respectively. The times and temperatures were varied with a constant pH of 5.8 and trypsin concentration of 0.5 Percent in TYSY medium. -17- The trypsin concentration was varied from 0 percent to 8.0 percent final concentration in two types of mixtures. One mixture contained one part toxin in TPSY to one part trypsin (double the final concentration) suspended in phosphate buffer; and the other contained one part toxin in TPSY, two parts ground fish, and one part trypsin (quadruple the final concentration) suspended in phosphate buffer. The second mix- ture was the high protein medium. These mixtures with varying trypsin concentrations were incubated at 45 C for 35 minutes at a pH of 5.8. The NaCl concentrations were adjusted by adding the desired amount of NaCl to suspensions of toxin in TPSY which had been dialyzed against deionized water for five days. The dialysate was checked daily for toxicity. The deionized water in the dialysis system was changed daily. Activation of the treated solutions was carried out at 45 C for 35 minutes at pH 5.8 to 6.0 with 0.5 percent trypsin. Toxin activation was attempted with various commercial trypsin preparations (Difco 1:250, Nutritional Biochemicals (NBC) 1:300, 2X crystaline trypsin, and 4XUSP pancreatin NBC) at 0.5 percent concentration and 37 C for one hour. A number of other enzymes were tried at pH levels determined by their optimum pH levels for activity and by the pH levels at which the toxin is stable. The concentration of the crude enzyme preparations was 2 to 4 percent. The effectiveness of the various conditions tested was determined by comparing the toxin titers obtained under -18... the various conditions. Toxin titers were determined by intraperitoneally injecting decreasing concentrations of the toxin into 12 to 15 gram Swiss Webster white mice and noting the lowest concentration which caused death within 48 hours. Toxin Production and Stability a§.§_Function of Time and Cell Development Three strains, VH, 517, and Kalamazoo, were used in observing toxin production and stability as a function of time and physiological development. A portion of the stock Spore suspension was heat shocked in a water bath at 60 C for 15 minutes in each case. At least nine 20 milliliter test tubes containing TPSY were inoculated with the heat shocked spores for each strain used such that the original Spore suspension became 10 percent of the total volume. The tubes containing the inoculum were incubated at 30 C. A single tube was removed from the incubator at each designated time. Two samples were removed from the tube; one for the direct microsc0pic count with a Petroff-Hausser Counter and the other for activation of the toxin in the cell suspension with trypsin and lysozyme for 35 minutes at 45 C. The lysozyme was added to break the cell wall and release any toxin which might be present within the cell or on the cell wall. The remainder of the culture in the tube was used to measure absorbancy with the Bausch and Lomb Spectronic 20 spectrophotometer at 645mu. The remaining cell suspension was then centrifuged at 4000 rpm for 10 minutes at 0 C in a Sorval Superspeed RC-z Automatic Refrigerated -19- Centrifuge. A sample of the cell-free supernatant was removed and incubated at 45 C for 35 minutes with 0.5 percent trypsin to activate the toxin which was in the supernatant. Both acti- vated toxin suspensions were intraperitoneally injected into 15 to 20 gram mice in graded dilutions to determine an approx- imate toxin titer. The pH levels of the cell suspensions were approximated with pH paper so as not to contaminate the pH meter. Two methods of disrupting the vegetative cell wall were tried in an attempt to release toxin from the cell without de- stroying the toxin in the previous eXperiment described. One involved the use of lysozyme and the other a mechanical homoge- nizer. The fi-l,4 bonds between muramic acid and N-actylglucosa- mine in many gram positive bacterial cell walls are cleaved by lysozyme thus disrupting the cell wall (White, Handler, and Smith, 1964). It was observed that when a cell suspension of ‘91. botulinum type E was incubated with 0.5 percent lysozyme and 0.5 percent trypsin the cells were disrupted (when observed under the phase microscope only debris was left) and there was a marked increase in titer over that incubated with just 0.5 percent trypsin. It was also observed that the titer of a toxin suspension (without cells) was unaltered when incubated with 0.5 percent lysozyme and 0.5 percent trypsin as compared with 0.5 percent trypsin alone. This information led to the con- clusion that lysozyme disrupted the vegetative cell walls and released toxin without destroying the toxin. When Q1. botulinum type E cell suspensions were mechan- ically homogenized for one minute at 4000 oscillations per -20.. minute in a Braun Model MSK Mechanical Cell Homogenizer, the toxin titer was markedly decreased from that of a sample of the same cell suspension which had not been homogenized. The Bronwill Homogenizer was cooled with a constant flow of C02 from a liquid C02 tank. Little apparent temperature change in the sample was noted. Because of these results lysozyme was used to disrupt the cells instead of mechanical homogeni- zation. Substrate Requirements for Optimum Toxin Production Two experiments were conducted in which the carbohy- drate sources in the media were varied. The basal medium used in both eXperiments contained 5.0 percent trypticase, 0.5 per- cent peptone, 1.0 percent yeast extract, 0.1 percent sodium thioglycolate, and 0.5 percent carbohydrate at pH 7.2. The media were dispensed into 20 milliliter test tubes and auto- claved. The following energy sources were used in the first experiment: sucrose, glucose, fructose, maltose, sorbitol, ribose, and glycerol. A control medium containing no carbo- hydrate was used. Acetate was also used in the second experi— ment. The spores of 26080 and VH strains were heat shocked at 60 C for 15 minutes. Two tubes from each medium were inoc- ulated, one with the 26080 strain and the other with the VH strain such that the inoculum was 10 percent of the final volume. The tubes were incubated at 30 C and were checked approximately every five hours for 7 days for turbidity and gas production. Toxin titers were determined after 24 hours by the previously described method. The cultures in all of the various media described above attained maximum growth in approximately the same time as the organisms in the medium containing sucrose as the carbohydrate source (TPSI). Incuba- tion for 24 hours in TPSY produced the highest titers. Con- sequently, toxin titers in these media were checked after 24 hours of incubation. The media were inoculated in the second experiment with heat shocked spores of strains 26080 and VH as in the first experiment. However, after 22 hours of incubation at 30 C each culture was then transferred (as a 10 percent inoculum) to fresh medium containing the same energy source. The cul- tures were transferred a total of five times. At the end of 24 hours of incubation after the final transfer, toxicity titers and absorbancies were determined for both strains in- cubated in the presence of each of the carbohydrates and the substitutes for carbohydrates. Sucrose and TrypticaSe Concentrations Sucrose concentration: The basal medium used to test toxin production as a function of sucrose concentration was the same as that used in the previous test (5.0 percent tryp- ticase, 0.5 percent peptone, 1.0 percent yeast extract, 0.1 percent sodium thioglycolate, and sucrose). The sucrose -22.. concentration was varied from 0 percent to 10 percent. The various media were dispensed into 20 milliliter test tubes and autoclaved. Each of the tubes was inoculated with heat shocked spores of 26080 or VH strains such that there were two tubes at each sucrose concentration, one inoculated with 26080 and one with VH. The cultures were incubated at 30 C for 24 hours. After 24 hours the toxin titers were determined, absorbancies recorded, and the pH levels were estimated with pH paper. Trypticase concentration: The basal medium used to determine toxin production as related to trypticase concentra- tion in the medium contained 1.0 percent yeast extract, 0.1 percent sodium thioglycollate, and either 5.0 percent or 0.5 percent sucrose. The trypticase concentration ranged from 0 percent to 10 percent. Again the media were dispensed into 20 milliliter test tubes, autoclaved, and inoculated with heat shocked spores of the 26080 or VH strains. The cultures were incubated for 24 hours at 30 C. The toxin titers were deter- mined and absorbancy readings taken after 24 hours of incuba- tion. Carbon and nitrogen determinations: Carbon and nitro- gen determinations were conducted on each of the media con- taining the various sucrose and trypticase concentrations. Com- plete carbon combustion to C02 with Van Slyke reagents, entrap- ment of C02 in Ba (0H)2, and back titration with HCl was used to determine total carbon content in the dry ingredients of each of the media (van Slyke, Plazin, and Weisiger, 1951; Van Slyke and Folch, 1940; van Slyke, Steele, and Plazin, 1951). -23- The Kjeldahl procedure was used to determine total nitrogen content in each of the media in the final prepared liquid form (American Instrument Company, 1961). Three samples were run for each level and mean values calculated. Masking Q: the Lysine More information about a toxic site (or sites) of the type B toxin was desired. Consequently an attempt was made to mask the lysine in the protein toxin so that trypsin would cleave the protein only at the carbonyl bond of the arginine residues. Masking of the lysine would permit comparisons be- tween toxin titers of lysine-masked and unmasked activated toxins. Redfield and Anfinsen (1956) and Anfinsen, Sela, and Tritch (1956) had success in masking the lysine residues of proteins including enzymes by dinitrophenylation of their -amino groups. They rendered the lysine residues unsuscep- tible to trypsin digestion in this manner and in the case of the enzymes they did not destroy the activity of these proteins. They used the procedure of Sanger (1945) to attach the 2,4- dinitrophenyl radical to the €-amino group of the lysine resi- dues in proteins. Sanger (1945) described the following procedure: 0.48 grams of oc-acetyl-l-lysine and 0.75 grams NaH003 were dissolved in three milliliters of water. To this solution was added a solution of 0.5 grams dinitrochlorobenzene in 10 milliliters of ethanol. The mixture was heated under reflux on a water -24- bath for 4 hours. The ethanol was removed by evaporation in vacuo and the residue dissolved in water, filtered to remove excess dinitrochlorobenzene and acidified with HCl while hot. To obtain the toxin for this study the organisms were grown in a dialysis sac. A dilute suspension of the heat shocked spores (35 ml.) was placed in dialysis tubing which was suspended in TPSI as described by Vinet and Fredette (1951) and Barron and Reed (1954). This culture was incubated at 30 C for 24 hours. Thus, all the molecules of greater than approximately 10,000 molecular weight which were produced by the cells would remain inside the tubing and those of the medium would remain outside the tubing. The suspension which was inside the tubing was centrifuged after 24 hours at 4000 rpm for 10 minutes to sediment the cells. The supernatant was re- moved and put into fresh dialysis tubing and dialyzed against phosphate buffer (pH 6) for 5 days. The phosphate buffer was changed each day. The molecules of molecular weight less than 10,000 should have been removed from the supernatant by dialysis, thus leaving only the molecules larger than molecular weight of 10,000(including the toxin) which had been produced by the organism within the tubing. The dialysis tubing technique per- mitted the toxin to be obtained in a more concentrated form in comparison to that produced in the TPSY medium without the use of dialysis tubing. Sanger's (1945) modified procedure was followed to mask the lysine residues of this partially purified type B toxin. Addition of sodium bicarbonate (NaHCOB) to the toxin naturally -25- caused an increase in pH above 7.0 which destroyed the toxicity of this protein. Consequently, only small amounts of NaHCOB were added such that the pH remained below 7.0. The water bath temperature of the reflux system was maintained between 40 and 45 C. Following the dinitrophenylation process, toxin titers were determined on the following six mixtures, each of which was incubated at 45 C for 35 minutes: (1) (2) (3) (4) (5) (6) toxin, not activated with trypsin, and not subjected to dinitrOphenylation; toxin, activated with trypsin, and not subjected to dinitrophenylation; toxin, plus dinitrochlorobenzene added only at the beginning of the 30 minute incubation period, not activated with trypsin; toxin, plus dinitrochlorobenzene added at the beginning of the 30 minute incubation period, activated with trypsin; toxin, not trypsin activated, but dinitrOphenylated; and toxin, trypsin activated, and dinitrophenylated. The volume of each portion of these mixtures was such that the toxin concentration was the same for each sample. Toxin titers were determined for two samples of each condition. RESULTS AND DISCUSSION Conditions for Activation 9f the Toxin 25 levels: The effect of pH upon the tryptic activa- tion of the type E toxin and upon the toxin itself are demon- strated in Figure l. The Optimum pH level for tryptic activa- tion of the toxin in trypticase-peptone-sucrose-yeast extract medium (TPSI) was 5.8. There was an increase in toxicity of approximately 25 to 50 fold upon tryptic activation at pH 5.8. Adjustment of pH below 5.5 and above 6.5 showed a marked deg crease in activation. A pH above 7.0 caused destruction of the toxicity of this protein. These findings parallel those of Duff, wright, and Iarinsky (1956) more closely than the data published by Dolman (1964). Temperature and time: The effect of temperature and time upon tryptic activation of the toxin was not dramatic. The results of varying temperatures and times of tryptic acti- vation are recorded in Table 1. Times and temperatures from one hour at 37 C to 30 minutes at 45 C allow similar activation. The observation that the titer of the toxin incubated with no activating enzyme at 37 C for 60 minutes was 2000; (3 indicates one mouse died and one lived of the two mice injected with the indicated dilution of the toxin suspension) and when incubated at 45 C for 30 minutes was 4000; (recorded in Table 4) may -26- -27- indicate that the shorter time at the higher temperature might be slightly superior from the standpoint of toxin stability during the activation incubation besides being time saving. An increase in time of incubation over one hour at 37 C did not increase the titer as was reported by Dolman (1964). Trypsin concentration: It was thought that higher trypsin concentrations than those used for the TPSY medium might be required for maximum toxin activation in a high pro- tein medium, such as one containing a large percentage of ground fish. An experiment was conducted to determine if this con— tention was valid. The results are tabulated in Table 2. The use of 0.5 percent trypsin in both TPSY and the fish media was as effective as any higher concentration tested. This data is in slight variance to that of Duff g§_al. (1956), in which 1.0 percent trypsin was optimum in beef infusion media. NaCl concentration: The effect of NaCl concentration in TPSY on toxin activation with trypsin is indicated in Table 3. The titer of a toxin suspension in TPSY, which had been dialyzed against water to remove the salts, could be increased by the~ addition of NaCl only to the original titer of the undialyzed suspension. This maximum titer was achieved when 4.0 percent NaCl had been added. When 5.0 percent NaCl was added, a marked decrease in titer was observed. Thus, provided a minimum ionic environment is achieved, the effect of ionic concentration is limited. Enzymes: The results of incubation of various com- mercial preparations of trypsin and of a number of other pro- teolytic enzymes with a type E toxin suspension in TPSY are -28.. recorded in Table 4. All the commercial preparations of trypsin which were tried were found to activate the toxin to approxi- mately the same titers. The protease (both endo- and exo-proteolytic) caused an increase in titer as great as that caused by trypsin (from 4000 MLD/ml to 100,000 MLD/ml). The proteinase (only endo- proteolytic) caused some increase in titer (from 4000 to 10,000 MLD's) but not as much as the protease. Therefore, at least part of the activation is due to the lytic action of enzymes (in this case proteinase) upon the internal peptide bonds Of the protein. Incubation of the toxin in the presence of a peptidase (only exo-proteolytic) resulted in no increase in titer as com- pared with the titer of the toxin incubated without an enzyme (Table 5). This information indicates that toxin activation (titer increase upon incubation with enzymes) is not caused by the removal of terminal amino acids of the protein toxin but rather that it is caused by the rupture of internal peptide bonds. Erepsin, a crude preparation of proteolytic enzymes, was not as effective as trypsin. Also, arginase, which splits arginine into ornithine and urea, had no effect on the toxicity of the protein, as might be expected. The Optimum conditions for activation observed in these experiments were a pH level of 5.8, a time and temperature re- lationship of 30 minutes and 45 C, a trypsin concentration of 0.5 percent, and a NaCl concentration of 4.0 percent or the -29- ionic concentration present in TPSY. The maximum titer increase achieved with the Optimum conditions determined for activation was approximately 50 fold. The magnitude of increase agrees more closely with that found by Duff 2£.§l- (1956) than with that found by Dolman (1964). Toxin Production versus Time and Cell Development Tables 6, 7, 8, and 9 and Figures 2, 3, 4, and 5 show the relationships of toxin production to time, the physiologi- cal state of the organism, and the pH of the culture for type E strains 517, Kalamazoo, and VH. The data in Table 6 and Figure 2 were collected in a preliminary study which was con- ducted to determine a rough indication of the relationship of physiological development of the cell and toxin production to time for strain 517. These data were obtained over a 48 hour period and demonstrated the marked decrease in toxin titer after 24 hours of incubation. The information in Table 7 and Figure 3 for the same strain, 517, is more extensive than that in Table 6 and Figure 2. However, the study was terminated at 36 hOurs of incubation and did not demonstrate the titer decrease. A number of observations and conclusions from the information in these tables will be presented in the following discussion. Demonstrable toxin titers were obtained for all three strains tested shortly after turbidity and gas production were noted and before sporulation occurred. The toxin titers of all the strains tested increased as the number of vegetative cells -30- increased and reached a peak after approximately 20 to 24 hours of incubation (Tables 6, 7, 8, and 9 and Figures 2, 3, 4, and 5). This titer peak occurred shortly (approximately four hours) after the number of vegetative cells plus sporangia had reached their maxima. The sporangia were just beginning to lyse and release free spores at this time. It was apparent from the data in Tables 7, 8, and 9 and Figures 3, 4, and 5, that the toxin was produced internally and then released by the vegetative cell into the medium. The data obtained from the VH strain (Table 9) showed that at five hours of incubation no toxin could be de- tected in the medium containing the intact cells. However, when the cells were disrupted, a titer of the released toxin of approximately 200 MLD/ml was observed. The titer of the toxin in the medium plus that from the disrupted cells was higher than that in the medium with the intact cells in all strains tested at all times, except after 36 hours of incuba- tion in the 51? strain (Table 7 and Figure 3), where the titers of the toxin in the medium with disrupted cells and that in the medium with intact cells was equal. From this information it was concluded that the toxin was produced steadily within the vegetative cell during the logarithmic growth phase similarly to, although more rapidly than, the production of toxin by types A and B 2;. botulinum as described by Kindler, Mager, and Grossowicz (1956) and Bonventre and Kempe (1959, 1960a, b). The type E data indicate that the toxin was released into the medium during the logarithmic growth phase before lysis occurred and also was released when the cell lysed to free its spore. -31- It was also concluded that, after the active (logarithmic) growth phase ended, little or no additional toxin was produced nor was it maintained within the cell because the vegetative cells which remained in the 51? strain after 36 hours released no detectable toxin upon disruption of their cell walls. This rapidly attained peak of toxin titers (24 hours) for 91. botulinum type E grown in TPSY medium was different from the toxin peak attained after three days of incubation observed by Segner, Schmidt, and Boltz (1964) in TPG and PE2 media. ‘91. botulinum type E produced toxin slightly more slowly in TPG and PE2 than in TPSY and the toxin in TPG and PE2 was much more stable than that in TPSY. The toxin titers, as re- corded in Tables 6, 7, 8 and 9, decreased steadily after 24 hours of incubation in TPSY at 30,0 while Segner gt_al. (1964) reported stable titers for 21 days in TPG and PE2 at 85 F (30 C). The final pH level after incubation at 30 C for 24 hours for both TPG and TPSY was 6.0. However, it was observed that, when a toxin suspension in TPSY was stored at 5 C, the toxin titer remained stable for at least three months. The differences between TPSY and TPG are that TPSY con- tains trypticase, peptone, sucrose and yeast extract, while TPG contains trypticase, peptone and glucose. When the sucrose in TPSY was replaced with glucose, as in TPG, the toxin was still unstable at 30 C. Therefore, yeast extract remained the substance in one medium which was not in the other. One could speculate a number of hypotheses to explain the instability of the toxin under these conditions. Possibly a metabolic by- product of 9;. botulinum type E, such as a proteolytic enzyme -32- which finds optimum conditions in the presence of yeast extract, could inactivate the toxin even though it was observed in the activation eXperiments that crude mixtures of a number of pro-‘ teolytic enzymes did not destroy toxicity when incubated in the presence of the toxin for one hour. Since Schantz (1964) reported that many oxidizing agents destroyed the toxicity of the botulinal toxin, it could also be speculated that an oxi- dizing agent might be supplied to the medium by the yeast ex- tract which could cause the instability of the toxin in TPSY. Any of a number of substances could be present in the TPSY medium which could cause a relatively slow conformational change in the protein toxin. The action of an enzyme, an oxidizing agent or some other reaction could be slowed when the tempera- ture was reduced to 5 C, thus making the toxin more stable at that temperature. It was apparent that sporulation had little effect on toxin production since the Kalamazoo strain (Table 8), which sporulated only slightly, produced as high or higher toxin titers as those strains which sporulated much more completely (Tables 7, 8, and 9). All three strains tested demonstrated relatively substantial toxin titers before sporulation occurred. It was concluded, therefore, that toxin production is £23 a part of the process of spore formation but that the toxin is a natural product of vegetative cell metabolism and growth. It was also observed that the 91. botulinum type E spores carried a small amount of toxin firmly attached to them. When a heat shocked stock spore suspension, incubated without lysozyme for 30 minutes, was injected intraperitoneally into -33- mice, no toxin was detected (recorded in Table 7 as titer at 0 time). However, if a similar spore suspension was incubated for 30 minutes with lysozyme, a titer of approximately 10 MLD was detected (recorded in Table 7 as titer at 0 time), while an identical amount of lysozyme injected alone caused no death. Substrate Requirements for Toxin Production Carbohydrate sources: Toxin titers and growth (as measured by absOrbancy) of 91. botulinum type E grown in basal media containing 5.0 percent trypticase, 0.5 percent peptone, 1.0 percent yeast, 0.1 percent sodium thioglycolate, and vari- ous carbohydrate sources or substitutes for a carbohydrate source are recorded in Table 10. Approximately equal amounts of growth and similar toxin titers were produced in media containing sucrose, glucose, and fructose as the carbohydrate sources. Maltose and sorbitol allowed approximately as much growth as sucrose, glucose, and fructose with only slightly reduced toxin titers. Therefore, the metabolic enzymes which catalyse the break down of sucrose to glucose and fructose (invertases), which cause the break down of maltose to glucose (maltase), and which cause the con— version of sorbitol to fructose (ketose reductase) must be present. Any of these carbohydrates can be used equally well for energy sources which permit growth and toxin production. Ribose allows less growth than the above carbohydrates, thus indicating the pentose shunt is present but not adequate in type E. The toxin titer observed in the medium containing -34- ribose appeared comparable to that produced by similar amounts of growth in media containing sucrose. Glycerol, a three car- bon source, also allows moderate growth and comparable toxin production. As might be expected of an anaerobe, only slight growth was observed in the medium containing acetate. However, toxin production did accompany this small amount of growth. The occurrence of growth and toxin production in the presence of acetate as the only substitute for a carbohydrate source, which was greater than the amount of growth and toxin production in the basal medium with no added carbohydrate, in- dicates that the citric acid cycle might be present but inade- quately functional in El. botulinum type E. The observations that toxin production occurred equally well with fructose as it did with glucose and sucrose, and better with fructose than it did with maltose, and the result that sorbitol, ribose, and glycerol allowed toxin production comparable to the amount of growth indicate that type E does not specifically require glucose for toxin production as did types A and B (Bonventre and Kempe, 1960b). The carbohydrate sources tested appeared to act only as energy sources and not as direct precursors to the toxin or as necessary ingredients of a toxin activation system. Trypticase and Sucrose Concentration versus Toxin Production and Growth The results of varying the sucrose and trypticase con- centrations in the growth medium for type E are recorded in -35- Tables 11 and 12. The ratio of nitrogen to carbon in the me- dium had no relation to the amount of toxin produced by 91. botulinum type E. Instead it was evident that minimum concen- trations of sucrose and trypticase were required for Optimum toxin production. Optimum growth and toxin titers were achieved in media with sucrose concentrations of 2.0 percent to 5.0 percent. However, sucrose concentrations of 0.5 percent and 1.0 percent, and 8.0 percent and 10.0 percent permitted similar, but slightly reduced, amounts of growth and toxin production. Markedly smaller amounts of growth and toxin production were observed in media containing less than 0.5 percent sucrose. Trypticase concentrations of 1.0 percent or more allowed Opti- mum toxin titers in medium containing only 0.5 percent sucrose. When the medium contained 5.0 percent sucrose, highest toxin titers were produced with 5.0 percent trypticase in the medium. The medium containing 5.0 percent sucrose, 5.0 percent trypticase, 1.0 percent yeast extract, and 0.1 percent sodium thioglycolate was the most desirable for the production of optimum toxin titers and optimum growth. Further studies on nutritional requirements for growth and toxin production are being undertaken in this laboratory in which growth and demonstrable toxin titers have been achieved in a chemically defined medium, Tissue Culture Medium 109 (Table 13). It would be desirable to determine the essential ingredients of this medium for both growth and toxin production. -36- Toxin Activation after Attempted Masking g: Lysine Because the activation process is so specific in that only trypsin can cause increased titers by its known action on the carbonyl peptide bonds of arginine and lysine, and because Gerwing, Dolman, and Ko (1965) found arginine to be the N- terminal residue of the tryptic activated toxic peptide, it was thought that it might be of some interest to determine if activation was a result of splitting both the arginine carbonyl and the lysine carbonyl bonds or if it was a result of splitting just one of these two types of bonds. Therefore, an attempt was made to mask the lysine. The results of this attempt on the trypsin activated and non- activated toxin titers are recorded in Table 14. The titer of the non-activated toxin which had been refluxed for four hours with dinitrochlorobenzene (DNCB) was the same as the titer of the non-activated toxin which had no DNCB added, thus indicating that the addition of DNCB did not detectably reduce or destroy the toxic capacity of this protein. The addition of DNCB to a toxin suspension, resulted in a slight reduction of the titer of the activated toxin in comparison to the titer of the acti- vated toxin to which DNCB had not been added (40,000 MLD instead of 100,000 MLD). This small difference in titers (only one dilution) can certainly not be a basis for any concluSions and can only warrant discussion because of the exact repetition of the titers for the six samples: the two samples of trypsin- activated toxin without DNCB with titers of 100,000- and -37- 200,000+ and four samples of trypsin-activated toxin in the presence of DNCB with titers of 40,000- and 100,000+. This slight reduction in titer may indicate that at least part of the titer increase upon digestion with trypsin is a result of the rupture of the peptide bond involving the carbonyl group of lysine. The slight decrease possibly may be due to only partial dinitrophenylation of lysine or it may indicate that rupture of the peptide bond involving the carbonyl group of arginine is the greater contributor to the activation process. Gerwing, Dolman, and KO (1965) described the resultant toxic peptide, after trypsin digestion, as having an N terminal arginine residue and containing seven lysine residues and two arginine residues, while two lysine and one arginine residues were split off. The amino acid which had been bonded to the N terminal arginine had to be either arginine or lysine so that it could be removed by trypsin. If rupture of the peptide bond involving the carbonyl group of lysine does account for only this small portion of the titer increase (instead of partial dinitrOphenyl- ation) one might eXpect that neither of the two removed lysine residues are adjacent to the N terminal arginine but rather that the one removed arginine might have been bound at that position. Thus upon its removal a toxic site is unmasked. However, if the slight titer decrease is due to only partial dinitrophenyla- tion, this hypothesis is not warranted, and it can only be sug- gested that at least some of the increase in titer upon activa- tion is caused by the splitting of the peptide bond involving the carbonyl group of lysine. SUMMARY AND CONCLUSIONS This research was conducted in the following five areas in an attempt to learn more about the toxin of Clostridium botulinum type E and its production. The five areas are: (l) optimum conditions for toxin activation, (2) toxin produc- tion as a function of time and cell development, (3) substrate requirements for toxin production and cell growth, (4) toxin production and cell multiplication as a function of trypticase (N) and sucrose (CHO) concentration in the media, and (5) toxin activation after attempted masking of lysine in the toxic pro- tein. The optimum conditions for Q1. botulinum type E toxin activation in a trypticase—peptone-sucrose-yeast extract (TPSI) medium were observed to be a pH level of 5.8, a time-temperature relationship of 45 C for 30 minutes, a trypsin concentration of 0.5 percent and a NaCl concentration of 4.0 percent or the ionic concentration which exists in TPSY. The enzymes which were used that caused activation were trypsin, protease, pro- teinase, and erepsin. The enzyme tested which caused no acti- vation was peptidase. The fact that a proteinase caused acti- vation but a peptidase did not would indicate that this titer increase is caused by a rupture of internal peptide bonds rather than terminal bonds. -38- -39- A number of observations were made of the relationship of toxin production to time and physiological state of the cell of 91. botulinum type E. It was observed that toxin titers increased as the number of vegetative cells increased, and that they reached a maximum after 20 to 24 hours of incubation, which was approximately 4 hours after the number of vegetative cells and sporangia combined reached a peak. The toxin titers decreased after 24 hours of incubation of the 21. botulinum type B in TPSY. It also appeared that toxin production had little relationship to sporulation of Cl. botulinum type E. It was apparent that the toxin was produced within the vegeta- tive cell during the logarithmic growth phase and then released into the medium. EXperiments were undertaken to determine some substrate requirements for toxin production. It was observed that when sucrose, glucose, fructose, maltose and sorbitol were the in- dividual carbohydrate Sources in TPSY, similar amounts of growth and toxin titers resulted. When ribose was the only carbohy- drate source reduced growth and lower toxin titers were observed, and when glycerol was the energy source even smaller amounts of growth and toxin were observed. Even though 9;. botulinum. type E is classified as an anaerobe, small amounts of growth and toxin were present when acetate was the only substitute for a carbohydrate source. It appeared that the carbohydrates tested were used by 9;. botulinum type E only as energy sources and not as precursors to the toxin or necessary ingredients for a toxin activation system as was the case with glucose for £1. botulinum types A and B. -40.. Optimum trypticase and sucrose concentrations in TPSY were also determined. A medium containing 5.0 percent tryp- ticase, 5.0 percent sucrose, 1.0 percent yeast extract and 0.1 percent sodium thioglycollate afforded optimal conditions for growth and toxin production. The data collected from the experiment involving mask- ing the lysine residues of the protein toxin with dinitro- chlorobenzene (DNCB) to prevent the action of trypsin upon the lysine and allow it only to rupture the carbonyl peptide bonds involving arginine is far from adequate to draw a conclusion or even prepare a hypothesis. Only a suggestion can be made that the rupture of the internal lysine carbonyl peptide bonds of the toxin of 21. botulinum type E by trypsin contributes at least partially to the activation process. LITERATURE CITED American Instrument Company. 1961 The determination of nitrogen by the Kjeldahl procedure including digestnxh distillation, and titration. Reprint No. 104 American Instrument Company. Silver Springs, Maryland. Anfinsen, C. B., Sela, M., and Tritch, H. 1956 A method for the specific proteolytic cleavage of protein chains. Archives of Biochemistry and Biophysics, 65, 156. Barron, A. L., and Reed, 0. B. 1954 Clostridium botulinum type A toxin and toxoid. Canadian Journal of Micro- biology, I, 108-117. Bonventre, P. F. 1957 The physiological basis of toxigenicity of Clostridium botulinum types A and B. Doctoral Thesis, university of Michigan, 134 pp. Bonventre, P. F., and Kempe, L. L. 1959 Toxicity enhancement of Clostridium botulinum types A and B culture fil- trates by proteolytic enzymes. Journal of Bacteriol- ogy, ZR. 892-893. Bonventre, P. F., andeempe, L. L. 1960a Physiology of toxin production by Clostridium botulinum types A and B. I. Growth, autolysis, and toxin production. Journal of Bacteriology, 22, 18-23. Bonventre, P. F., and Kempe, L. L. 1960b Physiology of toxin production by Clostridium botulinum types A and B. IV. Activation Of the toxin. Journal of Bacteriology, 12, 24-32. Boroff, D. A. 1955 Study of toxins of Clostridium botulinum. III. Relation of autolysis to toxin production. Journal of Bacteriology, 19, 363-367. Boroff, D. A., Raymaud, M., and Prevct, A. R. 1952 Studies of toxin of Clostridium botulinum type D. Journal of Immunology, 68, 503-5Il. FF Brooks, V. B. 1964 The pharmacological action of botulinum toxin, in Botulism: Proceedings 9: §_Symposium. U.S. Public Health Service Publication No. 999-FP-l. Cincinnati, Ohio. 105-111. -4l- -42- Buehler, H. J., Schantz, E. J., and Lmanna, C. 1947 The ele- mental and amino acid composition of Clostridium botulinum type A toxin. Journal of Biological Chem- istry, :82, 295-302. Burgen, A. S., Dickens, F., and Zatman, L. J. 1949 The action of botulinum toxin on the neuro-muscular junction. Journal of Physiology, 102, 10-24. Cartwright, T. E., and Lauffer, M. A. 1958 Temperature effects on botulinum A toxin. Proceedings of the Society for Experimental Biology and Medicine, 28, 327-330. Davies, J. B., Morgan, R. 3., Wright, E. A., and Wright, G. P. 1953 The results of direct injections of botulinum toxin into the central nervous system of rabbits. Journal of Physiology, 128, 618-623. Dixon, M., and Webb, E. 1958 The Enzymes. Academic Press, Inc" New York, New York. Dolman, C. E. 1957 Recent observations on type R botulism. Canadian Journal of Public Health, 48, 187-198. Dolman, C. E. 1964 Growth and metabolic activities of Clostgidium botulinum types, in Botulism: Egoceedings §:_§ Symposium. U.S. Public HeaIth Service Publication No. 999-FP-1. Cincinnati, Ohio. 43-68. Duff, J. T., Wright, G. G., and Yarinsky, A. 1956 Activation of Clostridium botulinum type E toxin by trypsin. Journal of Bacteriology, 22, 455-459. Gerwing, J., Dolman, C. E., and Bains, H. S. 1965 Isolation and characterization of a toxic moiety of low molecular weight from Clostridium botulinum type A. Journal of Bacteriology, 82, 1383-1386. Gerwing, J., and Dolman, C. E., and Ko, A. 1965 Mechanism of tryptic activation of Clostridium botulinum type E toxin. Journal of Bacteriology, 82, 1176-1180. Gunnison, J. B., Cummings, J. R., and Meyer, K. F. 1936 Clostridium botulinum type E. Proceedings of the Society for Experimental Biology and Medicine, 35, 278-280. Kindler, S. H., Mager, J., and Grossowicz, N. 1956 Toxin production by Clostridium parabotulinum type A. Journal of General Microbiology, 85, 394:403. Lamanna, C., and Glassman, H. N. 1947 The isolation of type B botulinum toxin. Journal of Bacteriology, 54, 575-584. -43- Lechowich, B. V. 1962 Personal communication. Lewis, K. H., and Hill, E. V. 1947 Practical media and con- trol measures for producing highly toxic cultures of Clostridium botulinum type A. Journal of Bacteriology, 51, 213-320. Mager, J., Kindler, S. H., and Grossowicz, N. 1954 Nutri- tional studies with Clostridium parabotulinum type A. Journal of General Microbiology, 22, 130-141. Redfield, B. B., and Anfinsen, C. B. 1956 The structure of ribonuclease. II. The preparation, separation and relative alignment of large enzymatically produced fragments. Journal of Biological Chemistry, 222, 385. Ross, H. E., warren, M. E., and Barnes, J. M. 1949 Clostridium welchii iota toxin: its activation b trypsin. Journal of General Microbiology, 2, 1 8-152. Sakaguchi, G., and Tohyama, Y. 1955a Studies on the toxin production of Clostridium botulinum type E. I. A strain of genus Clostridium having the action to pro- mote type E botulinal toxin production in a mixed culture. Japanese Journal of Medical Science and Biology, 53, 247-253- Sakaguchi, G., and Tohyama, Y. 1955b Studies on the toxin production of Clostridium botulinum type E. II. The mode of action of the contaminant organisms to promote toxin production of type E organisms. Japanese Journal of Medical Science and Biology, 53, 255-262. Sanger, F. 1945 The free amino groups of insulin. Biochemical Journal, 22, 507. Schantz, E. 1964 Purification and characterization of Clostridium botulinum toxins. Botulism: Proceedings .g§_§ S m osium. U.S. Public Health Service Publication No. 999-FP- . Cincinnati, Ohio. 105-111. Schantz, E. J., and Spero, L. 1957 The reaction of botulinum toxin type A with ketene. Journal of the American Chemical Society, Z2, 1623-1625. Segner, W. P., Schmidt, C. F., and Boltz, J. K. 1964 Growth characteristics of type E Clostridium botulinum in the temperature range 34 F - 50 F. IX. Toxin production and toxin stability in laboratory culture media. Con- tinental Can Company, Inc. Annual Report, Chicago, Illinois. Spero, L., and Schantz, E. J. 1957 The reaction of botulinum toxin type A with nitrous acid. Journal of the American Chemical Society, 12, 1625-1628. -44- Spero, L. 1958 The alkaline inactivation of botulinum toxin. Archives of Biochemistry and BiOphysics, 22, 484-491. Turner, A. W., and Rodwell, A. W. 1943a The epsilon toxin of Clostridium welchii type D. I. Proteolytic con- version of epsilon protoxin into epsilon toxin by trypsin and other proteases. Australian Journal of Experimental Biology and Medical Science, 22, 17-25. Turner, A. W. and Rodwell, A. W. 1943b The epsilon toxin of Clostridium welchii type D. II. Mechanism of its development in cultures through the action of extra- cellular proteinases upon epsilon protoxin. Australian Journal 2f Experimental Biology and Medical Science, 2;, 27-3 . Van Alstyne, D., Gerwing, J., and Tremaine, J. H. 1966 Amino acid composition of Clostridium botulinum type A toxin. Journal of Bacteriology, 2, 796-797. Van Slyke, D. D., and Folch, J. 1940 Manometric carbon de- termination. Journal of Biological Chemistry, 126, 509- Van Slyke, D. D., Plazin, J., and Weisiger, J. R. 1951 Reagents for the Van Slyke-Folch wet carbon combustion. Journal of Biological Chemistry, 141, 299. Van Slyke, D. D., Steele, B., and Plazin, J. 1951 Determina- tion of total carbon and its radioactivity. Journal of Biological Chemistry, 142, 769. Vinet, G., and Fredette, V. 1951 Apparatus for the culture of bacteria in cellophane tubes. Science, 124, 549- 550- White, A., Handler, P., and Smith, E. 1964 Principles of Biochemistry, third edition. McGraw-Hill Book Company, New York. APPENDIX 1 Tables and figures -u5- ’x! UR H m Titers of strains 517 and VB Clostridium botulinum type E toxin activated at various pH levels. -47- ~ 517 NOT AcnvflTED -5!7 "V/f up? r If r bl P FFLKPLlr 4 L, r r45, pH level 106 10”- 3 2 O O 1... l AHE\QAEV hmpdp 10 -48- Table 1. Titers in MLD/ml of Clostridium botulinum type E toxin after activation at various temperatures and times.8 fir: H temperatures (C) times 30 35 37 40 45 50 b 1 000- 10 min. ' 40,000+ 20 min. 40,000; 40,000+ 30 min. 38:888; 200,000; 40,000+ 9 40 min. 200,000; 100 000- 5° m1n° 2001000+ - - 100 000‘ - 100 000- 1 hr. 40,000+ 100,000+ zooioooib 100,000+ 200:000+ 100 000..b 2 hr' 200i000+ 4 hr. 100,000:b 100,000..b 8 hr' 200,000+ aSymbols: - Indicates that both animals died at that level; + Indicates that both animals lived at that level; ¥ Indi- cates that one animal died and one lived at that level. bThe activation was conducted with 1.0 percent trypsin, pH of 5.8 with the VB strain, while all the other activations were conducted with a pH of 5.8, 0.5 percent trypsin, with strain 517. -49- Table 2. Titers in MLD/ml of Clostridium botulinum type E toxin after activation using various trypsin con- centrations.a trypsin concentration (percent) TPSY TPSY + FISH 4 000- - 0 ‘ 10:000+ “00+ - 40,000- 0.5 200,000+ 100,000+ 100 000- - 1-0 200,000+ “0,009+ - 40 000- 2.5 200,000+ 1001000+ - 40 000- 5.0 200,000+ 100:000+ 8.0 100,000+ 8The activation was conducted at pH 5.8, and 45 C for 35 minutes with strain 517. -50- Table 3. Titers in MLD/ml of Clostridium botulinum type B dialyzed toxin after activation in media containing various NaCl concentrations. NaCl (percent) titer 0 40,000.b 100,000+ 0.5 1381888; 1.0 100,000+ 2-0 138:888: 3.0 133:333: 333033: 5.0 40,000+ 8The activation was conducted with 0.5 percent trypsin, at 45 C for 35 minutes at pH 5.8 - 6.0 with strain 517. bNondialyzed toxin 200,000i. -51- Table 4. Activation of strain 517 Clostridium botulinum type E toxin with various commercial preparations of trypsin and other enzymes. Conditions Enzymes tem . time pH conc. titer (c (mum) (Z) (MLD/ml) Trypsin 100 000- Dif l: 0 60 6.0 0. ’ °° 25 37 5 200,000+ Nutritional Biochemical _ Co. 1:300 37 60 6.0 0.5 100,000+ 100 000- ' Pancreatin 60 6.0 0. ’ 37 5 200,000+ 100 000- Cr talline 60 6.0 0. ’ ys 37 5 200,000+ 10 000- P t i e end - 4 0 .8 2.0 ’ ro e nas ( o ) 5 3 5 20,000+ Erepsin 45 30 5.8 2.0 40,000; 100 000- P tea e endo- and exo- 4 0 .8 1.0 ' ”O S ( ) 5 3 5 200,000+ Arginase 45 30 6.0 2.0 4,000: Ar inase 4 0 6. 4.0 ”:000' g 5 3 5 10,000+ 4 000- A 1 e ’4' 6o “’00 , rg nas 37 5 5 10,000+ Arginase 37 45 7.0 4.0 2,000: No enzyme 45 30 5.8 4,000: No enzyme 37 60 5.8 2,000? Table 5. Activation of strain VH Clostridium botulinum type E toxin with peptidase and trypsin. W temperature time pH concentration titer (C) (minutes) (percent) Peptidase 45 30 5.8 1.0 1,000+ 100 000- Trypsin 45 30 5.8 0.5 200:000+ No enzyme 45 30 5.8 400- 1,000+ -53- Table 6. Toxin production versus time and cell development for strain 517 Clostridium botulinum type E (Trial 1). direct microscopic count (x 107/m1) cell suspension in the presence of trypsin. time Absorb- vegetative sporangia free total titera (hours) ancy cells spores count MLD/ml O 000)4 0.1 0.1 8 1.10 32.0 32.0 ”0'000' 100,000+ 40 000- Q .4 16.0 .0 6.“ , 12 1 20 37 3 5 100,000+ 15 1.26 40.0 25.0 5.0 70.0 18 1.40 30.8 19.6 5.6 56.0 200,000: 200 000- . 0 0.0 6.4 10.0 6.4 ' 21 1 3 2 3 400,000+ 24 1.30 19.4 7.0 22.0 48.4 100,000: 36 1.30 4.0 0.5 61.0 65.4 20,000: 4 000- 4 1.2 0.6 42.0 42.6 ' 8 5 10,000+ aTiters were determined after activating toxin in the -54- Figure 2. Toxin production versus time and cell development for strain 517 Clostridium botulinum type E (Trial 1). -55- Am-wsmpoan mafia mHHoo o>apmpowobv access Haoo fiscamnommsm QHNOp copmbapom saunas» mo HE\QH=V hop-p 5 4 o m 1 0 - d 1 a: 4- 5 a .w m n P l e 1 t e .1- O t - - x 0 .w (fir L ( b-bhk F L I 2 0 O l 1 time (hours) -56- .haco Samnmnp mo monomopg on» :- sodmnmmmSm mop“ HHoo m :- zaxou on» wcaum>dpom Locum cos-Epmpov who: mama-en .demhnu cam oEmNOmmH ho monomopn on» ad godmnonSm HHoo on» :- Gaxou maapm>apom popmm con-anopmc ohms mama-em +ooo.ooo.- +ooo.ooo.- . . . . . uooo.ooa $8.81- 0 a 00 am on m o m m m an +ooo.oo~ +ooo.ooo.- . . . . . . nooo.oo- uooo.ooa o w on mm on m o m o H- mm 0 mm +ooo.oom +ooo.ooo.- . . . . . . nooo.oo- uooo.ooa o o on on 00 a o ma 0 m- mm 0 am +OOOQOOH . o o o o o o uooo.oa Moon on: o 8 00 mm 00 a o a- o a- mm o m- +ooo.o: +ooo.oom . . . . . uooo.o~ nooo.oo- o o oo mm o m- 0 ma mm o :- +OOOQOH - O o o o -ooo.: wooo oo- o a om mm m mm mm o o- Hooo.a ”000.0- A.@ 00.8- 0.8- a-.o m mmw no mafia-nus» man-nab o: a +2 Ho- -.a no.0 no.0 No.0 0 be m-e 8 4- ma Hapon monomm mawsmnomm mHHmo mosm Amazosv moan m>apmpowo> Inn0m9< 06-9 HENQAZM,pmpap AHE\m0H M% 92500 camoomosoHE poop-U III ‘I’ h .Am Hn-nev m was» saga-spon snap-ppmoao mam :«mppm pom meEQoaobmc HHmo cam 05-» mamno> :o-posoonm sauce .n ofinma Figure 3. -57- Toxin production versus time and cell development for strain 517 Clostridium botulinum type E (Trial 2). titer (MLD/ml of trypsin activated toxin suspenSion) -58- 106 E 1 t 3 + . o .N I )- u I X 10$:- 0 0 A 4 d L L . A J i- -< 10“, . t i I . l ‘ 1 1032 - ; 1 i k 102 _ - L x - cell number 1 - o - toxin titers of suspension with 4 - trypsin and with lysozyme i r 4 - toxin titers of suspension with - trypsin but without lysozyme lo 5L - 11$ 20 :30: 746* ’50 time (hours) 109 108 107 105 10 cell number (vegetative cells plus sporangia) .zano namahpp mo monommpa exp S- :o-mnommzm 00pm HHoo m Ca Saxon on» maapm>auom pmpmm won-apopoc who: mama-En .nawmanp cam oE»NOm»H mo monmmosm on» c- no-mnonSm HHoo on» ma s-xo» madam>apom nopmm cos-enmpoc ohms whoa-em -59- m.mm 0.: 0.0 0.0a ma +000.00- . . . . . 1000.0: m an 0 m a m 0 0m 00 0 0m +000.00: +000.000.- . . . . . u000.00m s000.00: m a: n m 0 a 0 00 00 0 am . +000.000.H . . . . . H000 00m 000.001- 0 00 0 N 0 m 0 on an 0 0m +000.00: . . . . . . :000.00N H000 00: m 00 0 H m m 0 0: on 0 0- H000.00m ”000.00: 0.-0 m.- 0.0 0.00 0a.0 m- +000.00: . . . . . u000.00m H000 00: 0 mm 0 H 0 mm NA 0 0- . Hmmm.ww- ”000.00m 0.Nm 0.~m 00.0 a +00- +000.: . . . m.a m.a Hm.0 0 DB m-e 8.HV annoy wosoam m-wsmnomm maamo mosm Amazon- oopm 11+ mpmpmpomm> Inp0mn< mad» hop-p Adafimwoa NM assoc o-WWomOpoHE poms-0 .m mama saga-0000 ends-nano-o namnpm commemamx on» now psoeaoao>o0 Haoo cam 05-» mamamb QOaposcoaQ saxoa .m manna -60- Figure 4. Toxin production versus time and cell deve1Opment for the Kalamazoo strain Clostridium botulinum type E. titer (MLD/ml of trypsin activated toxin suspension) 10 10 -61- _ 105 x - cell number I o - toxin titers of suspension with trypsin and with lysozyme A - toxin titers of suspension with . trypsin but without lysozyme 10"l 1? 2'5 3'? T0 30 time (hours) cell number (vegetative cells plus sporangia) -62- cH sonconSm mosh HHoo m 2H stOp msHpm>Hpom popum quHesopoc ohms whopHBQ 029 CH sonsmawa Haoo exp 2H :HxOp manm>Hpom nouns cosHsnopoc one: whopHBm .tho.chszp mo monommua on» .nHmahnp oEmNOth no oonmmmpm +000.00H +000.00m u000.0a -000.00H on mH 0m HH 0 H 0 H as 0 0m +000.00~ +000.000.H . . . . . u000.00H 1000.00: 00 0H om a 0 0 m m 00 0 00 4000.0: ”000.00H 00.nm 0m.m 0.Hm m.m 0A.0 0H +0oo-o: +000.00H .. . . . 1000.00 u000.0: 00 0H 0 HH m 0 H0 0 HH +000.0a +000.00H . . . . -000.0N u000.00 on an m NH 0 0H H0 0 HH +OOOQOH a o o 0 .000.3 H000 00 00 0H 0 0H 0a 0 0 +2 woom 00.a 00.H 0.0 mm.0 m NH.0 a mH.0 mH.0 HH.0 0 a MAB a A- ammo» monomm mesmpon mHHoo honm Amazon- n ovum oprMuowo> IppOmn< 05H» nopHp AHE\N0H xv assoc OHQOOmOpoHE poohHU .m 09%» ESQHHSpon echprmoao sHmppm mb 0:» com psmsQOHo>00 HHoo cam 08H» mango» :oHpozcopm stoe .m mfinme Figure 5. -63- Toxin production versus time and cell development for the VB strain Clostridium botulinum type B. titer (MLD/ml of trypsin activated toxin suspension) -64- 106 t 1 L 1 L- J F J L . L X x x 105 F' 6 - C x 3 L L . L . f 9 6 A 4 r a r- ‘ d J 10“. . L : L . L + F a F 1 ’ i 103 .4 F i * 1 J J x - cell number i o -itoxin titers of suspension with J trypsin and with lysozyme .4 - toxin titers of suspension with J trypsin but without lysozyme To 2'? ‘30 40”“ 50 time (hours) 109 10 10 105 10 cell number (vegetative cells plus sporangia) -65- Table 10. Growth and toxin production of Clostridium botulinum type E with various carbohydrate sources or substIEutes for carbohydrate sources in the media.a concentration 26080 VH CHO (z) (moles/ titerb absorb- titerb absorb- 10000c) (MLD/ml) ancy (MLD/ml) ancy sucrose 0.5 0.015 U 400,000+ 1.20 400,000: 1.20 400 000- 200 000- 1,000i000+ 0'70 4001000+ 0'80 100 000- 400 000- glucose 0.5 0.028 U 200:OOO+ 0085 1,000:OOO+ 1.20 200 000- 200 000- A 400:000+ 1'20 400:000+ 1'20 200 000- 200 000- fructose 0.5 0.028 U 400:000+ 1.20 400:000+ 1.25 400 000- 400 000- . 1,000:000+ 1°“0 1,0001000+ 1:30 - 40 000- maltose 0.5 0.015 U 100,000+ 1.15 100:000+ 1.30 2 000- 100 000- A 4:000+ 0'88 200:000+ 1'30 sorbitol 0.5 0.028 U égg'ggg; 1.40 200,000: 1.25 i 100 000- 40 000- A 2001000+ 1°20 100:000+ 1°20 40 000- 20 000- ribose 0.5 0.033 U 100:000+ 0.78 40:000+ 0.82 100 000- 10 000- A 2001000+ 0-69 20:000+ 0-65 100 000- 20 000- glycerol 0.5 0.045 U 200:000+ 0.60 ”O:OOO+ 0.32 - 10 000- A 20,000+ 0.74 20:000+ 0.59 l 000- 2 000- acetate 0.5 0.085 A 2:OOO+ 0.29 ”:000‘0' 0.16 aThe toxin titer obtained with no carbohydrate in the basal medium was 100$. bU Unadapted; A Adapted to the various media. -66- Table 11. Toxin production of Clostridium botulinum type E strain 26080 in media containing various sucrose concentrations. sucrosea N/C Absorb- titer (percent) N/C (mg/100 ml) Ratio ancy (MLD/ml) 100- 0 801/2496 0.3210 0.22 200+ 0.05 801/2410 0.3323 0.30 2,000: 10 000— 0.1 801/2534 0.3161 0.39 20:000+ 20 000- 0.2 801/2570 0.3116 0.75 401000+ 200 000- 0.5 801/2660 0.3011 1.20 400:000+ 1.0 801/2760 0.2902 1.20 200,000: 2.0 801/3060 0.2617 1.30 400,000: 5.0 801/4278 0.1872 1.40 400,000: 8.0 801/5220 0.1534 0.90 200,000; 200 000- 10.0 801/6138 0.1304 0.95 4001000+ 8Besides the sucrose the media contained 5.0 percent trypticase, 1.0 percent yeast extract, and 0.5 percent peptone. -67- Table 12. Toxin production of Clostridium botulinum type E strain 26080 in media containing various trypticase concentrations. trypticase N/C Absorb- titer (percent) N/C (mg/100 ml) Ratio ancy (MLD/ml) a 2,000- 0.0 107/570 0.1877 0.23 4,000+ a 40 000- 0.5 144/712 0.2022 0.74 100:000+ a 200 000- 1.0 224/900 0.2488 0.94 400:000+ 2.5a 433/1488 0.2909 1.00 200,000+ 5.0a 688/2418 0.2845 1.20 200,000: b 100 000- 1.0 214/2604 0.0821 1.30 200:000+ b 400 000- 5.0 664/4092 0.1630 1.40 1.000:000+ b 200 000- 10.0 1221/5952 0.2051 1.40 400:000+ aThese media also contained 0.5 percent sucrose and 1.0 percent yeast extract. bThese media also contained 5.0 percent sucrose and 1.0 percent yeast extract. -68- Table 13. Toxin production of Clostridium botulinum type E in tissue culture medium 109. Toxin titers (MLD/ml) for strains Medium concentration Minneapolis 517 . 4- 1X 10+ 2x 200- 10,000- 400+ 20,000+ -69- Table 14. Activation and non-activation toxin titer data for strain Minneapolis Clostridium botulinum type E subjected to dinitrophenylation in an attempt to mask the lysine in the toxin. titer conditions (MLD/ml) 200- toxin, no DNCB, no trypsin “00+ toxin, no DNCB, trypsin activation $389888; 9 200- toxin, DNCB (added same time as trypsin), “00+ no trypsin toxin, DNCB (added same time as trypsin), 40:000- trypsin activation 100,000+ toxin, DNCB (added 4 hours before trypsin), E00- no trypsin 00+ toxin, DNCB (added 4 hours before trypsin), 138.888; 3 trypsin activation G N STATE UNIV. //I///I/// /////I///I///I///I//I////I7//77//ii/?i??i“ 00076939