INFECTION AND IMMUNITY, May 1978, P. 352-359 0019-9567/78/0020-0352$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 20, No. 2 Printed in U.S.A.
Heat-Stable Enterotoxin from Escherichia coli: Factors Involved in Growth and Toxin Productiont W. M. JOHNSON,' H. LIOR,' AND K. G. JOHNSON2* National Enteric Reference Centre, Laboratory Centre for Disease Control, Health and Welfare Canada, Ottawa, Canada,' and Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada2 Received for publication 15 November 1977
Six enterotoxigenic strains of Escherichia coli produced variable levels of heatstable enterotoxin (ST) when grown under pH control at 8.5 in a simple synthetic medium containing neither amino acids nor vitamins. Bacterial growth and ST production were at levels as high as or higher than those observed in complex media. ST elaboration was detectable in the early logarithmic phase of growth and appeared to be related to disappearance of glucose in the growth medium. The results of this study did not suggest pH-dependent release of ST. Imposition of pH control in complex media resulted in increased growth rates, earlier detectable ST synthesis, and elevated levels of ST. In synthetic medium, attainment of the stationary growth phase was followed by a significant decrease in culture density and a concomitant increase in ST. Cellular autolysis experiments revealed that as much as 20% of the total ST activity was present in a cellassociated form. Enterotoxigenic strains of Escherichia coli quired for ST production in one strain of E. coli have been implicated as causative agents in in- (1), whereas certain carbon sources were sugfantile diarrhea (11, 24), diarrhea in neonatal gested as repressors of ST synthesis. The present study was undertaken to examine domestic animals (26, 28), and in so-called travelers' diarrhea (10, 19). Two distinct forms of certain growth parameters and their correlation exocellular enterotoxin, each capable of produc- to ST production in ST' strains of E. coli. ing a cholera-like illness, are elaborated by some MATERIALS AND METHODS enteropathogenic E. coli. The most well-studied Bacterial strains, media, and growth condienterotoxin, designated heat-labile enterotoxin (LT), is a high-molecular-weight, proteinaceous tions. E. coli strains 36-99, 77-1146, 77-217, 77-3753, material exhibiting many similarities to cholera 77-3899, and H-3698 were all of human origin and were toxin (9, 12, 22). Heat-stable enterotoxin (ST), associated with infantile diarrheal disease. The orgawere obtained from the National Enteric Refon the other hand, is a low-molecular-weight nisms Centre (Canada) and were maintained as frozen component which is non-immunogenic, and is erence stock cultures in the National Research Council Culnot neutralized by cholera antitoxin (9, 12, 22). ture Collection. Enterotoxigenic E. coli strains produce LT alone Cultures used for the study of ST production were (ST-/LT') or in combination with ST grown at 37°C in either Evans medium (9), a Casamino (ST+/LT+; 8, 10, 11, 24). Although enteropath- Acids-yeast extract-salts medium, or in a simple minogenic E. coli strains that produce only ST occur eral salts-citric acid-glucose (0.5%, wt/vol) medium less frequently (11, 19, 23, 24), their clinical (MSCG; 7). This medium contained (grams per liter): NH4CI, 3.12; KCI, 2.24; significance has been unequivocally established NaH2PO4 2H20, 4.68;citric acid, 3.15; MgCl2, 0.36; 10H20, 4.83; in a recent study wherein an ST+/ LT- strain Na2SO4 CaCl2, 0.033; and Na2MoO4, 0.03. Trace salts were was shown to produce diarrhea in humans (17). added (15 ml/liter of medium) from a stock trace salts Purification and characterization of ST have solution (total volume, 5 liters) containing: HCI, 50 ml; been hindered by the complexity of media in ZnO, 2.04 g; FeCl3 6H20, 27.0 g; MnCl2 4H20, 10.0 g; which ST+ organisms are grown and by insuffi- CuCl2 2H20, 0.85 g; CoCl2 6H20, 2.83 g; and H3BO4, cient knowledge about nutritional conditions fa- 0.31 g. Unless otherwise indicated, the pH of the voring ST production. Recent studies indicate medium was adjusted to 8.5 before autoclaving. Inocthat ST' strains of E. coli will produce ST in ula (1%, vol/vol) were prepared by washing 18-h culfresh sterile medium. simple synthetic media (1, 2, 21). Proline, serine, tures twice with the appropriate cultures (50 ml) were grown in 125-ml Erlenaspartic acid, and alanine were apparently re- Batch meyer flasks on a New Brunswick rotary shaker operated at 150 rpm. Fermentor cultures (10 liters) were t Issued as National Research Council of Canada no. 16546. 352
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HEAT-STABLE ENTEROTOXIN FROM E. COLI
grown in 14-liter New Brunswick fermentors equipped with a New Brunswick pH control unit. Bacterial growth was monitored by measuring the increase in absorbance of culture aliquots at 550 nm or by triplicate dry-weight determinations of doubly washed (distilled water) culture aliquots. Preparation of cell-free filtrates. Cell-free filtrates were prepared by centrifugation of culture aliquots at 27,000 X g for 15 min. Supernatant fractions were filter sterilized by passage through 0.22-,um membrane filters (Millipore Corp.) before biological assay. ST assay. ST was determined by the suckling mouse assay originally described by Dean et al. (6). Crude, sterile enterotoxin samples were mixed with 1 drop of 2% (wt/vol) Evans blue per ml and were introduced orally into Swiss-Webster suckling mice (1 to 3 days of age) with a 0.5-ml syringe, the canula of which was fitted with Teflon tubing (0.9 by 10 mm; 27). Each sample (total volume, 100 Ml) was assayed, using four mice per test. After 4 h of incubation at 22°C, the animals were killed with chloroform and the intestines from four mice were weighed together. The ratio of intestinal weight to remaining body weight (IW/BW) was then determined. Units of ST activity. Dose response curves relating the observed IW/BW to the various amounts of enterotoxin used in the suckling mouse assay were constructed. Activity was expressed in units as adopted by Jacks and Wu (13). One unit of ST activity was defined as the amount of enterotoxin producing an IW/BW ratio of 0.10 under standard assay conditions (see below). Efficiency of ST production. Efficiency of ST production was defined as units of ST per milliliter per millogram (dry weight) of bacterial mass. Heat stability of crude ST. The heat stability of crude ST was determined as follows. Sterilized culture filtrates were lyophilized, suspended at 40 mg/ml in distilled water, and dispensed in 2-ml portions. These portions were held for 30 min each at 22, 37, 56, 70, 85, and 100°C. After cooling, 2 drops of 2% (wt/vol) Evans blue were added to each. Activity was measured in triplicate, using 100 Al of each sample (four mice per test). Cellular autolysis studies. E. coli 36-99 was grown to mid-logarithmic phase (6 h) in MSCG medium in a New Brunswick fermentor. Aeration, agitation, and pH were maintained at 4 liters/min, 200 rpm, and 8.5, respectively. After being harvested by centrifugation, cells were washed twice with 50 mM sodium phosphate buffer (pH 8.0). Cell wash fractions were pooled, sterilized by passage through 0.22-gm filters, lyophiliWzed, and retained for analyses. Washed cells (6.6 g, dry weight) were suspended in 200 ml of 50 mM tris(hydroxymethyl)aminomethanehydrochloride buffer (pH 8.5) containing 1 mM MgCl2 and 5 Mg each of ribonuclease and deoxyribonuclease (Calbiochem) per ml. Before incubation at 37°C, duplicate 5-ml samples were removed and centrifuged at 48,000 x g for 15 min. Sedimented material was washed twice with distilled water, and dry weights were determined. Supernatant fractions were sterilized by passage through 0.22-,um filters and retained for analyses for protein, ST, and glucose 6-phosphate (G6-P) dehydrogenase contents. At designated intervals thereafter, portions were removed and similarly
353
treated. Analytical methods. Protein was estimated by the method of Lowry et al. (18), uring bovine serum albumin as the standard. G-6-P dehydrogenase (EC 1.1.1.49) was measured by a technique utilizing the reduction of nicotinamide adenine dinucleotide phosphate. G-6-P (18 Mmol), nicotinamide adenine dinucleotide phosphate (0.5 ,umol), tris(hydroxymethyl)aminomethane-hydrochloride buffer, (pH 8.0; 100 Mmol), and 0 to 900 Ml of sample were incubated at 22°C in a total volume of 1.5 ml. The reaction was followed by measuring the increase in absorbance at 340 nm in a recording Bausch and Lomb 200 UV spectrophotometer. One unit of G-6-P dehydrogenase was defined as the amount of enzyme catalyzing the formation of 1 nmol of reduced nicotinamide adenine dinucleotide phosphate per min at 22°C and pH 8.0. Glucose was estimated with the Glucostat reagent (Worthington Biochemicals Corp., Freehold, N.J.).
RESULTS Measurement of ST activity. The relationship between the amount of enterotoxin and the IW/BW ratio is depicted in Fig. 1. Various amounts of sterile culture filtrates (total volume, 100 u1) from 24-h Evans medium-grown batch cultures of E. coli 77-217 and 77-1146 were tested. For both strains, a linear response between dosage and IW/BW ratio was observed. Beyond a certain amount of enterotoxin, plateaus were evident. For several replicates from six ST-producing strains, linearity occurred in the region of an IW/BW of 0.1. Because this is
0'
0.15H
.0% 0).
C3 (n0) cy
0.10
005 40 60 80 100 Culture Filtrate (,. I) FIG. 1. Dose response curve for enterotoxic culture filtrates in the suckling mouse assay. Indicated amounts of culture filtrate from E. coli strains 77-217 (@-*) and 77-1146 (@- -0) were assayed in triplicate (four mice per test). 20
354
JOHNSON, LIOR, AND JOHNSON
considered to be a strongly positive reaction in the suckling mouse assay (13, 27), one unit of ST activity was designated as the amount of material producing an IW/BW ratio of 0.1. For each sample for which ST activity was quantified, construction of a dose response curve was mandatory. Growth of enterotoxigenic E. coli in defined medium. Because pH appears to play a central role in growth and ST production in enterotoxigenic E. coli (1, 21), batch growth experiments were conducted in which media were adjusted to various pH values. E. coli 3699 was grown for 24 h in Evans medium (pH 8.5) and in MSCG medium (pH 7.0 to 9.0). MSCG medium lacking glucose did not support the growth of any of the strains tested. As the data in Table 1 indicate, cultures grown in synthetic medium attained cell densities near those observed in Evans medium. Growth was maximal in media in which the initial pH was adjusted to 8.5. However, only those cultures grown in Evans medium contained detectable ST. Examination of medium pH at 24 h revealed that all synthetic-grown cultures were markedly acid, whereas the pH of cultures grown in Evans medium was well over 7.0. To clarify the possible influence of pH on growth and ST production, these parameters were more rigorously investigated in both media. Effect of pH on growth and ST production. The effect of pH on growth and ST production in Evans medium was compared with that in simple synthetic medium. (i) Evans medium. E. coli 36-99 was grown in a 14-liter New Brunswick fermentor containing 10 liters of Evans medium supplemented with 0.5% (wt/vol) glucose. Aeration and agitation were maintained at 4 liters/min and 200 rpm, respectively. At designated intervals during growth, portions of the culture were removed for dry-weight determinations. Culture filtrates from such samples were monitored for pH, ST activity, and glucose content. Growth was acTABLE 1. Growth of E. coli in simple synthetic Growth medium
MSCG
Evans
medium and Evans medium 1at pH at ST (U/mi) Initial pH OD40 24 ha
7.0 7.5 8.0 8.5 9.0
24 h 4.69 4.72 5.50 5.90 6.00
0.904 7.74 OD5fo, Optical density at 550 nm. b -, Not detectable. a
8.5
0.532 0.827 0.856 0.878 0.855
-b -
20
INFECT. IMMUN.
companied by a decrease in pH until glucose was depleted from the medium at 5 h (Fig. 2). After this point, the pH rose to 6.7 at 12 h. ST production was readily detectable in early logarithmic growth phase (3 h) but remained relatively unchanged well after the onset of the stationary growth phase. During the period of rapid ST elaboration between 2 and 4 h of growth, a large percentage of glucose remained in the medium. Because ST production did not appear to increase significantly after medium pH fell below neutrality, the effect of growth in Evans medium at constant pH was investigated. Parallel cultures were grown as described above. All conditions of aeration, agitation, and washed-inoculum size were identical, except that the medium pH of one fermentor was held constant at pH 8.5 by the automatic addition of 5 N KOH. An almost threefold increase in ST levels was observed in the pH-controlled culture (Fig. 3). ST accumulation in the pH-controlled culture continued to increase from the mid-logarithmic into the stationary growth phase, but ST levels remained relatively constant after the mid-logarithmic growth phase of the uncontrolled culture. ST production was detectable at an earlier stage in the growth cycle of the pH-controlled culture possibly because of the faster growth rate. Independent of pH control, glucose utilization occurred at essentially the same rate, with depletion occurring at 5 h for both cultures. (ii) Simple synthetic medium. Although batch cultures of E. coli 36-99 grown in the simple synthetic medium were devoid of detectable ST (Table 1), fermentor-grown cultures in which pH was held constant at 8.5 during the entire growth cycle yielded relatively high levels of enterotoxin (Fig. 4). Conditions of growth were identical to those used in the pH-controlled Evans medium fermentation. The data in Fig. 4 indicate that, although the growth rate was considerably slower than that observed in Evans medium, ST production was detectable in the early logarithmic growth phase and continued to rise throughout the rest of the experiment. As before, ST production increased with glucose depletion of the medium. In contrast to pHcontrolled fermentations in Evans medium, those effected in synthetic medium yielded higher bacterial dry weights (1.85 mg/ml in synthetic medium, as opposed to 1.30 mg/ml in Evans medium). In addition, once maximum cell densities had been attained in pH-controlled fermentations in synthetic medium, a rapid decline in bacterial dry weight was accompanied by elevation of ST levels. During the period when bacterial dry weight stabilized, ST levels doubled. Elevation of aeration levels resulted in in-
HEAT-STABLE ENTEROTOXIN FROM E. COLI
VOL. 20, 1978
355
.---4 I-@o
I
0 3:-
0
2H H 6
-J 4
GU
co I-
m-
TI M E (h) FIG. 2. Kinetics of growth and ST production in Evans medium for E. coli 36-99. ST is represented by the bar graph. (--4) Bacterial dry weight, (0 - 0) pH.
role of cellular autolysis suggested by the marked fall in bacterial dry weight observed in pH-controlled MSCG-grown cultures was investigated. E. coli 36-99 cells were grown and allowed to autolyse as described in Materials and Methods. Cellular autolysis, as shown by the release of cytoplasmic marker enzyme G-6-P dehydrogenase (5), the increase in non-sedimentable protein, and the loss of sedimentable material, was accompanied by the release of ST (Fig. 5). Maximal release of ST occurred after 4 h of autolysis at 37°C. Although half again as much G-6-P dehydrogenase was released between 5 and 24 h and bacterial dry weight continued to decrease, additional ST was not released. Analysis of the buffer wash (see Materials and Methods) revealed that this fraction was devoid of either ST or G-6-P dehydrogenase. The culTIME (h) FIG. 3. Influence of pH control on growth and ST ture filtrate (8.4 liters) from which these cells production for E. coli 36-99. Cultures were grown in were obtained contained 9,240 U of ST, whereas Evans medium with (A) and without (0) pH control the autolysate possessed 2,353 U. Thus, approx(see text). Bar graph, STproduction in pH-controlled imately 20% of the total ST in this experiment (O) and uncontrolled (J) cultures. Percent unused was cell associated and was released by cellular glucose in pH-controlled (A) and uncontrolled (0) autolysis. cultures. Efficiency of enterotoxin production. To ascertain whether growth and ST production of creased growth rates, although bacterial mass E. coli 36-99 in pH-controlled MSCG medium were unique phenomena, five additional strains and ST levels were unchanged. Autolysis-mediated release of ST. The were grown under identical conditions. As the
356
INFECTI. IMMUN.
JOHNSON, LIOR, AND JOHNSON
-30
-~~~~~~~~25-100
I
la 0
E Z 2.0
20 22 -80
'E
-C 0
iF
C
t0 10 I-
-60
~ ~ ~cn
/
o 0
0
-40
co
10~~~~~~~~~I
05 I 2
4
6
8 10 TIME (h)
12
24
FIG. 4. Growth and ST production of E. coli 36-99 in MSCG. Growth conditions were as described in the text. ST is represented by the bar graph. (0) Bacterial dry weight, (0) percent unused glucose.
E c0
E
.P
I
-S
a!ma t._
._5 cn
TIME(h) FIG. 5. Autolysis of E. coli 36-99 and release of ST. Conditions for cellular autolysis were as described in the text. (Bar graph) ST, (0- -0) G-6-P dehydrogenase, (O-O) bacterial dry weight, (-4*) solubilized bacterial protein. -
data in Table 2 indicate, these conditions permitted both growth and ST production in all strains tested, although considerable strain-tostrain variation in both factors was encountered.
Effect of temperature on enterotoxin activity. Heat stability of the enterotoxin derived from E. coli 36-99 grown in pH-controlled MSCG medium was determined as described in
HEAT-STABLE ENTEROTOXIN FROM E. COLI
VOL. 20, 1978
1.4
33.3
23.6
release of Vibrio cholerae toxin is inhibited by low pH (3,4), ST release in uncontrolled cultures grown in Evans medium appeared unrelated to pH. In fact, ST levels increased during the period when pH was decreasing and remained relatively unchanged thereafter (Fig. 2). Imposition of pH control in Evans medium resulted in elevated levels of ST at all stages of growth, suggesting that increased net synthesis rather than pH-
2.14
20.8
9.7
dependent release was the factor involved in ST
TABLEC 2. Growth and production of ST by enterotoxiigenic E. coli strains in simple synthetic
mediuma
Bacterial 1Phenotype
Strain
c'rgYt
ST
Efficiency (U of
(U/nil) ST/nml per
wt)l T-/ST+
LT-/ST+ LT-/ST+ LT+/ST+
36-99 77-217 77-1146 77-3753 77-3899 H-3698
mg
1.48 57.1 38.6 0.63 57.1 90.6 LT-/ST+ 1.27 9.5 7.5 LT+/ST+ All cult;ures were grown at constant pH (8.5) for 24 h in M[SCG medium supplemented with 0.5% (wt/vol) gliucose. Aeration and agitation were maintained at 4 liters/min and 200 rpm, respectively. b Establieshed by National Enteric Reference Centre (Canada). .a
OJ6 0'
0.14
/
._
0
.
OJ2
C ._
4,
C
357
0.10
____________________________ 20 4 6 80 20 40 60 100
Temperature of 30 min. heOt treatment (°C) FIG. 6. AFffect of heat treatment on E. coli 36-99 crude enterrotoxin. Conditions of the assay were as described il n the text. Materials and Methods. As the data in Fig. indicate, a 30-m heat treatment resulted in
6 a
stimulatio:,n rather than a decrease in activity.
DISCUSSION As a pre-lude to purification and characterization of ST'from enterotoxigenic E. coli, growth kinetics arad toxin production were examined in complex and simple synthetic media. Use of IpH control during fermentation produced a ariety of notable effects. In complex media, increased growth rates, earlier elaborav.
tion of enterotoxin, and greatly increased ST levels wer(e observed (Fig. 3). Although pH control was rnot required for bacterial growth in simnple syrithetic medium, it was absolutely essential for ST production. These observations would sugj gest that pH controls either the release of enterotcixin or its net synthesis. Although the
evolution. In contrast to previous results (1, 12), exocellular ST was detected well before the onset of
the stationary growth phase and usually before cultures attained the mid-logarithmic growth. These observations suggest that the factor(s) regulating expression of the ST+ plasmid is not a secondary metabolic product. In the initial study of ST production by enterotoxigenic E. coli, Alderete and Robertson (1) suggested that glucose serves as a catabolite repressor of ST synthesis. A more recent study (2) revealed that glucose completely represses ST synthesis by two enterotoxigenic strains of E. coli and that derepression can be effected by the addition of cyclic adenosine 3',5'-monophosphate to the growth media. Although glucose was required for bacterial growth in the synthetic medium used in our studies, ST elaboration was accompanied by the disappearance of glucose from the medium. However, incomplete repression of ST formation by glucose is indicated because relatively high levels of ST accumulation preceded glucose depletion of fermentations in both complex and simple synthetic media. These findings suggest that the composition of the growth media may influence the apparently repressive effect of glucose. Miller and Fung (20) have noted that the inhibitory effect of glucose on enterotoxin B synthesis in Staphylococcus aureus S-6 grown in defined media is less evident (or totally absent) compared with the response in complex media. Possibly, the continuous culture of enterotoxigenic E. coli under conditions of glucose limitation will provide unequivocal answers. Growth conditions for enterotoxigenic E. coli developed in this study differ in two fundamental respects from those previously described (1, 21). The first, pH control, has been discussed already. The second major difference is in nutritional simplicity of the growth medium. Growth studies in synthetic media evolved by Alderete and Robertson (1) indicate that proline, serine, aspartic acid, and alanine are required for growth and enterotoxin synthesis. Whereas no amino acid requirements for growth or ST elaboration were found in the defined medium of Mitchell et al. (21), their media contained a mixture of eight vitamins. Our studies indicate that neither vitamins nor amino acids are re-
358
JOHNSON, LIOR, AND JOHNSON
quired for either growth or ST synthesis by at least six enterotoxigenic E. coli strains. Moreover, and in contrast to the results of Alderete and Robertson (1), cell yields were as high or higher than those observed in Evans medium. Perhaps the apparent amino acid requirements described by Alderete and Robertson (1) are reflections of the nutritional specificities of the strains studied. More probably, the aforementioned amino acids serve as carbon sources in the absence of a more readily metabolized carbon source such as glucose. Heat stability of the enterotoxin, at least from E. coli 36-99, certainly was not affected by the growth conditions employed. Alderete and Robertson found that ST production and culture densities, under the growth conditions they prescribed (1), were maximal at approximately 8 h of growth and were essentially the same at 18 h. In contrast, and particularly with synthetic medium, we observed that ST levels continued to rise well after the onset of stationary phase, implying that cellular autolysis might be involved in the release of ST. Autolysis of mid-logarithmic cultures did reveal that a significant portion of the total ST activity was cell associated. Evidence exists for the synthesis of other heat-stable, low-molecular-weight enterotoxins by Klebsiella pneumoniae (15), Enterobacter cloacae (14), and Salmonella species (16, 25). In contrast to the autolysis-mediated ST release observed in this study, cell-free lysates from the above organisms apparently did not possess ST activity. If the growth conditions described here lead to a situation in which a portion of ST is cell associated, a potentially powerful tool for the study of the mechanism of ST release may be realized. That high-level growth and ST elaboration by enterotoxigenic strains of E. coli can be achieved under exceedingly simple nutritional conditions has been established. The logical extension of this work into continuous culture (in which the effect of parameters such as aeration, pH, and growth rate on ST production can be studied as independent phenomena) is in progress. Precise definition of the environmental factors controlling enterotoxin synthesis will hopefully lead to a more complete understanding of the pathogenesis of enterotoxigenic bacteria. ACKNOWLEDGMENTS The excellent technical assistance of B. E. Sinnott and E. R. Stephen is gratefully acknowledged. LITERATURE CITED 1. Alderete, J. F., and D. C. Robertson. 1977. Nutrition
and enterotoxin synthesis by enterotoxigenic strains of Escherichia coli: defined medium for production of heat-stable enterotoxin. Infect. Immun. 15:781-788.
INFECT. IMMUN. 2. Alderete, J. F., and D. C. Robertson. 1977. Repression of heat-stable enterotoxin synthesis in enterotoxigenic Escherichia coli. Infect. Immun. 17:629-633. 3. Callahan, L. T., III, and S. H. Richardson. 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. 4. Callahan, L. T., III, R. C. Ryder, and S. H. Richardson. 1971. Biochemistry of Vibrio cholerae virulence. II. Skin permeability factor/cholera enterotoxin production in a chemically defined medium. Infect. Immun. 4:611-618. 5. Cheng, K.-J., J. M. Ingram, and J. W. Costerton. 1970. Release of alkaline phosphatase from cells of Pseudomonas aeruginosa by manipulation of cation concentration and of pH. J. Bacteriol. 104:748-753. 6. Dean, A. G., Y. C. Ching, R. G. Williams, and L B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 7. Evans, C. G. T., D. Herbert, and D. W. Tempest. 1971. The continuous cultivation of micro-organisms. 2. Construction of a chemostat, p. 277-327. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 2. Academic Press Inc., London. 8. Evans, D. J., Jr., D. G. Evans, H. L. DuPont, F. 0rskov, and I. Orskov. 1977. Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype. Infect. Immun. 17:105-111. 9. Evans, D. G., D. J. Evans, Jr., and N. F. Pierce. 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of E. coli. Infect. Immun. 7:873-880. 10. Gorbach, S. L., B. H. Kean, D. G. Evans, D. J. Evans, Jr., and D. Bessudo. 1975. Travelers' diarrhea and toxigenic Escherichia coli. N. Engl. J. Med. 292:933-936. 11. Guerrant, R. L., R. A. Moore, P. M. Kirschenfeld, and M. S. Sande. 1975. Role of toxigenic and invasive bacteria in acute diarrhea of childhood. N. Engl. J. Med. 293:567-573. 12. Gyles, C. L. 1971. Heat-labile and heat-stable forms of the enterotoxin from E. coli strains enteropathogenic for pigs. Ann. N.Y. Acad. Sci. 196:314-322. 13. Jacks, T. M., and B. J. Wu. 1974. Biochemical properties of Escherichia coli low-molecular-weight, heat-stable enterotoxin. Infect. Immun. 9:342-347. 14. Klipstein, F. A., and R. F. Engert. 1976. Partial purification and properties of Enterobacter cloacae heatstable enterotoxin. Infect. Immun. 13:1307-1314. 15. Klipstein, F. A., and R. F. Engert. 1976. Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin. Infect. Immun. 13:373-381. 16. Koupal, L. R., and R. H. Diebel. 1975. Assay, characterization, and localization of an enterotoxin produced by Salmonella. Infect. Immun. 11:14-22. 17. Levine, M. M., E. S. Caplan, D. Waterman, R. A. Cash, R. B. Hornick, and M. J. Snyder. 1977. Diarrhea caused by Escherichia coli that produce only heatstable enterotoxin. Infect. Immun. 17:78-82. 18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 19. Merson, M. H., G. K. Morris, D. A. Sack, J. G. Wells, J. C. Feeley, R. B. Sack, W. B. Creech, A. Z. Kapikian, and E. J. Gangarosa. 1976. Travellers' diarrhea in Mexico: a prospective study of physicians and family members attending a congress. N. Engl. J. Med. 294:1299-1305. 20. Miller, R. D., and D. Y. C. Fung. 1973. Amino acid requirements for the production of enterotoxin B by
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24.
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Staphylococcus aureus S-6 in a chemically defined medium. Applied Microbiol. 25:800-806. Mitchell, I. deG., M. J. Tame, and R. Kenworthy. 1974. Conditions for the production of Escherichia coli Enterotoxin in a defined medium. J. Med. Microbiol. 7:395-400. Nalin, D. R., A. K. Bhattacharjee, and S. H. Richardson. 1974. Cholera-like toxin effect of culture filtrates of Escherichia coli. J. Infect. Dis. 130:595-601. Ryder, R. W., D. A. Sack, A. Z. Kapikian, J. C. McLaughlin, J. Chakraborty, A. S. M. Mizanur Rahaman, M. H. Merson, and J. G. Wells. 1976. Enterotoxigenic Escherichia coli and reovirus-like agent in rural Bangladesh. Lancet i:659-663. Sack, R. B., N. Hirschhorn, I. Brownlee, R. A. Cash, W. E. Woodward, and D. A. Sack. 1975. Enterotox-
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27. 28.
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igenic Escherichia coli-associated diarrheal disease in Apache children. N. Engl. J. Med. 292:1041-1045. Sandefur, P. D., and J. W. Peterson. 1976. Isolation of skin permeability factors from multure filtrates of Salmonella typhimurium. Infect. Immun. 14:671-679. Smith, H. W., and S. Halls. 1967. Observation by the ligated intestinal segment and oral inoculation method on Escherichia coli infections in pigs, calves, lambs, and rabbits. J. Pathol. Bacteriol. 93:499-529. Stavric, S., and D. Jeffrey. 1977. A modified bioassay for heat-stable Escherichia coli enterotoxin. Can. J. Microbiol. 23:331-336. Whipp, S. C., H. W. Moon, and N. C. Lyon. 1975. Heatstable Escherichia coli enterotoxin production in vivo. Infect. Immun. 12:240-244.
Vol. 20, No. 2 Printed in U.S.A.
Heat-Stable Enterotoxin from Escherichia coli: Factors Involved in Growth and Toxin Productiont W. M. JOHNSON,' H. LIOR,' AND K. G. JOHNSON2* National Enteric Reference Centre, Laboratory Centre for Disease Control, Health and Welfare Canada, Ottawa, Canada,' and Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada2 Received for publication 15 November 1977
Six enterotoxigenic strains of Escherichia coli produced variable levels of heatstable enterotoxin (ST) when grown under pH control at 8.5 in a simple synthetic medium containing neither amino acids nor vitamins. Bacterial growth and ST production were at levels as high as or higher than those observed in complex media. ST elaboration was detectable in the early logarithmic phase of growth and appeared to be related to disappearance of glucose in the growth medium. The results of this study did not suggest pH-dependent release of ST. Imposition of pH control in complex media resulted in increased growth rates, earlier detectable ST synthesis, and elevated levels of ST. In synthetic medium, attainment of the stationary growth phase was followed by a significant decrease in culture density and a concomitant increase in ST. Cellular autolysis experiments revealed that as much as 20% of the total ST activity was present in a cellassociated form. Enterotoxigenic strains of Escherichia coli quired for ST production in one strain of E. coli have been implicated as causative agents in in- (1), whereas certain carbon sources were sugfantile diarrhea (11, 24), diarrhea in neonatal gested as repressors of ST synthesis. The present study was undertaken to examine domestic animals (26, 28), and in so-called travelers' diarrhea (10, 19). Two distinct forms of certain growth parameters and their correlation exocellular enterotoxin, each capable of produc- to ST production in ST' strains of E. coli. ing a cholera-like illness, are elaborated by some MATERIALS AND METHODS enteropathogenic E. coli. The most well-studied Bacterial strains, media, and growth condienterotoxin, designated heat-labile enterotoxin (LT), is a high-molecular-weight, proteinaceous tions. E. coli strains 36-99, 77-1146, 77-217, 77-3753, material exhibiting many similarities to cholera 77-3899, and H-3698 were all of human origin and were toxin (9, 12, 22). Heat-stable enterotoxin (ST), associated with infantile diarrheal disease. The orgawere obtained from the National Enteric Refon the other hand, is a low-molecular-weight nisms Centre (Canada) and were maintained as frozen component which is non-immunogenic, and is erence stock cultures in the National Research Council Culnot neutralized by cholera antitoxin (9, 12, 22). ture Collection. Enterotoxigenic E. coli strains produce LT alone Cultures used for the study of ST production were (ST-/LT') or in combination with ST grown at 37°C in either Evans medium (9), a Casamino (ST+/LT+; 8, 10, 11, 24). Although enteropath- Acids-yeast extract-salts medium, or in a simple minogenic E. coli strains that produce only ST occur eral salts-citric acid-glucose (0.5%, wt/vol) medium less frequently (11, 19, 23, 24), their clinical (MSCG; 7). This medium contained (grams per liter): NH4CI, 3.12; KCI, 2.24; significance has been unequivocally established NaH2PO4 2H20, 4.68;citric acid, 3.15; MgCl2, 0.36; 10H20, 4.83; in a recent study wherein an ST+/ LT- strain Na2SO4 CaCl2, 0.033; and Na2MoO4, 0.03. Trace salts were was shown to produce diarrhea in humans (17). added (15 ml/liter of medium) from a stock trace salts Purification and characterization of ST have solution (total volume, 5 liters) containing: HCI, 50 ml; been hindered by the complexity of media in ZnO, 2.04 g; FeCl3 6H20, 27.0 g; MnCl2 4H20, 10.0 g; which ST+ organisms are grown and by insuffi- CuCl2 2H20, 0.85 g; CoCl2 6H20, 2.83 g; and H3BO4, cient knowledge about nutritional conditions fa- 0.31 g. Unless otherwise indicated, the pH of the voring ST production. Recent studies indicate medium was adjusted to 8.5 before autoclaving. Inocthat ST' strains of E. coli will produce ST in ula (1%, vol/vol) were prepared by washing 18-h culfresh sterile medium. simple synthetic media (1, 2, 21). Proline, serine, tures twice with the appropriate cultures (50 ml) were grown in 125-ml Erlenaspartic acid, and alanine were apparently re- Batch meyer flasks on a New Brunswick rotary shaker operated at 150 rpm. Fermentor cultures (10 liters) were t Issued as National Research Council of Canada no. 16546. 352
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HEAT-STABLE ENTEROTOXIN FROM E. COLI
grown in 14-liter New Brunswick fermentors equipped with a New Brunswick pH control unit. Bacterial growth was monitored by measuring the increase in absorbance of culture aliquots at 550 nm or by triplicate dry-weight determinations of doubly washed (distilled water) culture aliquots. Preparation of cell-free filtrates. Cell-free filtrates were prepared by centrifugation of culture aliquots at 27,000 X g for 15 min. Supernatant fractions were filter sterilized by passage through 0.22-,um membrane filters (Millipore Corp.) before biological assay. ST assay. ST was determined by the suckling mouse assay originally described by Dean et al. (6). Crude, sterile enterotoxin samples were mixed with 1 drop of 2% (wt/vol) Evans blue per ml and were introduced orally into Swiss-Webster suckling mice (1 to 3 days of age) with a 0.5-ml syringe, the canula of which was fitted with Teflon tubing (0.9 by 10 mm; 27). Each sample (total volume, 100 Ml) was assayed, using four mice per test. After 4 h of incubation at 22°C, the animals were killed with chloroform and the intestines from four mice were weighed together. The ratio of intestinal weight to remaining body weight (IW/BW) was then determined. Units of ST activity. Dose response curves relating the observed IW/BW to the various amounts of enterotoxin used in the suckling mouse assay were constructed. Activity was expressed in units as adopted by Jacks and Wu (13). One unit of ST activity was defined as the amount of enterotoxin producing an IW/BW ratio of 0.10 under standard assay conditions (see below). Efficiency of ST production. Efficiency of ST production was defined as units of ST per milliliter per millogram (dry weight) of bacterial mass. Heat stability of crude ST. The heat stability of crude ST was determined as follows. Sterilized culture filtrates were lyophilized, suspended at 40 mg/ml in distilled water, and dispensed in 2-ml portions. These portions were held for 30 min each at 22, 37, 56, 70, 85, and 100°C. After cooling, 2 drops of 2% (wt/vol) Evans blue were added to each. Activity was measured in triplicate, using 100 Al of each sample (four mice per test). Cellular autolysis studies. E. coli 36-99 was grown to mid-logarithmic phase (6 h) in MSCG medium in a New Brunswick fermentor. Aeration, agitation, and pH were maintained at 4 liters/min, 200 rpm, and 8.5, respectively. After being harvested by centrifugation, cells were washed twice with 50 mM sodium phosphate buffer (pH 8.0). Cell wash fractions were pooled, sterilized by passage through 0.22-gm filters, lyophiliWzed, and retained for analyses. Washed cells (6.6 g, dry weight) were suspended in 200 ml of 50 mM tris(hydroxymethyl)aminomethanehydrochloride buffer (pH 8.5) containing 1 mM MgCl2 and 5 Mg each of ribonuclease and deoxyribonuclease (Calbiochem) per ml. Before incubation at 37°C, duplicate 5-ml samples were removed and centrifuged at 48,000 x g for 15 min. Sedimented material was washed twice with distilled water, and dry weights were determined. Supernatant fractions were sterilized by passage through 0.22-,um filters and retained for analyses for protein, ST, and glucose 6-phosphate (G6-P) dehydrogenase contents. At designated intervals thereafter, portions were removed and similarly
353
treated. Analytical methods. Protein was estimated by the method of Lowry et al. (18), uring bovine serum albumin as the standard. G-6-P dehydrogenase (EC 1.1.1.49) was measured by a technique utilizing the reduction of nicotinamide adenine dinucleotide phosphate. G-6-P (18 Mmol), nicotinamide adenine dinucleotide phosphate (0.5 ,umol), tris(hydroxymethyl)aminomethane-hydrochloride buffer, (pH 8.0; 100 Mmol), and 0 to 900 Ml of sample were incubated at 22°C in a total volume of 1.5 ml. The reaction was followed by measuring the increase in absorbance at 340 nm in a recording Bausch and Lomb 200 UV spectrophotometer. One unit of G-6-P dehydrogenase was defined as the amount of enzyme catalyzing the formation of 1 nmol of reduced nicotinamide adenine dinucleotide phosphate per min at 22°C and pH 8.0. Glucose was estimated with the Glucostat reagent (Worthington Biochemicals Corp., Freehold, N.J.).
RESULTS Measurement of ST activity. The relationship between the amount of enterotoxin and the IW/BW ratio is depicted in Fig. 1. Various amounts of sterile culture filtrates (total volume, 100 u1) from 24-h Evans medium-grown batch cultures of E. coli 77-217 and 77-1146 were tested. For both strains, a linear response between dosage and IW/BW ratio was observed. Beyond a certain amount of enterotoxin, plateaus were evident. For several replicates from six ST-producing strains, linearity occurred in the region of an IW/BW of 0.1. Because this is
0'
0.15H
.0% 0).
C3 (n0) cy
0.10
005 40 60 80 100 Culture Filtrate (,. I) FIG. 1. Dose response curve for enterotoxic culture filtrates in the suckling mouse assay. Indicated amounts of culture filtrate from E. coli strains 77-217 (@-*) and 77-1146 (@- -0) were assayed in triplicate (four mice per test). 20
354
JOHNSON, LIOR, AND JOHNSON
considered to be a strongly positive reaction in the suckling mouse assay (13, 27), one unit of ST activity was designated as the amount of material producing an IW/BW ratio of 0.1. For each sample for which ST activity was quantified, construction of a dose response curve was mandatory. Growth of enterotoxigenic E. coli in defined medium. Because pH appears to play a central role in growth and ST production in enterotoxigenic E. coli (1, 21), batch growth experiments were conducted in which media were adjusted to various pH values. E. coli 3699 was grown for 24 h in Evans medium (pH 8.5) and in MSCG medium (pH 7.0 to 9.0). MSCG medium lacking glucose did not support the growth of any of the strains tested. As the data in Table 1 indicate, cultures grown in synthetic medium attained cell densities near those observed in Evans medium. Growth was maximal in media in which the initial pH was adjusted to 8.5. However, only those cultures grown in Evans medium contained detectable ST. Examination of medium pH at 24 h revealed that all synthetic-grown cultures were markedly acid, whereas the pH of cultures grown in Evans medium was well over 7.0. To clarify the possible influence of pH on growth and ST production, these parameters were more rigorously investigated in both media. Effect of pH on growth and ST production. The effect of pH on growth and ST production in Evans medium was compared with that in simple synthetic medium. (i) Evans medium. E. coli 36-99 was grown in a 14-liter New Brunswick fermentor containing 10 liters of Evans medium supplemented with 0.5% (wt/vol) glucose. Aeration and agitation were maintained at 4 liters/min and 200 rpm, respectively. At designated intervals during growth, portions of the culture were removed for dry-weight determinations. Culture filtrates from such samples were monitored for pH, ST activity, and glucose content. Growth was acTABLE 1. Growth of E. coli in simple synthetic Growth medium
MSCG
Evans
medium and Evans medium 1at pH at ST (U/mi) Initial pH OD40 24 ha
7.0 7.5 8.0 8.5 9.0
24 h 4.69 4.72 5.50 5.90 6.00
0.904 7.74 OD5fo, Optical density at 550 nm. b -, Not detectable. a
8.5
0.532 0.827 0.856 0.878 0.855
-b -
20
INFECT. IMMUN.
companied by a decrease in pH until glucose was depleted from the medium at 5 h (Fig. 2). After this point, the pH rose to 6.7 at 12 h. ST production was readily detectable in early logarithmic growth phase (3 h) but remained relatively unchanged well after the onset of the stationary growth phase. During the period of rapid ST elaboration between 2 and 4 h of growth, a large percentage of glucose remained in the medium. Because ST production did not appear to increase significantly after medium pH fell below neutrality, the effect of growth in Evans medium at constant pH was investigated. Parallel cultures were grown as described above. All conditions of aeration, agitation, and washed-inoculum size were identical, except that the medium pH of one fermentor was held constant at pH 8.5 by the automatic addition of 5 N KOH. An almost threefold increase in ST levels was observed in the pH-controlled culture (Fig. 3). ST accumulation in the pH-controlled culture continued to increase from the mid-logarithmic into the stationary growth phase, but ST levels remained relatively constant after the mid-logarithmic growth phase of the uncontrolled culture. ST production was detectable at an earlier stage in the growth cycle of the pH-controlled culture possibly because of the faster growth rate. Independent of pH control, glucose utilization occurred at essentially the same rate, with depletion occurring at 5 h for both cultures. (ii) Simple synthetic medium. Although batch cultures of E. coli 36-99 grown in the simple synthetic medium were devoid of detectable ST (Table 1), fermentor-grown cultures in which pH was held constant at 8.5 during the entire growth cycle yielded relatively high levels of enterotoxin (Fig. 4). Conditions of growth were identical to those used in the pH-controlled Evans medium fermentation. The data in Fig. 4 indicate that, although the growth rate was considerably slower than that observed in Evans medium, ST production was detectable in the early logarithmic growth phase and continued to rise throughout the rest of the experiment. As before, ST production increased with glucose depletion of the medium. In contrast to pHcontrolled fermentations in Evans medium, those effected in synthetic medium yielded higher bacterial dry weights (1.85 mg/ml in synthetic medium, as opposed to 1.30 mg/ml in Evans medium). In addition, once maximum cell densities had been attained in pH-controlled fermentations in synthetic medium, a rapid decline in bacterial dry weight was accompanied by elevation of ST levels. During the period when bacterial dry weight stabilized, ST levels doubled. Elevation of aeration levels resulted in in-
HEAT-STABLE ENTEROTOXIN FROM E. COLI
VOL. 20, 1978
355
.---4 I-@o
I
0 3:-
0
2H H 6
-J 4
GU
co I-
m-
TI M E (h) FIG. 2. Kinetics of growth and ST production in Evans medium for E. coli 36-99. ST is represented by the bar graph. (--4) Bacterial dry weight, (0 - 0) pH.
role of cellular autolysis suggested by the marked fall in bacterial dry weight observed in pH-controlled MSCG-grown cultures was investigated. E. coli 36-99 cells were grown and allowed to autolyse as described in Materials and Methods. Cellular autolysis, as shown by the release of cytoplasmic marker enzyme G-6-P dehydrogenase (5), the increase in non-sedimentable protein, and the loss of sedimentable material, was accompanied by the release of ST (Fig. 5). Maximal release of ST occurred after 4 h of autolysis at 37°C. Although half again as much G-6-P dehydrogenase was released between 5 and 24 h and bacterial dry weight continued to decrease, additional ST was not released. Analysis of the buffer wash (see Materials and Methods) revealed that this fraction was devoid of either ST or G-6-P dehydrogenase. The culTIME (h) FIG. 3. Influence of pH control on growth and ST ture filtrate (8.4 liters) from which these cells production for E. coli 36-99. Cultures were grown in were obtained contained 9,240 U of ST, whereas Evans medium with (A) and without (0) pH control the autolysate possessed 2,353 U. Thus, approx(see text). Bar graph, STproduction in pH-controlled imately 20% of the total ST in this experiment (O) and uncontrolled (J) cultures. Percent unused was cell associated and was released by cellular glucose in pH-controlled (A) and uncontrolled (0) autolysis. cultures. Efficiency of enterotoxin production. To ascertain whether growth and ST production of creased growth rates, although bacterial mass E. coli 36-99 in pH-controlled MSCG medium were unique phenomena, five additional strains and ST levels were unchanged. Autolysis-mediated release of ST. The were grown under identical conditions. As the
356
INFECTI. IMMUN.
JOHNSON, LIOR, AND JOHNSON
-30
-~~~~~~~~25-100
I
la 0
E Z 2.0
20 22 -80
'E
-C 0
iF
C
t0 10 I-
-60
~ ~ ~cn
/
o 0
0
-40
co
10~~~~~~~~~I
05 I 2
4
6
8 10 TIME (h)
12
24
FIG. 4. Growth and ST production of E. coli 36-99 in MSCG. Growth conditions were as described in the text. ST is represented by the bar graph. (0) Bacterial dry weight, (0) percent unused glucose.
E c0
E
.P
I
-S
a!ma t._
._5 cn
TIME(h) FIG. 5. Autolysis of E. coli 36-99 and release of ST. Conditions for cellular autolysis were as described in the text. (Bar graph) ST, (0- -0) G-6-P dehydrogenase, (O-O) bacterial dry weight, (-4*) solubilized bacterial protein. -
data in Table 2 indicate, these conditions permitted both growth and ST production in all strains tested, although considerable strain-tostrain variation in both factors was encountered.
Effect of temperature on enterotoxin activity. Heat stability of the enterotoxin derived from E. coli 36-99 grown in pH-controlled MSCG medium was determined as described in
HEAT-STABLE ENTEROTOXIN FROM E. COLI
VOL. 20, 1978
1.4
33.3
23.6
release of Vibrio cholerae toxin is inhibited by low pH (3,4), ST release in uncontrolled cultures grown in Evans medium appeared unrelated to pH. In fact, ST levels increased during the period when pH was decreasing and remained relatively unchanged thereafter (Fig. 2). Imposition of pH control in Evans medium resulted in elevated levels of ST at all stages of growth, suggesting that increased net synthesis rather than pH-
2.14
20.8
9.7
dependent release was the factor involved in ST
TABLEC 2. Growth and production of ST by enterotoxiigenic E. coli strains in simple synthetic
mediuma
Bacterial 1Phenotype
Strain
c'rgYt
ST
Efficiency (U of
(U/nil) ST/nml per
wt)l T-/ST+
LT-/ST+ LT-/ST+ LT+/ST+
36-99 77-217 77-1146 77-3753 77-3899 H-3698
mg
1.48 57.1 38.6 0.63 57.1 90.6 LT-/ST+ 1.27 9.5 7.5 LT+/ST+ All cult;ures were grown at constant pH (8.5) for 24 h in M[SCG medium supplemented with 0.5% (wt/vol) gliucose. Aeration and agitation were maintained at 4 liters/min and 200 rpm, respectively. b Establieshed by National Enteric Reference Centre (Canada). .a
OJ6 0'
0.14
/
._
0
.
OJ2
C ._
4,
C
357
0.10
____________________________ 20 4 6 80 20 40 60 100
Temperature of 30 min. heOt treatment (°C) FIG. 6. AFffect of heat treatment on E. coli 36-99 crude enterrotoxin. Conditions of the assay were as described il n the text. Materials and Methods. As the data in Fig. indicate, a 30-m heat treatment resulted in
6 a
stimulatio:,n rather than a decrease in activity.
DISCUSSION As a pre-lude to purification and characterization of ST'from enterotoxigenic E. coli, growth kinetics arad toxin production were examined in complex and simple synthetic media. Use of IpH control during fermentation produced a ariety of notable effects. In complex media, increased growth rates, earlier elaborav.
tion of enterotoxin, and greatly increased ST levels wer(e observed (Fig. 3). Although pH control was rnot required for bacterial growth in simnple syrithetic medium, it was absolutely essential for ST production. These observations would sugj gest that pH controls either the release of enterotcixin or its net synthesis. Although the
evolution. In contrast to previous results (1, 12), exocellular ST was detected well before the onset of
the stationary growth phase and usually before cultures attained the mid-logarithmic growth. These observations suggest that the factor(s) regulating expression of the ST+ plasmid is not a secondary metabolic product. In the initial study of ST production by enterotoxigenic E. coli, Alderete and Robertson (1) suggested that glucose serves as a catabolite repressor of ST synthesis. A more recent study (2) revealed that glucose completely represses ST synthesis by two enterotoxigenic strains of E. coli and that derepression can be effected by the addition of cyclic adenosine 3',5'-monophosphate to the growth media. Although glucose was required for bacterial growth in the synthetic medium used in our studies, ST elaboration was accompanied by the disappearance of glucose from the medium. However, incomplete repression of ST formation by glucose is indicated because relatively high levels of ST accumulation preceded glucose depletion of fermentations in both complex and simple synthetic media. These findings suggest that the composition of the growth media may influence the apparently repressive effect of glucose. Miller and Fung (20) have noted that the inhibitory effect of glucose on enterotoxin B synthesis in Staphylococcus aureus S-6 grown in defined media is less evident (or totally absent) compared with the response in complex media. Possibly, the continuous culture of enterotoxigenic E. coli under conditions of glucose limitation will provide unequivocal answers. Growth conditions for enterotoxigenic E. coli developed in this study differ in two fundamental respects from those previously described (1, 21). The first, pH control, has been discussed already. The second major difference is in nutritional simplicity of the growth medium. Growth studies in synthetic media evolved by Alderete and Robertson (1) indicate that proline, serine, aspartic acid, and alanine are required for growth and enterotoxin synthesis. Whereas no amino acid requirements for growth or ST elaboration were found in the defined medium of Mitchell et al. (21), their media contained a mixture of eight vitamins. Our studies indicate that neither vitamins nor amino acids are re-
358
JOHNSON, LIOR, AND JOHNSON
quired for either growth or ST synthesis by at least six enterotoxigenic E. coli strains. Moreover, and in contrast to the results of Alderete and Robertson (1), cell yields were as high or higher than those observed in Evans medium. Perhaps the apparent amino acid requirements described by Alderete and Robertson (1) are reflections of the nutritional specificities of the strains studied. More probably, the aforementioned amino acids serve as carbon sources in the absence of a more readily metabolized carbon source such as glucose. Heat stability of the enterotoxin, at least from E. coli 36-99, certainly was not affected by the growth conditions employed. Alderete and Robertson found that ST production and culture densities, under the growth conditions they prescribed (1), were maximal at approximately 8 h of growth and were essentially the same at 18 h. In contrast, and particularly with synthetic medium, we observed that ST levels continued to rise well after the onset of stationary phase, implying that cellular autolysis might be involved in the release of ST. Autolysis of mid-logarithmic cultures did reveal that a significant portion of the total ST activity was cell associated. Evidence exists for the synthesis of other heat-stable, low-molecular-weight enterotoxins by Klebsiella pneumoniae (15), Enterobacter cloacae (14), and Salmonella species (16, 25). In contrast to the autolysis-mediated ST release observed in this study, cell-free lysates from the above organisms apparently did not possess ST activity. If the growth conditions described here lead to a situation in which a portion of ST is cell associated, a potentially powerful tool for the study of the mechanism of ST release may be realized. That high-level growth and ST elaboration by enterotoxigenic strains of E. coli can be achieved under exceedingly simple nutritional conditions has been established. The logical extension of this work into continuous culture (in which the effect of parameters such as aeration, pH, and growth rate on ST production can be studied as independent phenomena) is in progress. Precise definition of the environmental factors controlling enterotoxin synthesis will hopefully lead to a more complete understanding of the pathogenesis of enterotoxigenic bacteria. ACKNOWLEDGMENTS The excellent technical assistance of B. E. Sinnott and E. R. Stephen is gratefully acknowledged. LITERATURE CITED 1. Alderete, J. F., and D. C. Robertson. 1977. Nutrition
and enterotoxin synthesis by enterotoxigenic strains of Escherichia coli: defined medium for production of heat-stable enterotoxin. Infect. Immun. 15:781-788.
INFECT. IMMUN. 2. Alderete, J. F., and D. C. Robertson. 1977. Repression of heat-stable enterotoxin synthesis in enterotoxigenic Escherichia coli. Infect. Immun. 17:629-633. 3. Callahan, L. T., III, and S. H. Richardson. 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. 4. Callahan, L. T., III, R. C. Ryder, and S. H. Richardson. 1971. Biochemistry of Vibrio cholerae virulence. II. Skin permeability factor/cholera enterotoxin production in a chemically defined medium. Infect. Immun. 4:611-618. 5. Cheng, K.-J., J. M. Ingram, and J. W. Costerton. 1970. Release of alkaline phosphatase from cells of Pseudomonas aeruginosa by manipulation of cation concentration and of pH. J. Bacteriol. 104:748-753. 6. Dean, A. G., Y. C. Ching, R. G. Williams, and L B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 7. Evans, C. G. T., D. Herbert, and D. W. Tempest. 1971. The continuous cultivation of micro-organisms. 2. Construction of a chemostat, p. 277-327. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 2. Academic Press Inc., London. 8. Evans, D. J., Jr., D. G. Evans, H. L. DuPont, F. 0rskov, and I. Orskov. 1977. Patterns of loss of enterotoxigenicity by Escherichia coli isolated from adults with diarrhea: suggestive evidence for an interrelationship with serotype. Infect. Immun. 17:105-111. 9. Evans, D. G., D. J. Evans, Jr., and N. F. Pierce. 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of E. coli. Infect. Immun. 7:873-880. 10. Gorbach, S. L., B. H. Kean, D. G. Evans, D. J. Evans, Jr., and D. Bessudo. 1975. Travelers' diarrhea and toxigenic Escherichia coli. N. Engl. J. Med. 292:933-936. 11. Guerrant, R. L., R. A. Moore, P. M. Kirschenfeld, and M. S. Sande. 1975. Role of toxigenic and invasive bacteria in acute diarrhea of childhood. N. Engl. J. Med. 293:567-573. 12. Gyles, C. L. 1971. Heat-labile and heat-stable forms of the enterotoxin from E. coli strains enteropathogenic for pigs. Ann. N.Y. Acad. Sci. 196:314-322. 13. Jacks, T. M., and B. J. Wu. 1974. Biochemical properties of Escherichia coli low-molecular-weight, heat-stable enterotoxin. Infect. Immun. 9:342-347. 14. Klipstein, F. A., and R. F. Engert. 1976. Partial purification and properties of Enterobacter cloacae heatstable enterotoxin. Infect. Immun. 13:1307-1314. 15. Klipstein, F. A., and R. F. Engert. 1976. Purification and properties of Klebsiella pneumoniae heat-stable enterotoxin. Infect. Immun. 13:373-381. 16. Koupal, L. R., and R. H. Diebel. 1975. Assay, characterization, and localization of an enterotoxin produced by Salmonella. Infect. Immun. 11:14-22. 17. Levine, M. M., E. S. Caplan, D. Waterman, R. A. Cash, R. B. Hornick, and M. J. Snyder. 1977. Diarrhea caused by Escherichia coli that produce only heatstable enterotoxin. Infect. Immun. 17:78-82. 18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 19. Merson, M. H., G. K. Morris, D. A. Sack, J. G. Wells, J. C. Feeley, R. B. Sack, W. B. Creech, A. Z. Kapikian, and E. J. Gangarosa. 1976. Travellers' diarrhea in Mexico: a prospective study of physicians and family members attending a congress. N. Engl. J. Med. 294:1299-1305. 20. Miller, R. D., and D. Y. C. Fung. 1973. Amino acid requirements for the production of enterotoxin B by
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24.
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Staphylococcus aureus S-6 in a chemically defined medium. Applied Microbiol. 25:800-806. Mitchell, I. deG., M. J. Tame, and R. Kenworthy. 1974. Conditions for the production of Escherichia coli Enterotoxin in a defined medium. J. Med. Microbiol. 7:395-400. Nalin, D. R., A. K. Bhattacharjee, and S. H. Richardson. 1974. Cholera-like toxin effect of culture filtrates of Escherichia coli. J. Infect. Dis. 130:595-601. Ryder, R. W., D. A. Sack, A. Z. Kapikian, J. C. McLaughlin, J. Chakraborty, A. S. M. Mizanur Rahaman, M. H. Merson, and J. G. Wells. 1976. Enterotoxigenic Escherichia coli and reovirus-like agent in rural Bangladesh. Lancet i:659-663. Sack, R. B., N. Hirschhorn, I. Brownlee, R. A. Cash, W. E. Woodward, and D. A. Sack. 1975. Enterotox-
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igenic Escherichia coli-associated diarrheal disease in Apache children. N. Engl. J. Med. 292:1041-1045. Sandefur, P. D., and J. W. Peterson. 1976. Isolation of skin permeability factors from multure filtrates of Salmonella typhimurium. Infect. Immun. 14:671-679. Smith, H. W., and S. Halls. 1967. Observation by the ligated intestinal segment and oral inoculation method on Escherichia coli infections in pigs, calves, lambs, and rabbits. J. Pathol. Bacteriol. 93:499-529. Stavric, S., and D. Jeffrey. 1977. A modified bioassay for heat-stable Escherichia coli enterotoxin. Can. J. Microbiol. 23:331-336. Whipp, S. C., H. W. Moon, and N. C. Lyon. 1975. Heatstable Escherichia coli enterotoxin production in vivo. Infect. Immun. 12:240-244.
