JOURNAL OF ULTRASTRUCTURE RESEARCH 58, 2 5 2 - 2 6 0 (1977)
Morphology of Vibrio cholerae during Enterotoxin Production under Anaerobic Conditions PRABHAVATHI B. FERNANDES,1 J. M. CLARK, AND H. L. SMITH, JR. Department of Microbiology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received June 8, 1976, and in revised form September 17, 1976 Pleomorphic cells were observed in cultures of Vibrio cholerae incubated u n d e r anaerobic conditions. The appearance of these pleomorphic cells coincided with the detection of large amounts of cholera enterotoxin in the culture filtrates. Many of these pleomorphic cells had morphologically defective cell efivelopes which appeared to lack a m u r e i n layer. They also contained intracellular membrane-bounded vesicles. The n a t u r e of these m e m b r a n e structures and t h e i r association with enterotoxin production is discussed.
Large numbers of Vibrio cholerae can be demonstrated in the lumen of the small intestine of individuals with cholera (26) but only those organisms adhering to the intestinal epithelial cells are apparently involved in the disease process (20). In this microenvironment adjacent to the epithelial cells the oxygen tension is most likely less than that of the well-aerated cultures employed to study toxin production in vitro (5). Studies on in vitro toxin production by V. cholerae have also employed incubation temperatures of 25 to 30°C (12) even though the temperature of the small intestine involved in the disease process is approximately 37°C. At this temperature, optimal growth is obtained aerobically in vitro (36), whereas the toxin yield is decreased as compared to that obtained at 30°C. We have examined the morphology and enterotoxin production of Vibrio cholerae under both anaerobic and aerobic conditions and at incubation temperatures of 30 and 37°C (15). Anaerobic conditions at 37°C resulted in production of enterotoxin at a high toxin to cell ratio and in conversion of the majority of the normal vibriolike cells into spherical forms of varying sizes. Although the ultrastructure of V. i Present address: Postdoctoral Fellow, Institute for Cancer Research, Burholme Avenue, Fox Chase, Philadelphia, Pa. 19111.
cholerae grown aerobically at 30 and 37°C has been well documented (7, 31), the morphology of organisms grown anaerobically has not been studied. This paper describes morphological observations on anaerobically grown cells. MATERIALS AND METHODS
Vibrio cholerae, strains 569B (Inaba) and B1307 (Ogawa), were obtained from Dr. J o h n Feeley, NIH, Bethesda, Maryland. We have described elsewhere (15) the m a i n t e n a n c e of stock cultures of these strains, culture media, incubation conditions, and methods for m e a s u r i n g growth and toxin production. These methods are briefly described here. The organisms were grown either aerobically or anaerobically in Syncase (17) or Peptone water (5) media. Aerobic incubation was carried out in a water-bath s h a k e r at either 30 or 37°C. Anaerobic growth was obtained by incubation in a flexible fiber chamber with a n atmosphere of 5% CO2, 88% N2, and 7% H2. For anaerobic growth the media were buffered with 0.025 M Tris with the pH adjusted to 8.0 and prereduced for 18 h in the anaerobic chamber before use. A fresh vial of the lyophilized stock cultures was used for each experiment. Cultures were reconstituted by adding 1 ml of the medium to be used and incubated at 30 or 37°C u n d e r aerobic or anaerobic conditions so t h a t the inoculum employed for the test cultures was in the logarithmic phase of growth. Estimations of growth were done turbidimetrically, employing viable counts, by weight of centrifuged cells dried to a constant weight and by the weight of m a t e r i a l precipitated from a culture sample by 5% (v/v) cold perchloric acid. S u p e r n a t a n t fluid from centrifuged samples of cultures grown under the various conditions was passed t h r o u g h a 0.45-t~m m e m b r a n e filter into con252
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
v. CHOLERAE MORPHOLOGY UNDER ANAEROBIC CONDITIONS
tainers to which EDTA had been added to give a final concentration of 0.005% and stored at 4°C (38). Enterotoxin activity of the filtrates was assayed by the passive hemagglutination inhibition (PHI) test of Finkelstein and Peterson (18) as modified by Callahan et al. (6). This PHI test does not distinguish between inactive and active toxin. Active enterotoxin was determined by the adult rabbit ileal loop assay using the method of Pierce and Wallace (35) with crude enterotoxin employed as a positive control. The presence of enterotoxin in the culture samples tested in the rabbit ileal loop assay was verified by specific neutralization with horse anti-choleragenoid prepared by Finkelstein (16) (NIH Lot No. 19) employed as antitoxin and by the heat lability (5) of the enterotoxin in the test filtrates which was inactivated after incubation at 56°C for 30 min. For electron microscopy the cells were harvested at appropriate intervals of growth by centrifugation at 5000g for 15 min and were fixed in 3% glutaraldehyde in 0.067 M cacodylate buffer with 1% sucrose at pH 7.3. The organisms were then postfixed in 1% osmium tetroxide, dehydrated with graded ethanol, and embedded in Epon 812 (24). Thin sections were cut with diamond knives on a Porter-Blum MT-2 ultramicrotome, picked up on copper grids, stained with uranyl acetate and lead citrate, and examined in a Hitachi HS-8 electron microscope at an accelerating voltage of 50 kV. RESULTS Since growth, morphology, and enterot o x i n p r o d u c t i o n by V. cholerae s t r a i n s 569B a n d B1307 g r o w n i n S y n c a s e o r P e p tone water media were similar, only the r e s u l t s o b t a i n e d w i t h s t r a i n 569B g r o w n i n Syncase medium will be reported here. The enterotoxin production of strain 569B g r o w n a e r o b i c a l l y o r a n a e r o b i c a l l y a t 30 o r 37°C is s h o w n i n T a b l e I. S i g n i f i c a n t amounts of toxin were produced under a e r o b i c c o n d i t i o n s a t 30°C, a s h a s b e e n r e p o r t e d b y o t h e r i n v e s t i g a t o r s (6, 37).
253
Only slight amounts of toxin were prod u c e d a t 37°C u n d e r a e r o b i c c o n d i t i o n s e v e n t h o u g h t h e a m o u n t o f g r o w t h a s det e r m i n e d b y d r y w e i g h t of c e n t r i f u g e d culture sediment was significantly greater t h a n t h a t p r o d u c e d a t 30°C. A n a e r o b i c inc u b a t i o n a t 30°C p r o d u c e d s i g n i f i c a n t a m o u n t s o f t o x i n o n l y a t 33 a n d 48 h r , w h i l e a n a e r o b i c i n c u b a t i o n a t 37°C p r o d u c e d s i g n i f i c a n t t o x i n a t 12, 33, a n d 48 h r . T h e r e w a s a m a r k e d d e c r e a s e i n t h e cell count of anaerobically grown cultures a f t e r 12 h r d u e to cell l y s i s u n d e r t h e s e c o n d i t i o n s . T h e r e w a s no d i f f e r e n c e i n g r o w t h o r t o x i n p r o d u c t i o n of a n a e r o b i c cultures grown in either stationary or shaken cultures. The maximum toxin prod u c t i o n is e x p r e s s e d a s m i c r o g r a m s p e r m i l l i l i t e r of c u l t u r e f i l t r a t e o c c u r r i n g a t 33 hr incubation under anaerobic conditions a t 37°C a n d a t 33 a n d 48 h r u n d e r a e r o b i c c o n d i t i o n s a t 30°C. T h e d a t a i n T a b l e I a p p e a r to i n d i c a t e t h a t t h e r e is s i g n i f i c a n t l y m o r e t o x i n p r o d u c e d a t 30°C a e r o b i c a l l y t h a n a t 37°C a n a e r o b i c a l l y . H o w e v e r , i f t o x i n y i e l d is c a l c u l a t e d on t h e b a s i s o f micrograms of toxin per milligram dry w e i g h t , 1.3 tLg/mg is o b t a i n e d for b o t h cult u r e s a t 33 h r . T o x i n p r o d u c t i o n t h e r e f o r e a p p e a r s to b e r e l a t e d to g r o w t h c o n d i t i o n s . W h e n t h e o r g a n i s m is g r o w i n g u n d e r a p p a r e n t l y o p t i m a l c o n d i t i o n s , i.e., 37°C a e r o b i c a l l y (36), o n l y s m a l l a m o u n t s o f t o x i n are produced. When the organisms are g r o w i n g u n d e r s u b o p t i m a l c o n d i t i o n s , i.e., a e r o b i c a l l y a t 30°C o r a n a e r o b i c a l l y , significant amounts of enterotoxin are released into the medium.
TABLE I ENTEROTOXIN PRODUCTION BY Vibrio cholerae STRAIN 569B GROWN IN SYNCASE MEDIUM
Hours of incubation
12 33 48
Aerobic culture 30°C
Anaerobic culture 37°C
30°C
37°C
PHI a
V/L ~
PHI
V/L
PHI
V/L
PHI
V/L
5.0 10.0 10.0
2.4 _+ 0.3 2.6 _+ 0.15 2.7 _+ 0.2
Morphology of Vibrio cholerae during Enterotoxin Production under Anaerobic Conditions PRABHAVATHI B. FERNANDES,1 J. M. CLARK, AND H. L. SMITH, JR. Department of Microbiology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received June 8, 1976, and in revised form September 17, 1976 Pleomorphic cells were observed in cultures of Vibrio cholerae incubated u n d e r anaerobic conditions. The appearance of these pleomorphic cells coincided with the detection of large amounts of cholera enterotoxin in the culture filtrates. Many of these pleomorphic cells had morphologically defective cell efivelopes which appeared to lack a m u r e i n layer. They also contained intracellular membrane-bounded vesicles. The n a t u r e of these m e m b r a n e structures and t h e i r association with enterotoxin production is discussed.
Large numbers of Vibrio cholerae can be demonstrated in the lumen of the small intestine of individuals with cholera (26) but only those organisms adhering to the intestinal epithelial cells are apparently involved in the disease process (20). In this microenvironment adjacent to the epithelial cells the oxygen tension is most likely less than that of the well-aerated cultures employed to study toxin production in vitro (5). Studies on in vitro toxin production by V. cholerae have also employed incubation temperatures of 25 to 30°C (12) even though the temperature of the small intestine involved in the disease process is approximately 37°C. At this temperature, optimal growth is obtained aerobically in vitro (36), whereas the toxin yield is decreased as compared to that obtained at 30°C. We have examined the morphology and enterotoxin production of Vibrio cholerae under both anaerobic and aerobic conditions and at incubation temperatures of 30 and 37°C (15). Anaerobic conditions at 37°C resulted in production of enterotoxin at a high toxin to cell ratio and in conversion of the majority of the normal vibriolike cells into spherical forms of varying sizes. Although the ultrastructure of V. i Present address: Postdoctoral Fellow, Institute for Cancer Research, Burholme Avenue, Fox Chase, Philadelphia, Pa. 19111.
cholerae grown aerobically at 30 and 37°C has been well documented (7, 31), the morphology of organisms grown anaerobically has not been studied. This paper describes morphological observations on anaerobically grown cells. MATERIALS AND METHODS
Vibrio cholerae, strains 569B (Inaba) and B1307 (Ogawa), were obtained from Dr. J o h n Feeley, NIH, Bethesda, Maryland. We have described elsewhere (15) the m a i n t e n a n c e of stock cultures of these strains, culture media, incubation conditions, and methods for m e a s u r i n g growth and toxin production. These methods are briefly described here. The organisms were grown either aerobically or anaerobically in Syncase (17) or Peptone water (5) media. Aerobic incubation was carried out in a water-bath s h a k e r at either 30 or 37°C. Anaerobic growth was obtained by incubation in a flexible fiber chamber with a n atmosphere of 5% CO2, 88% N2, and 7% H2. For anaerobic growth the media were buffered with 0.025 M Tris with the pH adjusted to 8.0 and prereduced for 18 h in the anaerobic chamber before use. A fresh vial of the lyophilized stock cultures was used for each experiment. Cultures were reconstituted by adding 1 ml of the medium to be used and incubated at 30 or 37°C u n d e r aerobic or anaerobic conditions so t h a t the inoculum employed for the test cultures was in the logarithmic phase of growth. Estimations of growth were done turbidimetrically, employing viable counts, by weight of centrifuged cells dried to a constant weight and by the weight of m a t e r i a l precipitated from a culture sample by 5% (v/v) cold perchloric acid. S u p e r n a t a n t fluid from centrifuged samples of cultures grown under the various conditions was passed t h r o u g h a 0.45-t~m m e m b r a n e filter into con252
Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
v. CHOLERAE MORPHOLOGY UNDER ANAEROBIC CONDITIONS
tainers to which EDTA had been added to give a final concentration of 0.005% and stored at 4°C (38). Enterotoxin activity of the filtrates was assayed by the passive hemagglutination inhibition (PHI) test of Finkelstein and Peterson (18) as modified by Callahan et al. (6). This PHI test does not distinguish between inactive and active toxin. Active enterotoxin was determined by the adult rabbit ileal loop assay using the method of Pierce and Wallace (35) with crude enterotoxin employed as a positive control. The presence of enterotoxin in the culture samples tested in the rabbit ileal loop assay was verified by specific neutralization with horse anti-choleragenoid prepared by Finkelstein (16) (NIH Lot No. 19) employed as antitoxin and by the heat lability (5) of the enterotoxin in the test filtrates which was inactivated after incubation at 56°C for 30 min. For electron microscopy the cells were harvested at appropriate intervals of growth by centrifugation at 5000g for 15 min and were fixed in 3% glutaraldehyde in 0.067 M cacodylate buffer with 1% sucrose at pH 7.3. The organisms were then postfixed in 1% osmium tetroxide, dehydrated with graded ethanol, and embedded in Epon 812 (24). Thin sections were cut with diamond knives on a Porter-Blum MT-2 ultramicrotome, picked up on copper grids, stained with uranyl acetate and lead citrate, and examined in a Hitachi HS-8 electron microscope at an accelerating voltage of 50 kV. RESULTS Since growth, morphology, and enterot o x i n p r o d u c t i o n by V. cholerae s t r a i n s 569B a n d B1307 g r o w n i n S y n c a s e o r P e p tone water media were similar, only the r e s u l t s o b t a i n e d w i t h s t r a i n 569B g r o w n i n Syncase medium will be reported here. The enterotoxin production of strain 569B g r o w n a e r o b i c a l l y o r a n a e r o b i c a l l y a t 30 o r 37°C is s h o w n i n T a b l e I. S i g n i f i c a n t amounts of toxin were produced under a e r o b i c c o n d i t i o n s a t 30°C, a s h a s b e e n r e p o r t e d b y o t h e r i n v e s t i g a t o r s (6, 37).
253
Only slight amounts of toxin were prod u c e d a t 37°C u n d e r a e r o b i c c o n d i t i o n s e v e n t h o u g h t h e a m o u n t o f g r o w t h a s det e r m i n e d b y d r y w e i g h t of c e n t r i f u g e d culture sediment was significantly greater t h a n t h a t p r o d u c e d a t 30°C. A n a e r o b i c inc u b a t i o n a t 30°C p r o d u c e d s i g n i f i c a n t a m o u n t s o f t o x i n o n l y a t 33 a n d 48 h r , w h i l e a n a e r o b i c i n c u b a t i o n a t 37°C p r o d u c e d s i g n i f i c a n t t o x i n a t 12, 33, a n d 48 h r . T h e r e w a s a m a r k e d d e c r e a s e i n t h e cell count of anaerobically grown cultures a f t e r 12 h r d u e to cell l y s i s u n d e r t h e s e c o n d i t i o n s . T h e r e w a s no d i f f e r e n c e i n g r o w t h o r t o x i n p r o d u c t i o n of a n a e r o b i c cultures grown in either stationary or shaken cultures. The maximum toxin prod u c t i o n is e x p r e s s e d a s m i c r o g r a m s p e r m i l l i l i t e r of c u l t u r e f i l t r a t e o c c u r r i n g a t 33 hr incubation under anaerobic conditions a t 37°C a n d a t 33 a n d 48 h r u n d e r a e r o b i c c o n d i t i o n s a t 30°C. T h e d a t a i n T a b l e I a p p e a r to i n d i c a t e t h a t t h e r e is s i g n i f i c a n t l y m o r e t o x i n p r o d u c e d a t 30°C a e r o b i c a l l y t h a n a t 37°C a n a e r o b i c a l l y . H o w e v e r , i f t o x i n y i e l d is c a l c u l a t e d on t h e b a s i s o f micrograms of toxin per milligram dry w e i g h t , 1.3 tLg/mg is o b t a i n e d for b o t h cult u r e s a t 33 h r . T o x i n p r o d u c t i o n t h e r e f o r e a p p e a r s to b e r e l a t e d to g r o w t h c o n d i t i o n s . W h e n t h e o r g a n i s m is g r o w i n g u n d e r a p p a r e n t l y o p t i m a l c o n d i t i o n s , i.e., 37°C a e r o b i c a l l y (36), o n l y s m a l l a m o u n t s o f t o x i n are produced. When the organisms are g r o w i n g u n d e r s u b o p t i m a l c o n d i t i o n s , i.e., a e r o b i c a l l y a t 30°C o r a n a e r o b i c a l l y , significant amounts of enterotoxin are released into the medium.
TABLE I ENTEROTOXIN PRODUCTION BY Vibrio cholerae STRAIN 569B GROWN IN SYNCASE MEDIUM
Hours of incubation
12 33 48
Aerobic culture 30°C
Anaerobic culture 37°C
30°C
37°C
PHI a
V/L ~
PHI
V/L
PHI
V/L
PHI
V/L
5.0 10.0 10.0
2.4 _+ 0.3 2.6 _+ 0.15 2.7 _+ 0.2
