CURRENT MICROBIOLOGYVol. 23 (1991), pp. 139-142
Current Microbiology © Springer-Verlag New York Inc. 1991
A Nonhemolytic Phospholipase C Produced by Pseudomonas cepacia Miriam K. Lonon and Anne Morris Hooke Department of Microbiology, Miami University, Oxford, Ohio, USA
A b s t r a c t . Pseudomonas cepacia Pc224c, a n o n h e m o l y t i c s t r a i n o r i g i n a l l y i s o l a t e d f r o m t h e s p u t u m o f a c y s t i c fibrosis p a t i e n t , p r o d u c e d an e x t r a c e l l u l a r , h e a t - l a b i l e p h o s p h o l i p a s e C a c t i v i t y , w h i c h w a s m e a s u r e d q u a n t i t a t i v e l y on the s y n t h e t i c s u b s t r a t e p - n i t r o p h e n y l p h o s p h o r y l c h o l i n e . C e l l - f r e e s u p e r n a t a n t s f r o m c u l t u r e s g r o w n to late log p h a s e in M O P S - m i n i m a l s a l t s - T r y p t o s e m e d i u m c o n t a i n e d specific a c t i v i t y at l e a s t 38 t i m e s g r e a t e r t h a n t h a t f r o m c u l t u r e s g r o w n in T r y p t o s e m i n i m a l m e d i u m , T r y p t i c S o y b r o t h , o r p e p t o n e m e d i u m . P r o d u c t i o n w a s i n h i b i t e d b y t h e p r e s e n c e o f i n o r g a n i c p h o s p h a t e in t h e m e d i u m a n d e n h a n c e d b y a e r a t i o n o f the c u l t u r e . T h e P L C o f P . cepacia is n o n h e m o l y t i c a n d d o e s n o t e x h i b i t l e c i t h i n a s e a c t i v i t y on e g g - y o l k agar.
Pseudomonas cepacia is a s e r i o u s o p p o r t u n i s t i c human pathogen, particularly for patients with cystic fibrosis ( C F ) [4, 12]. A l t h o u g h the t i s s u e d e s t r u c t i o n o f t e n a s s o c i a t e d with p u l m o n a r y i n f e c t i o n s in C F p a t i e n t s s u g g e s t s t h e a c t i o n o f a t o x i n ( s ) o r enz y m e ( s ) , P. cepacia, u n l i k e a n o t h e r o p p o r t u n i s t i c p a t h o g e n , P. aeruginosa, d o e s n o t p r o d u c e e x o t o x ins A o r S [9]. A n o t h e r e n z y m e t h a t m a y b e a v i r u l e n c e f a c t o r in p u l m o n a r y i n f e c t i o n s is p h o s p h o l i p a s e C ( P L C ) [5, 11]. T h e r e h a v e b e e n v a r y i n g r e p o r t s o f P L C a c t i v i t y b y P. cepacia [8, 10]. Recently, we detected small amounts of PLC activity in c o n c e n t r a t e d c u l t u r e s u p e r n a t a n t s o f P. cepacia Pc224c g r o w n in c h e m i c a l l y d e f i n e d m e d i u m [6], a n d V a s i l et al. h a v e c l o n e d f r o m P. cepacia a 4 . 2 - k b restriction fragment that codes for both hemolytic a n d P L C a c t i v i t i e s [14]. T h i s p a p e r d e s c r i b e s t h e preliminary characterization of a nonhemolytic PLC w e h a v e i d e n t i f i e d in c u l t u r e s u p e r n a t a n t s o f P . cepacia Pc224c. Materials and Methods Bacterial strains. Pseudomonascepacia Pc224c, a sputum isolate from a CF patient, was obtained from D.E. Woods (University of Calgary Health Sciences Centre, Calgary, Alberta). It was originally from the collection of J.D. Klinger (Gene-Trak Systems, Framingham, Massachusetts) and was maintained in 20% glycerol in Tryptic Soy broth (TSB) (Difco Laboratories, Detroit, Michigan) at - 20°C. Production and detection of PLC activity. P. cepacia Pc224c
was grown to late log phase in MOPS-minimal salts-Tryptose medium (MMST) (3 mM KC1, 12 mM [NH412SO4, 3.2 mM MgSO4, 0.02 mM FeSO4, 3 mM NaC1, 20 mM glucose, and 0.1% Tryptose [Difco] in 50 mM 3-[N-morpholino]pro-panesulfonic acid (MOPS; Sigma Chemical Company, St. Louis, Missouri], pH 7.4), or in Tryptose minimal medium (TMM) [3l, TSB, or peptone medium (1% peptone [Sigma]- 1% NaC1-1% glycerol) [ 14], at 37° C, with shaking at 200 rpm in a Lab-Line Orbit Environ-Shaker 18 (Lab-Line Instruments, Melrose Park, Illinois). Cell-free culture supernatants were assayed quantitatively for PLC activity on pnitrophenylphosphorylcholine (NPPC) [3]. Protein concentrations were determined with the Bio-Rad assay (Bio-Rad Laboratories, Richmond, California) and specific PLC activity (SA) was calculated as absorbance units at 410 nm per/zg total protein. Hemolytic activity. The organism was screened for hemolytic activity on blood agar (5% sheep, horse, rabbit, or human type O erythrocytes in Tryptic Soy agar). Concentrated supernatants (50 pA)from cells cultured in MMST were also assayed for hemolytic activity, by filling wells (5 mm diameter) punched in blood agar. Positive and negative controls were 50/xl commercially prepared PLC (Clostridium pe~fringens Type I, Sigma) and 50 ~1 Tris buffer. Production of lecithinase. The organism was streaked on egg yolk agar (Becton Dickinson Microbiology Systems, Cockeysville, Maryland) and incubated at 37°C for 72 h, when the plates were inspected for lecithinase activity. Supernatants from cultures grown in MMST were examined for lecithinase in a manner similar to that used to examine hemolysis. Concentration of supernatants. Culture supernatants were concentrated by precipitation with 0-30% saturated ammonium sulfate (SAS) at 4°C. The precipitates were pelleted at 17,000 g for
Address reprint requests to: Dr. Miriam K. Lonon, Department of Microbiology, Miami University, Oxford, OH 45056, USA.
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CURRENT MICROBIOLOGY Vol. 23 (1991)
30 min at 4°C, suspended in 1 ml 50 mM Tris buffer (pH 7.6), and dialyzed for 24 h against the same buffer.
Table 1. Effect of growth medium on the production of PLC by Pseudomonas cepacia Pc224c a
Kinetics of PLC production and the effects of phosphate. P. cepacia Pc224c was cultured in 200 ml of each of the following: MMST; MMST plus 10 mM K2HPO 4 added at time zero; MMST plus 10 mM K2HPO 4 added after 4 h incubation; MMST with 2% additional tryptose. Cultures were incubated with shaking (200 rpm) at 37°C for 8 h in a controlled environment incubator shaker (New Brunswick Scientific, Edison, New Jersey). Samples (0.5 ml) were taken at 1, 3, 4, and 5 h, and the remainder of the culture was harvested at 8 h. Supernatants were assayed for PLC activity on NPPC. The effect of inorganic phosphate (Pi) on PLC production was further examined by growing the organism in Anwar's chemically defined medium (ACDM) [1] with K2HPO 4 concentrations of 0.6, 1.2, 2.4, or 4.8 raM. Cultures were incubated in baffled flasks aerated at 200 rpm at 37°C for 24 h and, because PLC levels produced in ACDM were very low, supernatants were concentrated as described.
Growth medium
Absorbance at 410 nm
Specific activity b
MMST TMM TSB Peptone Positive control Negative control
0.27 0.00 0.03 0.01 0.1 t 0.00
3,50 0.00 0.09 0.03 0.01 0.00
Effects of aeration and mechanical agitation. Five 200-ml cultures of Pc224c in I-L flasks of MMST were inoculated and grown for 24 h at 37°C under one of the following aeration conditions: shaking at 200 rpm in a baffled flask; shaking at 200 rpm in a flask without baffles; incubation without shaking, but with filtered compressed air bubbling through the medium; shaking at 200 rpm in a sealed, evacuated flask, incubation without shaking or other aeration. The cultures were continued well into stationary phase, when the ceils were removed by centrifugation and the supernatants assayed for PLC activity on NPPC. Effect of heating on PLC activity. Concentrated culture supernatants from Pc224c were heated in a boiling water bath for 1 rain, immediately cooled and analyzed for PLC activity alongside unheated samples. Type I PLC from C. perfringens was used as a positive control for the assay and was also heated as a control for PLC heat lability.
Results and Discussion PLC production, hemolysis, and iecithinase reactions. The specific PLC activity of unconcentrated supernatants from cultures grown in MMST was at least 38 times that from cultures grown in any of the other media tested (Table 1). Both MMST and TMM are glucose-minimal salts media with 0.1% Tryptose, and the organism grows equally well in either. Tryptic soy broth is a complex medium commonly used for culturing a variety of microorganisms, and peptone medium was used by Vasil et al. for optimal hemolytic PLC production by the P. cepacia isolates they studied [14]. Our strain, when grown in MMST, produced over 100 times the specific PLC activity of cultures grown in peptone (Table 1). We screened 30 strains of P. cepacia from both clinical and environmental sources, and all produced PLC activity when cultured in MMST, but not in TMM (Table 2).
a Cultures were grown with aeration to late log phase at 37°C. Unconcentrated cell-free culture supernatants were assayed for PLC and total protein as described in Materials and Methods. b Specific activity = absorbance units at 410 nm/p.g total protein. The positive control was 8 ~g commercially prepared Type I PLC from C. perfringens. The negative control was 50 mM Tris buffer, pH 7.6.
Relatively small amounts of activity were produced by some, though not all, of the strains when grown in peptone (Table 2). Although PLC is often referred to as "lecithinase" (in spite of the fact that phospholipases of the C type may attack phospholipids other than lecithin (phosphatidylcholine) [2], we found no apparent correlation between PLC production and either hemolytic or lecithinase activity by P. cepacia (Table 2); Pc224c, in fact, produced neither. Nakazawa et al. likewise noted no association between hemolysis and lecithinase activity [9], and Vasil et al. found no association between hemolysis and PLC production [14], but in order to rule out the possibility that the lack of hemolytic activity by Pc224c was due to suppression of PLC production on blood agar, the organism was grown in MMST and the culture supernatant was examined for PLC activity on NPPC, for hemolysis on blood agar and for lecithinase activity on egg yolk agar. Supernatants from Pc224c contained no detectable hemolytic activity on any of the erythrocytes tested, and no lecithinase activity, even when concentrated. It appears, therefore, that the activity produced by P. cepacia Pc224c in MMST is due to a nonhemolytic PLC that is not a lecithinase.
Kinetics of PLC production and effects of inorganic phosphate concentration. The kinetics of production indicated that the enzyme was released into the supernatants of late log phase cultures and accumulated as the cultures continued into stationary phase (SA = 1.2) (Fig. 1). Inorganic phosphate (10 mM K2HPO4) inhibited PLC production in MMST when added at time zero (SA = 0.04) and interrupted it
P. cepacia
M . K . L o n o n and A. Morris Hooke: N o n h e m o l y t i c Phospholipase C of
141
T a b l e 2. P L C , h e m o l y t i c , a n d e g g y o l k a g a r r e a c t i o n s b y 30 strains of
Pseudomonas cepacia
E
0.5
o O
0.4
E r-
PLC Hemolysis b
c
Strain
TMM
MMST
Peptone
SRBC
Lecithinase c
Pc43-3 Pc61g Pc224c Pc275c
-
+ + + +
-
+
-
Pc445kk Pc535a Pc554d
-
+ + +
+ -
+ -
-
Pc704cc Pc706k
-
+ +
+ -
+
+ -
Pc710m Pc715j 90ee
-
+ + +
+ + -
-
+ + -
99bb 527i
-
+ +
+ +
-
+
22-20 22-22 48-36
-
+ + +
+ -
+ -
-
12544 34192 H1729-2
-
+ + +
+ -
-
+ +
K19-2 K30-6 K33-1
-
+ + +
+ -
-
+ + +
T a b l e 3. T h e e f f e c t o f i n o r g a n i c p h o s p h a t e c o n c e n t r a t i o n o n t h e p r o d u c t i o n o f P L C b y P. cepacia P c 2 2 4 c a
K41-6 K43-3 K53-2
-
+ + +
-
-
+ + +
K2HPO 4 concentration
K56-2 K63-2 K634 R5231-2
-
+ + + +
+ + -
+
+ + + -
D e t e r m i n e d q u a l i t a t i v e l y b y g r o w i n g t h e s t r a i n s in 100-txl cult u r e s in a 96-well m i c r o t i t e r p l a t e f o r 4 h a n d t h e n a d d i n g 100/xl N P P C r e a g e n t . P l a t e s w e r e i n c u b a t e d f o r 1 h at 37°C a n d i n s p e c t e d for the d e v e l o p m e n t of a yellow color. b T h e s t r a i n s w e r e g r o w n o n b l o o d a g a r c o n t a i n i n g 5% s h e e p r e d b l o o d cells ( S R B C ) . c T h e s t r a i n s w e r e g r o w n o n e g g y o l k a g a r f o r 72 h at 37°C, a n d the agar surrounding the colonies was inspected for opacity.
when added after enzyme production had begun (SA = 0.1). Adding tryptose in 20 times the normal amount resulted in complete suppression of PLC production, probably because that much tryptose contains sufficient Pi to inhibit production [13]. These results are similar to those reported by Stinson and Hayden, who performed comparable experiments with P. aeruginosa [13]. We also examined the effects of Pi in medium where it was the sole phosphate source. Increasing the Pi concentration from 0.6 to 4.8 mM in ACDM resulted in a reduction in specific PLC activity
c~ 6 v
Y
0.1
0.3
0.2
~." O
z-
0.1
0 I...
co Q.
c.~
0.01 0
I
2
3
4
5
6
T
I
7
8
0.0
TTme (hours) Fig. 1. E f f e c t o f Pi o n t h e k i n e t i c s o f P L C p r o d u c t i o n b y Pseudomonas cepacia P c 2 2 4 c . G r o w t h in M M S T w a s m o n i t o r e d as a b s o r b a n c e at 600 n m (©). S a m p l e s o f t h e d i f f e r e n t c u l t u r e s u p e r n a r a n t s w e r e a s s a y e d f o r P L C o n N P P C a n d p l o t t e d as a b s o r b a n c e at 410 n m . C u l t u r e s w e r e g r o w n in M M S T ( 0 ) ; M M S T p l u s 10 m M K z H P O 4 a d d e d at t i m e z e r o ([3); M M S T plus 10 m M K z H P O 4 a d d e d at 4 h ( A ) ; a n d M M S T plus 2 % t r y p t o s e (11).
Absorbance at 410 n m
Specific a c t i v i t y
4.8 m M 2.4 m M
0.44 0.53
0.037 0.041
1.2 m M 0.6 m M Positive control Negative control
0.74 0.88 0.11 0.00
0.048 0.065 0.014
C u l t u r e s w e r e g r o w n w i t h a e r a t i o n in A C D M s u p p l e m e n t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f K z H P O 4 o v e r n i g h t at 37°C. Cellf r e e c u l t u r e s u p e r n a t a n t s w e r e s u b j e c t e d to 0 - 3 0 % S A S p r e c i p i t a tion, d i a l y z e d , a n d a s s a y e d f o r t o t a l p r o t e i n a n d P L C a c t i v i t y . The positive control was 8 ~g commercially prepared Type I PLC f r o m C. perfringens. T h e n e g a t i v e c o n t r o l w a s 50 m M T r i s b u f f e r , p H 7.6.
from
0.065
(Table
to
0.037,
a
decrease
of
about
40%
3).
Effects of aeration, mechanical agitation, and heat. Table
4 shows
aeration appeared was or by
on
the effects PLC
to
enhance
provided bubbling
Shaking half the
by
by
PLC
agitation
air through
PLC
agitation
Pc224c.
production,
mechanical
in a flask without specific
of mechanical
production
the
culture
baffles
activity
(SA
whether (SA (SA
resulted =
and
Aeration
= =
it 1.9)
2.1).
in about
1.1). Agitation
142
CURRENT MICROBIOLOGYVol. 23 (1991)
Table 4. Effects of mechanical agitation and aeration on the production of PLC by P. cepacia Pc224c Treatment" Shaken in baffled flask Shaken in unbaffled flask Aerated by bubbling Shaken in evacuated baffled flask Static incubation
Specific activity 1.9 1.1 2.1 0.8 0.6
Cultures were grown in 200 ml MMST in 1-L flasks at 37°C; those cultures which were subjected to shaking were grown in an incubator shaker set at 200 rpm. Those grown without shaking were incubated in an environmental chamber. All were incubated until the cultures reached comparable optical densities (A600 of 1.5), and all but the static culture grew at approximately the same rate.
a l o n e , in a s e a l e d flask w i t h m o s t o f t h e air r e m o v e d , r e s u l t e d in s u b s t a n t i a l l y r e d u c e d P L C p r o d u c t i o n ( S A = 0.8). I n c u b a t i n g t h e c u l t u r e s t a t i c a l l y a l s o r e s u l t e d in l o w e r specific P L C a c t i v i t y ( S A = 0.6). T h e specific P L C a c t i v i t y o f Pc224c c o n c e n t r a t e d s u p e r n a t a n t s d e c r e a s e d f r o m 1.7 to 0 a f t e r h e a t i n g in a b o i l i n g w a t e r b a t h f o r 1 rain, as d i d t h a t o f the commercially prepared PLC (we-heated SA = 0.02).
Relationship to other lipolytic e n z y m e s . Vasil et al. also reported PLC activity from some nonhemolytic s t r a i n s o f P . c e p a c i a [14], b u t it is u n c l e a r w h e t h e r it is t h e s a m e as t h e a c t i v i t y r e p o r t e d in this p a p e r . The PLC activity we describe here does not appear to b e d u e to t h e 25 K d l i p a s e r e p o r t e d b y L o n o n et al., b e c a u s e c o n c e n t r a t e d s u p e r n a t a n t s f r o m s t r a i n 90ee, w h i c h c o n t a i n e d l a r g e a m o u n t s o f l i p a s e , d e m o n s t r a t e d little o r n o P L C a c t i v i t y on N P P C [6]. T h e k i n e t i c s o f p r o d u c t i o n a n d r e a c t i o n to h e a t i n g i n d i c a t e t h a t it is also d i f f e r e n t f r o m the egg y o l k r e a c t i v e e n z y m e a c t i v i t y m e a s u r e d in t h e m i c r o a s s a y w e r e c e n t l y d e s c r i b e d [7]. I t r e s e m b l e s t h e P L C p r o d u c e d b y o t h e r G r a m - n e g a t i v e o r g a n i s m s in t h a t it is e x t r a c e l l u l a r , is s e c r e t e d in l a t e log a n d s t a t i o n a r y p h a s e s , a n d is h e a t - l a b i l e [2, 3, I1]. It d i f f e r s from the PLC (heat-labile hemolysin) produced by P . a e r u g i n o s a [2, 3] in t h a t it is n o t h e m o l y t i c , n o r d o e s it a p p e a r t o h a v e l e c i t h i n a s e a c t i v i t y . The regulation of hemolysin and PLC product i o n b y P . c e p a c i a is n o t w e l l u n d e r s t o o d a n d m a y i n v o l v e m u l t i p l e g e n e s [14]. C l e a r l y , t h e e n z y m e w e d e s c r i b e h e r e is s u b j e c t to p h o s p h a t e r e g u l a t i o n , b u t there a r e a p p a r e n t l y o t h e r m e d i a - r e l a t e d f a c t o r s inv o l v e d as well. W e a r e c u r r e n t l y in the p r o c e s s o f purifying the nonhemolytic PLC from P. cepacia
Pc224c in o r d e r to c h a r a c t e r i z e t h e e n z y m e f u r t h e r a n d to initiate s t u d i e s o n its r o l e as a p o t e n t i a l v i r u l e n c e f a c t o r in P . c e p a c i a p u l m o n a r y i n f e c t i o n s .
ACKNOWLEDGMENTS The authors thank Patricia C.M. O'Brien for her critical reading of the manuscript. Miriam K. Lonon is the recipient of an Ohio Board of Regents Academic Challenge Post-Doctoral Fellowship. This work was also supported by a grant from the Cystic Fibrosis Foundation to M.K.L.
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