Journal of General Microbiology (1988), 134, 489-498. Printed in Great Britain
489
A Phospholipase C from the Dallas 1E Strain of Legionellapneumophila
Serogroup 5: Purification and Characterization of Conditions for Optimal
Activity with an Artificial Substrate
By WILLIAM B. B A I N E
Departments of Internal Medicine and Microbiology, Southwestern Medical School and
Southwestern Graduate School of Biomedical Sciences, The University of Texas Health Science
Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA
(Received 8 October 1987)
Phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5 was purified
from buffered yeast extract culture supernate by ion-exchange chromatography followed by
fractionation by manganous chloride and ammonium sulphate precipitation steps. Enzyme
activity was assayed by hydrolysis of p-nitrophenylphosphorylcholine and confirmed by release
labelled in the methyl groups
of radioactivity from tritiated L-a-dipalmitoylphosphatidylcholine
of choline. After SDS-PAGE, the purified preparation yielded a single band upon Coomassieblue staining. This protein migrated with an apparent M , of 50000-54000. Phospholipase C
activity was maximal at pH 28.4 and was enhanced in the presence of sorbitol and of several
nonionic detergents but was eliminated by SDS. EDTA, Cu2+,Fe2+and Zn2+inhibited enzyme
activity, whereas Ba2+, Ca2+, Co2+, Mg2+ and Mn2+ restored activity to EDTA-treated
material. No haemolytic activity was demonstrated with the purified enzyme.
INTRODUCTION
Phospholipase C (lecithinase, EC 3.1 .4.3) hydrolyses phosphatidylcholine (lecithin) to 1,2diacylglycerol and phosphorylcholine. This enzyme is recognized to be a virulence factor of
several bacterial pathogens, including Clostridiumperfringens (Mollby, 1978) and Pseudornonas
aeruginosa (Berka & Vasil, 1982).
Certain species of Legionella also produce phospholipase C, and several potential roles exist
for this enzyme in the pathogenesis of legionellosis (Baine et al., 1979; Baine, 1985). Hydrolysis
of phosphatidylcholine, which is a major component of alveolar surfactant (Klaus et al., 1961),
could impair pulmonary gas exchange. Phosphatidylcholine is also an important constituent of
eukaryotic membranes (Alberts et al., 1983). The cytolytic action of phospholipase C from C.
perfringens and other bacteria is well established (Mollby, 19719, and Legionella phospholipase C
might injure both lung tissue and inflammatory cells in the host. Lesser alterations of host-cell
membranes by phospholipase C may enhance bacterial adherence to target cells (Malmqvist et
al., 1984). Within phagocytes perturbation of phagosome membranes by hydrolysis of
phospholipid might contribute to the failure of phagosome-lysosome fusion that is observed in
mononuclear cells ingesting live L. pneumophila (Horwitz, 1983) and that presumably permits
unimpeded multiplication of the bacteria within the phagocyte.
Phospholipase C from Legionella might also affect the host by liberating active reaction
products. For example, phospholipase C from C. perfringens induces ornithine decarboxylase in
lymphocytes, apparently through the mediation of diacylglycerol (Otani et af., 1984). Since
diacylglycerol activates protein kinase C, an enzyme with broad regulatory functions
(Nishizuka, 1986; Waite, 1985), release of diacylglycerol by enzymic degradation of
phospholipid could have multiple pathophysiological consequences.
Abbreviation: N PPC, pnitrophenylphosphorylcholine.
0001-4236
0 1988 SGM
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W . B. BAINE
The Dallas IE strain, isolated in 1978 from a cooling tower in Dallas, Texas, is the type strain
for serogroup 5 of L. pneumophila (England et ul., 1980), although recent taxonomic studies
indicate that Dallas 1E may more appropriately be regarded as belonging to a separate species of
Legionella (Selander et al., 1985). Phospholipase C activity has been demonstrated in intact cells
of Dallas 1E and other strains (Baine, 1985), but purification of this enzyme from Legionella has
not previously been reported. Dallas IE is more haemolytic than the type strain of L.
pneumophilu, Philadelphia 1, when grown on agar that incorporates guinea-pig red cells (Baine,
1985), which are particularly rich in phosphatidylcholine (Turner et af., 1958). Accordingly,
Dallas 1E was chosen as the first strain of L. pneumophila from which to attempt isolation of
phospholipase C .
METHODS
Bacterial strain, media andgrowth conditions. A subculture of the Dallas 1E strain obtained from the Centers for
Disease Control, Atlanta, Ga, USA, was maintained by serial passage on buffered charcoal/yeast extract agar
(Feeley et al., 1979; Pasculle et al., 1980). Purity of cultures was monitored by typical growth on buffered
charcoal/yeast extract agar and failure to grow on buffered charcoal/yeast extract lacking a supplement of Lcysteine. HC1. Cultures in 25 ml portions of buffered yeast extract broth (Baine, 1985) were incubated at 21 "C in a
rotary water bath at 180 r.p.m. A 10 ml volume of an 18 h broth culture was used to inoculate a Microferm
Fermentor (New Brunswick) containing 8.0 1 buffered yeast extract broth in which the yeast extract content was
reduced to 2.5 g 1-I to minimize foaming. The fermenter culture was incubated at 37 "C with agitation at 200
r.p.m. and aeration at 1.0 1 min-'. After 48 h the stationary-phase culture was brought to 3.0 mM with NaN,, and
the cells were sedimented by centrifugation (4900 g , 30 min).
Phospholipase C assay. Phospholipase C activity was assayed by release of p-nitrophenol from pnitrophenylphosphorylcholine (NPPC) (Kurioka & Matsuda, 1976). Assay mixtures containing an appropriate
volume of test sample, 5-0 m~-CaCl,,5.0 mM-MnCl,, 3.0 mM-NaN,, 2.5 mM-NPPC (Sigma), and 0.50% (v/v)
Triton X-100 in 50 mM-HEPES (pH 7.5) at a final volume of 200 pl were placed in a water bath at 37 "C. The assay
conditions were chosen on the basis of results of preliminary studies with crude preparations. Substrate control
N,
buffer) for the test sample,
tubes substituting 50 mM-HEPES (pH 7.5) containing 3 ~ M - N ~ (HEPES/NaN3
sample control tubes substituting buffer for NPPC, and a blank tube substituting buffer for both the sample and
NPPC were processed in parallel. After incubation, the mixtures were diluted with water or buffer and the A410
measured. Net A410 attributable to release of p-nitrophenol was determined from the reading in the diluted
reaction mixture by subtracting the readings in the diluted sample and substrate control mixtures. The
corresponding concentration of pnitrophenol was calculated from a regression equation (HP-11C, HewlettPackard) for a standard curve of A410 constructed using p-nitrophenol solutions of known molarity. This
concentration was multiplied by the appropriate factor to calculate the molarity of p-nitrophenol present in the
undiluted reaction mixture.
Enzyme purification. All but a 20 ml sample of 7.8 1 of unfiltered culture supernate was applied onto a 5.0 cmdiameter column containing 120 ml DEAE-Sephadex (Pharmacia) previously equilibrated (Himmelhoch, 1971)at
4 "C with HEPES/NaN3 buffer. Eluate was collected by gravity flow at this and subsequent stages of ion-exchange
chromatography. The column bed was then washed with 100 ml HEPES/NaN, buffer and then with 50 ml0.1 MNaCl in the same buffer. Adsorbed material was then eluted with a linear gradient (BRL Gradient Former) from
0.1 to 0.3 M-NaCl in 180 ml HEPES/NaN, buffer followed by 130 mlO.3 M-NaC1 in the same buffer. The NaCl
concentration in eluate fractions (5.0 ml) was monitored with a CDM 2e conductivity meter (Radiometer,
Copenhagen).
Enzyme activity in each fraction was assayed as described above using 100 p1 samples of eluate with incubation
for 18 h. Sample control tubes were omitted since absorbance by the diluted eluate fractions was considered to be
negligible. Fractions encompassing the peak of activity were pooled, and 91 of the resulting 101 ml were dialysed
l ~HEPES/NaN, buffer (Heppel, 1955) in tubing (Spectrum Medical
at 4 "C for 2 h against 10 vols 52 m ~ - M n Cin
Industries) with a cutoff for proteins of M,12000-14000. Precipitated material was sedimented by centrifugation
(3600 g, 30 min), and 82 of the resultant 92 ml of supernate were brought to 40% saturation with ammonium
sulphate (Green & Hughes, 1955) in HEPES/NaN3 buffer. After 5 min at 4 "C, the precipitate was sedimented by
centrifugation as above, and 121 of the resultant 131 ml of supernate were dialysed at 4 "C against three changes
(2 h, 2 h, 18 h) of 10 vols HEPES/NaN, buffer.
Phospholipase C in 150 pl volumes of samples from successive stages of enzyme purification was assayed by
hydrolysis of NPPC as described above except that the manganous chloride supernate was assayed at 50.5 mMMnCI2.
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Phospholipase C from L. pneumophila
49 1
Protein concentrations were determined by the Bio-Rad Protein Assay microassay procedure, using bovine yglobulin as the standard.
SDS-PAGE. Samples were concentrated by vacuum dialysis against 50 mM-Tris/HCl (pH 6.8) in a collodion bag
apparatus (Schleicher & Schuell) and brought to 2% (w/v) SDS, 5 % (v/v) 2-mercaptoethanol, 10% (v/v) glycerol
and 0402% (w/v) bromophenol blue in 56 mM-Tris/HCl (pH 6.8). After heating at 100 "C for 3 min and
centrifugation (1 2000 g) to remove insoluble precipitates, the samples were subjected to SDS-PAGE (Hames,
1981) in 25 mM-Tris, 192 mM-glycine (pH 8.3) with a stacking gel of 3.8% (w/v) acrylamide in 125 mM-Tris/HCl
(pH 6-8) and a linear-gradient resolving gel of lO-l5% acrylamide in 375 mM-Tris/HCI (pH 8.8). Stacking was
achieved at 120 V with resolution at 200 V. Haemophilusinfluenzae type b major outer-membrane protein (39000)
(Gulig & Hansen, 1985) and proteins from M, kits (Bio-Rad), including myosin (200000), P-galactosidase
(1 16250), phosphorylase b (92500), BSA (66200), ovalbumin (45000), carbonic anhydrase (31 000), soybean
trypsin inhibitor (21 500) and lysozyme (14400) were used as M,standards. Gels were stained with Coomassie blue
R250 (Hames, 1981) or with a silver stain (Bio-Rad).
Confirmationofphospholipase C activity. Hydrolysis of authentic phosphatidylcholine, as distinct from NPPC, by
the putative phospholipase C was demonstrated by release of 3H-label from lecithin-bearing tritiated choline, as
previously described (Baine, 1985; Grossman et al., 1974). Dispersal of L-a-dipalmitoylphosphatidylcholinein
HEPES/NaN3buffer, 7.0 mM-CaCI,, 0.32% (v/v) Triton X-100 was accomplished by vortexing for 2 min followed
by sonication for 10 min using a Sonicator cell disrupter model W 185 F (Heat Systems-Ultrasonics) with the
[specific activity 27 Ci mmol-'
power setting at 9. ~-a-Dipalmitoyl-[choline-merhyl-~H]phosphatidylcholine
(1 TBq mmol-I), New England Nuclear] was then added to the unlabelled lecithin and dispersed by a second
sequence of vortexing and sonication. A 25 ml sample of the purified enzyme preparation was concentrated 60fold by vacuum dialysis against HEPES/NaN3 buffer. The final reaction mixtures contained 75 pg protein,
with 50 nCi ~-a-dipalmitoyl-[choline-meth~~-~H]phosphatidylcholine,
1.O mM-L-a-dipalmitoylphosphatidylcholine
50 mM-HEPES, 3-5mM-CaC1,, 3.0 mM-NaN, and 0.16% (v/v) Triton X-100 in a final volume of 200 p1. Substrate
control tubes contained HEPES/NaN3 buffer in place of enzyme. After 17 h incubation at 37 "C, the reaction
mixtures were acidified with 50 pl 6.84% (w/v) HCIO4 and diluted with 250 p1 distilled water. Unhydrolysed
lecithin was removed from the mixture by serial extraction with 2 ml volumes of petroleum ether (boiling range
37-58 "C), water-saturated diethyl ether and petroleum ether. Portions (200 PI) of the extracted aqueous phases
were transferred to vials containing 10 ml Handifluor (Mallinckrodt) for liquid scintillation counting (Minaxi
TRI-CARB 4000 series, Packard). Duplicate samples from substrate control tubes were also processed in parallel
omitting the extractions with hydrophobic solvents and yielded a mean and range of 5769 k 89 c.p.m. after
correcting for background counts. Serial extraction with petroleum ether, diethyl ether and petroleum ether
removed a mean and range of 56.1 _+ 6.8% of the label from substrate control tubes, suggesting that the lipid
solvents were only partially successful in extracting phosphatidylcholine from the aqueous suspension or that some
of the label was present on polar decomposition products of the tritiated lecithin. The proportion of tritiated
phosphorylcholine released into the acidified aqueous phase was calculated as follows after correcting for
background counts: (c.p.m. in extracted aqueous phase of test - c.p.m. in extracted aqueous phase of substrate
control)/(c.p.m. in unextracted substrate control).
Eflect of Substrateconcentration.Enzyme activity in the purified preparation was assayed by hydrolysis of NPPC
as described above, except that the substrate concentration varied from 5 to 100 mM-NPPC. The values for - K,,,
and V,,, were taken as the slope and ordinate intercept, respectively, of the linear regression of the data (HP-11C,
Hewlett-Packard) on a Woolf-Augustinsson-Hofstee plot, which is not strongly aq'ected by small errors in the
measurement of V at low substrate concentration (Segel, 1975). Reaction mixtures contained 1-1pg protein and
were incubated for 30, 61 and 170 min in three separate experiments.
pH optimum. Enzyme activity in the purified preparation as a function of pH was determined by hydrolysis of
NPPC in the standard assay, except that 5-0 mM-NPPC was used as substrate and the reaction was done in 0.3 MHEPES over a range from pH 6.51 to pH 8-46 with corresponding sample and substrate control tubes. The
reaction mixtures contained 1.6 pg protein and were incubated for 60 min. Measurements of p-nitrophenol
absorbance were made at each pH to give appropriate standard curves.
Diuulent cation requirements. Dialysed ammonium sulphate supernate was brought to 1.O mM-EDTA and then
dialysed at 4 "C over 6 h against three changes of 10 vols HEPES/NaN3 buffer. Phospholipase C activity of the
chelated preparation was determined by hydrolysis of NPPC as described for the standard assay, except that
5.0 mM-NPPC was used as substrate, and the usual combination of CaCI, and MnCI, was replaced by incremental
concentrations of the chloride salts of eight different divalent cations. The reaction mixtures contained 1.9 pg
protein and were incubated for 65 min.
Eflects of detergents. The effects of various detergents on enzyme activity in the purified preparation were
determined by hydrolysis of NPPC in 50 mM-HEPES (pH 7-5) as described for the standard assay, except that
10 mM-NPPC was used as substrate and 0.50% (v/v) Triton X-100 was omitted or replaced by one of six different
detergents. These were present at only 0.25% (w/v) to minimize the risk that differences in the effects of the
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492
W. B. BAINE
detergents might be obscured if they were present in excess. The reaction mixtures contained 1.7 pg protein and
were incubated for 80 min.
Eflects ofsugar alcohols. The effects of 40% (w/w) glycerol and of 33% (w/w) sorbitol on enzyme activity in the
purified preparation were determined by hydrolysis of NPCC as described for the standard assay after addition of
the appropriate sugar alcohol. The reaction mixtures contained 1.1 pg protein and were incubated for 170 min.
Testfor haemolyric activity. Petri dishes containing 20 ml dog-blood agar were prepared with 5% (v/v) sterile
defibrinated dog blood and 3.9% (w/v) tryptose agar (Difco) (Baine et al., 1979). Haemolytic activity in samples
from successive stages of enzyme purification was sought by inoculating 150 1-11volumes of each preparation into
wells 9 mm in diameter that had been bored into the agar. The plate was incubated for 18 h at 37 "C and then for
4 h at 4 "C with periodic inspection for zones of haemolysis around the wells.
HEPES/NaN3 buffer was brought to 86mM-NaC1, and a sample of the purified enzyme preparation was
brought to 53 mM-NaC1 to render the solutions isotonic (267 mOsm kg-l and 262 mOsm kg-*, respectively, by
freezing-point depression). Serial twofold dilutions of the purified enzyme preparation were made in the isotonic
buffer in a mictotitre plate. Duplicate rows of wells were supplemented with Tween 80, with or without CaCI2,and
with or without MnCl,. After addition of defibrinated dog blood, each well contained the purified preparation at
dilutions of 1 in 2 through 1 in 2048 and 2.5% (v/v) dog red cells and, as appropriate, 0.7% (v/v) Tween 80,
0.025 mM-CaC1, and 0.025 mM-MnC1, in isotonic HEPES/NaN, buffer in a final volume of 100 1-11. Control wells
contained isotonic buffer in place of the purified enzyme. The plate was incubated for 60 min at 37 "C and then for
18 h at 4 "C and periodically inspected for haemolysis.
RESULTS
Enzyme puriJication. Maximal NPPC-hydrolysing enzyme activity eluted from the DEAESephadex column in the range of 6.9-11.1 mS (110-207 mM-NaC1) (Fig. 1). Assessment of
protein concentration in the fractions by measurements of A 2 8 0 was confounded by absorbance
by a brown pigment (Baine & Rasheed, 1979; Baine et al., 1978) in the supernate.
The precision of the NPPC assay of phospholipase C activity in unconcentrated culture
10
1 28
9
- 24
8
7
-20
6
-16-E
+
5 4 -
4
2
5;
.-x
>
c)
- 12 2
.r(
c,
6
-8
2
1
A
30
40
50
60
Fraction no.
Fig. 1. Elution of L. pneumophila (Dallas 1E) NPPC-hydrolysing activity from DEAE-Sephadex
column by 100-300 mM-NaC1 gradient in 50 mM-HEPES (pH 7-50), 3 ~ M - N ~ under
N , gravity flow
with monitoring of A z s o( 0 )and conductivity (I).
Enzyme activity was measured asp-nitrophenol (net
A , , o ) (A)released from p-nitrophenylphosphorylcholine by 100 p1 samples of each 5 ml fraction.
Fractions 24-45 were pooled for manganous chloride fractionation.
10
20
-4
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Phospholipase C from L. pneumophila
493
Table 1. Purijication of phospholipase C from culture supernate of the Dallas IE strain of
L. pneumophila serogroup 5
Volume Total protein Total activity
Specific activity
Recoverytt
(U)*$
[U (mg protein)-']*$
(%>
(ml)
(ms)
Preparation
Culture supernate
Pooled DEAE-Sephadex fraction
nos 25-45
52 mM-manganous chloride
supernate
Dialysed 40% saturation
ammonium sulphate supernate
7800
80
82 & 17
1.0 f 0.2
100
101
4.6
74 & 33
16 f 7
91 k 4 0
92
1.9
98+ 2
51 f 1
119f 3
198
2.5
72+ 4
29 & 1
88+ 5
* One unit is defined as 1 nmol p-nitrophenol released by hydrolysis of NPPC min-l at 37 "C.
t Values represent the proportion of total activity in the initial supernate that was present in the volume of
material recovered at each step of purification. No adjustment is made for the samples that were retained at
each step and not further processed, as detailed in Methods.
$ Values are shown as means and ranges of duplicate assays.
supernate was limited by the prolonged period required to demonstrate significant hydrolysis of
this substrate by such a dilute solution of enzyme. However, the specific activity was increased
approximately 50-fold by ion-exchange chromatography and manganous chloride fractionation
of the supernate (Table 1). Ammonium sulphate fractionation increased the purity of the
preparation, as determined by SDS-PAGE (Fig. 2), but actually resulted in some loss of specific
activity. Assay of the supernate obtained after equilibration with 52 m-MnC1, was done at a
relatively high concentration of Mn2+, so the paradoxical decline in specific activity after
ammonium sulphate fractionation could have been an artifact of differing concentrations of
Mn2+in the assay mixtures. However, preliminary experiments (data not shown) indicated that
whereas enzyme activity could be removed from manganous-chloride-treated DEAE-Sephadex
eluates by ammonium sulphate at 60% saturation, recovery of enzyme activity from the resulting
precipitate was poor.
Significant enzyme activity was present in DEAE-Sephadex eluate fractions that were not
pooled for further processing (Fig. 1). Artifacts possibly contributing to the high apparent
recovery of enzyme during purification could include imprecision in the assay of weak
phospholipase C activity in unconcentrated supernate as well as removal of enzyme inhibitors
and alteration in relevant ion concentrations during purification.
After SDS-PAGE crude culture supernate stained with Coomassie blue revealed a faint band
with an M , of 50000 and a major band with an M , of 38000 (Fig. 2). After DEAE-Sephadex ionexchange chromatography, the 50 kDa band was the dominant protein by Coomassie-blue
staining. Further purification by treatment with manganous chloride and ammonium sulphate
yielded one visible band only by Coomassie-blue staining. In two SDS-PAGE gels this protein
migrated with an apparent M , of 50000 and 54000.
Hydrolysis of authentic phosphatidylcholine. Purified enzyme released a net of 33.7 %
[(4475 c.p.m. - 2533 c.p.m.)/(5769 c.p.m.)] & 1.0% (mean range) of the total amount of label
present in tritiated lecithin into the aqueous phase as presumed phosphorylcholine.
Phospholipase C kinetics. Phospholipase C activity increased with increasing substrate
concentration over the range from 5 to 100 mM-NPPC. The enzyme showed a low affinity for
NPPC, with an apparent K , of 35, 51 and 61 mM for this artificial substrate in three separate
experiments. The corresponding calculated values of V,,, for hydrolysis of NPPC were 4.3, 5.0
and 4.6 pmol l-l min-l at 37 "C.
pH optimumfor enzyme activity. Phospholipase C activity increased with increasing alkalinity
from pH 6.5 1 through pH 8.46 (Table 2). Accurate measurement of enzyme activity at lower pH
was impeded by inaccuracy in measuring very low concentrations ofp-nitrophenol at acid pH, at
which the molar extinction coefficient of this compound is low.
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494
W . B . BAINE
Fig. 2. SDS-PAGE of phospholipase C preparations from L. pneumuphilu (Dallas 1E) at different
stages of purification on 10-1 5 % linear polyacrylamide gradient gel stained with Coomassie blue. Lane
A, crude culture supernate (31 pg protein); lanes B (34 pg) and E (68 pg), pooled DEAE-Sephadex
eluate fractions 25-45; lanes C (16 pg) and G (31 pg), 52 mM-MnC1, precipitation supernate; lanes D
(19 pg) and I (38 pg), supernate of 40% saturation ammonium sulphate precipitation; lanes F and H,
blanks containing 2% (w/v) SDS, 5 % (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and 0.002% (w/v)
bromophenol blue in 62.5 mM-Tris/HCl (pH6.8). The positions of M, standards are noted.
Divalent cation requirements for enzyme activity. Addition of 1-10 mM-Ba2+,Ca2+,Mn2+or
Mg2+ to EDTA-treated purified enzyme produced a dose-dependent enhancement of
phospholipase C activity (Fig. 3). The more strongly oxidizing Zn2+,Cu2+and Fe2+(De Vries,
1961)at concentrations as low as 0.01-1 mM inhibited the enzyme. Although the electrochemical
potential of Co2+makes this ion a stronger oxidizer than Zn2+or Fe2+,Co2+was comparable to
Mg2+ in enhancing the activity of the chelated enzyme preparation.
Eflects of detergents upon enzyme activity. Little effect on enzyme activity was observed with
0.25% (w/v) Nonidet P-40, but the other nonionic detergents that were tested significantly
enhanced hydrolysis of NPPC (Table 2). The polysorbate detergents Tween 20 and Tween 80
were most effective, but Triton X- 100, a polyethylene glycol p-isooctylphenyl ether, also
significantly accelerated hydrolysis of the substrate in comparison with detergent-free control
mixtures. Sodium deoxycholate, a bile salt, had little effect on enzyme activity, whereas the ionic
detergent SDS was strongly inhibitory.
EJects of sugar alcohols upon enzyme activity. Sorbitol at 33 % (w/w) enhanced enzyme activity
over sugar-alcohol-free mixtures, but no effect was oberved with 40%(w/w) glycerol (Table 2).
Screenfor haemolytic activity. No lysis of red cells was apparent in the radial haemolysis assay
in dog-blood agar. No difference between control wells and those containing the purified
enzyme was apparent in the microtitre haemolysis assay.
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Phospholipase C from L. pneumophila
495
h
e
._
3 50
41a
0
Cation concn
(MI
Fig. 3. Effects of divalent cations on activity of EDTA-treated purified phospholipase C from L.
pneumophilu (Dallas IE). One unit (U) is defined as 1 nmol p-nitrophenol released by hydrolysis of
NPPC min-' at 37 "C. Values shown are the means and ranges of duplicate assays. Ions are grouped as
Bat+ (A), Ca2+( 0 )and Zn2+( 0 ) ;Cot+ (A), Fe2+ ( 0 )and Cu2+(m); and Mn2+ (0)and Mg2+ (A).
Table 2. Egect of pH, detergents and sugar alcohols upon activity of phospholipase C from the
Dallas 1E strain of L . pneumophila serogroup 5
Specific activity *
[U (mg protein)-']
PH
6.51
6.67
6.86
7.31
7.77
8.28
8-46
Detergent
None
Tween 20
Tween 80
Triton X-100
Nonidet P-40
Sodium deoxycholate
SDS
Sugar alcohol
None
Glycerol
Sorbitol
65 + 4
74 + 2 5
110
0
106 & 8
105 + 13
140 + 5
153 + 6
*
104 k 4
304 + 3 8
263 f 3
200 f 21
168 + 6 1
134 + 9
1.8 f 7.3
53 f 1
52 f 1
80 & 9
* One unit is defined as 1 nmol p-nitrophenol released by hydrolysis of NPPC min-' at 37 "C.Values are
means and ranges of duplicate assays.
DISCUSSION
SDS-PAGE indicated that an increase in purity of the phospholipase C from the Dallas 1E
strain of L. pneumophila was achieved at each step of the sequence of ion-exchange
chromatography, manganous chloride fractionation and ammonium sulphate fractionation.
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W . B. B A I N E
Nevertheless, despite an improvement in purity, the specific activity of the preparation
apparently declined after exposure to ammonium sulphate at 40 % saturation. Plausible
explanations for this paradox include the effect of a change in Mn2+concentration on enzyme
activity as well as the possibility that the enzyme was partially inactivated by transient exposure
to ammonium sulphate (Green & Hughes, 1955).
Recovery of the enzyme from culture supernates does not exclude the possibility that there are
cell-associated stores of this protein. However outer-membrane protein profiles of the Dallas 1E
strain published by Hindahl & Iglewski (1986) did not show a prominent band at 50-54 kDa. No
analogous information is available on proteins in the periplasmic space and cytosol.
Hydrolysis of NPPC is in common use as an assay for phospholipase C , but a
phosphodiesterase might also be able to release p-nitrophenol from the same artificial substrate
(Krug & Kent, 1981). Phospholipase C activity in the purified preparation from the Dallas 1E
strain was confirmed by release of label from authentic lecithin bearing tritiated choline. This
label is presumed to have been in the form of tritiated phosphorylcholine. However, if choline
were not extracted from the acidified mixture by petroleum ether and diethyl ether, hydrolysis of
the tritiated lecithin by a phospholipase D might also have been detected by this assay.
The observed specificity and activity of a given phospholipase are conditioned by the
identities of the fatty acid moieties and of the polar group present on the substrate (Dennis,
1983; El-Sayed et al., 1985). Furthermore, in contrast to simpler systems involving enzymes
acting upon water-soluble substrates, the target of a phospholipase rnay be present as monomer
or aggregated in micelles, reverse micelles, mixed micelles, or unilamellar or multilamellar
vesicles. This variability in the physical state of a phospholipid substrate can also affect the
apparent specificity and activity of a given enzyme (Mollby, 1978; Dennis, 1983).
Substituting NPPC for native phospholipid simplifies the system by eliminating the structure
and physical state of the lecithin target as variables. The soluble substrate also permits analysis
using classical Michaelis-Menten kinetics, which are inappropriate for native phospholipids
employed at levels above their critical micellar concentrations (Waite, 1985). However, data on
the activity of an enzyme upon this artificial substrate should not be extrapolated uncritically to
enzyme-substrate interactions with native phospholipids in uivo.
The high apparent K , for NPPC of the purified phospholipase C from the Dallas 1E strain
was comparable to that (0.2 M) reported for phospholipase C from Clostridium perfringens
(Kurioka & Matsuda, 1976). El-Sayed et ai. (1985) reported that the affinity of phospholipase C
from Bacillus cereus decreased when the fatty acid chain length in the substrate was less than six
carbons. The absence of fatty acids in NPPC may have contributed to the high K , observed
with this analogue of phosphatidylcholine.
The specific activity of purified phospholipase C from the Dallas 1E strain was maximal at
alkaline pH. L. pneurnophila is a nonfermentative organism (Hoffman, 1984), and its growth in
broth cultures (Pine et al., 1979) or within the phagosomes of human monocytes (Horwitz &
Maxfield, 1984)is accompanied by an increase in the pH of its environment. Such alkalinization
should favour activity of the phospholipase C.
Various bacterial phospholipases C are metalloenzymes susceptible to inactivation by EDTA
and to enhancement or inhibition by supplementation with various divalent cations (Kurioka &
Matsuda, 1976; Mollby, 1978; Dennis, 1983). Activity of the purified enzyme from the Dallas
1E strain was also potentiated by several divalent cations and inhibited by others. Although
Zn2+ enhances the activity of phospholipase C from C . perfringens at concentrations up to
1 0 - 4 ~the
, Dallas 1E enzyme was strongly inhibited by this cation.
Hydrolysis of native lecithin by phospholipase C may be markedly affected by the presence of
certain detergents which affect the physical state of the substrate (Dennis, 1983; El-Sayed &
Roberts, 1985). Although NPPC is freely soluble in water, the activity of phospholipase C from
C. perfringens upon this lecithin analogue is enhanced by strong ionic detergents, presumably
through interactions with the enzyme instead of the substrate (Kurioka & Matsuda, 1976). In
contrast, nonionic detergents enhanced the activity of the phospholipase C of L. pneurnophila,
whereas a strong ionic detergent was inhibitory.
Kurioka & Matsuda (1976) reported enhancement of hydrolysis of NPPC by phospholipase C
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Phospholipase C from L. pneumophila
497
from C. perfringens in the presence of high concentrations of sorbitol or glycerol. Sorbitol also
enhanced the activity of the purified enzyme from the Dallas 1E organism, but no effect of
glycerol on the reaction was documented.
Although it is possible that conditions exist under which this phospholipase C might damage
erythrocyte membranes (Mollby, 1978), no evidence of ability to lyse red cells was noted.
The substrate range and antigenicity of this phospholipase C remain to be determined, as does
the degree of variability of the enzyme among strains and species of Legionella. Finally, the
extent to which phospholipase C plays a role in the pathogenesis of legionellosis remains to be
elucidated.
This work was presented in part at the Twenty-sixth Interscience Conference on Antimicrobial Agents and
Chemotherapy, held in New Orleans, Louisiana, on September 28-October 1, 1986.
I thank Laurinda Ng, Prakuzy Sebastian, Colleen Kennedy and Edward Davis for very capable technical
assistance. I am grateful to Robert Munford, Leon Eidels and Eric Hansen for good advice and to Dr Hansen for a
generous gift of H. inzuenzae type b major outer-membrane protein.
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