From the Departments of Ecologie Microbienne des
Insectes et Interactions Insecte-Pathogène (EMIP) Unité
Mixte de Recherche 1133, Institut National de la Recherche
Agronomique-Université de Montpellier II, Place Eugène
Bataillon 34095 Montpellier, France and
Plasticité et
Synapse Glutamatergique, Unité Mixte de Recherche 5102, Centre
National de la Recherche Scientifique-Université de Montpellier
II, Place Eugène Bataillon, 34095 Montpellier, France
Received for publication, October 9, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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Xenorhabdus nematophila and
Photorhabdus luminescens are two related enterobacteriaceae
studied for their use in biological control and for synthesis of
original virulence factors and new kinds of antibiotics. X. nematophila broth growth exhibits different cytotoxic activities
on insect (Spodoptera littoralis, lepidoptera) immunocytes
(hemocytes). Here we report the purification of the flhDC-dependent
cytotoxin, a 10,790-Da peptide we have called Enterobacteriaceae of the genus Xenorhabdus and
Photorhabdus are potent pathogens of various insect
species (1), some strains of which are toxic for immunocompromised
humans (2, 3). The basis of this infectivity is still poorly
understood, although Photorhabdus luminescens was shown to
have an oral insecticidal activity (4) because of entomotoxic proteins
(5, 6). Other insecticidal toxins active after injection are also
produced by P. luminescens (7). Genes coding for similar
entomotoxins were cloned in Xenorhabdus nematophila (8), and
various cytotoxic factors were identified in bacterial broth growth of
this species (9, 10). Some of these factors are cytotoxic in
vitro for insect immunocompetent cells and also have hemolytic
activity on mammal red blood cells. Interestingly, all these cytotoxic and hemolytic activities are absent in P. luminescens broth
growth (10), suggesting differences in the mode of virulence of these two related entomopathogenic bacteria species. We know that these toxins have very little homology with known sequences and represent a
new class of toxins (8). The first aim of this work was to study the
mode of action of one of these new toxins on its cellular targets. In
X. nematophila the existence of toxins active both on red
blood cells and on hemocytes allows us to study the mode of action of
these molecules on mammal cells and on insect cells. Two different
hemolytic activities were identified that appear sequentially in the
course of bacterial growth (10). The earliest hemolytic activity
(activity C1) appears in broth growth when bacteria culture reaches the
stationary phase. It lyses sheep red blood cells
(SRBC)1 but is inactive on
rabbit red blood cells (RRBC) and is heat sensitive (30 min at
60 °C). It is under the control of flhDC, the flagellar
master operon of X. nematophila (11). The second hemolytic
activity (activity C2) appears late in the stationary phase, is heat
resistant (one hour at 100 °C), active on RRBC, inactive on SRBC,
and is not under the control of flhDC. Insertional inactivation of the
flhDC gene in X. nematophila leads both to loss
of C1 activity (C2 is maintained) and to a very attenuated virulence phenotype.
Because X. nematophila septicemia arises in the insect body,
it is obvious that this bacteria is able to escape defense reactions and especially phagocytosis. The means by which entomopathogenic bacteria escape the defense reactions of insects is totally unknown. Hemocytes, the free cells in hemolymph, are the major immunocompetent cells in insects. Phagocytosis is mainly achieved by macrophage-like cells that belong to the morphotype "granular hemocyte 1" (GH1) (12). As GH1 are one of the targets of the cytotoxic activities evidenced in X. nematophila broth growth (10), these
activities appear to be good candidates for supporting, at least in
part, the immunosuppressive effect. In the present work we report the purification of the flhDC-controlled lysin. The mode of action of this
lysin was studied on SRBC and on insect hemocytes. We show that insect
macrophages (GH1) were the most sensitive hemocytes to the lysin and
that this hemolysin was not recycled to react with multiple target
cells but works as a porin. Finally, the swollen appearance of
hemocytes incubated with flhDC-controlled lysin prompted us to check
for activity of this lysin on cell membrane permeability. We provide
evidence that monovalent cation channels and larger pores are opened in
the plasma membrane of the insect macrophages, leading to plasma
membrane depolarization and cell death through colloid-osmotic lysis
independent of Ca2+ movements.
Bacterial Strain, Production and Isolation of
Hemolysin--
X. nematophila (strain F1, phase variant I,
laboratory collection) were grown in Luria Bertani broth at 28 °C.
In these conditions the maximum production of
flhD-dependent cytotoxic activity (C1) (10) was reached in
20-h-old cultures. A purification of the factor responsible for this
activity was achieved from this C1 culture supernatant. Broth growth
was centrifuged (30 min at 12,000 × g). The
supernatant was immediately precipitated in 50% ammonium sulfate and
centrifuged, and the supernatant was precipitated in 70% ammonium
sulfate. The 70% pellet was dissolved and dialyzed against water,
concentrated by lyophilization, and dissolved in low ionic strength
buffer (phosphate buffer 10 mM, pH 7.8). This solution was
submitted to chromatography on HiTrap Q column (Amersham Biosciences),
and elution was achieved with NaCl. Positive fractions, as assayed by
measuring SRBC hemolysis and insect hemocyte lysis, eluted at 150 mM NaCl were pooled, lyophilized, dissolved in water, and
applied on a C18 reverse phase HPLC column. Active fractions, collected
in a single and isolated peak, were immediately lyophilized. Before
use, they were dissolved in phosphate-buffered saline (PBS) or HEPES
buffer. In this study PBS contained 1 mM CaCl2
and 2 mM MgCl2, unless otherwise stated. The
titer of the lysin solution was evaluated by its hemolytic activity on
SRBC (see below). Heat resistance was measured by incubating for 30 min
at 60 °C prior to testing for cytotoxic and hemolytic activities.
For trypsin resistance, fractions were incubated for 1 h at
37 °C with 30 units of trypsin (Sigma).
Insects, Hemocyte Monolayer Preparation, and Test for Cytotoxic
Activity--
Larvae of the common cutworm Spodoptera
littoralis (lepidoptera) were reared with a photoperiod of 12 h on artificial diet at 24 °C. Three-day-old sixth instar larvae
were selected and surface-sterilized with 70% (v/v) ethanol prior to
collection of hemolymph in test tubes filled with anticoagulant buffer
(62 mM NaCl, 100 mM glucose, 10 mM
EDTA, 30 mM trisodium citrate, 26 mM citric
acid; see Ref. 13) at 4 °C. After centrifugation, the hemocyte
pellet was rinsed in PBS and resuspended in the same saline. 20 µl of
hemocyte suspension were layered on glass coverslips. Hemocytes were
allowed to adhere on glass for 15 min in a moist chamber at 23 °C
and then gently rinsed with PBS before being used as monolayers.
Cytotoxic activity was tested on monolayers. Excess PBS was pipetted
off the coverslip, replaced by 20 µl of the solution of lysin in PBS,
and monolayers were incubated in a moist chamber at 23 °C. Hemocyte
mortality was checked by adding 2 µl of trypan blue dye (0.04% final
in PBS) and 5 min more of incubation. In a preliminary experiment,
results were expressed as a percentage of mortality in total hemocyte
population. In the other experiments, cytotoxic activity was expressed
as percentage of dead cells among the GH1 population.
When potassium channel inhibitors tetraethylammonium (TEA) and
tetrabutylammonium (TBA) were used, they were added to a solution of
the lysin in HEPES buffer (pH 7.2) at concentrations of 50-300 mM. Results were expressed as percentage of dead cells in
the GH1 population. Osmolarity was measured in an automatic
micro-osmometer (H. Roebling, Messtechnik, Berlin).
The role of Ca2+ ions was tested after extensive dialysis
of lysin against PBS without Ca-Mg. Monolayers were prepared in the same buffer after rinsing hemocytes several times in anticoagulant buffer (deprived of Ca-Mg and with EDTA).
Hemolytic Activity on SRBC and Titration of Lysin--
Sheep red
blood cells were provided by BioMérieux (Lyon, France) at
50% suspension. Before use, SRBC were extensively washed in PBS and
adjusted to 5% suspension in this buffer. Tests were performed by
using 50 µl of SRBC suspension to which 100 µl of the lysin
solution were added. Incubation lasted 2 h at 37 °C. Then the
suspension was centrifuged at 3,000 × g for 5 min. 130 µl of the supernatant were added to 770 µl of pure water, and absorbance was determined at 540 nm. One unit of hemolytic activity (1 HU) was defined as the OD measured after total hemolysis of 50 µl of
a 5% SRBC suspension in 900 µl of distilled water. Hemolytic titer
of a solution was calculated using the formula deduced from numerous
absorbance determinations with serial dilutions of lysin: Titer
(HU) = 210(OD-0.72). In some experiments, TBA
(final concentrations 100-300 mM) was added to red blood
cell suspensions in HEPES buffer before incubations with hemolysin.
Production of Red Blood Cell Ghosts--
Red blood cell ghosts
were obtained after 5 min of incubation of one volume of a 5% red
blood cell suspension in PBS and 5 volumes of distilled water.
Lysed red blood cells were centrifuged (10,000 × g, 5 min), rinsed two times in distilled water, and then four times in PBS.
For inhibition of hemolytic activity, lysin solutions were incubated
with large amounts of ghost suspensions for 2 h at 37 °C and
then centrifuged and tested for hemolytic activity.
Effect of Incubation Time and Increasing Target Cell
Concentration on Hemolytic Activity--
In a first series of
experiments, 100 µl of different concentrations of lysin in PBS (see
"Results") were incubated with 50 µl of a 5% suspension of SRBC
at 37 °C; absorbance of supernatant was determined from 0.5-24 h.
In a second series of experiments, a constant amount of lysin was
incubated for 2 h with increasing SRBC concentrations (5%, 10, and 20%), and the percentage of hemolysis was determined for each red
blood cell concentration.
Neutral Red Uptake--
Using the procedure described by Szabo
et al. (14), hemocyte monolayers were prepared in 24-well
tissue culture plates (106 cells per well) and incubated in
PBS for 30 min at 24 °C with or without lysin at a titer that
allowed vacuolation of the cells (max. 0.02 HU) but gave a low
percentage of lysis in time of the experiment (30 min). Data were
expressed as percentage of neutral red uptake values obtained in
controls (no lysin treatment).
Measurement of Cytosolic-free Ca2+
Concentration--
Intracellular calcium concentration
([Ca2+]i) was measured with
fluorescent indicator fura-2 (15). For this purpose, insect
hemocyte monolayer was prepared on either rectangular (20 × 7 mm)
or square (10 × 10 mm) glass coverslips. After plating, cells
were loaded with fura-2 after incubation for 30 min at room temperature
with the extracellular solution: 124 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1.5 mM
CaCl2, 1 mM MgSO4, 10 mM D-glucose (bubbled with
O2/CO2, 95:5) containing 5 µM of fura-2AM and 0.02% Pluronic.
[Ca2+]i was monitored either by
spectrofluorimetry or videomicroscopy. After rinsing, a rectangular
coverslip was inserted in the quartz cuvette of a Aminco-Bowman 2 spectrofluorimeter (SLM Instruments) with an angle of 45° respective
to the excitation beam. The toxin was applied directly in the quartz
cuvette containing the extracellular solution, magnetically stirred,
and thermostated at 25 °C. Fura-2 fluorescence was obtained by
excitation of the preparation alternatively at 340 and 380 nm and by
monitoring emissions (F340 and
F380) at 510 nm. The ratio of emissions at 510 nm (F340/F380) was
recorded every 0.5 s. Alternatively, a square coverslip was
transferred to the recording chamber mounted on an inverted microscope
(Leica, DMIRB). Fura-2 emission was obtained by exciting alternatively at 340 and 380 nm with a rotating filter wheel (Sutter Instruments). Fluorescent signals were collected with a CCD camera
(Hamamatsu), digitized, and analyzed with an image analysis
software ("Acquacosmos," Hamamatsu).
Measurement of Intracellular K+
Concentration--
To record K+ efflux from insect
hemocyte, intracellular K+ concentration was measured with
fluorescent K+-binding benzofuran isophtalate dye (or
PBFI). For this purpose, cells were plated on a square (10 × 10 mm) glass coverslip and incubated for 30 min at room temperature with 5 µM of PBFI-AM and 0.02% Pluronic diluted in the
extracellular solution. After rinsing, the coverslip was transferred to
the stage of an inverted microscope (Leica DMIRB). PBFI fluorescence
was obtained by exciting the preparation at 380 nm and was collected at
510 nm. Analysis and digitization were performed as described above.
Electrophysiology--
For electrophysiological recordings,
insect hemocyte monolayers were prepared on square (10 × 10 mm)
glass coverslips. After plating, a coverslip was transferred to the
recording chamber of an inverted microscope (IMT2, Olympus),
continuously superfused (flow rate: 5 ml/min) with the extracellular
solution described above and containing 10 mM HEPES (pH
7.4) at room temperature. Patch-clamp experiments were performed in the
cell-attached and the inside-out configurations with glass
microelectrodes (4-5 M Osmotic Protection--
Possible osmotic protection of insect
hemocytes and SRBC was tested with protectants of different sizes:
polyethylene glycol 6,000 and 4,000 and dextran 1,000, all at 30 mM, and raffinose (MW 504) and sucrose (MW 342), both at 50 mM. These protectants were added to the lysin solutions,
and hemocytes or red blood cells were incubated as described above. In
a series of experiments, after incubation and measurement of the
optical density of the supernatant (hemolysis), the red blood cell
pellet was resuspended and incubated 5 min more in PBS and measured
again for hemolysis. For cytolysis, two series of monolayers were
incubated with lysin and protectant. In one series, the percentage of
macrophage lysis was determined at the end of incubation. In the other
series, at the end of incubation monolayers were washed and incubated 5 min more in PBS. Then the percentage of lysis was determined and
compared with that obtained without rinsing the cells.
Electron Microscopy--
Hemocyte monolayers were incubated for
0.5 h with lysin diluted in PBS (titer 0.02 HU) or in PBS for
control, fixed in 5% glutaraldehyde, then in 1% osmium tetroxide, and
embedded in Epon. Ultra-thin sections were stained according to
Reynolds (17).
Purification of the Lysin--
Purification of the lysin was
achieved as described under "Experimental Procedures" and is
summarized in Table I. Matrix-assisted laser desorption ionization time-of-flight analysis of the C18 active
fraction gives only one peak with a MW of 10,790. The profile mass fingerprint (PMF) after trypsin digestion of this molecule was
determined (thanks to N. Galeotti, P. Marin, and E. Demay from Centre
CNRS INSERM de Pharmacologie et Endocrinologie, Montpellier, France). This PMF was used to search protein data bases, but the analysis did not yield any protein identification. We called this lysin
Vacuolation and Lysis of Hemocytes--
The effects of different
dilutions of
Cytolytic and hemolytic activities were lost after incubation of
In the second series of experiments, increasing SRBC concentrations
(5-20%) were incubated for 2 h in 0.2 or 0.05 HU of
In the last series of experiments, different dilutions of Effects of
Cytoplasmic concentration of K+ was monitored in
PBFI-loaded hemocytes. At each Channels Opened on Insect Macrophage Membrane by
Under symmetrical K+ conditions, equilibrium potential
(EK+) = 0 mV as calculated with the Nernst
equation. Under these conditions, currents mediated by K+
fluxes are expected to reverse when Vm = EK+ = 0 mV. Here, reversal of the currents
obtained in the presence of
In a second set of experiments, cell-attached recordings were performed
with electrodes filled with the extracellular solution. In the absence
of toxin, voltage-dependent ionic channels could not be
evidenced in GH1 because no microscopic currents could be recorded by
stepping Vcmd from
We next examined whether the effect of the toxin was selective for the
outside domain of the GH1 membrane. For this purpose, patch-clamp
recordings were performed in the inside-out configuration. Patches of
GH1 were held at Osmotic Protection--
Osmotic protection of insect hemocytes and
of SRBC was tested with potential protectants of different sizes.
Polyethylene glycols (PEG) 4,000 and 6,000 and dextran 1,000 were used
at a final concentration of 30 mM and raffinose and sucrose
at a final concentration of 50 mM. Protection of hemocyte
cytolysis (Fig. 7) and of SRBC hemolysis
(not shown) was obtained with all these protectants, depending on the
concentration of
We next examined whether protectants were either inhibiting the
insertion of the toxin in the plasma membrane or were blocking pores
formed by the toxin. For this purpose the following protocols with
insect hemocyte monolayers were designed. In a first series of
experiments, the percentage of GH1 lysis was determined in monolayers
incubated for 45 min in PBS (control 1), in PBS containing lysin (0.2 HU) (control 2), and in PBS containing lysin (0.2 HU) and PEG 4,000 as
protectant. In a second series of experiments, monolayers were
incubated under the same conditions; then hemocytes were rinsed in PBS
and incubated 5 min more in PBS. The percentage of GH1 lysis was
determined. The percentage of cell lysis obtained after washing
monolayers incubated with lysin and protectant was the same as the
percentage determined after incubation with lysin in the absence of
protectant (Table III).
In the last series of experiments, after incubation of SRBC with lysin
solution (0.5 HU) and protectant the optical density of supernatant
(hemolysis) was measured, and the red blood cell pellet was resuspended
in PBS. After 5 min more of incubation, red blood cells were pelleted
again and the OD of the supernatant measured. The sum of optical
densities of the two supernatants was close to the OD of the
supernatant of SRBC incubated with Effect of Potassium Channel Blockers on Hemocyte Cytolysis and Red
Blood Cell Lysis--
TEA and TBA were tested on cytotoxic and TBA on
hemolytic activities triggered by
In a second series of experiments, SRBC were incubated with different
dilutions of Two different cytotoxic activities (C1 and -2) on insect hemocytes
were evidenced in the culture medium of the entomopathogenic bacteria
X. nematophila (Ref. 10, this work, and Table II), with C1
being under the control of flhDC, the flagellar master operon (11). This flhDC-dependent activity was also
hemolytic for SRBC but did not lyse RRBC. SRBC hemolysis and insect
hemocyte cytolysis were equally sensitive to heat or trypsin treatments of the culture medium and were independent of Ca2+ ions. In
the present work the The total disappearance of cytotoxic activity after incubation with
SRBC ghosts and the results of osmotic protection and patch-clamp
experiments show that the plasma membrane is a target of Microspectrofluorimetry data suggest that the toxin-induced calcium
rise after a latency period resulted from Ca2+ influx from
the external medium. However, when the maximal increase of the ratio
340/380 nm has been obtained fura-2 emissions tend to display parallel
decreases. The more likely explanation is that the toxin induced
membrane disruption leading to leakage of fura-2 in the medium. Because
the toxin was able to induce cell death in the absence of external
calcium, it can be postulated that the increase in cytosolic
Ca2+ detected upon exposure of the cells to the toxin was
correlated neither with specific Ca2+ entry nor with
mobilization of Ca2+ from internal stores. More likely it
reflects cell lysis, as shown by light microscopic observation (Fig.
5D). On the other hand, Cell-attached patch-clamp experiments were designed to evaluate the
pore-forming activity of Taken together, these results show that the first effect of The cell burst was the most obvious effect of -Xenorhabdolysin (
X). We show that plasma membrane of insect hemocytes and of mammal red blood cells is the first target of this
toxin. Electrophysiological and pharmacological approaches indicate
that the initial effect of
X on macrophage plasma membrane is an
increase of monovalent cation permeability, sensitive to potassium
channel blockers. As a consequence, several events can occur
intracellularly, such as selective vacuolation of the endoplasmic reticulum, cell swelling, and cell death by colloid-osmotic lysis. These effects, inhibited by potassium channel blockers, are totally independent of Ca2+. However, the size of the pores
created by
X on macrophage or red blood cell plasma membrane
increases with toxin concentration, which leads to a rapid cell lysis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
resistance). According to the experiment,
electrodes were filled with various solutions: the extracellular
solution (as described above), potassium-rich solutions comprising 150 mM potassium gluconate and 50 mM HEPES (pH 7.4)
or 150 mM potassium chloride and 50 mM HEPES
(pH 7.4), a TEA-based solution containing 150 mM TEA
chloride and 50 mM HEPES, and a TBA-based solution composed
of 150 mM TBA chloride and 50 mM HEPES (pH
7.4). For both cell-attached and inside-out experiments, the toxin
X
was used at a concentration of 0.062 HU either diluted in the
intra-electrode solution or bath-applied, respectively. Recordings were
performed in the voltage clamp mode (Vcmd); command voltage refers to
the voltage applied in the recording electrode. Transmembrane voltage
(Vm) recorded in the cell-attached configuration equals
Vi
Vcmd, where Vi is the voltage of the
inner face of the patch (16). On graphs, only Vcmd is given because
Vi is not known. The voltage-dependence of the currents
recorded in the presence of
X in the cell-attached mode was studied
by stepping Vcmd from
80 to +80 mV with an increment of 20 mV. In
this protocol, each voltage step lasted 2 s. Single channel
currents were recorded with a patch-clamp amplifier (Axopatch 200 B,
Axon Instruments) and digitized (Digidata 1200 Interface, Axon
Instruments). Signals were filtered at 1 kHz and sampled at 10 kHz.
Continuous recording and analysis of the currents were performed with
John Dempster's softwares `WinCDR' and `WinWCP.'
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Xenorhabdolysin (
X). It was hemolytic for SRBC but not for RRBC
(Table II).
Purification of -Xenorhabdolysin from bacteria culture medium
Insect hemocyte cytolysis and red blood cell hemolysis
X are compared to X. nematophila culture
supernatant with C1 activity (10). Lysin concentration
was lower in tests for hemocytes (0.2 HU) than in tests for red blood
cells (0.85 HU). NT, not tested.
X on S. littoralis hemocytes were compared
with the effects of culture supernatant with C1 activity (10) under the
same experimental conditions (Table II). With
X solutions of 0.02 HU
or more, death of the hemocytes occurred by necrosis, as tested by
trypan blue uptake, in less than one hour. The main hemocyte types,
which are plasmatocytes (Pl) and Granular Hemocytes 1 (GH1, insect
macrophages), were unequally sensitive, with GH1 showing a higher
percentage of lysis than Pl did for the same
X titer (not shown).
Therefore, most of the numerations reported in this study were achieved
on GH1 alone. Before lysis, Pl and GH1 exhibited extensive vacuolation (Fig. 1, A and B),
suggesting a modification of plasma membrane permeability by the toxin.
Neutral red uptake quantification did not show any significant
difference between vacuolated cells and untreated hemocytes (not
shown). Transmission electron micrograph of
X-treated cells showed
the presence of numerous ribosomes on the cytoplasmic side of the
vacuole membrane. These vacuoles were dilated endoplasmic reticulum
cisternae and perinuclear cisterna (Fig.
2). Other cellular organelles,
especially mitochondria, Golgi apparatus, or lysosomes appeared only
very slightly altered, if any.
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Fig. 1.
Vacuolation of insect hemocytes by
X and inhibition by TBA. Hemocytes monolayers
were incubated for 30 min in PBS without toxin (A), in the
presence of 0.02 HU of
X (B), or with 0.02 HU of
X and
50 mM TBA (C). Arrowheads, vacuoles
in macrophages (arrow) or in plasmatocytes (double
arrow). Bar = 10 µm.
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Fig. 2.
X triggers endoplasmic
reticulum vacuolation in insect hemocyte (0.02 HU for 30 min).
Vacuoles are dilated vesicles of rough endoplasmic reticulum
(er) or nuclear cisterna (arrow). Mitochondria,
m. Nucleus, n. Ribosomes, arrowheads.
Bar = 1 µm.
X
or C1 broth growth at 60 °C for 30 min or incubation in the presence
of trypsin (Table II). The lytic effects on hemocytes and on SRBC were
still observed in a non-added calcium medium (Table II). The absence of
Ca2+ did not alter the difference in sensitivity between
plasmatocytes and granular hemocytes 1 to
X (not shown).
X Molecules Are Not Recycled--
To test for the possible
recycling of
X after a first exposure to cells, we conducted three
kinds of experiments. In the first series, a suspension of SRBC
was incubated with different dilutions of
X (Fig.
3). Incubations lasted up to 24 h
with a measurement of OD at different incubation times. In these
experiments we observed an increase of hemolysis up to 2 h, and
then the OD reached a plateau value. Such long term incubations were
not performed with insect hemocytes.
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Fig. 3.
Time course of
X-induced hemolysis of SRBC. 5% SRBC were
incubated with 0.03-1 HU of
X for 0.5-24 h and centrifuged;
optical density of supernatant was then determined. This result is
representative of three distinct experiments.
X. Fig.
4 shows that the percentage of hemolysis
elaborated decreased with increasing target cell concentration.
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Fig. 4.
Decrease in percent hemolysis with increase
in SRBC concentration. 5, 10, and 20% SRBC in PBS were incubated
for 1 h with 0.05 or 0.2 HU of X. Results were expressed as a
percentage of total hemolysis. Data are means of three distinct
experiments ± S.E.
X were
first incubated with SRBC ghosts, and then the supernatant was further
incubated with a 5% suspension of SRBC or with S. littoralis hemocyte monolayers. Neither hemolytic nor cytolytic activity was detected (Table II).
X on Hemocyte Ca2+ and K+
Ions Concentrations--
We have investigated whether potential
toxin-mediated modifications of membrane permeability could result in a
change in hemocyte Ca2+ and K+ cytosolic
concentrations. To record [Ca2+]i
changes, fura-2-loaded cells were exposed to increasing concentrations
of
X (0.032-0.25 HU), and the fluorescence ratio 340/380 of the
hemocyte monolayer was recorded at 510 nm in a spectrofluorimeter.
Toxin at all these concentrations led to cell death. At a concentration
of 0.25 HU, a transient rise in
[Ca2+]i could be recorded that was
apparently regulated quickly. Lower concentrations of toxin, ranging
from 0.062 to 0.032 HU, led to a dose- and time-dependent
increase in [Ca2+]i (Fig.
5A). In a Ca2+-
free external medium (no added Ca2+), the toxin at a
concentration of 0.25 HU had almost no detectable effects. Therefore,
this suggests that the toxin-induced
[Ca2+]i rise results from a
Ca2+ influx from the external medium (Fig. 5A).
The analysis of the fura-2 emissions at 510 nm, obtained by exciting at
340 (F340) and 380 nm (F380), indicates that
although the initial increase in fluorescence ratio in the presence of
X was effectively because of an increase in
[Ca2+]i as indicated by the
variations of fura-2 emissions in opposite directions (Fig.
5B), the decrease of fluorescence ratio more likely reflects
leakage and dilution of the probe in the medium because both F340 and
F380 decrease in parallel. The toxin at 0.062 HU elicits rapid and
unregulated increases in [Ca2+]i
in visually identified GH1 and plasmatocytes, as observed using
videomicroscopy (Fig. 5C). Observation in phase contrast microscopy of these cells during the experiment confirms that the cells
were lysed at the time of [Ca2+]i
rise (Fig. 5D). Because the toxin is able to induce cell
death in the absence of external calcium, it can be postulated that the
increase in [Ca2+]i detected upon
exposure of cells to the toxin is not responsible for its toxicity but
more likely reflects ionic disturbance across cell membrane and cell
lysis.
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Fig. 5.
Effect of X on
cytosolic Ca2+ and K+ concentrations.
A and B, after loading with fura-2 (30 min, room
temperature), hemocyte preparation plated on rectangular coverslips was
transferred to the recording chamber of a spectrofluorimeter.
Fluorescence ratio was collected every 0.5 s. A,
concentration-dependence (from 0.032 to 0.25 HU) of
X effect on
[Ca2+]i. B, analysis of
respective emissions recorded at 510 nm by excitation at 340 and 380 nm
after the application of
X at 0.25 HU. C and
D, effect of
X (0.062 HU) in visually identified GH1 and
plasmatocyte. C, time course of
[Ca2+]i increase after application
of the toxin in a GH1 and a plasmatocyte. D, phase contrast
microphotographs obtained (upper panel) prior to the
application of
X and (lower panel) at the time of
[Ca2+]i peak in these two cells.
Scale bar represents 20 µm. E, effect of
X
on [K+]i in visually identified GH1. Hemocyte monolayer
was loaded with PBFI (30 min, room temperature) and then used for
videomicroscopy. Potassium leakage was measured in identified GH1 after
successive applications of
X (0.02 HU). On all graphs,
vertical arrows indicate the time of application of
X.
Traces are representative of at least three distinct
determinations.
X (0.02 HU) application to the
medium, an immediate loss of K+ could be recorded that was
in part regulated up to the cell lysis. This lysis was evidenced by a
large decrease in PBFI fluorescence, revealing leakage of the probe in
the medium as illustrated in three visually identified GH1 (Fig.
5E).
X Are
Monovalent Cation-selective--
From the videomicroscopy data, one
can hypothesize that
X mediates to an ionic imbalance, leading to
cell death. This imbalance could initially be because of a
K+ efflux. Therefore, using patch-clamp recordings we have
examined whether
X could alter K+ ion permeability
through native GH1 membranes. In a first attempt to study membrane
modifications elicited by the toxin, conventional patch-clamp whole
cell recordings (WCRs) were undertaken in visually identified GH1. It
must be mentioned that experiments were conducted only within 90 min
following hemocyte plating. Indeed, the hemocytes would naturally
deteriorate after 90 min and give unreliable data. Stable WCRs were
almost impossible to get from GH1 because these cells sealed almost
immediately after obtaining a gigaohm seal and ruptured the
patch by applying a negative pressure (n = 20). By
contrast, plasmatocytes gave easy access to WCRs (n = 5). This discrepancy between the two cell populations could be
attributed to specific membrane properties. Indeed, GH1 have a rough
plasma membrane with invaginations and pseudopods, whereas
plasmatocytes have a smoother plasma membrane that allows easier access
to microelectrodes (not shown). This is the main reason why
cell-attached recordings were preferred to WCRs. In addition, to
examine the pore-forming activity of the
X and to avoid a lethal
exposure of the cells to this toxin,
X was applied directly within
the recording electrode. For this purpose, a fraction of the toxin was
diluted in the internal medium of the electrode and, just as in the
perforated patch technique using pore-forming antibiotics (16, 18),
toxin-induced modifications could be recorded underneath the electrode.
It must be emphasized that a concentration of 0.062 HU was chosen
because it gave a good activity without damaging the seal. Indeed, in
the presence of higher concentrations of the toxin, tight seals could
not be established. In a first set of experiments, we therefore
examined the selectivity of these
X pores for K+ ions.
For this purpose, recordings were performed under symmetrical concentrations of K+ obtained by filling electrodes with
K+-based solutions (K gluconate and KCl). In this respect,
we first tested K gluconate-containing solutions. In the absence of the toxin, no current could be recorded when stepping Vcmd from
80 to +80
mV (Fig. 6A). In the presence
of the toxin, channel formation could be observed after 3 to 20 min of
contact (n = 5) (Fig. 6B). These currents
reversed when Vcmd = 0 mV and displayed a linear voltage
dependence. The conductance of
X-generated channels was evaluated at
21 ± 2 pS (n = 4) from the current-voltage
relationship (Fig. 6C). The effect of the toxin was
time-dependent because after 30-45 min of contact
(n = 4), longer openings could be observed prior to the
rupture of the seal (Fig. 6D). Similar data were obtained
when recording electrodes were filled with KCl-based intracellular
solution (Fig. 6E), which suggests that
X generates pores
permeant to K+ ions. To confirm this possibility,
conventional K+ conductance blockers, i.e.
tetrasubstituted ammonium derivatives, tetraethylammonium chloride
(TEACl), and tetrabutylammonium chloride (TBACl), were further tested
for their ability to block currents recorded in the presence of
X.
When K+ was substituted for TBA+ ions, no
currents could be recorded from the patches when
X was applied
(n = 4) (Fig. 6F). A similar result was
obtained with TEA+ ions (data not shown). Therefore,
ammonium derivatives block
X-generated conductances. In addition,
because the counter anion of these compounds was Cl
ion
in both cases and because similar data were obtained with both KCl- and
K gluconate-filled electrodes, one can suggest that
X-formed pores
are not permeant for Cl
ions.
View larger version (34K):
[in a new window]
Fig. 6.
Patch-clamp recordings obtained from
GH1. A-H, cell-attached recordings performed in the
presence of X applied directly in the recording electrode at 0.062 HU. I, inside-out recording obtained from a patch of GH1
membrane and exposed to
X at 0.062 HU. A, recording
obtained with K gluconate-based (150 mM K gluconate, 50 mM HEPES, pH 7.4) filling solution without the toxin.
B and D, recordings obtained with K
gluconate-based (150 mM K gluconate, 50 mM
HEPES, pH 7.4) filling solution in the presence of the toxin.
D, the recording obtained 60 min after getting the seal.
C, averaged current-voltage relationships obtained from four
distinct experiments performed with K gluconate-based filling solution.
Data are presented as means ± S.E., graph.
E, recording obtained with KCl-based (150 mM
KCl, 50 mM HEPES, pH 7.4) filling solution with the toxin.
F, recording obtained with TBACl-based (150 mM
TBACl, 50 mM HEPES, pH 7.4) filling solution with the
toxin. G and H, recordings obtained with
extracellular filling solution without (G) and with the
toxin (H). I, inside-out recording was performed
under symmetrical conditions achieved with extracellular medium and at
a holding voltage of
60 mV. The arrow indicates the time
of application of
X (0.062 HU). Traces are representative
of at least three distinct determinations.
X was observed when Vcmd = 0 mV.
Therefore, this suggests that Vm = Vcmd in the presence of the
toxin in the recording electrode.
80 to +80 mV (Fig. 6G). In the presence
of the toxin, channel-like openings could be recorded at extreme
membrane potentials, i.e.
80 and +80 mV. No current could
be detected when Vcmd = 0 mV (Fig. 6H). This tends to
indicate that cations flowing through
X-generated pores are not
selective. Indeed, currents flowing through non-selective
cationic channels are expected to reverse when Vm = 0 mV.
60 mV under symmetrical conditions (extracellular
medium in the electrode and in the bath) and allowed to equilibrate for
5 min after excision. Exposing the inner face of the GH1 membrane to
X (0.062 HU, n = 4) resulted in the occurrence of
large inward currents followed by a rapid loss of the seal. This
indicates that
X may have an effect on both sides of the plasma
membrane of GH1 (Fig. 6I).
X solution. For the same
X concentration,
protectants were more efficient against cytolysis of insect macrophages
than against hemolysis of SRBC. Although a total inhibition of
cytolysis was obtained with PEG 4,000 at the highest toxin
concentration (1 HU), hemolysis was only reduced by one half by
PEG 4,000 at this concentration. Total hemolysis inhibition was
observed with PEG 6,000. No change in OD was recorded when the
different protectants were added to the supernatant obtained after
incubation of red blood cells with the toxin. This shows that there was
no direct effect of the protectants on hemoglobin absorbance.
View larger version (27K):
[in a new window]
Fig. 7.
Effect of protectants on
X-induced GH1 lysis. The columns represent the
percentages of GH1 lysis (trypan blue staining) after 1 h of
incubation of hemocyte monolayers in PBS (0) or in different
dilutions of
X supplemented with various protectants (PEG
and dextran, 30 mM; raffinose and
sucrose, 50 mM). Data are means of three
independent experiments ± S.E.
Dissociation of binding and cytolytic activity
X
(control 1), in
X solution (0.2 HU) (control 2), or
X solution in
PEG 4,000. In the first series of monolayers, percentage of GH1 lysis
was determined at the end of 45 min of incubation. In the second
series, monolayers were first incubated for 45 min and then rinsed in
PBS, incubated for 5 min more in this saline, and GH1 lysis was
determined. Data are presented as means ± S.E. of independent
experiments where each experimental value is determined in duplicate.
X in the absence of protectant
(Fig. 8). Because cytolysis and hemolysis
were restored after washing off the protectant, we conclude that the
observed inhibitions were not because of direct inactivation of
X by
PEG.
View larger version (23K):
[in a new window]
Fig. 8.
Dissociation of toxin binding and hemolytic
activity. 5% SRBC were incubated for 45 min with X (0.5 HU)
supplemented or not (control) with PEG and then centrifuged;
OD of supernatant was determined (1st
incubation). The red blood cell pellet was suspended and
incubated for 5 min more in PBS with neither toxin nor PEG added,
centrifuged again, and OD of the supernatant determined
(2nd incubation). There was an actual
osmotic protection in the first incubation. Removal of the first
supernatant washed off the protectant but not the lysin that was bound
to the red blood cell membrane. In these conditions hemolysis was
recovered. Experiments were carried out in quadruplicate, and similar
results were obtained.
X. In a first series of
experiments, they were added to solutions of
X giving almost 50%
(TEA) or 80% (TBA) GH1 mortality (from 0.04 to 0.2 HU). The cytolytic
activity on hemocytes was almost totally inhibited with 100 mM TEA or 50 mM TBA in the time of experiments
(not shown). In experiments conducted with lower toxin concentration
(0.01-0.02 HU) and a shorter incubation time, vacuolation of hemocytes
was also extremely reduced when TEA (100 mM) or TBA (50 mM) was added to the incubation medium (Fig. 1C
for TBA). The best protection achieved with TBA over TEA was attributed
to the larger size of the TBA molecule, which allows a better blockade
of the K+ channels (19).
X with 100-300 mM TBA. TBA solutions were able to inhibit the hemolytic activity depending on the
X and TBA
ratio, with the lower lysin concentration providing the better protection (not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
X, a 10.8-kDa toxin responsible for this
flhDC-dependent activity, was purified from X. nematophila broth growth with C1 activity. Culture medium
and the present purified toxin had the same heat and trypsin
sensitivity and the same range of specificity for insect hemocytes and
hemolytic activity on SRBC; RRBC was insensitive. After incubation of
broth growth or purified lysin solution with SRBC ghosts, hemolysis as
well as hemocyte cytotoxicity was lost. We conclude that the purified lysin is responsible for both SRBC hemolysis and insect hemocyte cytolysis. It is also responsible for the C1 activity previously evidenced in X. nematophila culture medium. Titration curves
show there was no linear relationship between lysin concentration and hemoglobin release (optical density). This suggests that
X has a
concentration-dependent binding affinity to its target, as
shown for
-toxin of Staphylococcus aureus (20).
Alternatively, possible interactions between lysin molecules could
occur as degrees of polymerization, depending on toxin concentration.
To our knowledge, an hemolysin secreted by an entomopathogenic
bacteria, which is also active on insect hemocytes, is purified here
for the first time.
X.
Furthermore, these results are consistent with data obtained with long
term incubations (up to 24 h) of
X with SRBC and with data
obtained with increasing target cell concentration. In these last
experiments, the amount of hemolysis did not increase later than 2 h of incubation, and the percentage of hemolysis decreased when target
cell concentration was increased. We conclude that once fixed on a
plasma membrane site,
X is not recycled to react with multiple
target cells, at least with red blood cells. According to Rowe and
Welch (21), these data show that
X looks like a pore-forming toxin
rather than a lysin with an enzymatic activity. Results of
microspectrofluorimetry, of patch-clamp studies, and of osmotic
protection experiments are consistent with such pore-forming activity.
X induced an immediate loss of
K+, which could be temporarily regulated by the cell until
lysis. These last observations are in accordance with the results of patch-clamp experiments.
X in native GH1 membranes. It must be
emphasized that no voltage-dependent channels could be detected in the absence of the toxin. However, this does not imply that
GH1 are "electrically" silent. Indeed, the activity of
Ca2+-activated K+ channels or second
messenger-operated channels, for instance, remains to be established in
these cells. Electrophysiological recordings to study
X actions had
to be adapted to the specific membrane properties of GH1 and to the
very high sensitivity of these cells to
X. For this purpose,
X
was directly applied in the recording electrode. This procedure enabled
us to evaluate the pore-forming activity of this toxin. Interestingly,
the bacterial toxin,
-toxin from S. aureus, has
previously been used for its pore-forming activity in the perforated
patch method (16). To observe microscopic currents due to channel
formation, low concentrations of
X had to be applied. Indeed, as
also evidenced for Helicobacter pylori (14), for instance,
high concentrations of
X could prevent tight seal formation between
the cell membrane and recording electrodes. In the presence of
X,
the activity of cation-selective channels could be evidenced. These
currents could be totally blocked by K+ channel antagonists
(TEA+ and TBA+). This tends to indicate that
X primarily alters membrane permeability by forming K+
permeable pores. Under physiological ionic concentrations,
non-selective cationic currents could be recorded in the presence of
the toxin. Under symmetrical K+ conditions, the conductance
of
X-generated pores was rather small (21 ± 2 pS). This is
probably because the toxin was applied at a low concentration and had a
small area of contact with the membrane of GH1. However, the effect of
the toxin on channel forming was time-dependent. Indeed, an
increase in currents was observed with time. This could be because of
an increase in the number of pores and/or the size of these pores, as
demonstrated for other porins (22).
X on the
insect hemocyte membrane was an increase in ionic permeability, mainly
for monovalent cations. Modification of ion permeability by bacterial
toxins in eukaryotic cells is well documented. Anion-selective channels
are formed in SF-9 insect cells by the
-endotoxin from Bacillus thuringiensis (23) and in HeLa cells by VacA from
H. pylori (14). Channels with weak discrimination among
different cations are formed in human macrophages by HlyA from
Escherichia coli (24). Alpha-X from X. nematophila was more specific because it created channels rather
selective for monovalent cations, as did the major cytolysin of
S. aureus (25). In our experiments, inhibition of the
current recorded in the presence of
X by the specific blockers of
potassium channels, TEA and TBA, showed that the cation channels opened
or created by
X could be rather specific for potassium. This
specificity has been evidenced for aerolysin from Aeromonas
hydrophila on baby hamster kidney (mammalian) cells (26).
TEA and TBA also inhibited both ER vacuolation and cell lysis, showing
that disturbance of potassium permeability induced by
X could be
sufficient to lyse the target cells. Furthermore, as in experiments
conducted with VacA toxin on HeLa cells (14), inhibition of insect
macrophage lysis by blockers was more effective at the lowest
X
doses. We cannot dismiss the possibility that
X activates unknown
endogenous channels rather than forming new ones. However, there is
evidence in favor of a pore-forming activity. First, the results
of osmotic protection experiments are consistent with the formation of
pores. Second, pore size increase with toxin concentration is well
documented for pore-forming molecules such as complement (pore sizes
ranging from 0.7 to 15 nm) (27) or E. coli toxin (0.6 to 1.3 nm) (28) and other RTX toxins (29). A total protection of insect
macrophage cytolysis was obtained with PEG 4,000 (pore radius: 1.9 nm)
(30) whatever the
X concentration, but protection of SRBC, at the
highest
X concentrations, was only obtained with PEG 6,000 (pore
radius: 2.9 nm). This suggests that the maximum size of the pores
formed in red blood cell plasma membrane would be larger than that of
pores made by the same lysin in insect macrophage plasma membrane. A
larger size of pore created in red blood cells than pore in nucleated
cells was already reported for ShlA from Serratia marcescens
(31), but the reason for this variability is unknown. Finally, our
results on osmotic protection led us to conclude that
X processes
through colloid-osmotic lysis (32). In colloid-osmotic lysis,
transmembrane channels allow only ions and small molecules
to pass freely across the cell membrane. Therefore, the osmotic
pressure generated by the high concentration of macromolecules inside
the cell causes a water influx that leads to cell swelling and
sometimes to cell burst. This colloid-osmotic process is consistent
with our results on Ca2+ and K+ movements
through hemocyte plasma membrane. Low doses of
X mediate a selective
leakage of K+ (and possibly other monovalent cations) from
hemocytes. However, low doses of
X do not enhance membrane
permeability for Ca2+ or larger molecules. In this respect,
intracellular Ca2+ entry was observed only upon cell lysis.
Therefore, Ca2+ entry was not a cause but a consequence of
cell lysis.
X from X. nematophila on hemocytes, but prior to lysis, insect hemocytes
showed extensive vacuolation of the cytoplasm. Vacuolation is a
non-classic pathway of toxicity of bacterial toxins. However, it is
achieved by cereulide from Bacillus cereus (33), VacA toxin
from H. pylori (34), aerolysin from A. hydrophila
(26), ShlA hemolysin from S. marcescens (31), and HlyA
hemolysin from Vibrio cholerae (35) in different mammalian
cell types in vitro. Among these toxins, only vacuolation
triggered by ShlA and HlyA is followed by a lysis of cells in culture.
Because all these toxins (cereulide, VacA, aerolysin, ShlA, and HlyA)
triggered the vacuolation of different cellular organelles, we
conducted two kinds of experiments to characterize which cell organelle
was subjected to vacuolation by
X. Neutral red is a supravital dye
of the endosome/lysosome system (36). Data obtained with neutral red
uptake experiments suggest that the vacuoles observed in hemocytes
after
X incubation do not belong to this endosome/lysosome system.
This conclusion is not in agreement with studies performed on mammal
cells with VacA, ShlA, or HlyA. Examination of
X-treated hemocytes
under electron microscopy lead us to conclude that the vacuoles are in
fact dilated cisternae of the ER. Vacuolation of ER by a bacterial toxin is reported here for the first time in non-mammalian cells. Another case of ER vacuolation by bacterial toxin was reported by
Abrami et al. (26) for aerolysin on baby hamster kidney
cells. For aerolysin (37-39), as for
X (this study), the first
target of the toxins is the plasma membrane, where they form channels selective for small cations before they trigger ER vacuolation. However, a fundamental difference between aerolysin and
X is their
respective effects on intracellular Ca2+ concentration.
Indeed, aerolysin induces Ca2+ release from intracellular
stores as well as a Ca2+ influx (39), but
X does not. In
this respect the activity of
X on its target cells appears different
from the different actions already studied and described for all other
bacterial toxins.
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ACKNOWLEDGEMENTS |
---|
We thank Alain Givaudan for helpful comments and suggestions and Marc Turiault, Leila Equinet, and Richard Hérail for assistance.
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FOOTNOTES |
---|
* This work was supported by grants from INRA, CNRS, and Fondation pour la Recherche Médicale (France) and Instituto de Cooperacao Cientifica e Tecnologica Internacional (Portugal).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Funded by a grant PRAXIS XXI (BD/13935/97) (Portugal). Present address: Secção de Biologia Celular e Molecular, Universidade dos Açores, 9501-801 Ponta Delgada, Açores, Portugal.
¶ Both authors contributed equally to this study.
** To whom correspondence should be addressed. Tel.: 33-4-67-14-46-72; Fax: 33-4-67-14-46-79; E-mail: brehelin@crit.univ-montp2.fr.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M210353200
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ABBREVIATIONS |
---|
The abbreviations used are:
SRBC, sheep red
blood cells;
X,
-Xenorhabdolysin;
ER, endoplasmic reticulum;
GH1, granular hemocyte 1;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HU, hemolysis unit;
OD, optical density;
PBFI, potassium-binding benzofuran isophtalate;
PBS, phosphate-buffered saline;
PEG, polyethylene glycol;
RRBC, rabbit
red blood cells;
TBA, tetrabutylammonium;
TEA, tetraethylammonium;
Vcmd, voltage clamp mode;
WCR, whole cell recording;
AM, acetoxymethyl ester.
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