Effects of pentamidine on polyamine level and biosynthesis in wild-type, pentamidine-treated, and pentamidine-resistant Leishmania


Basselin, M.; Badet-Denisot, M.A.; Lawrence, F.; Robert-Gero, M.

Experimental Parasitology 85(3): 274-282

1997


Polyamine biosynthesis was studied in wild-type promastigotes of Leishmania donovani and Leishmania amazonensis treated with pentamidine and in the parasites resistant to this drug. Treatment of wild-type clones with low pentamidine concentrations for 24 hr provoked a strong decrease in arginine, ornithine, and putrescine pools, while the level of intracellular spermidine remained unchanged. In these cells, the activity of the enzyme ornithine decarboxylase was found to be decreased. Compared to wild-type cells, resistant clones had a lower level of putrescine, higher pools of arginine and ornithine, and a similar spermidine content. Analysis by Western blot and DFMO-binding showed reduced amount of ornithine decarboxylase. Furthermore, in the resistant cells, the kinetic parameters of the enzyme spermidine synthase were markedly changed, showing increased affinity to putrescine and decreased affinity to pentamidine. Thus, it seems that polyamine biosynthesis pathway is a target of pentamidine in Leishmania and is altered in resistant clones.

EXPERIMENTAL
PARASITOLOGY
85,
274-282
(1997)
ARTICLE
NO.
PR964131
Effects
of
Pentamidine
on
Polyamine
Level
and
Biosynthesis
in
Wild
-Type,
Pentamidine-Treated,
and
Pentamidine-Resistant
Leishmania
MIREILLE
BASSELIN,
MARIE-ANGE
BADET-DENISOT,
FRANCOISE
LAWRENCE,
AND
MALKA
ROBERT-GERO
Institut
de
Chimie
des
Substances
Nature/les,
CNRS,
Avenue
de
la
Terrasse,
91198
Gif-sur-Yvette
Cedex,
France
BASSELIN,
M.,
BADET-DENISOT,
M
-A.,
LAWRENCE,
F.,
AND
ROBERT-GERO,
M.
1997.
Effects
of
pentamidine
on
polyamine
level
and
biosynthesis
in
wild
-type,
pentamidine-treated,
and
pentami-
dine-resistant
Leishmania.
Experimental
Parasitology
85,
274-282.
Polyamine
biosynthesis
was
studied
in
wild
-type
promastigotes
of
Leishmania
donovani
and
Leishmania
amazonensis
treated
with
pentamidine
and
in
the
parasites
resistant
to
this
drug.
Treatment
of
wild
-type
clones
with
low
pentamidine
concentrations
for
24
hr
provoked
a
strong
decrease
in
arginine,
ornithine,
and
putres-
cine
pools,
while
the
level
of
intracellular
spermidine
remained
unchanged.
In
these
cells,
the
activity
of
the
enzyme
ornithine
decarboxylase
was
found
to
be
decreased.
Compared
to
wild-type
cells,
resistant
clones
had
a
lower
level
of
putrescine,
higher
pools
of
arginine
and
ornithine,
and
a
similar
spermidine
content.
Analysis
by
Western
blot
and
DFMO-binding
showed
reduced
amount
of
orni-
thine
decarboxylase.
Furthermore,
in
the
resistant
cells,
the
kinetic
parameters
of
the
enzyme
sper-
midine
synthase
were
markedly
changed,
showing
increased
affinity
to
putrescine
and
decreased
affinity
to
pentamidine.
Thus,
it
seems
that
polyamine
biosynthesis
pathway
is
a
target
of
pentamidine
in
Leishmania
and
is
altered
in
resistant
clones.
©
1997
Academic
Press
ABBREVIATIONS:
Pent,
pentamidine;
DFMO,
alpha-difluormethylornithine;
ODC,
ornithine
decar-
boxylase;
dcSAM,
decarboxylated
S-adenosylmethionine;
MTA,
5'-methylthioadenosine;
DTT,
di-
thiothreitol;
EDTA,
ethylene
diamine
tetra
-acetic
acid;
PBS,
Dulbecco's
phosphate
-buffered
saline.
INTRODUCTION
Polyamines
(putrescine,
spermidine,
sperm-
ine)
are
polycationic
components
occurring
in
all
cells,
including
parasitic
protozoa
(Bacchi
1981;
Bacchi
and
Mc
Cann
1987).
Despite
nu-
merous
investigations
concerning
their
role
in
cellular
growth
and
differentiation,
their
physi-
ological
function
has
remained
an
enigma.
Their
intracellular
concentrations
may
be
regu-
lated
at
the
level
of
their
biosynthesis,
intercon-
version, degradation,
and
transport.
In
Leish-
mania,
the
synthesis
of
polyamines
starts
with
the
decarboxylation
of
the
amino
acid
ornithine
to
form
putrescine,
catalyzed
by
ornithine
de-
carboxylase,
a
key
enzyme
on
the
biosynthetic
pathway.
Putrescine
is
then
converted
into
sper-
midine
by
the
action
of
an
aminopropyltransfer-
ase
or
spermidine
synthase.
Finally,
spermidine
is
converted
either
into
spermine
by
spermine
synthase
or
into
dihydrotrypanothione
via
the
intermediate
glutathionylspermidine.
0014-4894/97
$25.00
Copyright
©
1997
by
Academic
Press
All
rights
of
reproduction
in
any
form
reserved.
DFMO,
an
irreversible
inhibitor
of
ODC,
in-
hibits
the
growth
of
L.
donovani
(Kaur
et
al.
1986)
and
L.
infantum
(Carrera-Ferrer
et
al.
1987;
Balana-Fouce
et
al.
1989,
1991b;
Carrera
et
al.
1994).
Polyamine
analogs
which
interfere
with
the
biosynthesis
and
function
of
poly
-
amines
have
been
reported
to
cause
suppression
of
both
antimony
-susceptible
and
antimony
-
resistant
L.
donovani
(Bauman
et
al.
1990).
Pentamidine,
a
drug
effective
against
Leish-
mania
interferes
with
the
synthesis
of
poly
-
amines
(Bachrach
et
al.
1979b)
and
reversibly
inhibits
the
S-adenosyl-L-methionine
decarbox-
ylase
activity
in
crude
extracts
from
rat
liver,
yeast,
Pseudomonas
aeruginosa
(Karnoven
et
al.
1985),
and
Trypanosoma
brucei
brucei
(Bi-
tonti
et
al.
1986b).
We
recently
demonstrated
that
pentamidine
shares
polyamine
transporters
to
enter
leishmanial
parasites
(Basselin
et
al.
1996)
and
that
the
polyamine
uptake
is
altered
in
pentamidine-resistant
Leishmania
(Basselin
et
al.,
in
press).
274
EFFECTS
OF
PENTAMIDINE
ON
Leishmania
275
To
examine
further
the
consequences
of
these
alterations,
we
compared
the
intracellular
con-
centrations
of
putrescine
and
spermidine
and
the
activities
of
ornithine
decarboxylase
and
spermidine
synthase
of
wild
-type,
pentamidine-
treated,
and
pentamidine-resistant
clones
of
L.
donovani
and
L.
amazonensis.
MATERIALS
AND
METHODS
Materials.
Pentamidine
isethionate
was
a
generous
gift
of
Roger
Bellon
Laboratories.
DL-difluoromethyl
[5-
14
C]orni-
thine
(60
mCi/mmol)
was
purchased
from
Amersham
(France).
Unlabeled
and
labeled
decarboxylated
S-adeno-
syl-[methy1-
14
C]methionine
(55
mCi/mmol)
were
pur-
chased
from
Dr.
A.
Shirahata
(Sakado,
Japan).
L-[2,3-
3
11]-
ornithine
(53.4
Ci/mmol),
monoclonal
antibody
directed
against
ornithine
decarboxylase
(mouse
IgG2b
isotype),
and
anti
-mouse
IgG
alkaline
phosphatase
conjugate
were
pur-
chased
from
Sigma
(France).
Coomassie
blue
reagent
for
protein
assays
was
from
Bio-Rad
(France).
Culture
medium
components
were
from
Gibco
(France)
and
serum
from
Flo-
bio
(France).
Stains
and
culture
conditions.
Leishmania
donovani
(strain
MHOM/IN/80/DD8)
originating
from
the
strain
col-
lection
of
the
World
Health
Organization's
(WHO)
refer-
ence
center
in
the
London
School
of
Hygiene
and
Tropical
Medecine
(United
Kingdom)
was
provided
by
Dr.
D.
Evans.
Leishmania
amazonensis
(strain
MHOM/BR/76/LTB-012)
originating
also
from
the
strain
collection
of
the
WHO
was
provided
by
Dr.
J.
L.
Lemesre
(Montpellier,
ORSTOM
Sci-
entific
Institute,
France).
Promastigotes
were
grown
at
26°C
in
a
semidefined
RPMI-1640
medium
containing
2
mM
glutamine,
25
mM
Hepes
(pH
7.4),
10%
heat
-inactivated
fetal
calf
serum,
streptomycin
at
5
pg/ml,
and
penicillin
at
5
U/ml.
IC
50
value
refers
to
the
concentration
that
inhibited
growth
rate
by
50%
after
3
days.
Selection
of
pentamidine-resistant
clones.
Cloned
wild
-
type
promastigotes
were
selected
for
pentamidine-resistance
by
stepwise
exposure
to
the
drug
until
final
concentrations
of
40
aM
for
L.
donovani
(Pent
R
40)
and
20
aM
for
L.
amazonensis
(Pent
R
20)
were
reached
(Basselin
et
al.
1996,
in
press).
Analysis
of
polyamines
by
a
high-performance
liquid
chromatographic
method.
Chromatography
of
polyamines
was
carried
out
as
described
previously
(Villanueva
et
al.
1987).
ODC
activity.
ODC
(EC
4.1.1.17)
activity
was
assessed
by
a
modified
method
of
Djurhuus
(1981)
by
determining
the
amount
of
[1,2-
3
H]putrescine
formed
from
[2,3-
3
11]-1,
ornithine.
Washed
cells
were
resuspended
(10
9
cells/ml)
in
a
solution
containing
50
mM
Tris—HC1
buffer,
pH
7.2,
0.1
mM
EDTA,
50
',LAI
pyridoxal
phosphate,
and
5
mM
DTT,
and
lysed
by
freeze
—thaw
cycles
in
a
methanol:dry
ice
bath.
Whole
cells
and
membranes
were
removed
by
centrifuga-
tion
at
12,000g
for
30
min
at
4°C.
The
reaction
mixture
in
a
final
volume
of
120
µl
contained
100
bd
of
cell
lysat,
0.5
[2,3-
3
M-L-ornithine
(53.4
Ci/mmol)
and
0.1-12
mM
L-ornithine.
After
incubation
at
37°C
for
1
hr,
the
reaction
was
stopped
by
transfer
of
the
samples
to
ice,
then
material
was
spotted
on
P
81
phosphocellulose
filter
papers
(What
-
man)
and
washed
with
0.1
M
ammonium
hydroxide.
The
radioactivity
retained
on
the
filters
was
counted.
Binding
of
[
14
CJDFM0
to
ODC
in
vitro.
Soluble
extracts
of
wild
-type
and
resistant
cells
were
incubated
with
3
piCi
of
[
14
C]DFMO
(60
mCi/mmol)
at
37°C
for
1
hr.
The
reac-
tions
were
stopped
by
addition
of
sodium
dodecyl
sulfate
lysis
buffer.
The
incorporation
of
radioactivity
into
proteins
was
then
determined
following
the
TCA/NaOH
treatment
described
below.
Electrophoresis
and
immunoblotting.
Standard
methods
were
used
to
prepare
10%
sodium
dodecyl
sulfate—
polyacrylamide
gel
(Laemmli
1970).
The
resolved
proteins
were
electrotransferred
to
nitrocellulose
sheets
for
1
hr
at
100
V.
antibody
analysis
was
performed
as
described
by
Sambrook
et
al.
(1989).
The
primary
antibody
used
was
mouse
anti-ODC
at
Vio
dilution.
Immunocomplexes
were
detected
with
alkaline
phosphatase-conjugated
rabbit
anti
-
mouse
IgG.
DFMO
uptake
and
incorporation.
Cells
were
incubated
with
1
piCi/0.5
ml
[
14
C]DFMO
at
27°C
for
various
times
from
30
min
to
24
hr.
Samples
were
drawn
at
intervals
and
washed
with
cold
PBS.
The
pelleted
cells
were
extracted
with
5%
cold
trichloroacetic
acid
(30
min,
0°C). The
soluble
fraction
(uptake)
was
removed
and
the
pellet
hydrolyzed
in
1
M
NaOH
for
1
hr
at
80°C
(incorporation).
An
aliquot
of
each
fraction
was
mixed
with
liquid
scintillation
fluid
and
counted
for
radioactivity.
Total
uptake
represents
the
sum
of
radioactivity
in
the
soluble
and
insoluble
fractions.
Spermidine
synthase
activity.
Spermidine
synthase
(EC
2.5.1.16)
activity
was
determined
by
measuring
the
forma-
tion
of
[methyl-
14
C]MTA
from
[methyl-
14
C]dcSAM
by
the
method
of
Raina
et
al.
(1983).
Washed
cells
were
resus-
pended
(10
9
cells/ml)
in
a
solution
containing
25
mM
po-
tassium
phosphate,
pH
7.5,
1
mM
EDTA,
5
mM
DTT,
and
broken
by
freeze
—thaw
cycles.
The
enzyme
activity
was
measured
in
the
supernatant
after
centrifugation
at
12,000g
for
30
min
at
0°C.
To
determine
the
K
for
dcSAM
and
putrescine,
the
velocities
of
the
enzyme
reaction
were
mea-
sured
in
the
presence
of
1-20
µM
dcSAM
plus
1
mM
pu-
trescine
and
50-500
µM
putrescine
plus
30
piM
dcSAM,
respectively.
Determination
of
intracellular
drug
concentration.
The
intracellular
drug
amount
was
determined
by
HPLC
as
de-
scribed
earlier
(Basselin
et
al.
1996).
The
intracellular
con-
centration
was
calculated
based
on
previous
work
showing
that
the
cell
volume
is
22
and
100
lim
3
,
respectively,
for
L.
donovani
and
L.
amazonensis:
1
mg
of
protein
is
equivalent
to
2.72
x
10
8
cells
for
L.
donovani
or
1.43
x
10
8
cells
for
L.
amazonensis
(Phelouzat
et
al.
1992;
Berman
et
al.
1987).
Protein
determination.
Protein
concentration
was
mea-
sured
by
the
dye
-binding
method,
with
bovine
serum
albu-
min
as
standard
(Bradford
1976).
276
BASSELIN
ET
AL.
RESULTS
Intracellular
levels
of
polyamines
and
of
their
biosynthetic
precursors.
We
have
previ-
ously
shown
that
putrescine
and
spermidine
up-
takes
are
lowered
in
pentamidine-treated
and
pentamidine-resistant
clones
with
respect
to
wild
-type
ones
(Basselin
et
al.
1996,
in
press).
To
see
to
what
extent
this
diminished
uptake
influences
the
intracellular
pool
of
free
poly
-
amines
and
their
biosynthetic
precursors
argi-
nine
and
ornithine,
the
level
of
these
molecules
was
determined
in
wild
-type,
pentamidine-
treated
and
pentamidine-resistant
promastigotes
(Table
I).
According
to
HPLC
analyses
(Basselin
et
al.
1996)
when
cells
were
treated
with
high
pent-
amidine
concentration
(100
iLM)
for
a
short
time
(2
hr),
the
intracellular
pentamidine
con-
centration
was
197
iLM
in
L.
donovani
and
100
iLM
in
L.
amazonensis.
Under
these
conditions,
the
putrescine
level
increased
and
the
levels
of
arginine
and
ornithine
decreased,
suggesting
in-
creased
ODC
activity,
catalyzing
the
synthesis
of
putrescine
from
ornithine.
However,
when
the
cells
were
submitted
to
a
long-time
treat-
ment
(24
hr)
with
low
pentamidine concentra-
tion
(10
iLM
for
L.
donovani
and
1µM
for
L.
amazonensis),
leading
to
intracellular
pentami-
dine
concentrations
of
39
iLM
in
L.
donovani
and
6.2
iLM
in
L.
amazonensis
(Basselin
et
al.
1996),
not
only
the
intracellular
arginine
and
ornithine
pools continued
to
decrease
but
the
level
of
putrescine
was
also
severely
lowered
(by
56%
in
L.
donovani
and
by
72%
in
L.
ama-
zonensis).
These
results
indicate
that
intracellu-
lar
accumulation
of
pentamidine
for
a
longer
period
induces
severe
alterations
in
ODC
activ-
ity
and/or
synthesis
and
unavailability
of
bio-
synthetic
precursors.
Interestingly,
under
both
conditions,
spermidine
level
remained
stable.
This
suggests
that
the
synthesis
of
this
poly
-
amine
is
not
altered
by
pentamidine
or
that
pu-
trescine
is
rapidly
transformed
into
spermidine.
We
described
earlier
that
pentamidine-
resistant
clones
have
stimulated
arginine
uptake
from
the
external
medium
(Basselin
et
al.
1996,
in
press).
In
spite
of
this
fact,
in
these
cells,
the
putrescine
level
is
very
low
(68%
less
in
L.
donovani
and
84%
less
in
L.
amazonensis)
compared
to
that
seen
in
their
respective
wild
-
type
clones.
Again
the
spermidine
level
does
not
vary.
Since
the
resistant
cells
multiply
(al-
though
with
a
much
longer
generation
-time;
Basselin
et
al.,
in
press),
it
seems
that
the
low
intracellular
putrescine
level
does
not
alter
cell
-
viability.
ODC
activity.
To
examine
if
the
decreased
putrescine
pool
was
a
direct
consequence
of
a
diminution
in
the
enzymatic
conversion
of
or-
nithine
to
putrescine,
the
level
of
ODC
was
as-
sessed.
As
shown
in
Table
II,
ODC
activity
in
the
two
species
was
different
due
probably
to
TABLE
I
Intracellular
Concentrations
of
Pentamidine,
Polyamines,
and
Their
Biosynthetic
Precursors
in
Leishmania
Intracellular
concentration
(RM)
Pentamidine
Arginine
Ornithine
Putrescine
Spermidine
L.
donovani
Wild
-type
1620
1537 1387
1888
Pent
100
/..LM
2
hr
197
1103
936
2172
2005
Pent
10
/..LM
24
hr
39
535
33
602
1922
Pent
R
40
2757
1855
434
1955
L.
amazonensis
Wild
-type
119
1021
986
846
Pent
100
/..LM
2
hr
100
84
1000
1161
895
Pent
1µM
24
hr
6.2
49
392
273
909
Pent
R
20
1161
1273
154
839
Note.
Promastigotes
in
the
exponential
phase
of
growth
were
processed
as
described
under
Materials
and
Methods.
The
results
(mean
values
for
two
different
experiments)
are
expressed
in
1.,LM
±
20%.
EFFECTS
OF
PENTAMIDINE
ON
Leishmania
277
strain
variations.
Cells
treated
with
100
iLM
pentamidine
for
2
hr
(corresponding
to
intracel-
lular
pentamidine
concentration
of
197
and
100
iLM
for
L.
donovani
and
L.
amazonensis,
re-
spectively)
expressed
twofold
higher
ODC
ac-
tivity
than
the
untreated
control.
After
a
24
-hr
treatment
with
low
pentamidine
concentration,
levels
of
ODC
decreased
in
both
strains
below
the
control
values.
Under
these
conditions
the
intracellular
pentamidine
concentration
was
39
and
6.2
iLM
for
L.
donovani
and
L.
amazonen-
sis,
respectively
(Basselin
et
al.
1996).
In
resistant
clones,
the
apparent
maximal
ve-
locity
(V
max
)
was
reduced
compared
to
that
of
wild
-type
cells
by
2.5-
to
4
-fold
for
L.
donovani
and
L.
amazonensis,
respectively,
whereas
ap-
parent
K
m
values
were
similar,
indicating
that
ODC
from
sensitive
and
resistant
cells
have
identical
affinities
for
ornithine.
With
a
crude
extract
as
enzyme
source,
pent-
amidine
at
100
and
500
iLM
in
the
assay
stimu-
lated
ODC
activity
by
20
and
50%
for
L.
dono-
vani
and
by
44
and
140%
for
L.
amazonensis.
Putrescine
and
spermidine
had
no
effect
up
to
8
mM
on
enzyme
activities
from
control
and
re-
sistant
cells,
but
ODC
from
the
resistant
cells
was
more
sensitive
to
inactivation
by
DFMO.
Expression
of
ODC
in
wild
-type
and
resistant
promastigotes.
In
order
to
determine
whether
TABLE
II
Kinetic
Parameters
of
ODC
Apparent
kinetic
parameters
V
ma
,
(nmol/mg/hr)
(mIVI)
L.
donovani
Wild
-type
28.2
±
1.1
3.2
±
0.4
Pent
100
I.LM
2
hr
58.1
±
1.5
2.9
±
0.5
Pent
10
I.LM
24
hr
16.4
±
0.5
2.8
±
0.5
Pent
R
40
11.3
±
1.2
2.5
±
0.3
L.
amazonensis
Wild
-type
12.7
±
1.6
2.7
±
0.3
Pent
100
I.LM
2
hr
21.1
±
1.3
2.9
±
0.4
Pent
1
RM
24
hr
5.3
±
1.2
2.3
±
0.6
Pent
R
20
3.1
±
0.6
2.5
±
0.4
Note.
The
ODC
activities
were
measured
in
cell
-free
ex-
tract.
The
kinetic
parameters
were
determined
from
the
Lin-
eweaver-Burk
plot.
These
results
represent
the
mean
values
±
SD
for
three
different
experiences.
the
diminution
in
ODC
activity
in
resistant
cells
can
be
attributed
to
decreased
levels
of
ODC
protein,
the
amounts
of
ODC
in
wild
-type
and
resistant
promastigotes
of
L.
donovani
were
compared
by
titration
with
DFMO
which
binds
irreversibly
and
specifically
to
active
ODC
(Metcalf
et
al.
1978)
and
by
Western
blot
using
monoclonal
mouse
ODC-antibody.
Equal
amounts
of
soluble
proteins
(800
p,g)
of
both
clones
were
incubated
with
DFMO
and
the
rate
of
[
14
C]DFMO
incorporation
was
mea-
sured.
Results
indicate
that
1
U
of
enzyme/mg
of
protein
(1
U
represents
1
nmol
of
pentami-
dine/30
min/37°C)
is
inactivated
by
binding
of
126
and
57
pmol
of
DFMO/mg
of
protein
for
wild
-type
and
resistant
cells.
Assuming
that
one
molecule
of
DFMO
is
needed
to
inactivate
each
subunit
of
ODC,
this
indicates
that
1
U/mg
of
protein
is
equivalent
to
about
9.6
and
4.3
p,g
of
active
ODC
protein/mg
of
protein
for
wild
-type
and
resistant
cells,
respectively.
So
the
amount
of
enzymatically
active
ODC
present
in
crude
extract
in
resistant
cells
is
twice
lower
than
that
in
wild
-type
ones.
To
verify
the
specificity
of
the
monoclonal
antibody,
purified
ODC
from
Escherichia
coli
and
the
bacterial
crude
extract
were
used
as
positive
controls
in
analyses
by
Western
blot
(Fig.
1,
lanes
c
and
d)
as
their
ODC
proteins
contain
about
the
same
number
of
amino
acids
as
the
ODC
of
the
parasite:
732
for
E.
coli
and
707
for
L.
donovani
(Hanson
et
al.
1992).
Other
proteins
with
lower
molecular
weight also
re-
acted
with
the
monoclonal
antibody.
These
proteins
probably
correspond
to
degration
products
of
E.
coli
ODC
(lanes c
and
d).
Figure
1
(lanes
a
and
b)
revealed
a
cross
-reactivity
between
anti-ODC
of
L.
donovani
and
mouse
(462
amino
acids)
and
showed
a
single
band
of
around
70
kDa,
corresponding
to
the
76,000
±
4000-ODC
subunit
determined
from
SDS-gel
electrophoretograms
of
DFMO-
resistant
cell
extracts,
affinity
labeled
with
[
3
H]DFMO
(Coons
et
al.
1990).
This
band
is
less
intense
in
pentamidine-resistant
L.
dono-
vani
(lane
b)
that
in
the
wild
-type
(lane
a),
in-
dicating
a
lower
amount
of
ODC
protein
in
the
resistant
clone.
278
BASSELIN
ET
AL.
1m
.
1 1
1 1
1
di(
94
kD
+1111(
68
kD
du<
43
kD
-4111(
30
kD
a
b
c
d
e
FIG.
1.
Western
blot
analysis
of
wild
-type
and
pentami-
dine-resistant
L.
donovani
promastigotes
with
mouse
anti-
ODC
(
1
/
1
0
dilution).
The
same
amount
of
proteins
of
each
sample
is
loaded
on
the
gel.
(a)
Wild
-type,
(b)
pentamidine-
resistant,
(c)
purified
ODC
from
Escherichia
coli
(Sigma),
(d)
crude
extract
of
ODC
from
E.
coli,
and
(e)
molecular
weight
standards.
Quantification
of
ODC
was
assessed
by
scan-
ning
densitometry
of
the
blots
and
showed
that
the
amount
of
ODC
in
wild
-type
is
2.4
-fold
higher
than
in
resistant
parasites.
Effect
of
DFMO
on
cell
viability
and
its
up-
take.
DFMO
inhibited
the
growth
of
wild
-type
promastigotes
with
an
IC
50
of
90
and
110
µM
and
of
resistant
ones
with
710
and
865
µ/V/
for
L.
donovani
and
L.
amazonensis,
respectively.
Thus,
resistant
clones
were
eightfold
more
re-
sistant
to
DFMO
than
the
wild
-type
clones.
A
modification
of
DFMO
uptake
could
be
at
the
origin
of
a
decreased
sensitivity
to
this
mol-
ecule.
Both
the
rate
of
[
14
C]DFMO
uptake
and
the
plateau
were
2-
to
2.7
-fold
lower
in
resistant
cells
reaching
a
plateau
of
half
the
value
reached
by
the
wild
-type
clones
(Fig.
2).
Ac-
cording
to
published
cell
volume
of
L.
donovani
(Phelouzat
et
al.
1995),
the
intracellular
DFMO
concentration
in
wild
-type
and
resistant
clones
were
calculated
to
be
389
and
217
µM,
respec-
tively,
after
22
hr,
whereas
the
extracellular
concentration
was
33
µN/.
This
represents
a
12
-
and
6.5
-fold
accumulation
of
the
drug
within
Total
uptake
(cpm/pg
protein)
400
,
300
200
a
,
a
100
10
15
20
25
Time
(h)
FIG.
2.
Uptake
of
[
14
C]DFMO
into
wild
-type
and
pent-
amidine-resistant
L.
donovani.
Promastigotes
of
L.
dono-
vani
were
incubated
with
33
1.,LM
[
14
C]DFMO
for
different
times
and
then
processed
as
described
under
Materials
and
Methods.
Results
are
expressed
as
cpm/pg
protein.
Wild
-
type
(■)
and
pentamidine-resistant
(❑).
these
cells,
indicating
active
transport
in
con-
trast
with
the
results
published
for
T.
brucei
brucei
(Bitonti
et
al.
1986a)
where
DFMO
en-
ters
by
passive
diffusion.
Spermidine
synthase
activity.
Spermidine
synthase
catalyzes
the
conversion
of
putrescine
to
spermidine.
An
increase
of
the
activity
of
this
enzyme
could
have
contribute
to
the
lower
pu-
trescine
level
in
pentamidine-treated
and
pent-
amidine-resistant
cells.
To
measure
this
activity,
the
cell
extract
was
diluted
100
fold:
spermine
synthase
activity
was
no
more
detectable
under
this
condition.
In
cell
-
free
extract
as
enzyme
source,
pentamidine
is
a
non
competitive
inhibitor
of
spermidine
syn-
thase
with
respect
to
putrescine
and
dcSAM.
Its
apparent
affinity
is
different
for
the
two
wild
-
type
strains.
In
both
resistant
clones,
the
K,
of
pentamidine
is
decreased
only
for
the
putrescine
binding
site
of
the
enzyme
(Table
III),
indicat-
ing
a
decreased
affinity
for
this
inhibitor.
In
Leishmania
grown
in
the
presence
of
drug
for
24
hr,
enzyme
activity
was
lowered
in
the
case
of
L.
amazonensis
where
the
apparent
Vmax
and
IC'
values
were
reduced
by
three-
and
six
-fold,
respectively.
In
both
resistant
cells,
spermidine
synthase
activity
was
decreased
more
particularly
in
L.
donovani.
The
affinity
for
putrescine
was
in-
creased
in
both
clones,
whereas
that
for
dcSAM
was
modified
only
in
L.
amazonensis.
EFFECTS
OF
PENTAMIDINE
ON
Leishmania
279
TABLE
III
Kinetic
Parameters
of
Spermidine
Synthase
Apparent
kinetic
paramaters
Putrescine
dcSAM
Pe"'
K
Pe"'
L.
donovani
Wild
-type
111
185
134
148
2.5
97
Pent
10
piM
24
hr
83
139
144
2.9
Pent
R
40
30
53
562
42
3.2
66
L.
amazonensis
Wild
-type
248
180
343
526
10.5
357
Pent
1µM
24
hr
86
33
473
12.5
Pent
R
20
104
60
767
245
4.3
331
Note.
The
spermidine
synthase
activities
were
measured
in
cell
-free
extract.
The
kinetic
parameters
were
determined
from
the
Lineweaver—Burk
plot.
These
results
represent
the
mean
values
±
10%
for
two
different
experiences.
The
results
are
expressed
in
pmol/pig/hr
for
V
max
and
I.LM
for
K„,
and
K
1
for
pentamidine.
DISCUSSION
In
an
earlier
work,
we
have
shown
that
pent-
amidine
enters
into
leishmanial
promastigotes
via
polyamine
transporters
(Basselin
et
al.
1996)
and
that
the
establishment
of
pentami-
dine-resistance
involves
altered
polyamine
up-
take
(Basselin
et
al.,
in
press).
The
aim
of
this
work
was
to
examine
further
the
effect
of
pent-
amidine
on
the
level
of
intracellular
polyamines
and
on
their
biosynthesis
in
pentamidine-treated
and
pentamidine-resistant
Leishmania.
The
polyamines
studied
were
putrescine
and
sper-
midine.
According
to
the
literature,
spermine
is
present
only
in
trace
amounts
in
different
pro-
mastigotes
of
Leishmania
(Bachrach
et
al.
1979a;
Coombs
and
Sanderson
1985;
Keithly
and
Fairlamb
1987;
Balana-Fouce
et
al.
1991a),
undetectable
by
our
analytical
method.
The
differences
of
the
intracellular
contents
of
putrescine,
ornithine,
arginine
as
well
as
the
differences
in
the
ODC
and
spermidine
synthase
activities
between
the
two
wild
-type
strains
are
not
unexpected,
as
such
variations
among
spe-
cies
were
frequently
reported
(Bachrach
et
al.
1979a;
Schnur
et
al.
1979;
Balana-Fouce
et
al.
1991a;
Carrera
et
al.
1994).
Our
work
clearly
indicates
that
pentamidine
treatment
and
resistance
developed
to
this
drug
have
no
effect
on
the
intracellular
level
of
sper-
midine.
Such
stability
was
also
observed
by
Coons
et
al.
(1990)
and
by
Balana-Fouce
et
al.
(1991a)
with
DFMO
and
by
Balana-Fouce
et
al.
(1991a)
with
berenil
treatment.
Maintenance
of
the
intracellular
spermidine
concentration
seems
to
be
an
important
requirement
for
cell
viability.
Another
interesting
observation
is
that
even
a
sixfold
diminution
of
the
intracellular
putres-
cine
level
below
the
control
value
does
not
af-
fect
cell
viability
but
may
contribute
to
de-
creased
growth
rate.
In
the
pentamidine-
resistant
cells,
the
putrescine
level
is
severely
reduced,
due
to
lower
amount
of
ODC
in
addi-
tion
to
decreased
uptake
and
increased
efflux
of
this
polyamine
(Basselin
et
al.,
in
press).
These
results
suggest
also
that
the
regulation
of
the
two
polyamines
is
different
and
independent.
The
response
of
the
wild
-type
cells
of
the
two
species
to
pentamidine
treatment
seems
to
be
time
and
concentration
dependent.
An
intracel-
lular
pentamidine
concentration
of
100
ptiV/
or
higher
for
a
short
time
stimulates
ODC
activity
leading
to
increased
putrescine
production
and
to
a
decrease
of
the
concentration
of
the
precur-
sors,
arginine
and
ornithine.
Thus,
it
seems
that
the
cells
can
overcome
even
a
strong
but
short
pentamidine
pulse.
However,
a
longer
treatment
even
with
lower
pentamidine
concentrations
leads
to
significantly
decreased
putrescine/
spermidine
ratio,
decreased
ODC
activity,
and
decreased
concentration
of
biosynthetic
precur-
sors.
These
cells
are
no
more
dividing
and
are
280
BASSELIN
ET
AL.
dying
if
the
treatment
is
prolonged.
It
seems
that
the
establishment
of
pentamidine
resistance
re-
quires
adaptation
to
this
low
putrescine
concen-
tration
and
to
altered
membrane
properties.
The
highly
stimulated
arginine
uptake
and
concen-
tration
in
the
resistant
cells
is
difficult
to
ex-
plain,
but
as
it
is
not
directly
used
for
putrescine
formation,
it
is
probably
involved
in
Krebs
cycle
or
in
the
respiratory
chain.
The
catabolism
of
arginine
through
y-guanidinobutyramide
pathway
(Bera
1987)
produces
succinate
which
enters
the
cycle
or
the
electron
transport
chain.
Thus,
in
resistant
cells,
ornithine
pool
is
not
significantly
elevated
although
ODC
activity
is
reduced,
so
it
is
probably
converted
to
a-keto-
glutarate
via
glutamate
(Simon
et
al.
1983)
and
enters
also
the
tricarboxylic
acid
cycle.
Estab-
lishment
of
an
intracellular
amino
acid
pool
pro-
vides
a
means
for
the
storage
of
metabolic
in-
termediates
capable
of
generating
energy
and
contributes
to
the
maintenance
of
ionic
balance.
The
second
enzyme
in
polyamine
biosynthe-
sis
affected
by
pentamidine
in
wild
-type
and
in
resistant
cells
is
spermidine
synthase
which
has
never
been
characterized
before
in
Leishmania.
Its
activity
in
pentamidine-treated
and
pentam-
idine-resistant
cells
is
reduced
probably
partly
due
to
lower
availability
of
the
substrate
putres-
cine.
Bachrach
et
al.
(1979b)
found
also
that
this
drug
inhibited
spermidine
synthesis
from
r
14
c1putrescine
in
promastigotes
of
L.
mexi-
cana,
L.
tropica,
and
L.
donovani
after
a
5
-hr
incubation
period.
To
explain
the
stability
of
intracellular
level
of
free
spermidine
in
resistant
cells
in
spite
of
decreased
uptake,
increased
ef-
fl
ux
(Basselin
et
al.,
in
press),
increased
affinity
to
the
substrate
and
decreased
affinity
to
pent-
amidine,
several
mechanisms
are
possible
such
as
the
altered
conversion
of
spermidine
to
spermine
and/or
to
dihydrotrypanothione,
or
a
rapid
glutathionylspermidine
deconjugation
to
free
spermidine
and
dihydrotrypanothione
as
re-
ported
for
Crithidia
fasciculata
(Shim
and
Fair
-
lamb
1988).
The
interconversion
of
spermine
to
spermidine
is
unlikely
as
the
enzymes
involved
in
this
way,
polyamine
oxydase
and
spermine
acetyl
transferase,
were
not
yet
detected
in
Leishmania;
furthermore,
pentamidine
is
a
known
competitive
inhibitor
of
these
enzymes
in
vitro
(Libby
and
Porter
1992).
Data
presented
in
this
paper
show
that
poly
-
amines
biosynthesis
is
among
the
targets
of
pentamidine
in
Leishmania.
Changes
in
poly
-
amine
levels
might
be
one
of
the
reasons
of
the
overall
efficacy
of
the
antileishmanial
action
of
this
molecule
since,
as
suggested
by
Bacchi
(1981),
this
agent
might
in
fact
act
by
compet-
ing
with
polyamines
for
binding
to
nucleic
acid,
particularly
kinetoplast
DNA.
Polyamine
bio-
synthesis
is
altered
in
resistant
clones;
this
seems
to
be
the
consequence
and
not
the
prime
cause
of
the
resistance.
ACKNOWLEDGMENTS
We
thank
Victor
Villanueva
for
polyamines
analyses.
This
work
was
partly
supported
by
an
INSERM
Grant
921304.
M.
Basselin
is
the
recipient
of
a
Ph.D.
fellowship
from
the
Ministere
de
la
Recherche
et
de
la
Technologie.
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