Nitric Oxide-Mediated Proteasome-Dependent Oligonucleosomal DNA Fragmentation in Leishmania amazonensis Amastigotes

Abstract

Resistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages through the l-arginine-nitric oxide (NO) metabolic pathway. Here we investigate the cell death process induced by NO for the intracellular
protozoan Leishmania amazonensis. Exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite NaNO2 or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages
resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis techniques (including
electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and in situ recognition of DNA strand breaks
by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] assay) revealed a rapid and extensive
fragmentation of nuclear DNA in both axenic and intracellular NO-treated amastigotes of L. amazonensis. Despite some similarities to apoptosis, the nuclease activation responsible for characteristic DNA degradation was not under
the control of caspase activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine
proteases. In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or calpain
inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data strongly suggest that NO-induced
oligonucleosomal DNA fragmentation in Leishmania amastigotes is, at least in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways
leading up to cell death might ultimately allow the identification of new therapeutic targets.

Full text

INFECTION AND IMMUNITY, July 2002, p. 3727–3735 Vol. 70, No. 7
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.7.3727–3735.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Nitric Oxide-Mediated Proteasome-Dependent Oligonucleosomal
DNA Fragmentation in Leishmania amazonensis Amastigotes
Philippe Holzmuller,
1
Denis Sereno,
1
Mireille Cavaleyra,
1
Isabelle Mangot,
1
Sylvie Daulouede,
2
Philippe Vincendeau,
2
and Jean-Loup Lemesre
1
*
UR 008 Pathogénie des Trypanosomatidés, Institut de Recherche pour le Développement, 34032 Montpellier Cedex 1,
1
and
Laboratoire de Parasitologie, Université Victor Segalen Bordeaux II, 33076 Bordeaux Cedex,
2
France
Received 4 September 2001/Returned for modification 15 November 2001/Accepted 21 March 2002
Resistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages
through the L-arginine–nitric oxide (NO) metabolic pathway. Here we investigate the cell death process
induced by NO for the intracellular protozoan Leishmania amazonensis. Exposure of amastigotes to moderate
concentrations of NO-donating compounds (acidified sodium nitrite NaNO
2
or nitrosylated albumin) or to
endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages
resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis
techniques (including electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and
in situ recognition of DNA strand breaks by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-
biotin nick end labeling] assay) revealed a rapid and extensive fragmentation of nuclear DNA in both axenic
and intracellular NO-treated amastigotes of L. amazonensis. Despite some similarities to apoptosis, the
nuclease activation responsible for characteristic DNA degradation was not under the control of caspase
activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine proteases.
In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or
calpain inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data
strongly suggest that NO-induced oligonucleosomal DNA fragmentation in Leishmania amastigotes is, at least
in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways
leading up to cell death might ultimately allow the identification of new therapeutic targets.
Leishmania spp. are obligate intracellular protozoan para-
sites of macrophages that are responsible for a wide range of
human diseases, including self-healing skin lesions, diffuse cu-
taneous and mucosal lesions, or fatal visceral infections. In the
Leishmania life cycle, two principal parasite forms exist: (i) the
amastigote form that develops inside mononuclear phagocytes
of a vertebrate host and (ii) the motile promastigote form that
develops in the vector gut. The various possible outcomes of
leishmanial infection have been associated with expansion of
specific T helper lymphocyte populations (52). Immune control
of leishmaniasis involves a dominant Th1 response, leading to
macrophage activation and elimination of intracellular para-
sites through the induction of nitric oxide synthase (NOS II)
and NO synthesis from L-arginine. This prototypical model has
been largely evidenced in murine leishmaniasis (17, 22, 33).
Human activated macrophages can also induce antileishmanial
activity via the L-arginine NO pathway (44, 58, 60). Studies on
human cutaneous leishmaniasis reveal that Leishmania killing
is associated with NO production (40). Moreover, the capacity
of canine macrophages to eliminate intracellular amastigotes
through a NO-dependent mechanism has been documented
(46, 55, 59).
NO-dependent cytostatic and/or cytotoxic activities by acti-
vated macrophages on various parasites have been clearly
demonstrated (20, 26, 29, 33, 57). In Leishmania infections,
NO-generating creams applied topically to lesions revealed a
modest efficacy in mouse models, whereas they were able to
cure human patients (15, 65). Moreover, studies have shown
that successful chemotherapy in a murine and canine model of
visceral leishmaniasis depended on upregulation of the L-argi-
nine NO pathway (14, 58). However, the cellular and molecu-
lar mechanisms whereby NO exerts its cytotoxic activity are not
yet well understood. NO toxicity results from complex phe-
nomena involving several molecular targets. In antitumoral
models, impairment of essential cellular functions, including
mitochondrial respiration, DNA synthesis by blocking ribonu-
cleotide reductase (31), and enzyme inactivation through Fe/S
center alterations or S nitrosylation, have been documented
(16, 19). Recent studies found that several parasite targets may
be affected by NO toxicity in Leishmania parasites. These tar-
gets include metabolic enzymes, such as GAPDH (glyceralde-
hyde-3-phosphate dehydrogenase) and aconitase (29, 32) or
cysteine proteinase (51).
Apoptosis represents an important mechanism by which ex-
ogenous or endogenous NO mediates toxicity in mammalian
cells (1, 36, 41, 42, 63). We investigated the genomic DNA
status of NO-treated Leishmania amazonensis amastigotes.
Several apoptosis detection methods (appearance of the char-
acteristic DNA ladder banding pattern on agarose gels and in
situ TUNEL [terminal deoxynucleotidyltransferase-mediated
dUTP-biotin nick end labeling] and flow cytofluorometry as-
says) revealed that Leishmania cell death was associated with
extensive nuclear DNA fragmentation. Cell-permeable
caspase inhibitors did not modify NO-mediated nuclear DNA
* Corresponding author. Mailing address: UR 008 Pathogénie des
Trypanosomatidés, IRD (Institut de Recherche pour le Développe-
ment), 911 Ave. Agropolis, BP 5045, 34032 Montpellier Cedex 1,
France. Fax: 33(0)4-67-41-63-30. Phone: 33(0)4-67-41-62-20. E-mail:
lemesre@melusine.mpl.ird.fr.
3727

fragmentation. In contrast, endonuclease activation could be a
consequence of Ca
2⫹
-sensitive calpain and/or proteasome ac-
tivity. These data strongly suggest that NO can induce damage
in the unicellular pathogen Leishmania by a coordinated pro-
cess of intracellular protein degradation mediated by the pro-
teasome, which may lead to nuclear DNA fragmentation. An
apoptosis-like cell death pathway could represent an important
and highly regulated mechanism used for the clearance of
Leishmania within infected macrophages stimulated to pro-
duce NO endogenously or during treatments with NO-releas-
ing drugs.
MATERIALS AND METHODS
Reagents. Fetal calf serum (FCS) was obtained from Dutscher S.A. RPMI
1640 medium (lot 0MB0174) was purchased from BioWhittaker Europe, and
L-glutamine (lot 3414) was from BioMedia. Penicillin-streptomycin (10,000
IU/ml to 10,000 UG/ml; lot 3037222) and phosphate buffer saline (Ca
2⫹
and
Mg
2⫹
free) were obtained from Life Technologies. Bacterial lipopolysaccharide
(LPS), gamma interferon (IFN-␥; mouse recombinant), bovine serum albumin
(BSA; fraction V, lot 59H0696), and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-di-
phenyltetrazolium bromide; lot 66H5033) were purchased from Sigma. RNase
was obtained from Eurogentec, proteinase K was from Promega, and agarose
was purchased from Eurobio. YOPRO-1 iodide was provided by Molecular
Probes. Nitro-L-arginine, inhibitory peptides Z-DEVD-CMK and Z-VAD-FMK,
E-64d [(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester],
calpain inhibitor I, lactacystin, proteasome inhibitor II, and MG-115 were pur-
chased from Alexis Biochemicals. All other cellular-grade and molecular biolo-
gy-grade chemicals were obtained from Sigma. Cell-free MAA/20 culture me-
dium for axenically grown amastigotes was defined according to the method of
Lemesre et al. (29, 30). Nitrosylated albumin was prepared as previously de-
scribed (38).
Animals. Female BALB/c mice (Iffa Credo, Saint-Germain-sur-l’Arbresle,
France) were housed under conventional conditions and given water and chow
ad libitum. The use of the animals conformed to institutional guidelines.
Parasites and in vitro cultures. A cloned line of L. amazonensis (MHOM/BR/
76/LTB-012) was used in all experiments. L. amazonensis axenically grown amas-
tigote forms were maintained at 32 ⫾1°C by weekly subpassages in MAA/20
medium. From a starting inoculum of 5 ⫻10
5
amastigote forms/ml, a cell density
of ca. 2 ⫻10
7
parasites/ml was obtained on day 7. Axenically grown amastigote
forms appeared homogeneous, round to ovoid, without apparent flagella, and
nonmotile. Axenically grown amastigotes from various Leishmania species
clearly resembled intracellular amastigotes with regard to their ultrastructural,
biological, biochemical, and immunological properties (5, 6, 13, 24, 29, 30, 53).
Cell concentrations were determined by daily counts with a hemocytometer at a
⫻400 magnification after adequate dilution in phosphate-buffered saline (PBS)
containing 0.25% glutaraldehyde.
Treatment with the NO donors sodium nitrite (NaNO
2
) and nitrosylated
albumin (NO-BSA). Late-log-phase amastigote forms were washed three times
in PBS–0.01 M (pH 4.5) before treatment. Parasites (10
7
/ml) were resuspended
in the same buffer containing 5 mM sodium nitrite. After 20 min, 2 h, 4 h, 6 h,
and 12 h of incubation at 32 ⫾1°C in the dark, respectively, parasites were
washed in Ca
2⫹
-Mg
2⫹
-free PBS (0.01 M, pH 7.2) before analysis. Control amas-
tigotes were treated in the same way but in the absence of sodium nitrite.
Albumin or nitrosylated albumin (4 mg/ml) was added to the parasite cultures.
After incubation for 20 min, 4 h, or 12 h at 32 ⫾1°C, the amastigotes were
washed three times in Ca
2⫹
-Mg
2⫹
-free PBS (0.01 M, pH 7.2) before analysis.
Assessment of NO donors’effect on amastigote survival. NO-treated and
untreated amastigotes (2 ⫻10
8
/ml) were resuspended in MAA/20 medium for
each incubation time. The MTT-based microassay was used to estimate NO-
induced death as described before (53). Tetrazolium bromide has the property of
being reduced by parasitic dehydrogenases into a dark blue insoluble formazan
product, the amount of which depends on the number of viable amastigotes.
Briefly, parasites (100 ␮l) were seeded in a 96-well microplate. After a 1-h
incubation, 10 ␮l of MTT (10 mg/ml) was added to each well, and the plates were
further incubated for 4 h. The enzyme reaction was then stopped by the addition
of 100 ␮l of 50% isopropanol–10% sodium dodecyl sulfate. The plates were
incubated for an additional 30 min while being agitated at room temperature
before the optical density value at 570 nm was determined with a Titer-Tech
96-well scanner (Labsystems Multiscan EX).
In vitro macrophage infections. Mouse peritoneal macrophages were washed
with prewarmed RPMI 1640 medium supplemented with 10% heat-inactivated
FCS, 2 mM glutamine, 100 U of penicillin/ml, and 100 ␮g of streptomycin/ml and
cultured overnight in 16-well Lab-Tek tissue culture slides (Nalge Nunc Inter-
national). Nonadherent cells were removed by two washes with prewarmed
RPMI medium, and then the macrophages were infected with stationary-phase
extracellular amastigotes at a parasite/macrophage ratio of 3:1 for2hat34°C
with 5% CO
2
. Noninternalized parasites were removed by gentle washing. In-
fected macrophages were then cultured in the presence or absence of the fol-
lowing reagents: LPS (10 ng/ml), gamma interferon (IFN-␥; 100 U/ml), and a
combination of LPS and IFN-␥. Controls were made by adding 1 mM nitro-L-
arginine, a competitive inhibitor of NOS II, or 1 mM nitro-L-arginine plus 2 mM
L-arginine to revert the inhibition.
Culture supernatants were collected for nitrite production measurement 48 h
later. Macrophages were then washed with prewarmed RPMI, fixed with meth-
anol, and stained with Giemsa for parasite counts or processed for the TUNEL
technique (see below). The percent parasite index (PI) inhibition compared to
the untreated controls was calculated as follows: percent PI ⫽100 ⫺[(mean
number of amastigotes per macrophage ⫻percent infected macrophages in
treated wells)/(mean number of amastigotes per macrophage ⫻percentage of
infected macrophages in untreated wells)] ⫻100. The results were taken as the
mean of duplicate experiments.
Measurement of nitrite production. NO
2⫺
accumulation in the medium over
48 h for the leishmanicidal assay was used as an indicator of NO production and
was assayed by the Griess reaction (50). Briefly, 60 ␮l of Griess reagent A (1%
sulfanilamide in 1.2 N HCl) and 60 ␮l of Griess reagent B [0.3% N-(1-naphth-
yl)ethylenediamine] were added to 100 ␮l of each supernatant in triplicate wells
in a 96-well plate. Plates were read at 540 nm in an enzyme-linked immunosor-
bent assay plate reader (Labsystems Multiskan EX). NaNO
2
in RPMI was used
to construct a standard curve for each plate reading.
In situ TUNEL assay. Slides with infected macrophages or axenic amastigotes
were fixed for 20 min with PBS containing 4% paraformaldehyde, washed twice
with 0.01 M PBS (pH 7.2), and stored at ⫺20°C until use. DNA fragmentation
was analyzed in situ by using a colorimetric apoptosis detection system (Promega,
Madison, Wis.) according to the manufacturer’s instructions. After performance
of the TUNEL protocol, preparations were analyzed under a microscope at
⫻1,000 magnification. Apoptotic nuclei appeared dark brown. Other nuclei were
not colored.
DNA agarose gel electrophoresis. Cell pellets were incubated in lysis buffer (10
mM Tris, 10 mM EDTA, 0.5% Triton X-100 [pH 7.4]) for 30 min at 4°C.
Qualitative analysis of DNA fragmentation was performed as previously de-
scribed (7) by agarose gel electrophoresis of DNA extracted from 5 ⫻10
8
axenically grown amastigotes. DNA was then visualized under UV light after the
gels were stained with ethidium bromide.
Flow cytofluorometry analysis with YOPRO-1. The percentage of apoptotic
cells was quantitated by flow cytofluorometric analysis by using the DNA inter-
calatant YOPRO-1 as previously described (25). Briefly, 10
6
L. amazonensis
amastigotes were incubated with 10 ␮M YOPRO-1 for 10 min. Cells were
immediately analyzed with a FACScan flow cytometer (Becton Dickinson, Ivry,
France) by using an argon-ion laser tuned to 488 nm. Green cell fluorescence,
gated on forward scatter and side light scatter, was collected by using a (525 ⫾
10)-nm band-pass filter (FL1) and displayed by using a logarithmic amplification.
Statistical analysis. Statistical significance was analyzed by Student’sttest. All
experiments were performed at least twice.
RESULTS
Kinetics of NO-mediated effect on L. amazonensis amasti-
gote viability. Treatment of L. amazonensis extracellular amas-
tigotes with both NO donors led to a dramatic loss of parasite
viability with regard to morphological changes such as de-
creased cell volume under microscopic examination (data not
shown), as well as a decrease in dehydrogenase activities as
determined by the MTT micromethod (Fig. 1). Exposure of
axenically grown amastigotes to NO generated from acidified 5
mM NaNO
2
resulted in a time-dependent cell death (Fig. 1A).
Significant toxicity could be detected as early as 20 min after
treatment with the chemical (ca. 40% of amastigote mortality).
Survival gradually decreased; ⬍10% of the microorganisms
were viable after a 12-h exposure to NO generated from
3728 HOLZMULLER ET AL. INFECT.IMMUN.

NaNO
2.
Incubation of amastigotes in PBS (pH 4.5) in the
absence of added NaNO
2
did not significantly affect parasite
viability in the first few hours, and fewer than 25% amastigotes
were dead after 12 h (Fig. 1A).
About 30% of amastigotes exposed to 4 mg of NO-BSA/ml
were killed within the first 20 min of incubation, reaching ca. 65
and 90% after 4 and 12 h of incubation, respectively (Fig. 1B).
The addition of albumin alone at 4 mg/ml failed to affect
amastigote viability, even after 12 h of incubation (Fig. 1B),
and no striking morphological change was observed under the
microscope (not shown).
The relevance of the observations above would remain ques-
tionable unless similar effects could be observed in intracellu-
lar amastigotes of L. amazonensis exposed to endogenous NO
produced by activated macrophages. Mouse peritoneal macro-
phages infected with axenically cultured amastigotes and acti-
vated for 24 and 48 h with IFN-␥and LPS led to a time-
dependent intracellular killing of Leishmania as determined by
the reduction of PIs of ca. 55% at 24 h and 90% at 48 h (Fig.
2B), concomitant with NO
2
⫺
accumulation in culture fluids
(Fig. 2A). NO
2
⫺
production and antileishmanial activity both
decreased in the presence of 1 mM nitro-L-arginine, a compet-
itive inhibitor of NOS II. They were almost totally restored by
the addition of 2 mM L-arginine (Fig. 2). Inhibition of the PI
did not exceed 20% when incubation conditions did not induce
NO production (Fig. 2B).
NO-mediated apoptosis-like DNA fragmentation of L. ama-
zonensis amastigotes. NO-mediated DNA fragmentation ex-
hibiting features of apoptosis was first assessed by using extra-
cellular amastigotes incubated 5 mM NaNO
2
and monitoring
the genomic DNA status of treated versus untreated parasites.
As shown in Fig. 3 (lanes 5, 7, and 9), nuclear DNA fragmen-
tation into oligonucleosomal-sized fragments (720, 360, and
180 bp), a typical feature of apoptotic cells, was readily visible
in the case of NO-treated amastigotes. Untreated amastigotes
did not show any DNA fragmentation, even after6hofincu-
bation in acidic conditions (Fig. 3, lanes 2, 4, 6, and 8). Inter-
estingly, NO-mediated nuclear DNA fragmentation (720-bp
multimer) could be detected as early as after 2 h of contact
with the NO donor (Fig. 3, lane 5). After6hofcontact with the
chemical, the entire genomic DNA of amastigotes was mostly
fragmented into oligonucleosome-sized DNA of 360 and 180
bp (Fig. 3, lane 9), the end products of DNA alteration in
apoptosis.
Endonuclease activity was also evaluated by the TUNEL
assay. As shown in Fig. 4C, nuclei of axenically grown amasti-
gotes exposed to 5 mM NaNO
2
were intensely stained dark
FIG. 1. Effects of 5 mM acidified sodium nitrite (A) or 4 mg of
nitrosylated albumin/ml (B) on the death of L. amazonensis amasti-
gotes. Parasite viability was assessed by the MTT micromethod as
described by Sereno and Lemesre (53). Statistical significance was
determined by the Student’sttest. Results are expressed as the mean
and standard deviation of three independent experiments made in
triplicate. Statistical significance: ⴱ,P⬍0.05; ⴱⴱ,P⬍0.01; ⴱⴱⴱ,P⬍
0.001.
FIG. 2. NO-dependent cytotoxicity against L. amazonensis in acti-
vated mouse macrophages. Macrophages were infected with L. ama-
zonensis amastigotes at a cell/parasite ratio of 1:3 in RPMI 1640 me-
dium supplemented with 10% FCS. Infected cells were treated with
LPS, IFN-␥, LPS plus IFN-␥, LPS plus IFN-␥plus nitro-L-arginine (a
competitive inhibitor of NOS II), and LPS plus⫹IFN-␥plus nitro-L-
arginine plus L-arginine (to reverse NOS II inhibition). NO
2
⫺
levels in
supernatants (A) and PI inhibitions in cells (B) were determined 24
and 48 h later. The results represent the mean and standard deviation
of two independent experiments made in duplicate. Statistical signifi-
cance: ⴱ,P⬍0.05; ⴱⴱ,P⬍0.01; ⴱⴱⴱ,P⬍0.001.
VOL. 70, 2002 DNA FRAGMENTATION IN LEISHMANIA AMASTIGOTES 3729

brown after a 4-h incubation in contrast to those of untreated
parasites (Fig. 4A). Corresponding controls incubated without
terminal deoxynucleotidyl transferase (TdT) enzyme in order
to determinate the specificity of the reaction did not show any
staining (Fig. 4B and D). Interestingly, kinetoplast DNA of
NO-treated and untreated amastigotes was also stained. In-
deed, replication of kinetoplast DNA involves the release of
catenated minicircles from the network with subsequent DNA
3⬘-OH tail production stained by biotinylated dUTP via the
TdT enzyme. Similarly, the in situ TUNEL technique revealed
that the nuclear DNA fragmentation of intracellular amasti-
gotes also occurred in Leishmania-infected stimulated macro-
phages (Fig. 5). Moreover, a strong correlation between nu-
clear DNA fragmentation, increased NO
2
⫺
levels, and
antileishmanial activity was observed (Fig. 2 and 5). Labeled
amastigote nuclei could only be visualized inside activated
macrophages producing NO (Fig. 5D and F). Nuclei of intra-
cellular amastigotes, which we can visualized after Giemsa
staining, were free of label in nonactivated macrophages (Fig.
5A) and in macrophages stimulated with LPS alone (B), with
IFN-␥alone (C), or with LPS and IFN-␥plus 1 mM nitro-L-
arginine (E). In the absence of TdT enzyme, corresponding
negative controls did not exhibit any staining, thus demonstrat-
ing the specificity of the reaction (Fig. 5G and H).
Quantitation and time course of NO-mediated cell death of
L. amazonensis amastigotes. The impermeable DNA interca-
lated YOPRO-1 has been previously used for monitoring the
apoptosis of Trypanosoma cruzi epimastigotes (3, 25). We show
here that it could also selectively differentiate viable, necrotic
(sodium dodecyl sulfate-treated), and apoptotic amastigotes
(Fig. 6). Trivalent antimonial (potassium antimonyl tartrate at
50 ␮g/ml) was used as a positive apoptosis inducer as described
by Sereno et al. (54). Antimonial-mediated apoptotic cells
(Fig. 6C and D) were easily distinguished from living (Fig. 6E
and F) and necrotic cells (Fig. 6A and B) by using the com-
FIG. 3. DNA fragmentation analysis. Agarose gel electrophoresis of untreated amastigote (suspended in PBS [pH 4.5], lanes 2, 4, 6, and 8) and
NO-treated amastigote (suspended in PBS [pH 4.5] plus 5 mM NaNO
2
; lanes 3, 5, 7, and 9) DNA (5 ␮g) is shown. Exposure times: 20 min (lanes
2 and 3), 2 h (lanes 4 and 5), 4 h (lanes 6 and 7), and 6 h (lanes 8 and 9); molecular weights are indicated beside lanes 1 and 10. The experiment
was done four times.
FIG. 4. In situ analysis of L. amazonensis extracellular amastigote
death exhibiting features of apoptosis. (A and B) Untreated (PBS [pH
4.5], 4 h) amastigotes (A) and, as negative control, untreated parasites
incubated without TdT (B). (C and D) NO-treated amastigotes (5 mM
NaNO
2
, 4 h) incubated with (C) or without (D) TdT. DNA fragmen-
tation analysis was determined by the TUNEL method and analyzed
under a microscope at ⫻1,600 magnification. The experiment was
done twice.
3730 HOLZMULLER ET AL. INFECT.IMMUN.

bined analysis of their different patterns of FSC-H properties
and YOPRO-1 staining (FL1-H).
The time course of the apoptosis-like changes was deter-
mined by evaluating the percentage of cells that exhibited the
maximum fluorescence seen in NO-treated amastigotes. Amas-
tigotes incubated in 0.01 M PBS (pH 4.5) for 20 min, 2 h, and
4 h (not shown) and 6 h (Fig. 6E and F) displayed a homoge-
neous population of living cells with a low fluorescence back-
ground. In contrast, a new cell population with a high FL1
fluorescence corresponding to apoptotic cell-like cells ap-
peared in NO-treated amastigotes. This population increased
as a function of exposure time to the NO donor, representing
ca. 14% at 20 min (not shown), 19.9% at 2 h (Fig. 6G and H),
46.9% at 4 h (Fig. 6I and J), and 88.9% at 6 h (Fig. 6K and L).
Interestingly, the time courses of YOPRO-1 staining and of
viability loss were quite similar (Fig. 1 and 6). Altogether, these
data indicated that NO directly and quickly induced extracel-
lular amastigote death through a process that mimics apopto-
sis.
Effects of apoptosis inhibitors. In higher eukaryote cells,
apoptosis often occurs downstream of the death signal by pro-
tease activation, leading to endonuclease ignition (21). In these
instances, cysteine proteases of the caspase family, Ca
2⫹
-sen-
sitive calpains, or proteasomes can initiate cell death. We used
here cytofluorometric analysis with YOPRO-1 staining to test
the relevance of these pathways with apoptosis inhibitors.
Amastigote treatment with apoptosis-blocking agents did not
alter the viability of untreated amastigotes during the time
course experiments (not shown). Whereas caspase inhibitors
reversed the apoptosis of murine peritoneal macrophages in-
duced by 4 mM butyrate as recently described (47), inhibitors
of caspase 3 (inhibitory peptide Z-DEVD-CMK at 5 ␮M),
caspase 1 (inhibitory peptide Z-VAD-FMK at 5 ␮M), or cys-
teine proteases (E-64d at 20 ␮M] had no effect on the cell
death induced by NO (data not shown). In contrast, a signifi-
cant time-dependent inhibitory effect was observed on the NO-
mediated apoptosis-like phenomenon with specific irreversible
proteasome inhibitors (10 ␮M lactacystin and 20 ␮M calpain
inhibitor I) (Fig. 7). Treatment with reversible proteasome
inhibitors (10 ␮M proteasome inhibitor II or 10 ␮M MG-115)
exhibited a less potent and an inversely time-dependent inhib-
itory activity. In the first2hoftreatment, all of the proteasome
FIG. 5. In situ analysis of the apoptosis-like process of L. amazonensis amastigotes in mouse macrophages. Cells were infected with amastigote
forms at a parasite/cell ratio of 3:1 in RPMI 1640 medium supplemented with 10% FCS. Infected cells were activated for 48 h. (A) Untreated
macrophages; (B) macrophages activated with LPS; (C) macrophages activated with IFN-␥; (D) macrophages activated with LPS plus IFN-␥;
(E) macrophages activated with LPS plus IFN-␥plus nitro-L-arginine (a competitive inhibitor of NO synthase type II); (F) macrophages activated
with LPS plus IFN-␥plus nitro-L-arginine plus L-arginine (to reverse inhibition of NOS-II). (G and H) Negative controls, either activated
macrophages (G) or nonactivated macrophages (H), were incubated without TdT. DNA fragmentation analysis was determined by the TUNEL
technique and analyzed under a microscope at ⫻1,000 magnification (A to H). A magnification of ⫻1,600 was used to identify labeled amastigotes
in panel I. The experiment was done twice.
VOL. 70, 2002 DNA FRAGMENTATION IN LEISHMANIA AMASTIGOTES 3731

inhibitors used exerted an inhibitory effect ranging from 20 to
40%. After a 4-h incubation with proteasome inhibitor II and
MG-115, NO-induced DNA changes were reduced by ca. 25%,
whereas lactacystin and calpain inhibitor I produced marked
inhibitions of 47 and 62%, respectively. These percentages
reached ca. 50 and 64%, respectively, after6hofincubation
(Fig. 7). All of these data support the view that the proteasome
pathway could be involved in promoting apoptosis-like changes
in NO-exposed amastigotes.
DISCUSSION
In this study we showed that the leishmanicidal effect of NO
seemed to be the consequence of the induction of a pro-
grammed cell death-like process. NO donors induced a pattern
of DNA fragmentation typical of an apoptotic process in ax-
enic amastigotes of L. amazonensis. This apoptotic phenome-
non was confirmed by both the TUNEL technique and flow
cytometry. This typical aspect of apoptosis was also demon-
strated in the Leishmania intracellular parasite stage inside
NO-producing macrophages, by using the combination IFN-␥
and LPS, which is mainly used as a potent inducer of NOS II.
NO also seems to be able to induce necrosis, as well as
apoptosis (34). Interestingly, NO-mediated apoptosis has been
extensively studied in mammalian cells (1, 2, 12, 18, 28, 36, 63).
Recent reports have shown that unicellular organisms may kill
themselves by an evolutionarily conserved programmed cell
death pathway (i.e., apoptosis) (4). This pathway has been
described in several lower organisms, including the slime mold
Dictyostelium discoideum (11) and several parasitic protozoa.
The effect of chloroquine on a sensitive strain of the malaria
parasite P. falciparum led to an oligonucleosomal DNA frag-
mentation, suggesting that apoptosis may be involved in the
action of chloroquine on the parasite (45). Trypanosoma brucei
brucei and Trypanosoma brucei rhodesiense procyclic forms,
upon treatment with the lectin concanavalin A, died by a pro-
cess similar to apoptosis in mammals (61, 62); changes in
cytoskeletal organization, cytosol vacuolization, and nuclear
DNA fragmentation were reported. Apoptosis-like changes
have also been reported for T. cruzi in response to conditioned
medium or antibiotic G418 (3). A shift in the distribution of
EF-1␣to a nuclear localization was reported as one of the
changes associated with cell death (8). In leishmaniasis, a heat
FIG. 6. Analysis of the NO-induced apoptosis-like process in extracellular amastigotes by a cytofluorometry YOPRO-1 differential staining
technique. Parasites were incubated for6hin0.01 M PBS (pH 7.2) (negative control, not shown), treated for 10 min by 0.1% sodium dodecyl
sulfate (necrosis control, A and B), and treated for 24 h with 50 ␮g of potassium antimonyl tartrate/ml (apoptosis control, C and D). Amastigotes
were, respectively, incubated in 0.01 M PBS (pH 4.5) as an internal control (a 6-h incubation is depicted in E and F) and treated with 5 mM NaNO
2
for2h(GandH),4h(IandJ),or6h(KandL).Theapoptotic cell percentage (R2), corresponding to both reduced forward scatter and high
fluorescence intensity, is indicated in each experimental condition. M1 shows the peak of fluorescence intensity in panels B, D, F, H, J, and L. The
experiment was done three times in duplicate.
3732 HOLZMULLER ET AL. INFECT.IMMUN.

shock was able to induce in promastigotes of L. amazonensis a
DNA cleavage into an oligonucleosomal ladder characteristic
of cells dying by apoptosis (39). In the same way, luteolin and
quercetin, two plant-derived flavonoids, inhibited cell cycle
progression in L. donovani promastigotes, leading to apoptosis
(37). Recently, we demonstrated that the antileishmanial tox-
icity of trivalent antimonials is associated with parasite oligo-
nucleosomal DNA fragmentation, a finding indicative of the
occurrence of late events in the overall apoptosis process (54).
However, the activation of an apoptosis-like machinery in the
parasite stage directly in contact with macrophage antimicro-
bial agents such as NO (i.e., amastigotes) has never been in-
vestigated. In the present study, we demonstrated that both
axenically grown and intracellular L. amazonensis amastigotes,
when submitted to NO action, commit cell suicide through a
pathway that is referred to as apoptosis.
In the last few years it has been established that protease
activation—in cysteine proteases of the caspase family in par-
ticular—is an integral component of the apoptotic cell death
metabolic pathway (64). We report here that cell-permeable
caspase inhibitors had no effect on the oligonucleosomal DNA
fragmentation induced by NO on L. amazonensis amastigotes.
This result correlated with observations made on T. brucei
brucei exposed to reactive oxygen species (48). The inhibitors
used are known, at least, to block caspases that are directly and
indirectly responsible for the activation of endonuclease activ-
ity (i.e., caspases 3 and 1, respectively). NOS II induction is
triggered and regulated by a series of signaling pathways in-
ducing NF-␬B (56). Inhibitors of the proteasome and caspase
3 abrogate the induction of NOS II in macrophages by block-
ing activation of the transcription factor NF-␬B (9, 23). These
inhibitors cannot be used in culture models relying on NOS II
induction. Thus, our experiments of inhibition were restricted
to axenically grown amastigotes. Surprisingly and in contrast to
data obtained from African trypanosomes by Ridgley et al.
(48), specific irreversible proteasome inhibitors (lactacystin
and calpain inhibitor I) markedly inhibited the NO-induced
oligonucleosomal DNA fragmentation of Leishmania extracel-
lular amastigotes, suggesting that an NO-mediated apoptosis-
like process needed proteasome activity to work. This diver-
gence with the observations made by Ridgley et al. (48) might
be explained by the status of cell maturation of the parasite
considered (35). Moreover, evidence is now accumulating that
noncaspases, including cathepsins, calpains, granzymes, and
the proteasome complex, also have roles in mediating and
promoting cell death (27, 43). Recent studies have otherwise
pointed out in two Leishmania species the existence of an
active proteasome, one similar to the proteasomes of other
eukaryotes (10, 49). This potent proteasome might be involved
in the NO-mediated apoptosis-like process. This finding sug-
gests the existence of an ancestral noncaspase protease cell
death pathway.
Finally, NO-mediated cytostatic and cytotoxic effects on ax-
enically grown amastigotes were thus a consequence of the
activation of a cell death program exhibiting features of apo-
ptosis. A proteasome-dependent apoptosis-like machinery is
also present and active in the clinically relevant stage of the
parasite (i.e., amastigote) and can be induced in the host cell by
the L-arginine–NO pathway. However, this apoptotic event
might limit the host’sinflammatory and specific immune re-
sponses, leading to a decrease in NO-dependent parasite kill-
ing and favoring the persistence of some parasites. Neverthe-
less, NO donors have been used to cure murine and human
cutaneous leishmaniasis (15, 65). The investigation of an apo-
ptotic process in cells from NO-treated lesions might merit
further research.
ACKNOWLEDGMENT
We thank Phil Agnew for reviewing the manuscript and for many
useful suggestions.
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Editor: B. B. Finlay
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