Jean-Loup Lemesre

Antimonial-Mediated DNA Fragmentation in Leishmania infantum Amastigotes

ABSTRACT The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes ofLeishmania infantum at low concentrations (10 μg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 μg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.

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.

Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death.

The Silent Information Regulator (SIR2) family of genes have been cloned from a variety of species ranging from bacteria to man. In previous studies, we reported the characterization of a Leishmania major gene encoding a protein with extensive homology to yeast SIR2p and expressed by different Leishmania species and parasite developmental stages and thus termed LmSIR2. Unlike the yeast SIR2p, LmSIR2p is mainly localized within the cytoplasm. In the present study, sequencing of a homologue encoding gene in another Leishmania species, Leishmania infantum, revealed 93% overall amino acid identity with L. major SIR2 gene. Further, using L. infantum as a recipient for a plasmid vector (pTEX) which allows overexpression of LmSIR2p led to the accumulation of the protein in the parasite cytoplasm of both promastigote and amastigote forms and a striking increase in the survival of amastigotes, the vertebrate stage of the parasite, when maintained under normal axenic culture conditions. This phenotype was also observed when L. infantum parasites were transfected with a cosmid vector (CLHyg), isolated from a L. infantum cosmid library, carrying the L. infantum SIR2 gene (CLHyg-LiSIR2). In contrast, no effect was observed on survival of the promastigote forms (insect stage) under similar culture conditions. However, when the glucose was used as a unique source of energy under starvation conditions, the viability of promastigotes was significantly enhanced. Moreover, we showed that amastigote forms in the stationary phase of culture died with a feature of apoptosis as revealed by the appearance of YOPRO-1 positive cells and that expression of LmSIR2 protein substantially delays this phenomenon. Taken together, these results demonstrate the existence of SIR2-related proteins encoding genes in different Leishmania species and suggest that LmSIR2p could participate among other factors in the control of cell death.

Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro

Leishmaniasis is a significant cause of morbidity and mortality in tropical and subtropical areas. These protozoan parasites exist as a flagellate extracellular promastigote form in their sandfly vectors. In the mammalian hosts, only the nonflagellate amastigote form persists, surviving and dividing in the phagolysosome of macrophages. Leishmania promastigotes have often been quantified in vitro by an enzymatic method that involves the conversion of a tetrazolium salt, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), into a colored, insoluble formazan product, the amount of which depends on the number of viable parasites present (Mossman 1983; Cole 1986; Rabinovitch et al. 1986; Berg et al. 1994). The use of amastigote forms has been hampered by difficulties in obtaining sufficient amounts of the purified mammalian stage. Toward that end, we and other investigators have recently developed culture systems for the in vitro growth of large amounts of extracellular amastigote forms that are free of host-cell contamination (Bates 1993; Lemesre, patent PCT/FR 94/00577). In the present paper we report the use of an MTT-based microassay to quantify the proliferation and the viability of axenically grown amastigote forms of L. amazonensis. The efficiency of the method was compared with that of another conventional method, cell counting. A general procedure was used in the current study to generate large quantities of each of the parasite stages. Active and dividing populations of axenically grown amastigote forms of L. amazonensis (MHOM/BR/76/ LTB-012) were maintained at 32 ± 1 °C by weekly subpassage in a cell-free medium called MAA (medium for axenically grown amastigotes) in 25-cm flasks (Lemesre et al. 1994; Lemesre, patent PCT/FR 94/00577). From a starting inoculum of 5 × 10 amastigote forms/ ml a cell density of about 5 × 10 parasites/ml was obtained on day 7. Promastigote cultures were maintained at 25 ± 1 °C in RPMI 1640 medium (Gibco BRL) buffered with 25 mM HEPES and 2 mM NaHCO3 (pH 7.2) and supplemented with 20% heat-inactivated fetal calf serum (FCS). Initial parasite concentrations were 5 × 10 flagellates/ml of medium. For verification of the correlation between the MTT-based assay and the number of parasites, increasing concentrations of latelog-phase parasites, ranging from 2.5 to 40 × 10 cells/ well, were distributed in triplicate in 100 ll of media in 96-well flat-bottom microtrays. Plates were then incubated with 10 ll of MTT (10 mg/ml) for 4 h at 25° or 32 ± 1 °C, depending upon the parasite stage. The enzyme-substrate reaction was stopped by the addition of 100 ll of a solution of 50% isopropanol and 10% sodium dodecyl sulfate (SDS; pH 5.4). Microplates were further incubated for 30 min at room temperature. The optical density (OD) was determined at 570 nm with a Titertech 96-well scanner. To evaluate the possibility of using the MTT-based assay to achieve growth kinetics curves as is possible with cell-counting experiments, microorganisms were seeded at two inocula (1 and 2 × 10 parasites/well) and the dehydrogenase activities were measured daily as described above. Cell concentrations were determined by daily counting of parasites in a Thoma chamber at 400x magnification after adequate dilution in 0.01 M phosphate-buffered saline (PBS, pH 7.2). Morphological aspects of parasites were determined daily by microscope examination to detect the possible occurrence of differentiation phenomena in 96well plates. As shown in Fig. 1, for densities higher than or equal to 2.5 × 10 parasites/well a linear correlation between the OD signal and the parasite number was revealed when both amastigote and promastigote forms were used. Amastigote forms of L. amazonensis showed lower levels of dehydrogenase activities than did their corresponding promastigote forms. This difference was Parasitol Res (1997) 83: 401–403  Springer-Verlag 1997

Leishmania major: Cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family

In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (eg logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti‐LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [35S]methionine‐labeled promastigote antigens after pulse chase experiments, using anti‐LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted‐secreted antigens (ESA). Moreover, immunoflurescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.

Efficacy of second line drugs on antimonyl-resistant amastigotes of Leishmania infantum.

In a previous paper we have demonstrated that the induction, by direct drug pressure, of a resistance to Sb(III) antimony at physiological concentration in the amastigote stage of the parasite, led to a high cross-resistance to Sb(V) species in the form of Glucantime. In this paper, further chemoresistant clones were characterized. Axenic amastigotes of Leishmania infantum were adapted to survive in culture medium containing 4, 20, 30 and 120 microg/ml of potassium antimonyl tartrate Sb(II). These mutants were 12, 28, 35 and 44-fold more resistant to Sb(III) than the parental wild-type clone. They were able to resist at concentrations of Glucantime Sb(V) as high as 160 microg/ml when growing in THP-1 cells. We have investigated the efficacy of second line drugs in clinical use (pentamidine and amphotericin B) on the antimony-resistant mutants. Amphotericin B was toxic for both wild-type and chemoresistant mutants at concentrations ranging from 0.05 to 0.15 microM. Pentamidine which is extensively used when the first course of antimonial pentavalent compounds is unsuccessful, was more toxic for all the chemoresistant organisms than for the wild-type clone. In the same way, chemoresistant amastigotes growing within THP-1 cells were more susceptible to pentamidine than the wild-type clone. Our results showed that the resistance of the mutants was restricted to the antimony containing drugs and did not led to a cross-resistance against the other clinically relevant drugs. These results confirmed that these two drugs (pentamidine and amphotericin B) are good candidates to treat pentavalent antimonial unresponsiveness.

Effective humoral and cellular immunoprotective responses in Li ESAp-MDP vaccinated protected dogs

Cell-mediated and humoral immunity were explored in LiESAp-MDP vaccinated protected dogs versus susceptible placebo dogs 2 months and 8 months post-vaccination. As previously described, a strong and long-lasting cell-mediated immunity, critical for protection against Leishmania infantum was exclusively revealed in vaccinated dogs as confirmed by a positive response to the intradermal inoculation of leishmanin and by a significant higher anti-leishmanial activity of canine monocytes-derived macrophages. Moreover, our results support the view that cooperation of humoral antibody with cell-mediated immunity might be important in developing protective immunity in LiESAp-MDP vaccinated dogs. Anti-LiESAp serum samples were found functionally active in vitro, promoting (i) early killing of pretreated promastigotes and amastigotes, (ii) strong inhibitory effect on the in vitro growth of both parasitic developmental stages of L. infantum and (iii) most importantly, a significant inhibition of pretreated promastigotes in vitro infectivity in canine macrophages. However, anti-LiESAp antibody response was not implicated in the promastigotes-amastigotes differentiation process. In these experiments, we have added additional support to the concept that antibodies to Leishmania may be important in developing protective immunity.

Phenotypical characteristics, biochemical pathways, molecular targets and putative role of nitric oxide-mediated programmed cell death in Leishmania

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania, both in vitro and in vivo. We investigated the type of cell death process induced by NO for the intracellular amastigote stage of the protozoa Leishmania. Specific detection methods revealed a rapid and extensive cell death with morphological features of apoptosis in axenic amastigotes exposed to NO donors, in intracellular amastigotes inside in vitro - activated mouse macrophages and also in activated macrophages of regressive lesions in a leishmaniasis-resistant mouse model. We extended our investigations to the dog, a natural host-reservoir of Leishmania parasites, by demonstrating that co-incubation of infected macrophages with autologous lymphocytes derived from dogs immunised with purified excreted-secreted antigens of Leishmania resulted in a significant NO-mediated apoptotic cell death of intracellular amastigotes. From the biochemical point of view, NO-mediated Leishmania amastigotes apoptosis did not seem to be controlled by caspase activity as indicated by the lack of effect of cell permeable inhibitors of caspases and cysteine proteases, in contrast to specific proteasome inhibitors, such as lactacystin or calpain inhibitor I. Moreover, addition of the products of two NO molecular targets, cis-aconitase and glyceraldehyde-3-phosphate dehydrogenase, also had an inhibitory effect on the cell death induced by NO. Interestingly, activities of these two enzymes plus 6-phosphogluconate dehydrogenase, parasitic enzymes involved in both glycolysis and respiration processes, are overexpressed in amastigotes selected for their NO resistance. This review focuses on cell death of the intracellular stage of the pathogen Leishmania induced by nitrogen oxides and gives particular attention to the biochemical pathways and the molecular targets potentially involved. Questions about the role of Leishmania amastigotes NO-mediated apoptosis in the overall infection process are raised and discussed.

Experimental studies on the evolution of antimony-resistant phenotype during the in vitro life cycle of Leishmania infantum: implications for the spread of chemoresistance in endemic areas

Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasites life cycle.

Lower Nitric Oxide Susceptibility of Trivalent Antimony-Resistant Amastigotes of Leishmania infantum

ABSTRACT We previously documented the induction of Leishmania amastigote apoptosis by trivalent antimony (SbIII) and nitric oxide (NO). We demonstrate here that SbIII-resistant amastigotes were resistant to NO toxicity when delivered extracellularly by NO donors or intracellularly via macrophage activation. Shared biochemical targets for SbIII and NO resistance in Leishmania are discussed.