Philippe Holzmuller

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.

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.

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.

Identification of total and differentially expressed excreted-secreted proteins from Trypanosoma congolense strains exhibiting different virulence and pathogenicity.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south-east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted-secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host-parasite interactions. We used a combined proteomic approach (one-dimensional SDS-PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.

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.

Transcriptomics and proteomics in human African trypanosomiasis: Current status and perspectives

Human African trypanosomiasis, or sleeping sickness, is a neglected vector-borne parasitic disease caused by protozoa of the species Trypanosoma brucei sensu lato. Within this complex species, T. b. gambiense is responsible for the chronic form of sleeping sickness in Western and Central Africa, whereas T. b. rhodesiense causes the acute form of the disease in East Africa. Presently, 1.5 million disability-adjusted life years (DALYs) per year are lost due to sleeping sickness. In addition, on the basis of the mortality, the disease is ranked ninth out of 25 human infectious and parasitic diseases in Africa. Diagnosis is complex and needs the intervention of a specialized skilled staff; treatment is difficult and expensive and has potentially life-threatening side effects. The use of transcriptomic and proteomic technologies, currently in rapid development and increasing in sensitivity and discriminating power, is already generating a large panel of promising results. The objective of these technologies is to significantly increase our knowledge of the molecular mechanisms governing the parasite establishment in its vector, the development cycle of the parasite during the parasites intra-vector life, its interactions with the fly and the other microbial inhabitants of the gut, and finally human host-trypanosome interactions. Such fundamental investigations are expected to provide opportunities to identify key molecular events that would constitute accurate targets for further development of tools dedicated to field work for early, sensitive, and stage-discriminant diagnosis, epidemiology, new chemotherapy, and potentially vaccine development, all of which will contribute to fighting the disease. The present review highlights the contributions of the transcriptomic and proteomic analyses developed thus far in order to identify potential targets (genes or proteins) and biological pathways that may constitute a critical step in the identification of new targets for the development of new tools for diagnostic and therapeutic purposes.

Secretome of Animal Trypanosomes

Animal trypanosomosis is one of the most severe constraints to agricultural development in sub‐Saharan Africa and is also an important disease of livestock in Latin America and Asia. The causative agents are various species of protozoan parasites belonging to the genus Trypanosoma, among which T. congolense and T. evansi are the major pathogenic species. The extracellular position of trypanosomes obliges us to consider both the parasite and its excreted/secreted factors in the course of the physiopathologic process. The advent of proteomics led us to propose a comparative approach of the proteome (i.e., the whole parasite content) and the secretome (i.e., naturally excreted/secreted molecules) of T. congolense and T. evansi with particular attention to common and specific molecules between strains of differing virulence and pathogenicity. The molecular identification of differentially expressed trypanosome molecules correlated with either the virulence process or the pathogenicity will provide new potential molecular targets for improved field diagnosis and chemotherapy of animal trypanosomosis.

Serodiagnosis of bovine trypanosomosis based on HSP70/BiP inhibition ELISA

Animal trypanosomosis is a serious constraint to livestock productivity in tropical and sub-tropical countries. The pathogenic trypanosomes in bovidae are Trypanosoma congolense, T. vivax, T. brucei and T. evansi. Current serological tests to detect trypanosome infections are based on the use of whole trypanosome lysates; their potential is limited by antigen instability, lack of reproducibility and lack of test specificity due to the antibodys long persistence after treatment. The development of new tests based on recombinant technology that could be standardized and applied on a large scale at low cost would be very helpful. The major invariant antigen recognized by T. congolense infected cattle belongs to the heat shock protein (HSP) 70 family and is closely related to mammalian Immunoglobulin Binding Protein (BiP). To improve the initial ELISA based on a recombinant fragment of HSP70/BiP, we developed an inhibition ELISA using an anti-BiP monoclonal antibody and a full-length fusion protein expressed in E. coli. Here we report on the development of the test and provide an initial assessment of its performance using sets of sera from experimental infections and from naturally infected cattle maintained in tsetse infested areas of Africa. The HSP70/BIP-based inhibition ELISA shows a good sensitivity in cattle experimentally infected with T. congolense, with an improved sensitivity in secondary infections. One major advantage, particularly for its further application in national laboratories, is that one single set of reagents and one single procedure are sufficient to apply on different mammalian host species infected with different trypanosome species.