Leishmania (Mundinia) spp.: from description to emergence as new human and animal Leishmania pathogens.

During the last 20 years, Leishmania (Mundinia) spp. have emerged as new causative agents of human and animal leishmaniases. We provide a historical view of these parasites, from their initial description to their emergence as pathogens, to help avoiding future confusion in species assignation of these newly emerging pathogens.

Synthesis of aminophenylhydroxamate and aminobenzylhydroxamate derivatives and in vitro screening for antiparasitic and histone deacetylase inhibitory activity.

A series of aminophenylhydroxamates and aminobenzylhydroxamates were synthesized and screened for their antiparasitic activity against Leishmania, Trypanosoma, and Toxoplasma. Their anti-histone deacetylase (HDAC) potency was determined. Moderate to no antileishmanial or antitrypanosomal activity was found (IC50 > 10 µM) that contrast with the highly efficient anti-Toxoplasma activity (IC50 < 1.0 µM) of these compounds. The antiparasitic activity of the synthetized compounds correlates well with their HDAC inhibitory activity. The best-performing compound (named 363) express a high anti-HDAC6 inhibitory activity (IC50 of 0.045 ±â€¯0.015 µM) a moderate cytotoxicity and a high anti-Toxoplasma activity in the range of known anti-Toxoplasma compounds (IC50 of 0.35-2.25 µM). The calculated selectivity index (10-300 using different human cell lines) of the compound 363 makes it a lead compound for the future development of anti-Toxoplasma molecules.

In vitro susceptibility to antimonials and amphotericin B of Leishmania infantum strains isolated from dogs in a region lacking drug selection pressure.

The aim of this study was to evaluate the susceptibility to anti-leishmanial agents of 24 strains isolated from dogs living in the urban area of Alger lacking drug selection pressure. Two different Leishmania infantum zymodemes, MON-1 and MON-281, were identified in these dogs. The in vitro susceptibility to the main forms of antimonial and amphotericin were assessed on promastigote and amastigote life stages in culture. The results obtained for both parasite life stages were concordant whatever the molecule tested. Moreover, our data showed that isolates belonging to the relatively rare zymodeme of L. infantum, MON-281, were less susceptible to antimony than MON-1, when at the same time there was no significant difference for amphotericin B.

The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target.

Preliminary study on the galectin molecular diversity in Moroccoan Phlebotomus papatasi sandfly population.

CONTEXT: Galactose binding protein (PpGalec) plays an important role in the specificity of Phlebotomus papatasi sandfly for Leishmania major. The molecular diversity of this ligand is currently unknown but might have some influence on the ability of PpGalec to efficiently recognize L. major in natural sandfly populations. OBJECTIVE: To explore the molecular diversity of the P. papatasi Galectin gene (PpGalec) in natural sandfly population of Morocco. RESULTS & CONCLUSIONS: Sequence variations of PpGalec was analyzed in 31 P. papatasi specimens collected from endemic and non-endemic zoonotic cutaneous leishmaniasis foci of Morocco. Among the 211 amino acid positions analyzed, 11 are subjected to mutation. Interestingly, we observe that one mutation directly affect an amino acid known to be involved in the substrate recognition by galectin. The repercussion of this polymorphism on the capacity of the galectin to efficiently bind the L. major Lipophosphoglycane (LPG) awaits further investigations.

Bacterial flora as indicated by PCR-temperature gradient gel electrophoresis (TGGE) of 16S rDNA gene fragments from isolated guts of phlebotomine sand flies (Diptera: Psychodidae).

In this study, we tested the capacity of Temperature Gradient Gel Electrophoresis (TGGE)-based fingerprinting of 16S rDNA PCR fragments to assess bacterial composition in a single isolated sand fly gut. Bacterial content was studied in different life stages of a laboratory-reared colony of Phlebotomus duboscqi and in a wild-caught Phlebotomus papatasi population. Our study demonstrates that a major reorganization in the gut bacterial community occurs during metamorphosis of sand flies. Chloroflexi spp. was dominant in the guts of pre-imaginal stages, although Microbacterium spp. and another as yet unidentified bacteria were detected in the gut of the adult specimen. Interestingly, Microbacterium spp. was also found in all the adult guts of both species. We demonstrate that the analysis of bacterial diversity in an individualized sand fly gut is possible with fingerprinting of 16S rDNA. The use of such methodology, in conjunction with other culture-based methods, will be of great help in investigating the behavior of the Leishmania-bacterial community in an ecological context.

Malformations of the genitalia in male Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae).

Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae) is a major vector of Leishmania major (Kinetoplastida: Trypanosomatidae), a causative agent of zoonotic cutaneous leishmaniasis. Morphological characters of sand fly genitalia are key indicators for species identification. Various anomalies affecting male genitalia have been previously described. We take advantage of a large sand flies survey conducted in 32 stations in Central and Southern Morocco to systematically quantify the prevalence and spatial distribution of malformations affecting the genitalia of P. papatasi. Among 597 examined males, 122 were abnormal (20.4%). Malformations were widespread and largely concerned the number of spines in the lateral lobes and in the styles. Asymmetrical anomalies in lateral lobes were common. Correspondence analysis of our results highlighted the symmetrical anomalies observed in the lateral lobes, and abnormal styles of the male genitalia were found to be associated with environmental disturbances since they were prevalent in sewage dumps.

Leishmania infantum: tuning digitonin fractionation for comparative proteomic of the mitochondrial protein content.

Leishmania infantum belongs to the Kinetoplastidae that is characterized by a specific mitochondrial DNA, the kinetoplast. This parasite is responsible for both benign cutaneous leishmaniasis and severe visceral leishmaniasis in humans. Molecular determinants of such differences in pathogenesis are not well understood, and the parasites as well as their hosts may contribute to the disease phenotype. Factors that help parasite to adapt its metabolism to nutritional conditions encountered in different location might play pivotal roles in controlling parasite development in these various host environments. Thus, we have decided to initiate studies aimed to compare the mitochondrial protein content of L. infantum. To avoid the drawback caused by the most abundant proteins such as tubulin and proteins of the cytoskeleton present in whole cell extract, we have decided to fractionate the subcellular components of the cells. Using both cytosolic and mitochondrial markers, we have improved a protein pre-fractionation protocol using digitonin that allowed us to generate an enriched mitochondrial fraction.

Advances and perspectives in Leishmania cell based drug-screening procedures.

Efforts for the development of new therapeutics, essential for the control of leishmaniasis rely mainly on screening of potentially effective compounds in pathogen growth/multiplication assays, both in vitro and in vivo. Screenings designed to closely reflect the situation in vivo are currently labor-intensive and expensive, since they require intracellular amastigotes and animal models. Screenings designed to facilitate rapid testing of a large number of drugs are not performed on the clinically relevant parasite stage, but the promastigotes. The ability to select transgenic Leishmania expressing reporter proteins, such as the green fluorescent protein (GFP) or the luciferase, opened up new possibilities for the development of drug screening tests. In this review we will focus on available methodologies for direct drug screening purposes against the mammalian stage of the parasite, with emphasis on the future developments that could improve sensitivity, reliability, versatility and the throughput of the intracellular model screening.

Looking for putative functions of the Leishmania cytosolic SIR2 deacetylase.

During the past few years, the silent information regulator SIR2 protein family has attracted great interest due to its implication in an organism's life span extension. They bear diverse subcellular localization and play a role in transcriptional silencing and DNA repair. The biochemical reaction catalysed by these enzymes (nicotinamide adenine dinucleotide-dependant deacetylase/adenosine diphosphate-ribosyl transferase) is supposed to be linked to metabolism. Members of this protein family were described in parasitic organisms, but little information is available on potential functions of such enzymes in these organisms. In this article, we review recent information on structure and peculiar functions of SIR2s in eukaryotes, with emphasis on parasitic protozoa, particularly the Trypanosomatidae. Through the enzyme localization and the diverse substrates and by-products of the enzymatic reactions, we approach the potential pathways in which the Leishmania cytosolic SIR2 protein can be involved.

Conversion of Trypanosoma cruzi Tc52 released factor to a protein inducing apoptosis.

In this study Tc52, a Trypanosoma cruzi released protein, which exerts an immunoregulatory activity, was converted to a molecular form with altered biological function. Indeed, the genetic fusion of Tc52 to a carrier protein, the Shistosoma japonicum glutathione S-transferase (Tc52-Sj26), was shown to induce apoptosis in spleen cells from BALB/c or CBA mice and the human T-cell leukemic cell line (CEM). Cell death by apoptosis was evidenced by the following criteria: (1) increased binding of Annexin V to rTc52-treated spleen cells; (2) the presence of an ordered cleavage of the DNA backbone; (3) double labeling showed increased number of T cells undergoing apoptosis upon incubation with rTc52; (4) the use of a CEM cell line and TUNEL assay allowed to show in situ DNA fragmentation. Surprisingly, intraperitoneal injections of rTc52 to BALB/c mice, which were then infected with T. cruzi, resulted in increased parasiteamia levels and is congruent to 2.5 times increase of macrophages number. Since native Tc52 could not trigger, apoptosis of T cells we could hypothesized that the fusion of Tc52 with Sj26 led to conformational changes resulting in apoptosis inducing properties of rTc52. The possible in vivo physiopathological implications of these finding were discussed.

Lower nitric oxide susceptibility of trivalent antimony-resistant amastigotes of Leishmania infantum.

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.

Differential infectivity and immunopathology in murine experimental infections by two natural clones belonging to the Trypanosoma cruzi I lineage.

Immunopathology of Chagas' disease in Balb/c mice infected with 2 Trypanosoma cruzi clones, belonging to the T. cruzi I lineage and presenting different in vitro virulence (P/209 cl1 > SO34 c14) was compared. In the acute phase, evading mechanisms such as parasite-induced lymphocyte polyclonal activation and T cell immunosuppression were higher in mice infected with the clone giving a higher parasitaemia (P/209 cl1). A similar increase of non-specific isotypes was observed in both infections with IgG2a prevalence. Interestingly, CD8+ cell hypercellularity and lymphocyte immunosuppression were observed during the chronic phase (245 days post-infection) in mice infected by the most virulent clone. In the same way, the parasite-specific antibody response was more intense in P/209 cl1-infected mice over the acute phase. During the chronic phase this response remarkably dropped down in SO34 cl4-infected mice exclusively. Finally, P/209 cl1-infected mice presented a more severe inflammation and tissue damage in heart and quadriceps than SO34 cl4-infected mice. This comparative study showed differences between the two clones: a higher virulence in vivo being clearly associated with a greater ability to induce evasion mechanisms and severe tissue damage.

In vitro antileishmanial activity of nicotinamide.

Our study represents the first report demonstrating the antileishmanial activity of nicotinamide (NAm), a form of vitamin B(3). A 5 mM concentration of NAm significantly inhibited the intracellular growth of Leishmania amastigotes and the NAD-dependent deacetylase activity carried by parasites overexpressing Leishmania major SIR2 (LmSIR2). However, the transgenic parasites were as susceptible as the wild-type parasites to NAm-induced cell growth arrest. Therefore, we conclude that NAm inhibits leishmanial growth and that overexpression of LmSIR2 does not overcome this inhibition. The mechanism of the inhibition is not defined but may include other in vivo targets. NAm may thus represent a new antileishmanial agent which could potentially be used in combination with other drugs during therapy.

Identification of antibodies to Leishmania silent information regulatory 2 (SIR2) protein homologue during canine natural infections: pathological implications.

Dogs are the domestic reservoir of zoonotic visceral Leishmaniasis caused by Leishmania infantum in the Mediterranean basin and thus constitute an important health problem in both human and veterinary medicine. Until vaccines become available, conventional measures such as epidemiological surveillance including reservoir control will be among the practical options for prevention and containment of the disease. We have recently characterised novel Leishmania sp. genes encoding parasite proteins named (LmS3a: homologous to mammalian ribosomal protein S3a; LmSIR2: homologous to the silent information regulatory 2 protein family; LimTXNPx: homologous to the peroxiredoxin family with N-terminal mitochondrial leader sequence) that may contribute to the host immune dysfunction in murine experimental Leishmaniasis. In the present study we have investigated the humoral responses against the parasite antigens in groups of L. infantum-infected dogs with different clinical status: symptomatic and asymptomatic with DTH positive or negative test. The determination of immunoglobulin (Ig) isotypes revealed high levels of total IgG in both symptomatic and asymptomatic animals when compared to IgM. Furthermore, the IgG2 appeared to be the predominant subclass of Ig present in the sera of infected animals particularly in the case of symptomatic dogs. The IgG subclass reactivity analysis revealed a broad specific recognition range of parasite recombinant antigens. Interestingly, differential profiles of IgG1 and IgG2 antibody reactivity were observed in asymptomatic and symptomatic dogs. The LmSIR2 protein was found to be a highly reactive molecule with IgG2 from most of the asymptomatic and symptomatic animals. Considering the fact that LmSIR2 secreted by the parasites can be bound and taken up by neighbouring cells, the latter could be a target for anti-LmSIR2 antibodies and this may contribute to the immunopathological alterations and host tissue damage. The implications of these observations in the pathogenesis of Leishmaniasis are 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 parasite's life cycle.

Antimonial-mediated DNA fragmentation in Leishmania infantum amastigotes.

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 of Leishmania infantum at low concentrations (10 microg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 microg/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.

In vitro growth of Leishmania amazonensis promastigotes resistant to pentamidine is dependent on interactions among strains.

The in vitro growth of promastigote cells of Leishmania amazonensis was found to strongly depend on interactions among strains that differed in their pentamidine resistance. In particular, the growth of resistant strains was reduced when they shared the same environment with a less-resistant strain.

DNA transformation of Leishmania infantum axenic amastigotes and their use in drug screening.

Protocols for DNA electroporation in Leishmania promastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 microM (0.12 microg/ml), 55 microg/ml, 95 microg/ml, and 0.12 microg/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferase-expressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.