Characterisation of atovaquone resistance in Leishmania infantum promastigotes

Atovaquone, an antiparasitic agent, could possibly represent an alternative therapy after relapse following classical treatment for visceral leishmaniasis. Atovaquone-resistant strains were selected in vitro by stepwise drug pressure to study the mechanism of resistance in Leishmania. Characteristics of a promastigote strain resistant to 250 microg/ml of atovaquone were compared with those of the wild type (WT) strain. Resistant strains were shown to have a high level of resistance (45 times). They were stable in drug-free medium for 6 months, and showed no cross-resistance with other antileishmanial drugs. Rhodamine uptake and efflux were studied. They were not modified in the resistant strain, indicating the absence of P-glycoprotein overexpession. The effect of atovaquone on membrane lipidic composition was determined in both WT and atovaquone-resistant promastigotes. Analysis of lipid composition of the atovaquone-resistant strain showed that sterol biosynthesis was decreased in atovaquone-resistant parasites. Cholesterol was found to be the major membrane sterol as opposed to the WT strain. Cholesterol, due to its ordering effect, could decrease membrane fluidity and subsequently block the passage of atovaquone through the membrane. Increased membrane cholesterol content and altered drug membrane fluidity resulted from possible decrease of ergosterol biosynthesis by atovaquone, incorporation of cholesterol by promastigotes in the culture medium, solubilisation of atovaquone by cholesterol and co-passage of the two compounds or influence of dimethylsulfoxide. These results indicate that different cellular alterations may participate in the resistant phenotype, by altering drug membrane permeability.     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.     

Heme metabolism in promastigotes of Leishmania donovani

Promastigotes of Leishmania donovani (Dd-8 strain) showed presence of important key enzymes of heme synthesizing (d-aminolevulinic acid synthase and ferrochelatase) and degrading (heme oxygenase and biliverdin reductase) systems, classical leishmanicidal drugs viz allopurinol, amphotericin B, pentamidine and CDRI compound 93/202 inhibited the heme oxygenase activity of the parasite, whereas, -aminolevulinic acid synthase activity practically remained unaffected. The Km, Vmax ad pH values of heme oxygenase of promastigotes were found to be 1666 M hemin, 625 nmol of bilirubin formed h-1 mg protein-1 and 7.5 respectively. The findings suggest the presence and importance of heme metabolism in the de novo synthesis of different hemoproteins of the Leishmania parasite as well as the detoxification and its defence against biological insults.     

Leishmania infantum (Protozoa, kinetoplastida): transmission from infected patients to experimental animal under conditions that simulate needle-sharing

Morillas-Marquez, F., Martin-Sanchez, J., Acedo-Sanchez, C., Pineda, J. A., Macias, J., and Sanjuan-Garcia, J. Leishmania infantum (Protozoa, kinetoplastida): Transmission from infected patients to experimental animal under conditions that simulate needle-sharing. Experimental Parasitologym100, 71-74.     

Stage-specific proteinases of Leishmania mexicana mexicana promastigotes

Four distinct bands of cysteine proteinase activity were detected when stationary-phase populations of Leishmania mexicana mexicana were subjected to gelatin-SDS-PAGE. The highest mobility band contained at least three isoforms separable by mono Q anion exchange chromatography. These high mobility activities were distinct from all the major amastigote enzymes. Stationary-phase promastigote populations also contained two acid-activable precursor forms of the promastigote-specific band. It is suggested that these promastigote-specific activities occur in the infective metacyclic stage of the parasite and may have a role in parasite survival upon inoculation into a mammal.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major

The genus Leishmania includes many pathogenic species which are genetically very distant. The possibility of genetic exchange between different strains is still an important and debated question. Very few genetic hybrids (i.e., offspring of genetically dissimilar species) have been described in Leishmania. In this study, we report the first example of genetic hybrids occurring between two divergent Leishmania species, Leishmania infantum and Leishmania major. These two species have distinct geographical distributions and are transmitted by different vector species to different mammalian reservoir hosts. These hybrid strains were isolated in Portugal from immunocompromised patients and characterized by molecular and isoenzymatic techniques. These approaches showed that these chimeric strains probably contained the complete genome of both L. major and L. infantum. We believe this is the first report of genetic hybrids between such phylogenetically and epidemiologically distant species of Leishmania. This raises questions about the frequency of such cross-species genetic exchange in natural conditions, modalities of hybrid transmission, their long term maintenance as well as the consequences of these transfers on phenotypes such as drug resistance or pathogenicity.     

Detection of cysteine-proteinases in Leishmania amazonensis promastigotes using a cross-reactive antiserum

Rabbit serum against the cysteine-proteinases papain has been employed for the cellular localization of cysteine-proteinases of in Leishmania amazonensis promastigotes. By immunocytochemistry, immune complexes were found in the plasma membrane and in the flagella pocket of the parasite. The antiserum immunoprecipitated major iodinated proteins with molecular masses of 66, 45, 28 and 24 kDa and a wide partitioning of the Triton X-114 detergent phase. The presence of cysteine-proteinase at the cell surface membrane was also suggested by the detection of proteolytic activity in living cells (19.0 microg azocasein min(-1) 10(-7) promastigotes (1.0 S.D. )).     

Activity of an engineered synthetic killer peptide on Leishmania major and Leishmania infantum promastigotes

This study was undertaken to analyze the effect of an engineered, killer decapeptide (KP) on Leishmania major and Leishmania infantum promastigotes. The KP was synthesized on the basis of the sequence of a recombinant, single-chain anti-idiotypic antibody acting as a functional internal image of a yeast killer toxin. The evaluation of in vitro inhibitory activity of KP on L. major and L. infantum, release of intracellular green fluorescent protein (GFP) molecules by L. major, DNA fragmentation, and ultrastructural analysis (TEM) of L. infantum upon KP treatment were performed. KP presented antiproliferative and leishmanicidal activity with LC(50)/1 day of 58 and 72 microM for L. major and L. infantum, respectively. A dose-dependent decrease in proliferation and increase of killing of promastigotes was seen after KP treatment. No DNA fragmentation in L. infantum promastigotes or release of intracellular GFP molecules on peptide treatment of a GFP expressing L. major clone was demonstrated. Moreover the plasma-membrane was not disrupted, but, by TEM analysis, intracellular damage was observed.     

Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.     

Rapid transport of phospholipids across the plasma membrane of Leishmania infantum

The internalization of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) in Leishmania infantum promastigotes was studied. We observed a rapid inward redistribution of NBD-PC, -PE, and -PS across the plasma membrane at 28 and 4 degrees C. This internalization was shown to be independent of the endocytic activity of parasites. Rapid inward movement was coupled to an energy-dependent transporter because it was almost inhibited by depletion of cellular ATP and was blocked after pretreatment with N-ethylmaleimide (NEM). In contrast, NBD-SM traverses the plasma membrane by passive flip-flop. By comparing this pattern of phospholipid transbilayer movement with those known from other eukaryotic cells, candidate lipid transporters are discussed.     

Molecular detection of Leishmania (Sauroleishmania) tarentolae in human blood and Leishmania (Leishmania) infantum in Sergentomyia minuta: unexpected host-parasite contacts

The detection of atypical Kinetoplastida in vertebrate hosts and vectors might suggest unexpected host-parasite contacts. Aside to major vectors of Leishmania (Leishmania) infantum in Italy (e.g. Phlebotomus perniciosus and Phlebotomus perfiliewi), the sand fly fauna also includes Sergentomyia minuta, herpetophilic and proven vector of Leishmania (Sauroleishmania) tarentolae, in which records of blood meal on mammals and detection of L. infantum DNA are increasing. This study was conducted in Central Italy aiming to molecularly detect potential atypical Leishmania host-vector contacts. Detection of Leishmania spp. DNA was performed by polymerase chain reaction (SSU rRNA, ITS1 targets) on field-collected sand fly females (N = 344), blood samples from humans (N = 185) and dogs (N = 125). Blood meal identification was also performed on engorged sand flies. Leishmania spp. DNA was found in 13.1% sand flies, 3.7% humans and 14.4% dogs. Sequence analysis identified L. infantum in S. minuta (4.4%), P. perniciosus (9.1%), humans (2.2%) and dogs (14.4%). Leishmania tarentolae was detected in S. minuta (12.6%), P. perfiliewi (6.6%) and human (1.6%) samples. Of 28 S. minuta examined for blood meal, 3.6 and 21.4% scored positive for human and lizard DNA, respectively. These results indicate the importance of one-health approach to explore new potential routes of transmission of leishmaniasis involving S. minuta.     

Application of kDNA as a molecular marker to analyse Leishmania infantum diversity in Portugal

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinical forms in both immunocompetent and immunocompromised humans. Efficient monitoring and evaluation of epidemiology with discriminatory molecular markers are required. We investigated the genetic diversity of L. infantum in Portugal by polymerase chain amplification and restriction fragment length polymorphism analysis of kinetoplastid DNA, as molecular marker. We analysed 120 Portuguese isolates of L. infantum plus 16 other non-Portuguese isolates (as a reference group) from humans, dogs and sand flies. The Portuguese population showed a high degree of polymorphism with a total of 13 profiles identified. The predominant profile was A, which was only detected in the Portuguese samples.

The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained were stable during long-term growth in vitro and in laboratory animals.     

Genome-wide gene expression profile induced by exposure to cadmium acetate in Leishmania infantum promastigotes

Leishmania infantum is the etiological agent of visceral leishmaniasis in Mediterranean areas. The life cycle of the protist is dimorphic and heteroxene, as promastigotes develop inside the gut of sand-fly vectors and amastigotes multiply inside mammalian phagocytic cells. In previous studies, we analyzed the expression profiles of these stages and the modulation of gene expression triggered by temperature increase and acidification, both of which are crucial in the differentiation of promastigotes to amastigotes. Differential expression profiles of translation initiation and elongation factors were detected. Here we report that the presence of 1 mM cadmium acetate in the culture medium leads to a shock response consisting of growth arrest, morphological changes, the absence of motility, and the up-regulation of genes that code for: a heavy metal transporter, trypanothione reductase, a haloacid-dehalogenase-like hydrolase, and a metalloexopeptidase from the M20 family, among others. This response is probably controlled by the differential expression of regulatory genes such as those encoding initiation factors 4E, eukaryotic translation initiation factor 3 subunits 8 and 2α, and elongation factor 1β. The initiation factor 2α gene is induced in anomalous environments, i.e., those outside of the protist's normal life-cycle progression, for example, in response to the presence of cadmium ions, acidification without temperature increase, and vice versa. Our results suggest that the regulation of gene expression is a key component of the shock response.     

Heterogeneity among zymodemes of Leishmania infantum from HIV-positive patients with visceral leishmaniasis in south Italy

Abstract Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortaient patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.     

In vitro sodium nitroprusside-mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L(-1) SNP for 24 h showed about 75% growth inhibition, and 97-100% when the cultures were treated with >2 mmol L(-1) SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25-8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP-mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP-mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal-independent oxidation of biomolecules to free radical intermediates.     

Chemiluminescence activity in whole blood phagocytes of dogs naturally infected with Leishmania infantum

Dogs are the domestic reservoir of Leishmania infantum, a vector-borne intracellular protozoan agent of human visceral leishmaniasis. The role of polymorphonuclear leukocytes (PMNs) in the immune defence against this parasite has been poorly studied. We have investigated the function of peripheral blood PMNs in naive beagle dogs that have been naturally exposed to phlebotomine vectors in an area highly endemic for canine leishmaniasis, and found infected by Leishmania at the end of the transmission season. Whole blood phagocyte oxidative metabolism was assessed by a rapid method that determines a luminol-amplified chemiluminescence (CL) emission. This was evaluated using either a soluble stimulant, phorbol mirystate acetate (PMA), or phagocytic stimuli, such as zymosan unopsonized (ZYM) or opsonized with autologous serum (OPZ). In blood samples taken 2 months after exposure to Leishmania transmission, data on CL emission revealed a significant decrease of reactive oxygen intermediates (ROI) production in the presence of both PMA and ZYM, compared with blood samples obtained from dogs before exposure. On the contrary, no variations in CL emission were detected in presence of OPZ. Our data indicate that immunological changes occur early in canine leishmaniasis and confirm that the role of PMNs and their products need to be clarified. Copyright © 2000 John Wiley & Sons, Ltd.