Alteration in glycerol and metalloid permeability by a single mutation in the extracellular C‐loop of Leishmania major aquaglyceroporin LmAQP1

The Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of antimonite [Sb(III)], an activated form of Pentostam or Glucantime. Downregulation of LmAQP1 provides resistance to trivalent antimony compounds and increased expression of LmAQP1 in drug‐resistant parasites can reverse the resistance. Besides metalloid transport, LmAQP1 is also permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. LmAQP1 also plays a physiological role in volume regulation and osmotaxis. In this study, we examined the role of extracellular C‐loop glutamates (Glu143, Glu145 and Glu152) in LmAQP1 activity. Alteration of both Glu143 and Glu145 to alanines did not affect either the biochemical or physiological properties of the protein, suggesting that neither residue is critical for LmAQP1 activity. Alteration of Glu152 to alanine, aspartate and glutamine affected metalloid transport in the order, wild‐type > E152Q > E152D > E152A. In fact, axenic amastigotes expressing E152A LmAQP1 accumulated negligible levels of either arsenite [As(III)] or Sb(III). Alteration of Glu152 significantly affected volume regulation and osmotaxis, suggesting that Glu152 is critical for the physiological activity of the parasite. More importantly, alteration of Glu152 to alanine did not affect glycerol permeability. Although the metalloids, As(III) and Sb(III), are believed to be transported through aquaglyceroporin channels as they behave as inorganic molecular mimic of glycerol, this is the first report where metalloid and glycerol transport can be dissected by a single mutation at the extracellular pore entry of LmAQP1 channel.     

Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.     

Involvement of protein tyrosine kinases and phosphatases in uptake and intracellular replication of virulent and avirulent Leishmania donovani promastigotes in mouse macrophage cells

In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related to the ultimate virulence mechanism associated with intracellular replication of this pathogen. These data collectively suggest that protein tyrosine kinase mediated entry of L. donovani promastigotes into macrophages is not a virulence-associated event.     

Biochemical characterization of Leishmania major aquaglyceroporin LmAQP1: possible role in volume regulation and osmotaxis

The Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of trivalent metalloids, arsenite and antimonite. We have earlier shown that downregulation of LmAQP1 provides resistance to trivalent antimony compounds whereas increased expression of LmAQP1 in drug-resistant parasites can reverse the resistance. In this paper we describe the biochemical characterization of LmAQP1. Expression of LmAQP1 in Xenopus oocytes rendered them permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. The transport property of LmAQP1 was severely affected when a critical Arg230, located inside the pore of the channel, was altered to either alanine or lysine. Immunofluorescence and immuno-electron microscopy revealed LmAQP1 to be localized to the flagellum of Leishmania promastigotes and in the flagellar pocket membrane and contractile vacuole/spongiome complex of amastigotes. This is the first report of an aquaglyceroporin being localized to the flagellum of any microbe. Leishmania promastigotes and amastigotes expressing LmAQP1 could regulate their volume in response to hypoosmotic stress. Additionally, Leishmania promastigotes overexpressing LmAQP1 were found to migrate faster towards an osmotic gradient. These results taken together suggest that Leishmania LmAQP1 has multiple physiological roles, being involved in solute transport, volume regulation and osmotaxis.     

Myriocin,a Serine Palmitoyltransferase Inhibitor,Blocks Cytokinesis in Leishmania (Viannia) braziliensis Promastigotes

We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log‐phase promastigotes treated with 1 μM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 μM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 μM myriocin‐treated parasites, respectively. High percentage of myriocin‐treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 μM myriocin, respectively). Transmission electron microscopy following treatment with 1 μM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3‐ketodihidrosphingosine to myriocin‐treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid‐free mutant, where addition of ethanolamine rescue wild‐type parasite characteristics.     

Identification of a protein kinase A regulatory subunit from Leishmania having importance in metacyclogenesis through induction of autophagy

cAMP‐mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP‐binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP‐dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP‐dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation‐induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP‐binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.     

Delayed in vitro Utilization of Glucose by Leishmania tropica Promastigotes*

Leishmania tropica promastigotes do not utilize glucose provided in the medium until late log phase. Rapid depletion of glucose from the medium, however, occurs during late log and stationary phases. At about the same time, the cells show maximal rates of glucose uptake as well as peak levels of phosphofructokinase and pyruvate kinase activities. The glucose analog, 2-deoxy-D-glucose inhibits glucose transport. Incorporation of this analog in the growth medium results in inhibition of growth. The hexokinase of L. tropica phosphorylates 2-deoxy-D-glucose. Pyruvate kinase is activated by fructose-1, 6-diphosphate and adenosine monophosphate.     

Localization and induction of the A2 virulence factor in Leishmania: evidence that A2 is a stress response protein

Leishmaniasis is a vector‐borne infectious disease with a wide range of pathologies depending on the species of Leishmania. Leishmania parasites are transmitted by the sand fly vector as promastigotes; within the mammalian host, Leishmania parasites differentiate into amastigotes and replicate in macrophages. The A2 protein from Leishmania donovani is expressed predominantly in amastigotes and therefore likely plays a role in survival in the mammalian host. In the present study, we have determined that the A2 protein colocalized with the Leishmania endoplasmic reticulum binding protein, BiP, was induced by stress and complexed with BiP following heat shock. The A2 gene in Leishmania major is a non‐expressed pseudogene, and we present evidence that ectopic expression of a transfected A2 gene in L. major enhanced its viability following heat shock. A2 may therefore play a role in protecting L. donovani from stress associated with infection in visceral organs, including the fever typically associated with visceral leishmaniasis. Interestingly, when comparing A2 protein localization, we also observed that the Leishmania secreted acid phosphatase SAcP protein was transported out of the parasite‐containing phagolysosome and was located throughout the macrophage cytoplasm in vesicles, providing the first example of a secreted Leishmania‐derived protein exiting the parasite‐containing phagolysosome.     

Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation

Leishmania mexicana has a large family of cyclin‐dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2‐related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co‐expression of a CRK3 transgene during rapamycin‐induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3^T178E mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.     

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb‐S; and antimony resistant Sb‐R). MIL‐R was easily induced in both strains using the promastigote‐stage, but a significant increase in MIL‐R in the intracellular amastigote compared to the corresponding wild‐type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain‐specific genetic changes were discovered in MIL‐adapted parasites, including deletions at the LdMT transporter gene, single‐base mutations and changes in somy. The most obvious lipid changes in MIL‐R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL‐R parasites, with more genetic changes occurring in Sb‐R compared with Sb‐S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb‐R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.     

Valosin‐containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress

Valosin‐containing protein (VCP)/p97/Cdc48 is one of the best‐characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin‐dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)‐mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin‐selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development.     

FRB domain of human TOR protein induces compromised proliferation and mitochondrial dysfunction in Leishmaniadonovani promastigotes

Visceral leishmaniasis (VL) or Kala-azar, the second-largest parasitic killer worldwide, is caused by Leishmania donovani. The drugs to treat VL are toxic and expensive. Moreover, their indiscriminate use gave rise to resistant strains. The high rate of parasite proliferation within the host macrophage cells causes pathogenesis. In the proliferative pathway, FRB domain of TOR protein is ubiquitously essential. Although orthologues of mTOR protein are reported in trypanosomatids and Leishmania but therein depth molecular characterization is yet to be done. Considerable protein sequence homology exists between the TOR of kinetoplastidas and mammals. Interestingly, exogenous human FRB domain was shown to block G1 to S transition in mammalian cancer cells. Thus, we hypothesized that expression of human FRB domain would inhibit the proliferation of Leishmaniadonovani. Indeed, promastigotes stably expressing wild type human FRB domain show 4.7 and 1.5 folds less intra- and extra-cellular proliferations than that of untransfected controls. They also manifested 2.65 times lower rate of glucose stimulated oxygen consumption. The activities of all respiratory complexes were compromised in the hFRB expressing promastigotes. In these cells, depolarized mitochondria were 2-fold more than control cells. However, promastigotes expressing its mutant version (Trp²⁰²⁷-Phe) has shown similar characteristics like untransfected cells. Thus, this study reveals greater insights on the conserved role of TOR in the regulation of the respiratory complexes in L. donovani. The slow growing variant of FRB expressing promastigotes will have great potential to be exploited as a prophylactic agent against leishmaniasis.     

Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice

Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨ_m) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches.     

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in ‘sub G0/G1’ phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.     

As(III) and Sb(III) uptake by GlpF and efflux by ArsB in Escherichia coli

The toxicity of the metalloids arsenic and antimony is related to uptake, whereas detoxification requires efflux. In this report we show that uptake of the trivalent inorganic forms of arsenic and antimony into cells of Escherichia coli is facilitated by the aquaglyceroporin channel GlpF and that transport of Sb(III) is catalyzed by the ArsB carrier protein; everted membrane vesicles accumulated Sb(III) with energy supplied by NADH oxidation, reflecting efflux from intact cells. Dissipation of either the membrane potential or the pH gradient did not prevent Sb(III) uptake, whereas dissipation of both completely uncoupled the carrier protein, suggesting that transport is coupled to either the electrical or the chemical component of the electrochemical proton gradient. Reciprocally, Sb(III) transport via ArsB dissipated both the pH gradient and the membrane potential. These results strongly indicate that ArsB is an antiporter that catalyzes metalloid-proton exchange. Unexpectedly, As(III) inhibited ArsB-mediated Sb(III) uptake, whereas Sb(III) stimulated ArsB-mediated As(III) transport. We propose that the actual substrate of ArsB is a polymer of (AsO)(n), (SbO)(n), or a co-polymer of the two metalloids.     

Calcineurin B‐like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis

Potassium transporters and channels play crucial roles in K⁺ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K⁺ channel in Arabidopsis roots that is involved in K⁺ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B‐like proteins CBL1/CBL9. The present study showed that another calcineurin B‐like protein (CBL10) may also regulate AKT1 activity. The CBL10‐over‐expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low‐K⁺ conditions. In addition, the K⁺ content of both CBL10‐over‐expressing lines and akt1 mutant plants were significantly reduced compared with wild‐type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two‐hybrid, BiFC and co‐immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1‐mediated inward K⁺ currents. Furthermore, the results from the yeast two‐hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL‐interacting protein kinase‐independent regulatory mechanism of calcineurin B‐like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Evidence for Rosettes as an Unrecognized Stage in the Life Cycle of Leishmania Parasites

ABSTRACT. Leishmania parasites, which afflict 12 million people in 88 countries, exist as promastigotes transmitted by insect vectors and as amastigotes residing in mammalian macrophages. Promastigote cells arranged in rosettes have also been described but universally disregarded as a distinct stage in the life cycle. We present evidence that only rosettes of Leishmania major promastigotes express cell surface poly‐α2,8 N‐acetyl neuraminic acid (PSA) and PSA containing de‐N‐acetyl neuraminic acid (NeuPSA). Expression of rosette‐specific PSA antigens was mosaic, with individual promastigotes expressing PSA, NeuPSA or both. A 50 kDa protein was detected by Western blot analysis of a detergent‐insoluble cell fraction with both PSA and NeuPSA‐reactive antibodies. Frequencies of rosette formation as well as cell surface PSA/NeuPSA expression were temperature dependent. Rosettes also engaged in an unusual swarming behavior, congregating into extended clusters. Distinct structures resembling cellular fusion bodies were formed in and released from rosettes. The results indicate that rosettes are an unrecognized stage in the life cycle of Leishmania. We hypothesize that rosettes initiate mating in Leishmania during which PSA/NeuPSA expression plays an important role. Recognizing rosettes as a distinct form of the Leishmania life cycle opens new possibilities for treatment or prevention of disease and, possibly, in vitro genetic recombination without passage of cells through insect vectors.     

New Antiparasitic Bis‐Naphthoquinone Derivatives

A series of bis‐naphthoquinone derivatives prepared by condensation of aryl aldehydes with lawsone were tested for antiparasitic activities against Toxoplasma gondii and Trypanosoma brucei parasites. Monofluorophenyl derivative 1a , 3,4‐difluorophenyl analog 1c and furyl compound 1l exhibited significant activity against T. gondii cells and appear to be new promising drug candidates against this parasite. The 3,4,5‐trifluorophenyl derivative 1g and the isovanillyl derivative 1j displayed selective activity against Leishmania major amastigotes.