Megasome biogenesis in Leishmania amazonensis : a morphometric and cytochemical study

Megasomes are large lysosomes found in the amastigote stage of Leishmania species belonging to the mexicana complex. The biogenesis of megasomes was investigated by transmission electron microscopy during the transformation of promastigotes into the amastigote form of L. amazonensis maintained in axenic cultures. Mainly small vacuoles with low electron density were found in the promastigote and early intermediate forms. Morphometrical analysis showed an increase in the volume density of these structures during the transformation process. Cysteine proteinase was localized in this structure by immunocytochemical assay. Membrane-bounded structures filled with electron-dense material were also found in significant amounts from the 2nd day on. These structures were relatively abundant, both in axenic and lesion-derived amastigotes, but not in stable long-term axenic amastigote culture. A three-dimensional reconstruction of lesion-derived amastigotes and axenic amastigotes of L. amazonensis demonstrated that megasomes comprise almost 5% of the total cell volume. In addition, the development of other organelles was examined during the transformation process. Received: 18 May 2000 / Accepted: 31 August 2000     

A radioiodinated peptidyl diazomethane detects similar cysteine proteinases in amastigotes and promastigotes ofLeishmania (L.) mexicana andL. (L.) amazonensis

Cysteine proteinase activities were examined in lesion amastigotes as well as in stationary-phase promastigotes ofLeishmania (L.) mexicana andLeishmania (L.) amazonensis isolates. Enzyme detection in gelatin gels revealed that amastigotes of threeL. (L.) mexicana isolates (M379, IOC-0561, and IP) shared similar proteinases, including the multiple low-molecular-weight (25–35 kDa) cysteine proteinases. High cysteine proteinase activity was also observed inL. (L.) amazonensis amastigotes, but the banding profile was different in two of the isolates examined. Promastigotes displayed fewer low-molecular-weight proteinase bands, and these were much less intense as compared with those of lesion amastigotes. Independently of theLeishmania isolates and developmental stages examined, incubation of the parasites for 2 h with 0.2 M radioiodinatedN-benzyloxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[¹²⁵I]-AlaCHN₂) markedly and selectively labeled bands comigrating with the 28- and 31-kDa cysteine proteinases. Under reducing conditions, labeling was associated with four similar polypeptides (29–34 kDa), which were also detected when incubation with Z-Tyr[¹²⁵I]-AlaCHN₂ was carried out after cell lysis. Labeling was completely abolished if lysates were first incubated with 20 M E-64 and then exposed to the¹²⁵I-tagged inhibitor, thus confirming the specificity of the compound toward cysteine proteinases.     

Depletion of secondary lysosomes in mouse macrophages infected withLeishmania mexicana amazonensis: A cytochemical study

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes ofL. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reppeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5° C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.     

Antileishmanial activity and ultrastructural alterations of Leishmania (L.) chagasi treated with the calcium channel blocker nimodipine

In a search for novel antileishmanial drugs, we investigated the activity of the calcium channel blocker nimodipine against Leishmania spp. and explored the ultrastructural damages of parasites induced by nimodipine after a short period of incubation. Nimodipine was highly effective against promastigotes and intracellular amastigotes of Leishmania (L.) chagasi, with 50% inhibitory concentration values of 81.2 and 21.5 μM, respectively. Nimodipine was about fourfold more effective than the standard pentavalent antimony against amastigotes and showed a Selectivity Index of 4.4 considering its mammalian cells toxicity. Leishmania (L.) amazonensis and Leishmania (L.) major promastigotes were also susceptible to nimodipine in a range concentration between 31 and 128 μM. Ultrastructural studies of L. (L.) chagasi revealed intense mitochondria damage and plasma membrane blebbing, resulting in a leishmanicidal effect as demonstrated by the lack of mitochondrial oxidative metabolism. The amastigote-killing effect suggests other mechanism than macrophage activation, as no upregulation of nitric oxide was seen. This calcium channel blocker is an effective in vitro antileishmanial compound and if adequately studied could be used as a novel drug candidate or as a novel drug lead compound for drug design studies against leishmaniasis.     

Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, l-glutamine, and HEPES at 37oC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host–parasite relationship as well as the process of pathogenesis.     

A new experimental culture medium for cultivation of Leishmania amazonensis: its efficacy for the continuous in vitro growth and differentiation of infective promastigote forms

Parasites from the genus Leishmania cause a variety of disease states in humans and other mammals in tropical and subtropical regions, which include cutaneous, mucocutaneous and visceral leishmaniasis. The elaboration of a culture medium for the in vitro cultivation of Leishmania spp., which promotes the growth and differentiation of the parasites, is an important tool for diagnosis, biochemical, biological and immunological studies in the genus. Herein, we have reported the development of a rapid, inexpensive and reliable monophasic culture medium. The novel medium, designated PBHIL, promoted an excellent parasite growth, generating high quantities of promastigotes with long-term viability, and was able to induce cellular differentiation of L. amazonensis promastigotes to the amastigote-like forms (93%). Additionally, we reported the influence of this novel medium on the biochemical characteristics of L. amazonensis and on the interaction of this parasite parasites with mammalian macrophages.     

Subcellular localization of an extracellular serine protease in<Emphasis Type="Italic"> Leishmania</Emphasis> (<Emphasis Type="Italic">Leishmania</Emphasis>)<Emphasis Type="Italic"> amazonensis</Emphasis>

Extracellular proteolytic activity was detected in a Leishmania (L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH₄)₂SO₄ precipitation followed by affinity chromatography on aprotinin–agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. (L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.     

Activity of the julocrotine, a glutarimide alkaloid from Croton pullei var. glabrior, on Leishmania (L.) amazonensis

The antiproliferative effect of julocrotine, an alkaloid isolated from Croton pullei var. glabrior (Euphorbiaceae), was studied in the macrophage amastigote and promastigote stages of the protozoan Leishmania (L.) amazonensis, which causes cutaneous leishmaniasis in the New World. Julocrotine showed a dose-dependent effect against the amastigote and promastigote forms, where 79 μM julocrotine inhibited promastigote growth by 54%, with an IC50 of 67 μM. To analyze the antiamastigote activity of the drug, murine peritoneal macrophages infected with L. amazonensis promastigotes were treated with different concentrations of julocrotine. An 80% inhibition of amastigote development was observed using 79 μM julocrotine for 72 h, with an IC50 of 19.8 μM. In addition, ultrastructural observation of the parasites showed a significant reduction in the number of amastigotes in the parasitophorous vacuoles and morphological changes in promastigotes, such as swelling of the mitochondrion, chromatin condensation, presence of membranous structures near the Golgi complex, and some vesicle bodies in the flagellar pocket. A colorimetric assay (MTT), which measures cytotoxic metabolic activity, showed that macrophages maintain their viability after treatment with the drug. These results suggest that julocrotine effectively inhibits the growth of parasites and does not have any cytototoxic effects on the host cell.     

Identification and enzymatic activities of four protein disulfide isomerase (PDI) isoforms of Leishmania amazonensis

Leishmania parasites primarily infect cells of macrophage lineage and can cause leishmaniasis in the skin, mucosal, and visceral organs, depending on both host- and parasite-derived factors. The protein disulfide isomerases (PDIs) are thiol-disulfide oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds of proteins in cells. Although four Leishmania PDI genes are functionally inferred from homology in the genome sequences, only two of them have been expressed as active proteins to date. The functional relationship among various PDI enzymes remains largely unclear. In this study, we expressed and partially characterized all four L. amazonensis PDIs encoding 52-, 47-, 40-, and 15-kDa proteins. Homology analysis showed that the sequence identity between L. amazonensis (New World) PDIs and their counterpart PDI sequences from L. major (Old World) ranged from 76% to 99%. Kinetic characterization indicated that while the 15-, 40-, and 47- kDa PDI proteins displayed both insulin isomerase and reductase activities, the 52-kDa protein had only isomerase activity with no detectable reductase activity. All four PDI proteins were recognized by sera from L. amazonensis-infected mice and were sensitive to inhibition by standard PDI inhibitors. This study describes the enzymatic activities of recombinant L. amazonensis PDIs and suggests a role for these proteins in parasite development.     

Leishmania expressed lipophosphoglycan interacts with Toll‐like receptor (TLR)‐2 to decrease TLR‐9 expression and reduce anti‐leishmanial responses

Two different Toll‐like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR‐2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR‐9 has been shown to promote a host‐protective response. However, whether there is a relationship between the interaction of LPG and TLR‐2, on one hand, with the effect of TLR‐9, on the other hand, remains unknown. In this study, we report that in‐vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR‐9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti‐LPG as well as anti‐TLR‐2 antibodies prevents this reduction of TLR‐9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR‐9 expression, which is shown to be mediated by transforming growth factor (TGF)‐β and interleukin (IL)‐10. Finally, in‐vitro treatment of macrophages with anti‐LPG and/or anti‐TLR‐2 antibodies before infection reduces the number of amastigotes in macrophages and co‐treatment of mice with anti‐TLR‐2 antibodies and cytosine–phosphate–guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)‐γ‐predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR‐2 reduce anti‐leishmanial responses via cytokine‐mediated decrease of TLR‐9 expression.     

Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis elicits immunity against visceral leishmaniasis

Leishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to partially rectify their defective heme‐biosynthesis renders them inducible with δ‐aminolevulinate to develop porphyria for selective photolysis, leaving infected host cells unscathed. Delivery of released “vaccines” to antigen‐presenting cells is thus expected to enhance immune response, while their self‐destruction presents added advantages of safety. Such suicidal L. amazonensis was found to confer immunoprophylaxis and immunotherapy on hamsters against L. donovani. Neither heat‐killed nor live parasites without suicidal induction were effective. Photodynamic vaccination of hamsters with the suicidal mutants reduced the parasite loads by 99% and suppressed the development of disease. These suppressions were accompanied by an increase in Leishmania‐specific delayed‐type hypersensitivity and lymphoproliferation as well as in the levels of splenic iNOS, IFN‐γ, and IL‐12 expressions and of Leishmania‐specific IgG2 in the serum. Moreover, a single intravenous administration of T cells from vaccinated hamsters was shown to confer on naïve animals an effective cellular immunity against L. donovani challenges. The absence of lesion development at vaccination sites and parasites in the draining lymphnodes, spleen and liver further indicates that the suicidal mutants provide a safe platform for vaccine delivery against experimental visceral leishmaniasis.     

Furazolidone is a selective in vitro candidate against Leishmania (L.) chagasi: an ultrastructural study

The current treatment for leishmaniasis is unsatisfactory due to toxic side effects, high cost, and problems with drug resistance. Various approaches have been used to identify novel drug candidates to treat Leishmania sp. parasites including the use of re-purposed drugs. Furazolidone is a nitrofuran derivative with antiprotozoal and antibacterial activity and is used for the treatment of giardiasis. In the present work, we determined the in vitro antileishmanial activity of furazolidone and its ability to induce ultrastructural alterations of parasites. Promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) amazonensis were highly susceptible to furazolidone, with IC₅₀ values ranging between 0.47 and 0.73 μg/mL. Furazolidone was also very effective against L. chagasi intracellular amastigotes, and despite mammalian cytotoxicity, the selectivity index was 8.0 in human monocytes. The drug also had limited toxicity in mice erythrocytes. Furazolidone demonstrated specific activity against Leishmania, a potential consequence of the lack of macrophage nitric oxide activation. As determined by electron transmission microscopy, drug treatment induced severe damage to the parasite mitochondria and nucleus. This older oral drug is an effective agent for the treatment of L. (L.) chagasi in vitro and is a novel candidate for further experimental studies.     

The effect of phospholipase A2 from <Emphasis Type="Italic">Crotalus durissus collilineatus</Emphasis> on <Emphasis Type="Italic">Leishmania (Leishmania) amazonensis</Emphasis> infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.     

Modulation of phagolysosome biogenesis by the lipophosphoglycan of Leishmania

Promastigotes of the protozoan parasite Leishmania are inoculated into the mammalian host by an infected sandfly and are phagocytosed by macrophages. There, they differentiate into amastigotes, which replicate in phagolysosomes. A family of glycoconjugates, the phosphoglycans (PGs), plays an important role in the ability of promastigotes to survive the potentially microbicidal consequences of phagocytosis. Lipophosphoglycan (LPG), an abundant promastigote surface glycolipid, has received considerable attention over the past several years. Of interest for this review, lipophosphoglycan confers upon Leishmania donovani promastigotes the ability to inhibit phagolysosome biogenesis. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-harboring phagosomes from interacting with late endosomes and lysosomes. Thus, similar to several other pathogens, Leishmania promastigotes hijack the host cell's cytoskeleton early during the infection process. Here, we review this phenomenon and discuss the potential underlying mechanisms.     

Subverted transferrin trafficking in Leishmania- infected macrophages

The intracellular fate of human transferrin (HTf) in macrophages infected by Leishmania was investigated. Binding of HTf-gold complexes at 4 °C was competitively inhibited by native holoHTf but not by apoHTf. Infected and uninfected macrophages displayed rather distinct HTf trafficking. Pulse-chase experiments using uninfected macrophages loaded with 15-nm gold-conjugated bovine serum albumin (BSA) and then incubated with 5-nm gold-conjugated HTf revealed a remarkable segregation of these tracers in distinct compartments. Nevertheless, Leishmania-infected macrophages presented extensive particle colocalization at both 60 min and 18 h. Light and electron microscopy immunolabeling indicated that HTf was delivered to the parasitophorous vacuole, formed patches on the amastigote surface, and was endocytosed via the flagellar pocket. Double-staining assays showed the colocalization of biotinylated HTf and its receptor in association with the parasitophorous vacuole. To approach the Tf-binding sites of amastigotes we performed HTf-fluorescein isothiocyanate (FITC) assays. Staining was diffuse at 4 °C and punctate at 35 °C, and only the former was sensitive to ethidium bromide, indicating an eventual temperature-dependent endocytic process. Within parasites, HTf was found in cysteine-proteinase-rich structures, suggesting that the protein can be endocytosed by intracellular amastigotes and sorted to the parasite endosomal-lysosomal compartments rather than being recycled. The treatment of infected macrophages with holoHTf, but not apoHTf, promoted the parasite's intracellular survival. These results suggest that Leishmania amastigotes can exploit and subvert the host-cell endocytic system and indicate the role of Tf-carried iron in the outcome of leishmanial infection. Received: 20 May 1998 / Accepted: 18 June 1998     

Eosinophil chemotactic activity in Leishmania amazonensis promastigotes

 Tissue eosinophilia was observed in the subcutaneous tissue of mice shortly after their inoculation not only with living but also with lysed promastigotes of Leishmania amazonensis. Intraperitoneal inoculation of lysed promastigotes from five different Leishmania species (L. donovani, L. chagasi, L. tropica, L. amazonensis, and L. braziliensis) induced eosinophil accumulation in the mouse peritoneum. This eosinophil infiltration was also detected in C5-deficient AKR mice, indicating complement independent eosinophil chemotaxis by the parasite. The induced eosinophils were hypodense, suggesting activation of the cells. Finally, we demonstrated in vitro eosinophil chemotactic activity in the promastigote lysates using purified eosinophils and blind well chambers. These results suggest the presence of an eosinophil chemotactic factor in Leishmania, a protozoan parasite. Received: 6 November 1995 / Accepted: 31 January 1996     

Cell structure and cytokinesis alterations in multidrug-resistant Leishmania (Leishmania) amazonensis

Multidrug-resistant Leishmania (Leishmania) amazonensis may be obtained by in vitro selection with vinblastine. In order to determine whether this phenotype is linked to structural alterations, we analyzed the cell architecture by electron microscopy. The vinblastine resistant CL2 clone of L. (L.) amazonensis, but not wild-type parasites, showed a cytokinesis dysfunction. The CL2 promastigotes had multiple nuclei, kinetoplasts and flagella, suggesting that vinblastine resistance may be associated with truncated cell division. The subpellicular microtubule plasma membrane connection was also affected. Wild-type parasites treated with vinblastine displayed similar alterations, presenting lobulated and multinucleated cells. Taken together, these data indicate that antimicrotubule drug-selected parasites may show evidence of the mutation of cytoskeleton proteins, impairing normal cell function.     

Efficacy of the tubercidin antileishmania action associated with an inhibitor of the nucleoside transport

Tubercidin (TUB) is an adenosine analog with potent antiparasite action, unfortunately associated with severe host toxicity. Prevention of TUB toxicity can be reached associating nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside transport, specifically target to the mammal cells. It was demonstrated that this nucleoside transport inhibitor has no significant effect in the in vitro uptake of TUB by Schistosoma mansoni and Trypanosoma gambiense. Seeking to evaluate if the association of these compounds is also effective against leishmania, we analyzed the TUB–NBMPR combined treatment in in vitro cultures of promastigote forms of Leishmania (L.) amazonensis, Leishmania (L.) chagasi, Leishmania (L.) major, and Leishmania (V.) braziliensis as well as in cultures of amastigote forms of L. (L.) amazonensis, mice macrophages infected with L. (L.) amazonensis, and in vivo tests in BALB/c mice infected with L. (L.) amazonensis. We demonstrated that TUB–NBMPR combined treatment can be effective against leishmania cells protecting mammalian cells from TUB toxicity.     

A new approach to the phylogeny ofLeishmania: species specificity of glycoconjugate ligands for promastigote internalization into murine macrophages

TwoLeishmania donovani glycoconjugate ligands for the internalization receptor on BALB/c peritoneal macrophages [fucose-mannose ligand (FML) and phosphate mannogalactan ligand (PMGL)] were shown to be species-specific in a comparative phagocytosis-inhibition test. Promastigotes ofL. donovani Sudan (LD1S),L. infantum, L. d. donovani, L. major (Jericho and Sudan),L. tropica, L. chagasi, L. mexicana venezuelensis, L. m. mexicana, L. m. amazonensis, L. m. pifanoi, L. m. garnhami, L. braziliensis braziliensis, L. m. amazonensis (Josefa),L. enrietti orL. adleri were incubated with macrophages in the presence of 10 g/ml FML and PMGL purified fromL. donovani (LD1S). Parasite internalization was determined and compared with that obtained in control experiments. Specific inhibition of phagocytosis ranged from 83% (L. donovani LD1S) to 7% (L. m. amazonensis). We could distinguish groups ofLeishmania consistently with their geographic distribution and the clinical aspects of the disease. Analogous experiments withL. m. amazonensis glycoconjugates showed reciprocal results, with inhibition ranging from 76% (L. m. amazonensis) to 8% (L. donovani LD1S).L. chagasi remained separated from the Old World kalaazar agents. Possible phylogenetic implications of these observations are discussed.