Hydrolysis of L-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases.

The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatin-sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed L-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite's cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to L-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.     

Leishmania amazonensis: uptake and hydrolysis of 3H-amino acid methyl esters by isolated amastigotes

Intracellular and isolated amastigotes of Leishmania amazonensis can be destroyed by L-amino acid methyl esters known to disrupt mammalian lysosomes. To evaluate the mechanism(s) involved in the leishmanicidal activity, we examined the uptake and hydrolysis of tritiated esters by isolated amastigotes. After incubation with the labeled compounds, parasites were recovered, were washed on filters, and their radioactivity was determined. Alternatively, amastigotes were separated from the medium by centrifugation through oil, and the radioactivity associated with free or esterified amino acids was measured after thin-layer chromatography. The results showed that the methyl esters of Trp, Leu, and Met, which are leishmanicidal, accumulated in and were rapidly hydrolysed by the amastigotes. [3H]Leu derived from [3H]Leu-OMe remained associated with the amastigotes even after a 1-hr chase in label-free medium, but the ester species was rapidly lost upon washing of the parasites. In contrast, the esters of Ile and Ala, which are not leishmanicidal, were only slowly hydrolysed, and most of the radioactivity was lost upon washing. We have previously shown that certain amino acid esters and weak bases protect Leishmania from damage by leucine methyl ester (Leu-OMe). In the present experiments, these compounds reduced, in concentration-dependent fashion, the hydrolysis of [3H]Leu-OMe and the accumulation of [3H]Leu in the amastigotes. Overall, the results indicate that, as in lysosomal disruption, leishmanicidal activity is associated with ester hydrolysis and amino acid accumulation in the parasites. The nature and location of the parasite esterolytic enzymes requires additional investigation.     

Leishmania amazonensis: specific labeling of amastigote cysteine proteinases by radioiodinated N-benzyloxycarbonyl-tyrosyl-alanyl diazomethane

Living Leishmania amazonensis amastigotes were incubated with radioiodinated N-benzyloxycarbonyl-L-tyrosyl-L-alanyl diazomethane (Z-Tyr-AlaCHN2), an irreversible inhibitor of mammalian cathepsins B and L. Parasite lysates were subjected to electrophoresis in gelatin-containing sodium dodecyl sulfate-acrylamide gels to detect regions of proteolytic activity, and the distribution of the inhibitor was ascertained by autoradiography. Of the three main bands of proteolysis associated with cysteine proteinases, two, with apparent molecular weights of 28 and 31 kDa, were shown to be labeled. The third enzyme activity, detected at the 35-kDa region in substrate gels, was only faintly labeled. The distribution of labeled bands was similar when lysates of untreated parasites were electrophoresed and the gels incubated with the radioiodinated inhibitor. Under reducing conditions, the inhibitor bound to polypeptides of 29, 31, 32, and 34 kDa, of which the first and the last were the most intensely labeled. Polypeptides with the same apparent molecular weights were labeled when amastigote lysates were incubated with the 125I inhibitor. Uptake of radioactivity by the parasites was time and concentration-dependent and more than 80% of the total counts could be precipitated with trichloroacetic acid. Radioactivity associated with the amastigotes was quite stable after they were pulsed with labeled inhibitor and chased for up to 24 hr in inhibitor-free medium. Both total uptake and labeling of cysteine proteinases were markedly reduced in parasites preincubated with Z-Phe-AlaCHN2 prior to exposure to Z-Tyr(125I)-AlaCHN2. However, more radioiodinated inhibitor was taken up by parasites preincubated with cold inhibitor and chased in inhibitor-free medium, suggesting de novo synthesis or processing of inactive enzyme precursors.     

Leishmania amazonensis: acidic organelles in amastigotes

Leishmania amastigotes are intracellular protozoan parasites which exclusively invade cells of the macrophage series and multiply within phagolysosomes. Recent studies showed that intracellular and isolated amastigotes of L. amazonesis are killed by amino acid esters which appear to be trapped within as yet unidentified, possibly acidified, "lysosome-like" parasite compartments and cleaved by hydrolytic enzyme(s) (M. Rabinovitch, V. Zilberfarb, and C. Ramazeilles, 1986, Journal of Experimental Medicine 163, 520-535). In the present study, we have localized acidic compartments of Leishmania amastigotes using as a probe the weak base 3-(2,4 dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP). This indicator, which can be detected within cells by light and electron microscopy using immunocytochemical immunocytochemical methods, mainly accumulates within megasomes and in dense inclusion vacuoles. With the help of quantitative assays to titrate cell-associated DAMP, it was found that (a) its uptake is temperature dependent and thus probably requires an energy supply, (b) the proton ionophore monensin partially inhibits the trapping of DAMP, and (c) monensin greatly increases its efflux from cells. These results, as well as those obtained by quantitative ultrastructural immunocytochemistry of cells incubated with DAMP in the absence or presence of monensin, show that megasomes and inclusion vacuoles have a low pH probably maintained by an active process. Furthermore, confirming the report of H. F. Hassan and G. H. Coombs (1987, Molecular and Biochemical Parasitology 23, 285-296) megasomes were found to display acid phosphatase activity at both light and electron microscope levels. This, together with the demonstration that megasomes are acidified, suggests that these organelles may be targets for amino acid derivatives.     

Leishmania amazonensis: biosynthesis of polyprenols of 9 isoprene units by amastigotes

The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol.     

Effect of thiadiazine derivatives on intracellular amastigotes of Leishmania amazonensis

Current therapy for leishmaniasis is not satisfactory. We describe the in vitro antiproliferative effects of new thiadiazine derivatives against Leishmania amazonensis. The compounds were found to be active against the amastigote form of the parasite, inhibiting parasite growing, from 10 to 89%, at a concentration of 100 ng/ml. This activity suggests that thiadiazine derivatives could be considered as potential antileishmanial compounds.     

Effects of hyperbaric oxygen on Leishmania amazonensis promastigotes and amastigotes

In the present study, we evaluated the effects of hyperbaric oxygen (HBO) exposure in both Leishmania amazonensis life stages (promastigotes and amastigotes) and on macrophage cultures infected with the parasite. HBO treatment protocols, which can be tolerated by humans and animals, induced irreversible metabolic damage and affected parasite morphology, growth and ability to transform. The observation that the antioxidant N-acetylcysteine (NAC) prevents some of these deleterious effects indicated an involvement of oxidative stress during parasite HBO exposure. In addition, HBO exposed L. amazonensis-infected macrophage cultures showed reduction of the percentage of infected cells and of the number of intracellular parasites per cell. Thus, the demonstration that HBO, a therapy used in the management of different diseases, is toxic for both L. amazonensis life stages and can alter macrophage susceptibility to the infection encourages further studies of this therapy in animal models of Leishmania infection.     

LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-β-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.     

Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism

Macrophages infected with amastigotes of Leishmania major and treated with IFN-gamma in vitro develop potent antimicrobial activities that eliminate the intracellular parasite. This antileishmanial activity was suppressed in a dose dependent fashion by NG-monomethyl-L-arginine (NGMMLA), a competitive inhibitor of nitrite, nitrate, nitric oxide and L-citrulline synthesis from L-arginine. Excess L-arginine added to infected macrophage cultures reversed the inhibitory effects of NGMMLA. Addition of arginase to culture media inhibited intracellular killing by IFN-gamma-treated cells. Similar effects were seen with macrophages obtained from BCG-infected C3H/HeN mice. Increased levels of nitrite, an oxidative product of the L-arginine-dependent effector mechanism, was measured in cultures of infected IFN gamma-treated macrophages as well as infected BCG-activated macrophages. Nitrite production correlated with development of antileishmanial activity. Nitrite production and microbicidal activity both decreased when in vivo or in vitro-activated macrophages were cultured in the presence of either arginase or NGMMLA. Nitric oxide synthesized from a terminal guanidino nitrogen atom of L-arginine and a precursor of the nitrite measured, may disrupt Fe-dependent enzymatic pathways vital to the survival of amastigotes within macrophages.     

Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.     

Effect of amidine derivatives on nitric oxide production by Leishmania amazonensis promastigotes and axenic amastigotes.

The effects of pentamidine isethionate (reference drug) and N,N'-diphenyl-4-methoxy-benzamidine (test compound) on NO. production by Leishmania amazonensis promastigotes and axenic amastigotes were investigated by measuring nitrite, a by-product of nitric oxide released into culture supernatants. The NO. production by infective promastigotes was inhibited by OCH(3)-amidine in about 23.53% and by pentamidine in only 3.78%. In axenic amastigotes, the inhibition of NO. production by OCH(3)-amidine was significantly higher (52.94%; p=0.01) than that by pentamidine, which inhibited this radical production nonsignificantly (25.29%; p=0.1). The mechanism of amidine derivatives, as an antimicrobial agent, is unknown. However, other amidines, such as a diamidine (pentamidine), contain chemical structures shared by the guanidino group of the nitric oxide synthase substrate L-arginine, suggesting the possibility of an interaction with this enzyme or electronic factors (substituent constant) that alter physical and chemical properties significant for biological activity.     

Roles of cysteine proteinases of trypanosomes and Leishmania in host-parasite interactions

Trypanosomes and Leishmania contain an abundance of stage-regulated cysteine proteinases encoded by several gene families. Analysis of parasites rendered defective in cysteine proteinase function, either through genetic manipulation or through the use of specific inhibitors, has revealed roles for the enzymes in parasite virulence, in modulation of the host's immune response and in parasite differentiation.     

Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.     

A strategy for the identification of T-cell epitopes on Leishmania cysteine proteinases

In this study computational analysis was used to compile sequence alignments, construct a dendrogram and calculate physical data in order to predict potential T-cell epitopes of the Leishmania cysteine proteinase. Using multiple alignment of human and Leishmania proteinase sequences deposited on data bank sequences, it was possible to predict that the extreme C-terminus of cysteine proteinase (Cyspep, 355-444) contained three peptides (pI 361-370, pII 415-422 and pIII 431-444) with charge score, hydrophobicity and isoelectric points compatible for human leucocyte-associated antigen (HLA) class II binding. The prediction was confirmed in vitro through the ability of synthetic peptides corresponding to the predicted regions to stimulate peripheral blood mononuclear cells of patients with leishmaniasis.     

Life in vacuoles--nutrient acquisition by Leishmania amastigotes

Leishmania have a digenetic life cycle, involving a motile, extracellular stage (promastigote) which parasitises the alimentary tract of a sandfly vector. Bloodfeeding activity by an infected sandfly can result in transmission of infective (metacyclic) promastigotes to mammalian hosts, including humans. Leishmania promastigotes are rapidly phagocytosed but may survive and transform into non-motile amastigote forms which can persist as intracellular parasites. Leishmania amastigotes multiply in an acidic intracellular compartment, the parasitophorous vacuole. pH plays a central role in the developmental switch between promastigote and amastigote stages, and amastigotes are metabolically most active when their environment is acidic, although the cytoplasm of the amastigote is regulated at near-neutral pH by an active process of proton extrusion. A steep proton gradient is thus maintained across the amastigote surface and all membrane processes must be adapted to function under these conditions. Amastigote uptake systems for glucose, amino acids, nucleosides and polyamines are optimally active at acidic pH. Promastigote uptake systems are kinetically distinct and function optimally at more neutral environmental pH, indicating that membrane transport activity is developmentally regulated. The nutrient environment encountered by amastigotes is not well understood but the parasitophorous vacuole can fuse with endosomes, phagosomes and autophagosomes, suggesting that a diverse range of macromolecules will be present. The parasitophorous vacuole is a hydrolytic compartment in which such material will be rapidly degraded to low molecular weight components which are typical substrates for membrane transporters. Amastigote surface transporters must compete for these substrates with equivalent host transporters in the membrane of the parasitophorous vacuole. The elaboration of accumulative transporters with high affinity will be beneficial to amastigotes in this environment. The influence of environmental pH on membrane transporter function is discussed, with emphasis on the potential role of a transmembrane proton gradient in active, high affinity transport.     

Complement-mediated serum cytotoxicity for Leishmania major amastigotes: killing by serum deficient in early components of the membrane attack complex

Leishmania major, the agent of Oriental sore, is an obligate intracellular parasite of macrophages in mammalian hosts. Man's immune defense against this organism requires participation of specifically sensitized lymphocytes and activated macrophages. Recent studies, however, have demonstrated that as little as 1/120 concentration of normal human serum is highly cytotoxic for the amastigote form of L. major. Initiation of the lethal process occurs rapidly, requiring only 30 sec of parasite exposure to serum, and is mediated by antibody-independent activation of the alternate complement pathway. The molecular mechanism of cytotoxicity is not known, but may require participation of the membrane attack complex, C5b-9. We investigated this possibility by treating amastigotes with human sera genetically deficient in complement components C5, 6, 7, 8, or 9. We then measured viability of treated parasites by amastigote-promastigote conversion. Our results were quite unexpected: not only did C9-deficient serum kill organisms, but sera singly deficient in each of the preceding components C6 to C8 were also cytotoxic. The degree of cytotoxicity was related both to serum concentration and to the point in the complement cascade at which deficiency occurred. Sera lacking C6 or C7 were less cytotoxic than those deficient in C8, which were less toxic than those deficient in C9. Cytoxicity of deficient sera was abolished by heating serum to 56 degrees C for 30 min. These findings indicate that an incomplete membrane attack complex may mediate cytotoxicity for L. major amastigotes. Moreover, our results raise important questions regarding the mechanism by which the complex is assembled on the surface of a living, unicellular eukaryotic organism.     

Mimicry of apoptotic cells by exposing phosphatidylserine participates in the establishment of amastigotes of Leishmania (L) amazonensis in mammalian hosts

Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFbeta1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c x C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFbeta1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.     

In vitro killing of Babesia bovis by the ornithine analog alpha-monofluoromethyldehydroornithine methyl ester

The effects of the 2 ornithine decarboxylase inhibitors alpha-difluoromethylornithine (DFMO) and alpha-monofluoromethyldehydroornithine methyl ester (delta MFMO-ME) on growth of Babesia bovis blood stages in vitro were tested. The DFMO had no apparent effects on in vitro growth of B. bovis nor on the morphology of the parasite at concentrations up to 20 mM. In contrast, delta MFMO-ME had cytotoxic effects on B. bovis at 0.5 mM which were more pronounced at 5 mM. delta MFMO-ME caused both a decrease in percentage parasitized erythrocytes and a degeneration of parasites after 12 h exposure, and the magnitude of both effects was dose-dependent. The effects of delta MFMO-ME were not reversible for B. bovis precultured for 12 h (5 mM) or 24 h (0.5 mM) in drug before culturing the parasite in drug-free medium. Unexpectedly, 300 microM putrescine did not reverse the effects of delta MFMO-ME on B. bovis blood stage, raising the possibility that inhibition of ornithine decarboxylase is not responsible for these effects.