Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.     

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.     

Modulation of macrophage mannose receptor affects the uptake of virulent and avirulent Leishmania donovani promastigotes.

The effect of oxidants and the anti-inflammatory steroid dexamethasone on the attachment and internalization of virulent and avirulent Leishmania donovani promastigotes by the macrophage mannosyl fucosyl receptor was examined. Oxidants and dexamethasone are known to down- and upregulate the expression of the mannose receptor. Macrophages, when treated with 500 microM H2O2 at 37 C for 30 min, stimulate about 45% inhibition in uptake of an avirulent strain (UR6), and 30 and 25% inhibition for virulent strains AG-83 and GE-I, respectively. Treatment of macrophages with dexamethasone for 20 hr resulted in a stimulation in uptake of the parasite. When UR6 was used, a 3-fold increase in uptake was observed compared with the controls. Parasite uptake was also inhibited by the H2O2-generating system, glucose/glucose oxidase; inhibition was blocked by catalase. Treatment of macrophages either with H2O2 or dexamethasone did not affect the binding of the advanced glycosylation end product-bovine serum albumin (AGE-BSA), the ligand for AGE receptor of macrophages. Similarly, indirect evidence also shows that both types 1 and 3 complement receptors (CR1, CR3) are not affected by these treatments, indicating that, besides the mannosyl fucosyl receptor, other receptors are minimally altered in the identified condition. These results suggest that the up- and downregulation of the mannose receptor of macrophages may play a role in affecting L. donovani infection.     

Leishmania chagasi: homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model

Homogenous metacyclic promastigotes of Leishmania chagasi were isolated by buoyant density from in vitro heterogeneous cultures and used for biochemical characterization of isoforms of the major surface protease (MSP). Compared to stationary phase promastigotes, metacyclic cells had three times more MSP, produced 3-fold higher parasite loads in a mouse model in vivo, and were more resistant to complement-mediated lysis in vitro. These metacyclic L. chagasi expressed both the virulence-associated 59-kDa, and the constitutively expressed 63-kDa, isoforms of MSP.     

Effects of DL-alpha-difluoromethylornithine on Leishmania donovani promastigotes

Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been demonstrated to be an effective agent against a variety of parasitic protozoa but not against Leishmania spp. In this report, we show that Leishmania donovani promastigotes in continuous culture are sensitive to the growth inhibitory and cytotoxic effects of DFMO. Incubation of the promastigotes with DFMO obliterates intracellular putrescine pools and depletes spermidine concentrations, which correlates with the onset of growth inhibition. The effects of DFMO on the growth and the intracellular polyamine pools can be reversed completely by the addition of 10 microM putrescine to the culture medium. These results suggest that the treatment of leishmaniasis may be amenable to chemotherapeutic manipulation by DFMO.     

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.     

The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages

The interaction between the macrophage and the parasite plays a central role in the continued success of Leishmania infection. The promastigote surface ligand, and its complementary macrophage membrane receptor, involved in attachment and phagocytosis are likely to exert considerable influence over the outcome of a new infection. In this study, we report experiments pertaining to one such parasite membrane protein. Initial examination of promastigote surface proteins by radiolabeling and two-dimensional-polyacrylamide gel electrophoresis revealed an abundant polypeptide with an apparent m.w. of 63,000. Lectin-binding studies indicated that it was a glycoprotein containing mannose, N-acetyl glucosamine, and N-acetyl galactosamine residues. Monospecific antiserum raised against this glycoprotein, gp63, decorated the entire promastigote plasmalemma. Univalent antibody fragments from this antiserum blocked the interaction between promastigotes and macrophages by inhibiting attachment. Anti-gp63-inhibition reduced parasite/macrophage binding to 30 to 35% of the control binding level. Additional evidence of the involvement of gp63 in attachment to macrophages was provided by studies that made use of gp63-containing proteoliposomes. These vesicles were avidly phagocytosed by macrophages. Uptake of the gp63-containing liposomes was suppressed by greater than 90% by both anti-gp63 F(ab) fragments and the oligosaccharide mannan, indicating that their phagocytosis was receptor dependent. These results demonstrate that the abundant glycoprotein gp63 plays an important role in attachment of promastigotes to macrophages, and attachment via this parasite ligand is sufficient to trigger phagocytosis.     

Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form.

Leishmania sp. protozoa contain an abundant surface protease (gp63) that is important for the virulence of the parasite. We found that the average amount of gp63 expressed by Leishmania donovani chagasi promastigotes increases 6-11-fold as they develop from a less infectious form in logarithmic phase to a highly infectious form during stationary phase of cultivation in vitro. The predominant gp63 RNA switches from a 2.7 to a 3.0 kilobase (kb) RNA during the transition from log to stationary phase. Sequence analysis of gp63 cDNAs reveals that three different classes of gp63 RNAs, containing unique 3'-untranslated regions (3' UTRs), are expressed during growth to stationary phase. The predominant 2.7-(log) and 3.0-kb (stationary) class gp63 RNAs possess nearly identical coding regions, but they diverge in their 3' UTRs. A third class, consisting of 3.1- and 2.6-kb (constitutive) gp63 RNAs, is expressed at low levels throughout cultivation. This latter class encodes a gp63 with an additional 41 amino acids at its C terminus, replacing a potential signal for attachment of a glycolipid membrane anchor with a sequence that could be a transmembrane region. These findings are consistent with the regulated expression of different gp63 genes, resulting in different amounts of gp63 protein, during the promastigote's in vitro development to an infectious form.     

The major surface protein of Leishmania promastigotes is a protease

The major surface protein of Leishmania promastigotes is evolutionarily conserved and is found in isolates of L. donovani, L. major, L. tropica, L. mexicana, and L. braziliensis. The data provided in this communication demonstrate that in L. major this integral membrane protein is a protease, which we now designate promastigote surface protease. The enzyme has an alkaline pH optimum and is active both in its detergent-solubilized form and at the surface of living or fixed promastigotes. A water-soluble form of promastigote surface protease is obtained following digestion with the phospholipase C responsible for the release of the variant surface glycoprotein of Trypanosoma brucei. Possible biological functions of promastigote surface protease during the life cycle of Leishmania parasites are discussed.     

Identification of a surface membrane proton-translocating ATPase in promastigotes of the parasitic protozoan Leishmania donovani.

ATPase activities were measured in surface membranes and mitochondria isolated from promastigotes of the parasitic protozoan Leishmania donovani. The two enzymes were differentiated on the basis of pH optima, inhibitor sensitivity and by immunochemical methods. The surface-membrane (SM-) ATPase had an activity of 100 nmol/min per mg of protein, which was optimal at pH 6.5. The enzyme was Mg2+-dependent, partially inhibited by Ca2+, and unaffected by Na+ or K+. The SM-ATPase was inhibited by orthovanadate, NN'-dicyclohexylcarbodi-imide, and N-ethylmaleimide [IC50 (concentration causing half-maximal inhibition) 7.5, 25 and 520 microM respectively]; however, it was unaffected by ouabain, azide or oligomycin. The SM-ATPase demonstrated a Km of 1.05 mM and a Vmax. of 225 nmol/min per mg of protein. Moreover, fine-structure cytochemical results demonstrated that the SM-ATPase was localized to the cytoplasmic lamina of the parasite SM. A method was devised for the isolation of SM-derived vesicles. These were used to demonstrate the proton-pumping capacity of the SM-ATPase. Cumulatively, these results constitute the first demonstration of a surface-membrane proton-translocating ATPase in a parasitic protozoan.     

Mechanism of complement-independent and antibody-mediated killing of Leishmania donovani promastigotes

Immune serum raised against flagellar fraction of Leishmania donovani isolate UR6 has profound lethal effect on the in vitro growth of the parasite. Lethal effect of immune serum was also examined using two other isolates of L. donovani, namely DD8 and AG83. It was observed that immune serum is equally effective against UR6 and DD8 but has no effect on AG83 promastigotes. Parasite killing is mediated by Leishmania-specific antibodies in the absence of complement or any other factors present in rabbit serum. Results indicate that the lethal effect of immune serum is due to impairment in membrane function leading to inhibition in uptake of essential nutrients needed for growth and survival of parasites.     

Effects of long-term in vitro cultivation on Leishmania donovani promastigotes

Promastigotes of Leishmania donovani that had been subcultured in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophages infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macrophages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tobie's medium, and in the quantities of some of their antigens.     

Metabolic variation during development in culture of Leishmania donovani promastigotes

The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3-6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase.     

Leishmania chagasi: in vitro differentiation of promastigotes monitored by flow cytometry

A sequential development from a less infective to an infective stage of Leishmania promastigotes growing in culture has been previously reported. The aim of this work was to investigate whether freeze-fracture electron microscopy and flow cytometry would be able to provide some reliable morphological markers of in vitro differentiation of Leishmania chagasi promastigotes. The flow cytometry technique discriminates between the L. chagasi promastigotes from the different stages of their in vitro differentiation. The "forward scatter" intensity of the parasite, very high 15 hr after seeding when the parasites were very condensed and with a high DNA content per particle, strongly decreased during the culture course. Parallel experiments have shown a striking correlation between forward scatter intensity, growth curves, and infectivity of promastigote populations. By contrast, freeze-fracture techniques showed that in either less infective or infective promastigote plasma membranes, the intramembrane particles density in protoplasmic fracture faces (about 2800/micron 2) and in exoplasmic fracture faces (about 1000/micron 2) was independent of the time of cultivation. The amount of filipin lesions, which reflects the cholesterol content within the plasma membrane, was also constant throughout the culture course. Both data suggest that the architecture of the plasma membrane is an intrinsic characteristic of the promastigote stage. This study shows that whereas freeze-fracture electron microscopy does not provide markers for the differentiation of Leishmania promastigotes, flow cytometry may on the other hand be of value as a screening test for promastigote populations allowing the characterization of their developmental stages in in vitro cultures.     

Active cutaneous leishmaniasis in Brazil, induced by Leishmania donovani chagasi

L.d. chagasi was isolated from active cutaneous leishmaniasis in both human and canine infections in an endemic area in Rio de Janeiro, Brazil. Both isolates were identified by molecular and immunological characterization of the parasite using three different methods: electrophoretic mobility of isoenzymes; restriction endonuclease fragment analysis of kDNA and serodeme analysis using monoclonal antibodies. This seems to be the first well documented case in the New World of a "viscerotropic" Leishmania inducing a case of cutaneous leishmaniasis. This observation emphasizes that the diagnosis of the etiologic agent of human or canine visceral leishmaniasis based solely upon clinical and epidemiological criteria may lead to erroneous conclusions.     

Interaction of sitamaquine with membrane lipids of Leishmania donovani promastigotes

Sitamaquine is an 8-aminoquinoline which is active by the oral route for the treatment of life-threatening visceral leishmaniasis caused by Leishmania donovani, with an IC50 of 29.2 microM against the promastigote form in vitro. At high concentration (100 microM), sitamaquine affected parasite motility, morphology and growth in a way that was only partially reversible. As a first approach to determine its mechanism of action, we describe the interaction of sitamaquine with parasite membrane components, representing the first barrier to be crossed by the drug. Analysis of the physicochemical interactions of sitamaquine with monolayers of phospholipids and sterols at the air-water interface showed that these interactions only occurred in the presence of anionic phospholipids. Thus, electrostatic interactions between positively charged sitamaquine and the negative polar headgroups are a pre-requisite for subsequent hydrophobic interactions between the sitamaquine aromatic ring and the alkyl chains of phospholipids leading to drug insertion into the monolayer.     

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.