Leishmania braziliensis: localization of glycoproteins in promastigotes

Two species of glycoproteins from Leishmania braziliensis promastigotes of apparent molecular weights of 53,000 (glycoprotein 53) and 47,000 (glycoprotein 47) were localized. Four lectins with different sugar specificities bound to the blotting sheet to which the electrophoretically separated materials were transferred. Concanavalin A and Ricinus communis agglutinin bound to the band of glycoprotein 53 and the lectin from Dolichos biflorus bound to the band of glycoprotein 47. Wheat germ agglutinin bound to the bands of both glycoproteins. Histochemical examinations using fluorescence labeled lectins demonstrated that the glycoproteins 53 and 47 were located on the cell surface and in the cytoplasm of promastigotes, respectively. The results are consistent with the result of agglutination test.     

Leishmania braziliensis: cell surface differences in promastigotes of pathogenic and nonpathogenic strains

A comparative study of cell surface characteristics of pathogenic and nonpathogenic promastigotes of Leishmania braziliensis, NR and LBY strains, respectively, was carried out by means of concanavalin A agglutination and labeling with concanavalin A-fluorescein isothiocyanate, concanavalin A-ferritin, and cationized ferritin. Cytochemical examination showed cell surface differences in lectin receptors and negative charge moieties in the two strains of L. braziliensis. The pathogenic NR strain agglutinated with low concentrations of concanavalin A and presented abundant lectin-binding and cationized ferritin-binding surface labeling. The nonpathogenic LBY strain neither agglutinated when incubated with concanavalin A, bound lectins, or cationized ferritin at the cell surface.     

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.     

Preliminary results in favor of the existence of a major surface antigen, specific for Leishmania braziliensis braziliensis

The comparative surface antigen study of 10 bolivian strains and 1 brazilian reference strain from L. b. braziliensis sub-species shows an important homogeneity within this group. All the strains tested so far present similar antigenic patterns with a major antigen at 72 kD. On the contrary, the comparative analysis of surface antigens of L. b. braziliensis with those of L. b. guyanensis, L. b. panamensis, L. mexicana amazonensis and L. donovani chagasi shows a large antigenic heterogeneity between the Leishmania sub-species and species we studied. The 72 kD antigen was only detected on L. b. braziliensis surface.     

Energetic metabolism of axenic promastigotes of Leishmania (Viannia) braziliensis

Leishmania spp are protozoans capable of carbohydrates degradation and as energy source they can use glucose, aminoacids or lipids from the environment. The products of the metabolic pathways such as organic acids may be used as an index of their energetic metabolic profile. Therefore, in this study a metabolic profile comparison was made between promastigotes from one reference strain (MHOM/BR/1975/M2903) and two different isolates of Leishmania (Viannia) braziliensis (MHOM/BR/2003/IMG3 and MHOM/BR/2005/RPL5). The parasites culture was performed in complete Grace’s culture media seeded in 24-well plates at 26 °C. During the growth curve performance samples were collected from the logarithmic and stationary phases of culture and therefore analyzed by high performance liquid chromatography (HPLC) and spectrophotometry assays to determine the concentrations of glucose, lactate, citrate, α-ketoglutarate, succinate, fumarate, malate, oxaloacetate and β-hydroxybutirate which are indicative of the energetic pathways. It was possible to detect an increase in the glucose from the stationary phase from the M2903 strain when compared to the logarithmic phase while in the IMG3 and RPL5 isolates there was a decrease (p < 0.05). The spectrophotometric and chromatographic results indicated that the logarithmic phase which presents higher energy consumption due to the intense replication rate have the energetic pathways intensified. It was also possible to note some metabolic differences between the analyzed parasites which may indicate possible adaptations of the parasite when facing different environmental and physiological conditions during its life cycle and that these differences may help in the understanding of the diversity of the host-parasite relationship from Leishmania parasites.     

Leishmania braziliensis: protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro

When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin and lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces.     

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host–parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1β, and tryparedoxin peroxidase. Data mining showed that ~ 5% of identified proteins present a classical secretion signal whereas ~ 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.     

Evidence of transferrin binding sites on the surface of Leishmania promastigotes.

A glycoprotein of 78,000 molecular mass (78 kDa), associated with the membrane of Leishmania infantum promastigotes, was identified and immunopurified by monoclonal antibody (mAb) LD9 produced against isolated membrane preparations. mAb LD9 was subsequently found to bind to human transferrin, also of 78 kDa. Binding of LD9 to transferrin was completely abolished when the mAb was preabsorbed by Leishmania membranes, thereby indicating that the 78-kDa Leishmania membrane-associated glycoprotein and transferrin have common antigenic epitope(s). The 78-kDa Leishmania membrane-associated protein was released in soluble nonaggregated form by mild treatment with acetic acid saline. Anti-transferrin polyclonal antibodies, recognized both the membrane-associated and the soluble form of the 78-kDa glycoprotein. The 78-kDa soluble form was characterized further as an iron-containing protein. The above data combined with iron uptake by promastigotes as demonstrated by the Prussian blue reaction indicate that the 78-kDa Leishmania membrane-associated glycoprotein is transferrin. The binding of 125I-human transferrin to Leishmania-purified membrane preparations was then investigated. The results indicate the presence of a high affinity saturable binding site (Kd = 2.2 10(-8) M) that is specific for transferrin. We suggest that the 78-kDa glycoprotein recognized by mAb LD9 is transferrin that binds to the surface of Leishmania promastigotes via a transferrin receptor.     

The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid.

Promastigotes of the protozoan parasite Leishmania major were biosynthetically labeled with myristic acid. Solubilization and phase separation in the non-ionic detergent Triton X-114 shows that the label is not incorporated into soluble hydrophilic proteins, but is incorporated into a few insoluble proteins. The bulk of the incorporated fatty acid is associated with a heterogeneous phosphorylated glycolipid and a few amphiphilic integral membrane proteins. Among these, the major surface protein of Leishmania promastigotes, p63, is predominantly labeled. Upon digestion with Bacillus cereus phospholipase C, amphiphilic p63 is shown to lose its myristic acid label and to acquire concomitantly the characteristic electrophoretic mobility and solubility behavior of hydrophilic p63. These data show that the amphiphilic character of the major surface protein of Leishmania promastigotes is due to a covalently attached phospholipid. We propose that this phospholipid provides the sole hydrophobic moiety anchoring the protein to the pellicular membrane of the protozoan parasite.     

Solubilization and partial purification of a cell surface component of Leishmania braziliensis

A simple procedure that allowed the extraction and partial purification of a component of 65,000 mol.wt. from the surface of Leishmania braziliensis promastigotes is described. Iodinated cell surface membrane fractions were solubilized using Triton X-100 followed by Nonidet P-40. The macromolecular components were then freed of the detergents by passage of the extracts through a column of DE52 cellulose. The component of 65,000 mol.wt. was eluted from the column with 1 m NaCl. This component in whole parasites was immunoprecipitated by sera from patients with cutaneous, mucocutaneous, and diffuse cutaneous leishmaniasis and kala-azar. None of the major surface determinants reacted with sera from normal individuals with antibovine rabbit serum. The relevance and the possible applications on the immunoprophylaxis of the disease are briefly discussed.     

Granulocyte-macrophage colony-stimulating factor is a growth-factor for promastigotes of Leishmania mexicana amazonensis

In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonesis. Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.     

Infectivity and virulence of Leishmania donovani promastigotes: a role for media,source, and strain of parasite

Transformation of promastigotes of Leishmania donovani strain AG83 from amastigotes derived from an infected animal was studied in three media, Schneider's Drosophila medium (SDM), Medium 199 (M199), and biphasic M199 (B-M199) with 10% fetal bovine serum. The media, SDM and B-M199, both supported a more efficient transformation of promastigotes in comparison with M199. Infectivity studies in hamsters and BALB/c mice showed that promastigotes isolated in B-M199 were several folds more infective than those obtained from M199. Comparison of the infectivity and virulence of promastigotes of AG83, with a recent isolate of kala-azar, SL94, harvested under similar conditions, revealed greater infectivity of SL94 for both macrophages and animal models. The present study demonstrates that the medium used for the conversion of amastigotes to promastigotes plays a major role in determining the infectivity of the freshly transformed L. donovani promastigotes in hamsters and BALB/c mice. The source and the strain of the parasite also influence the outcome of L. donovani infection.     

The PSA-2 glycoprotein complex of Leishmania major is a glycosylphosphatidylinositol-linked promastigote surface antigen

Polyclonal rabbit antiserum to the Triton X-114 phase material of Leishmania major, which comprises the surface and internal integral membrane proteins of the parasite, was used to screen a lambda gt11 genomic expression library. A recombinant clone producing a Mr 123,000 beta-galactosidase fusion protein was isolated. Antibodies affinity-purified on this fusion protein recognized a complex of three surface-oriented proteins of promastigotes of L. major of Mr 94,000, 90,000, and 80,000 that we have termed the promastigote surface Ag 2 (PSA-2) complex. The DNA sequence of the insert in this clone predicted the 3' end of an open reading frame encoding a hydrophobic C-terminus. The inferred C-terminal sequence was suggestive of a glycosylphosphatidyl-inositol membrane anchoring mechanism. Phosphatidylinositol-specific phospholipase C treatment of the native PSA-2 proteins caused a shift in their electrophoretic mobility with an apparent reduction in the molecular weight of the PSA-2 complex. After phospholipase C treatment these proteins also displayed the cryptic cross-reacting determinant recognized by antibodies to the Trypanosoma brucei variant surface Ag. Moreover, PSA-2, which previously partitioned in the detergent phase after Triton X-114 phase separation, became water-soluble after phospholipase C treatment. Immunoprecipitation of the PSA-2 proteins with sera directed to lectin-binding proteins indicated that these polypeptides may be differentially glycosylated. Finally, these PSA-2 proteins were recognized by sera from some patients with cutaneous leishmaniasis.     

Sensitivity of Leishmania braziliensis promastigotes to meglumine antimoniate (glucantime) is higher than that of other Leishmania species and correlates with response to therapy in American tegumentary leishmaniasis

The first line drugs for the treatment of leishmaniasis are antimonial derivatives. Poor clinical response may be credited to factors linked to the host, the drug, or the parasite. We determined the sensitivity of Leishmania sp. promastigotes and amastigotes by counting parasites exposed to increasing concentrations of meglumine antimoniate (Glucantime). Leishmania braziliensis promastigotes were significantly more sensitive than those belonging to other species. The sensitivity of L. braziliensis isolates from patients with unfavorable clinical outcome, such as therapeutic failure or relapse, was significantly lower than those from patients who had clinical cure. Poor clinical response to therapy (therapeutic failure or relapse) was also associated with inadequate antimonial therapy. We also found a significant and positive correlation between promastigotes and intracellular amastigotes with regard to their in vitro susceptibilities to meglumine antimoniate. Our data provide evidence for an association between the sensitivity of promastigotes to antimonials in vitro and clinical response to therapy in American tegumentary leishmaniasis. The high sensitivity of the local L. braziliensis to meglumine antimoniate in vitro provides an explanation for the good clinical response of cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil, even when low-dose regimens are employed.     

Infectivity and attenuation of Leishmania donovani promastigotes: association of galactosyl transferase with loss of parasite virulence

During the course of long-term in vitro cultivation of Leishmania donovani parasites, the promastigotes were found to lose their infectivity. Lectin agglutination studies revealed that there is an up-regulation of terminally exposed galactose residues on the noninfective promastigotes. The enzyme galactosyl transferase was absent in the infective form but present in the non-infective form of the parasites. An association between galactosylation and loss of infectivity was also observed. We propose that the enzyme galactosyl transferase is developmentally regulated and induces galactosylation of surface carbohydrates.     

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.