Metacyclogenesis of Leishmania spp: species-specific in vitro transformation, complement resistance, and cell surface carbohydrate and protein profiles.

Metacyclic (stationary) and logarithmic (log) forms of promastigotes of Leishmania donovani and Leishmania major were characterized in several ways. The highly active metacyclic forms were larger with more protein and less carbohydrate. The flagellum increased in length 2.4 times in L. major as compared to 1.8 times in L. donovani. Resistance to complement-mediated lysis by normal human serum of in vitro grown Leishmania promastigotes was related to the species, the growth phase in culture, and also the temperature. Metacyclic forms of both species had a much increased resistance to killing by normal serum at different temperatures. Differences in membrane-exposed carbohydrates were detected by fluorescein-conjugated lectins. Peanut agglutinin and Ulex agglutinin I differentiated log and stationary phase promastigotes of L. major. Higher amounts of acid phosphatase were demonstrated in the metacyclic phase. Differences in polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides of approximately 51 and 114 kDa were found exclusively in metacyclic promastigotes of both species, whereas 38- and 23-kDa polypeptides were lost or reduced during transformation from log to metacyclic phase promastigotes of L. donovani. In addition, a 75-kDa polypeptide was expressed only in metacyclic promastigotes of L. major.

Leishmania donovani: characterization of a 38-kDa membrane protein that cross-reacts with the mammalian G-protein transducin.

We investigated the presence in Leishmania donovani promastigotes of proteins with homology to the G-proteins known to mediate signal transduction in other organisms. [alpha 32P]GTP binding experiments revealed the presence in the promastigote membrane of GTP-binding sites with high affinity and specificity. Experiments with antisera directed against mammalian G-proteins showed that the promastigotes possess a 38-kDa protein (p38) which strongly reacts with an antiserum directed against a decapeptide containing the C-terminal sequence of transducin, the G-protein that mediates visual signal transduction. The interaction of p38 with the antiserum is specifically blocked by the decapeptide antigen. p38 is enriched in plasma membranes and is absent in cytosol and in a mitochondria-enriched fraction. p38 was also detected in two other Leishmania species, L. mexicana and L. major. The migration of p38 upon sucrose gradient centrifugation of detergent extract of L. donovani membranes corresponded to Mr of approximately 70,000, indicating that p38 is part of an oligomeric structure. The findings suggest that p38 may be a component of a transmembrane signal transduction system in Leishmania.

Mucocutaneous leishmaniasis due to Leishmania (Leishmania) infantum: biochemical characterization.

A vegetative mass in the right nasal cavity of a 62-year-old man from Palma de Mallorca, Spain, was found to be due to Leishmania. The organism was isolated in culture and characterized by in situ hybridization, Southern blot hybridization, and isoenzyme analysis; it was thus demonstrated to be the most common enzyme variant 1 (MON 1) of Leishmania (Leishmania) infantum.

An improved serodiagnostic procedure for visceral leishmaniasis.

The enzyme-linked immunosorbent assay (ELISA) is a sensitive and specific serodiagnostic method for leishmaniasis. In this report, we describe how this versatile assay can be improved by the use of protein A or protein G conjugates for the specific detection of Leishmania antibody in the sera of patients with visceral leishmaniasis. In direct comparisons with anti-immunoglobulin conjugate, enzyme-linked protein A gave significantly higher absorbance values for positive sera without a corresponding increase in absorbance values for sera from normal individuals or from patients with other diseases known to cross-react with leishmaniasis. The effect was to increase the distance between positive and negative values, which aided in the interpretation of the results. This also permitted visual distinction between positive sera and negative or weakly reactive sera. The assay was effective using either blood or serum as the source of primary antibody. A further advantage of protein A over anti-Ig conjugate was its ability to detect specific antibody in dog as well as human sera. Finally, we demonstrated the usefulness of the protein A ELISA with a recombinant leishmania antigen, gp63.

Lipophosphoglycan and secreted acid phosphatase of Leishmania tropica share species-specific epitopes.

Several species-specific monoclonal antibodies (T11, T13-T15) which only react with Leishmania tropica, recognize phosphorlated carbohydrate epitopes on lipophosphoglycan and the structurally related molecule, phosphoglycan, which is shed by promastigotes into spent culture medium. During immunoaffinity isolation of [32P]orthophosphate-labeled phosphoglycan on monoclonal antibody T15 conjugated to Sepharose 4B, a high-Mr component (approx. 200,000) was co-purified. The latter material is metabolically labeled with [35S]methionine and [3H]glucosamine. This glycoprotein was separated from phosphoglycan by chromatography on lentil lectin resin. The glycoprotein exhibited a L-tatrate-sensitive acid phosphatase activity, typical of secreted acid phosphatase (EC 3.1.3.2) from Leishmania. Monospecific antibodies to Leishmania donovani-secreted acid phosphatase selectively precipitated the L. tropica enzyme from immunoaffinity purified mixtures of the two antigens, and monoclonal antibodies to lipophosphoglycan precipitate the pure enzyme. Species-specific monoclonal antibodies to L. major lipophosphoglycan also recognized both L. tropica antigens. Treatment of the acid phosphatase with periodate or phosphodiesterase I abolished binding by the monoclonal antibodies to the pure enzyme. These results demonstrate that the two major secreted glycoconjugates of Leishmania tropica, the lipophosphoglycan and the acid phosphatase, share species-specific phosphorylated carbohydrate epitope(s).

The leishmanial lipophosphoglycan: a multifunctional molecule.

A striking characteristic of leishmanial parasites is their ability to avoid destruction in hostile environments throughout their life cycle. To survive, the parasites must have evolved specialized molecules. One such molecule that has received considerable attention is an unusual glycoconjugate called lipophosphoglycan. The macromolecule is the major cell surface glycoconjugate of all Leishmania promastigotes. This minireview summarizes current information on the structure and possible functions of this intriguing molecule.

Characterization of two proteins from Leishmania donovani and their use for vaccination against visceral leishmaniasis.

Two proteins from Leishmania donovani, dp72 and gp70-2, have been previously utilized to specifically serodiagnose patients with visceral leishmaniasis. The proteins were shown by ELISA and Western blotting with monoclonal and polyclonal antibodies to be present in both stages of the parasite. Antibodies to gp70-2 recognize in promastigotes multiple discrete bands of similar m.w. which are common to several isolates of L. donovani. The total amount of Ag and number of bands observed per isolate is not constant. Lectin blots with Con A show gp70-2 to be a glycoprotein. Dp72 shows pronounced microheterogeneity between isolates of L. donovani. The Brazilian isolates examined appear to possess a lower m.w. form (64,000 or 68,000) of this molecule. No reactions were observed with dp72 and lectins in Western blots; and neither tunicamycin, N-glycanase, endoglycosidase H nor F affected the migration of [35S]-methionine-labeled protein on SDS-PAGE. A mAb against dp72 also cross-reacted in Western blots with a 60-kDa protein in Leishmania major, Leishmania aethiopica, and Leishmania tropica. No reaction was observed between the purified promastigote surface protease (gp63) and either monoclonal or polyclonal antibodies produced to dp72 or gp70-2. The ability of the pure proteins to provide protection against a challenge by L. donovani amastigotes was examined. BALB/c mice were immunized with gp70-2 and/or dp72 by using Corynebacterium parvum as an adjuvant. Mice immunized with gp70-2 were not protected; however, mice receiving dp72 showed a 81.1% reduction in the liver parasitemia compared with the adjuvant controls.

Inhibitory effects on protein kinase C activity by lipophosphoglycan fragments and glycosylphosphatidylinositol antigens of the protozoan parasite Leishmania.

Fragments of the lipophosphoglycan of Leishmania donovani were generated by phospholipase C digestion and mild acid hydrolysis. The fragments were purified and examined for inhibitory activity on protein kinase C isolated from rat brains. On a molar basis, the 1-O-alkylglycerol portion of LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not as effective. In addition, several glycolipid antigens from L. major, which contain short carbohydrate chains attached to phosphatidylinositol, were also efficient inhibitors of the enzyme. These results are consistent with the hypothesis that protein kinase C may be a key target for the parasites to overcome within host macrophages.

Structure of the phosphosaccharide-inositol core of the Leishmania donovani lipophosphoglycan.

The phosphosaccharide-inositol core of the lipophosphoglycan of Leishmania donovani was generated by treatment of the glycoconjugate with mild acid and digestion with phosphatidylinositol-specific phospholipase C. The core was purified and examined by one- and two-dimensional 1H-1H NMR and by methylation analysis. From the results, the carbohydrate core was elucidated as a phosphosaccharide attached to the inositol residue of the lyso-alkylphosphatidylinositol anchor of lipophosphoglycan as follows: PO4----6GalP(alpha 1----6)GalP(alpha 1----3)Galf(alpha 1----3)ManP(alpha 1----3)ManP(alpha 1----4)GlcNP(alpha 1----6)myo-inositol. The presence of an internal galactofuranose residue is highly unusual and the ManP(alpha 1----4)GlcNP(alpha 1----6)myo-inositol sequence is homologous to the respective portion of the glycosylphosphatidylinositol anchors reported for both the Trypanosoma brucei variant surface glycoprotein and the rat brain Thy-1 glycoprotein.

Inhibition of Leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate ligands.

Leishmania donovani, the agent of human visceral leishmaniasis, is an intracellular parasite that must be recognized and internalized by host macrophages to complete its biological cycle. In a search for possible ligands for macrophage surface receptors, glycoconjugates were obtained from Leishmania promastigotes by aqueous, phenol-aqueous, and alkaline extraction. A fucose-mannose glycoproteic ligand, a lipopeptidephosphoglycan, and a phosphate mannogalactan ligand were purified from promastigotes and analyzed for their chemical contents, with special attention to their glycidic moieties. Sugars that were identified as components of these glycoconjugates were tested for their capacity to inhibit promastigote internalization by BALB/c peritoneal macrophages in vitro. Neutral hexoses showed little inhibitory activity; fucose, charged monosaccharides, and a mannose polymer showed the highest activity. Two of the glycoconjugates (fucose-mannose glycoproteic ligand and phosphate mannogalactan ligand) purified from promastigotes were potent inhibitors of internalization, 75% inhibition being obtained at concentrations of 6 to 10 micrograms/ml. The simultaneous presence of both ligands in low concentrations yielded an increase in inhibitory activity above that found for each ligand alone, indicating that promastigotes may use at least two receptor sites for penetration into macrophages. These ligands are specific inhibitors of L. donovani promastigote phagocytosis, since 10 micrograms of each ligand per ml interfered neither with internalization of yeast cells nor with phagocytosis of Leishmania adleri promastigotes.

Purification and characterization of an 80-kilodalton membrane protein from Leishmania donovani.

Visceral leishmaniasis is caused by the protozoan parasite Leishmania donovani. We previously described the development of 16 monoclonal antibodies specific for L. donovani. The epitope recognized by one of these monoclonal antibodies, D13, is present at high density on nearly all isolates of L. donovani and, along with two other monoclonal antibodies, has been used to develop a sensitive and specific competitive assay for serodiagnosis of visceral leishmaniasis. In this report, we characterize the antigens recognized by D13 by immunoprecipitation of [35S]methionine-labeled promastigotes as two proteins (apparent molecular mass, 72 and 80 kilodaltons). Pulse-chase studies showed no evidence of a precursor-product relationship for the two proteins. We purified the 80-kilodalton protein (p80) to homogeneity by detergent solubilization of promastigote membranes, immunoaffinity chromatography, and ion-exchange chromatography. The epitope on p80 recognized by D13 was completely destroyed by proteolysis but was not affected by periodic acid treatment. P80 did not bind to the radioiodinated lectins concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Its apparent molecular mass was not affected by tunicamycin. Thus, it does not appear to be glycosylated. This protein is highly immunogenic and may prove useful for immunoprophylaxis and serodiagnosis of visceral leishmaniasis.

The identification of membrane glycoconjugates in Leishmania species.

The membrane glycoconjugates of 8 different species of Leishmania were compared by lectin blotting. Five different lectins with various sugar specificities were examined: concanavalin A, Lens culinaris, Ricinus communis, soybean agglutinin, and peanut agglutinin. Concanavalin A and Lens culinaris reacted with every Leishmania tested. The patterns observed for these 2 lectins, as well as the various species of parasites, were different. However, a common 41,000-52,000 and a 160,000-185,000 Mr component was present in almost all the parasite isolates examined. Ricinus communis only recognized a nondiscrete galactose-containing glycoconjugate similar to Leishmania-excreted factor. Soybean and peanut agglutinins reacted with a few low molecular weight parasite components. Soybean agglutinin reacted with all the Leishmania species tested, whereas peanut lectin only recognized 3 isolates. The latter lectin bound to discrete components migrating with the dye front and with Mr's of 35,000 and 52,000. Increased glycosylation was noted on avirulent L. major promastigotes and was associated with the appearance of several new peanut agglutinin-binding glycoproteins.

Isolation and identification of coenzyme Q from Leishmania donovani.

Crystalline coenzyme Q (CoQ) was obtained from Leishmania donovani promastigotes by extraction of the unsaponifiable material with hexane, chromatography on a Florisil column and crystallization from ethanol. Crystalline CoQ in ethanol revealed an ultraviolet-peak, at 275 nm, which disappeared upon reduction with NaBH4. Polarographic and mass fragmentographic measurements were characteristic of CoQ homologs. Reversed-phase thin-layer chromatography revealed that it is CoQ9.

An enzyme-linked lectin assay for the study of lectin receptors of Leishmania.

An enzyme-linked lectin assay (ELLA) based on the ELISA assay, using intact formalin-fixed promastigotes to coat poly-L-lysine-treated microtiter plates is described. The assay was used to study the lectin receptors of Leishmania donovani chagasi, L. donovani donovani and L. mexicana amazonensis. ConA, RCA, WGA, and PNA receptors were found in the three parasites. SBA receptors were found to be as frequent as the other receptors in L. donovani chagasi but not in the other two parasites which showed little SBA binding. Trypsin treatment of the two L. donovani subspecies did not remove any of the lectin receptors studied.

Structure of the major carbohydrate fragment of the Leishmania donovani lipophosphoglycan.

The major carbohydrate fragment from the lipophosphoglycan of Leishmania donovani was generated by mild acid hydrolysis (0.02 N HCl, 5 min, 100 degrees C) and purified by chromatography on DE-52 cellulose and thin layer. By a combination of analyses including gas-liquid chromatography-mass spectrometry and 1H NMR, the structure of the fragment was elucidated as PO4----6Gal(beta 1----4)Man. Approximately 16 of these phosphorylated disaccharide units occur in the overall glycoconjugate structure. NMR analysis of an alkaline phosphatase treated phosphorylated tetrasaccharide generated from lipophosphoglycan showed that the phosphorylated disaccharide units are linked together via alpha-glycosidic linkages. Complete characterization of the phosphorylated disaccharide units of lipophosphoglycan provides the first example of a defined carbohydrate anchored in membranes by a derivative of phosphatidylinositol.

Structure of the lipid moiety of the Leishmania donovani lipophosphoglycan.

The lipid moiety of the lipophosphoglycan of Leishmania donovani had been isolated and characterized as a novel lyso-alkylphosphatidylinositol. Treatment of lipophosphoglycan with either 10% NH4OH or a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus liberated a monoalkylglycerol substituent. Structural characterization of the monoalkylglycerol by gas-liquid chromatography-mass spectrometry indicated the presence of two saturated, unbranched hydrocarbons: a C24 alkyl chain comprising 78% of the lipid with the remaining 22% as a C26 alkyl chain. Periodate sensitivity demonstrated that the alkyl side chain is linked to the C-1 position of the glycerol backbone. Treatment of lipophosphoglycan with nitrous acid released 1-O-alkylglycerophosphorylinositol due to an unacetylated glucosamine residue linked to the inositol of the lyso-alkylphosphatidylinositol. Quantitative analysis of the organic solvent-soluble product of nitrous acid deamination of lipophosphoglycan confirmed the expected ratio of inositol:phosphate:1-O-alkylglycerol as 1:1:1. These results suggest that L. donovani anchors its lipophosphoglycan with a unique lipid component.