Promastigote Surface Research Articles

Kinetics of Entry of Virulent and Avirulent Strains of Leishmania donovani into Macrophages: A Possible Role of Virulence Molecules (gp63 and LPG)

Specific receptors may be involved in the process of attachment of Leishmania donovani promastigotes to macrophage surfaces and their subsequent internalization. Two virulent strains of Indian L. donovani (AG83 and GE-I) were found to enter into macrophages much faster than the avirulent ones (UR6). These virulent promastigotes express surface glycoprotein (gp63) and lipophosphoglycan (LPG) to a greater extent than avirulent strains. We examined their interaction with macrophages as a function of time by preblocking the macrophage receptors with the exogenous addition of gp33 or LPG. In experiments where gp63 was used as the blocking agent, the entry of one virulent strain (GE-I) was affected. In other experiments where LPG was used, the entry of another virulent strain (AG83) was affected. Entry of the avirulent strain (UR6) was unaffected by either of these treatments. Exposed LPG or gp63 on the surface of promastigotes thus appear to expedite their recognition and entry into the host cell. To assess the role of gp63 further in the entry of Leishmania into the macrophages, an avirulent UR6 strain was transfected with the gp63 gene cloned from L. amazonensis. The transfected UR6 as expected expressed more GP63 at a faster rate and entered into the macrophages like the virulent strain when compared to the nontransfected UR6 or UR6 transfected with vector alone. Thus, the expression of the gp63 gene is involved in the recognition and intracellular entry of visceral Leishmania into the macrophages in addition to the cutaneous species demonstrated previously.

C-reactive protein binds to a novel ligand on Leishmania donovani and increases uptake into human macrophages.

C-reactive protein (CRP) is a major acute phase protein of man, with serum concentrations increasing dramatically following stimulation of hepatocytes by inflammatory cytokines. However, the role of CRP in inflammation and resistance to infection is still poorly understood. Here, the specificity of CRP binding to the surface of Leishmania donovani, an obligate intracellular parasite of mononuclear phagocytes, is described. CRP is shown to bind to promastigotes at the infectious metacyclic stage of development, at concentrations found in normal human serum. The presence of CRP on the surface of promastigotes substantially increases uptake into human monocyte-derived macrophages. Unusually, CRP does not bind via its characteristic ligand, phosphorylcholine. We show that CRP binds to the lipophosphoglycan (LPG) component of the promastigote cell surface, a molecule implicated in both uptake and survival of these parasites within the macrophage, and also to the major secreted protein of promastigotes, secreted acid phosphatase. Using mAb to LPG with known ligand specificities, we define a novel ligand for CRP as the repeating phosphorylated disaccharide units that form the backbone of LPG.

Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response.

Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. Significant protection was also obtained in the genetically susceptible BALB/cH-2k and BALB/c mice. One of the PSA-2 genes was cloned and expressed in both Escherichia coli and Leishmania mexicana promastigotes. Vaccination with the recombinant PSA-2 purified from E. coli did not confer protection, in contrast to the L. mexicana-derived recombinant PSA-2, which provided excellent protection. CD4+ T cells isolated from the spleens of vaccinated mice produced large amounts of gamma interferon but no detectable interleukin 4 upon stimulation with PSA-2 in vitro. Limiting dilution analysis showed a marked increase in the precursor frequency of PSA-2-specific gamma interferon-secreting CD4+ T cells. No substantial change in precursor frequency was observed for interleukin 4-secreting T cells in the vaccinated mice. A CD4+ PSA-2 specific T-cell line generated from splenocytes of a vaccinated mouse produces a cytokine pattern consistent with a TH1 phenotype. Intravenous injection of this line into naive mice reduced significantly the parasite burden upon challenge infection. Taken together, the data suggest that vaccination with PSA-2 induces a TH1 type of immune response which protects mice from L. major infection. Moreover, a single recombinant PSA-2 polypeptide derived from a genomic clone can also vaccinate, provided that the structural form of the antigen is near native.

The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle

The promastigote surface antigen 2 (PSA-2) complex comprises a family of antigenically similar polypeptides of M r 96 000, 80 000 and 50 000, anchored to the membrane with glycosylphosphatidylinositol. Although PSA-2 was initially detected only in promastigotes, Northern blot analysis indicated that mRNA transcripts are also present in amastigotes. Unlike the situation in promastigotes, where at least four major transcripts (2.6–5.3 kb) were detected, only one major (2.6 kb) and two minor transcripts were present in amastigotes. A cDNA clone encoding a member of the PSA-2 family expressed in amastigotes was isolated using DNA probes. The predicted protein sequence of M r 40 000 is distinct from promastigote sequences, but shows significant similarity to previously described members of the family from L. major and L. amazonensis. Antibodies to the carboxyl terminal sequence conserved in all L. major PSA-2 studied to date, as well as antibodies affinity purified on the amastigote cDNA-derived polypeptide recognized a major M r 50 000 amastigote polypeptide. Immuno-electron microscopy localized both promastigote and amastigote PSA-2 to the cell surface. The expression of PSA-2 polypeptides during the transformation of amastigotes into promastigotes was ordered in a time-dependent manner, with the promastigote M r 80 000 polypeptide appearing first, followed by the M r 96 000 polypeptide. In contrast to the glycosylphosphatidylinositol anchor of promastigote PSA-2, which could be hydrolysed by phosphatidylinositol-specific phospholipase C, the amastigote form was resistant to this enzyme.

Detection of serum antibodies against Leishmania 94 kDa antigen in visceral and cutaneous leishmaniosis due to Leishmania infantum.

Leishmania promastigotes polypeptides are analyzed by immunoblotting with sera from patients infected with different Leishmania species and presenting visceral or cutaneous infections. These sera recognize Leishmania polypeptides in several molecular masses. The major findings of this study are as follow. 1) The Leishmania 94 kDa antigen, which is specifically recognized by all sera from L. infantum-infected patients with visceral infection, is recognized by some sera from L. infantum-infected patients presenting cutaneous infection. 2) All patients with cutaneous infections due to L. tropica, L. amazonensis, or L. guyanensis do not develop anti-94 kDa antibodies, whatever the Leishmania species used as antigens. 3) Difference in electrophoretic mobilities is seen between the 94 kDa antigen identified by sera from Leishmania infantum-infected patients, and the antigen both recognized by the Concavalin A lectin and a rabbit antiserum raised against deglycosylated Promastigote Surface Protease.

Biochemical and immunochemical characterization of a P-type ATPase from Leishmania donovani promastigote plasma membrane

An ATPase on the plasma membrane of Leishmania donovani has been characterized. An antiserum, generated against ATPase active bands from native gels, was specific for a 105 kDa protein in promastigotes. However, in plasma membrane preparations a 70 kDa protein is also recognized, suggesting proteolysis of the intact 105 kDa protein or the presence of a second similar ATPase. [γ- 32P]ATP phosphorylates two proteins (105 kDa and 70 kDa) in promastigotes and plasma membranes. Both proteins form a transient phosphorylated intermediate, characteristic of a P-type ATPase. Immunostaining of permeabilized parasites shows diffuse staining of the surface of promastigotes and amastigotes, which is consistent with a plasma membrane protein. The antiserum immunoprecipitates a 70 kDa [ 14C]DCCD binding protein from whole cells and plasma membranes of promastigotes. Furthermore, the antiserum immunoprecipitates a 105 kDa and 70 kDa protein which can be subsequently phosphorylated. These results indicate the presence of a 105 kDa P-type ATPase on the L. donovani plasma membrane which is similar to the mammalian and fungal cation pumps.

Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species

Antibodies raised against a Leishmania major recombinant promastigote surface antigen 2(PSA-2) fragment recognized three major polypeptides of approximate M r 96 000, 80 000 and 50 000 in promastigotes of three Israeli isolates of L. major including the cloned line LRC-L137-V121, but detected a different array of polypeptides in other L. major isolates. The pattern was different both in number of polypeptides detected and their molecular weight. The antibodies to L. major PSA-2 also recognized polypeptides in L. tropica, L. donovani and very weakly in L. mexicana promastigotes and in Crithidia lucilliae. The number and size of the polypeptides was different in each species. In addition to the membrane-bound PSA-2 polypeptides we identified water-soluble forms of PSA-2 released in promastigote culture supermatants. Peptide maps of the various L. major PSA-2 membrane polypeptides showed they were different from each other. N-terminal amino acid sequence of the three polypeptides expressed by L. major PSA-2 gene and shown that it produces a M r35 000 polypeptide recognized by polymorphic family. Because of the extensive sequence similarity between the PSA-2 genes it has been difficult to assign protein products to individual genes. As a first step towards solving this problem, we have transfected into L. mexicana a genomic clone of a L. major PSA-2 gene and shown that it produces a M r 35 000 polypeptide recognized by monoclonal and polyclonal antibodies to L. major PSA-2.

Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression.

BALB/c mice are susceptible to cutaneous leishmaniasis upon infection with Leishmania major while C57BL/6 are not. There is a major promastigote surface protease (PSP or gp63) which is available in both native and recombinant forms, and for which the primary amino acid sequence is known. Immunization with PSP has been shown to offer some protection against challenge with the live organism. Therefore, we attempted to develop a peptide vaccine with PSP peptides. In the first experiments, recall proliferative responses to PSP were measured using a set of 15mer peptides spanning the entire PSP molecule which allowed designation of major determinant regions in BALB/c, C57BL/6, and CBA mice. Several of these determinants were promiscuous and shared almost the identical core amino acid residues in the different strains. Immunization with major determinant peptides was recalled vigorously with L. major soluble antigen as well as with PSP. The response to peptide was almost entirely Th1 as measured by a localized ELISA assay for single-cell production of IFN-gamma. A similar assay for IL-5, which overcomes problems of sensitivity and inhibition by lymphokines produced by Th1 cells, indicates very little production of Th2 cells even by BALB/c. It was found that if a major responsive peak was examined by recall with overlapping peptides, the highest, central peptide gave a mainly Th1 response while the boundary, less efficient peptides gave more of a Th2 response. Possible reasons for this were discussed. These results point to the importance of selecting the exactly appropriate peptide in considering a vaccinogen that might protect susceptible individuals. Even the choice of a somewhat immunogenic peptide within the determinant envelope might actually exacerbate infection by steering the response in a Th2 direction.

Reversion to virulence in Leishmania major correlates with expression of surface lipophosphoglycan

An attenuated clone of Leishmania major was produced by chemical mutagenesis with N-methyl- N′-nitro- N-nitrosoguanidineand was biochemically characterized to determine the reason(s) for its loss of virulence. We found that the degree of virulence of L. major did not correlate with either the levels of lipophosphoglycan (LPG) expression of promastigote surface protease (PSP) or with the enzymatic activity of the molecule. In contrast, the levels of lipophosphoglycan (LPG) expressed by the attenuated clone were found to be at least 6-fold less than those of virulent L. major. When the attenuated L. major was injected into BALB/c mice and allowed to revert to virulence, the degree of reversion to virulence that the parasites underwent correlated directly with the amount and form (metacyclic) of LPG expressed by the parasites. Thus, these results further implicate LPG as an important Leishmania virulence factor.

The lysosomal gp63-related protein in Leishmania mexicana amastigotes is a soluble metalloproteinase with an acidic pH optimum

Leishmania mexicana amastigotes express a lysosomal protein, which is antigenically related to the promastigote surface metalloproteinase (gp63). It is shown that the purified gp63-related protein from amastigote is also an active metalloproteinase. The pH-optimum of the enzyme is acidic, similar to lysosomal cysteine proteinases, but distinct from the neutral to basic pH-optimum of the promastigote surface proteinase. This study appears to be the first report on a metalloproteinase with a lysosomal localization.

Leishmania major-specific, CD4+, major histocompatibility complex class II-restricted T cells derived in vitro from lymphoid tissues of naive mice.

Several studies indicate that the outcome of experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm) is determined by immunological events occurring shortly after infection. These events lead to outgrowth of either protective CD4+ T cells in the C57BL/6 mouse, which cures, or exacerbative cells in the BALB/c mouse, which succumbs to disease. Potential factors influencing the outgrowth of protective or exacerbative T cells include antigen-presenting cells (APC), cytokines, and parasite antigens. An in vitro system, in which one could precisely control the factors shaping early events in the T cell response to Lm, would be very useful. To this end, we have examined the in vitro response of naive lymphocytes to Lm promastigotes. The data presented here show that Lm-specific CD4+ T cell receptor alpha/beta + T cells can be generated in vitro from spleen and lymph node cell populations of naive mice. Furthermore, they can be obtained from the CD44low (unprimed) population of T lymphocytes, indicating that in vitro priming occurs. The ability to generate these T cells is dependent on the presence of live parasites and is not due to a parasite-derived nonspecific T cell mitogen. Restimulation, as assayed by proliferation, requires APC bearing syngeneic I-A. Optimal restimulation of the in vitro derived T cells is achieved only when live promastigotes are used. The T cells do not proliferate in response to a frozen-and-thawed lysate of promastigotes, yet they exhibit mild reactivity to lysates prepared from heat-shocked promastigotes. Furthermore, they do not recognize two predominant antigens on the promastigote surface, lipophosphoglycan and gp63. T cells derived in vitro with Lm show crossreactivity with live L. donovani, less crossreactivity with live L. mexicana, and no crossreactivity with live Bacillus-Calmette-Guerin or live Brugia malayi microfilariae. Finally, these early T cells, whether derived from healing C57BL/6 or nonhealing BALB/c mice, produce interleukin 2 (IL-2), IL-4, and interferon gamma.

Molecular variation in Leishmania

The surface coat of the protozoan parasite Leishmania affords remarkable protection in the harsh environments encountered within the insect vectors and vertebrate hosts. It also provides specificity for the interaction of these parasites with the cells in the sandfly gut and with the human macrophage. Surprisingly few molecules have been identified on the Leishmania surface. The major surface molecules of both promastigotes and amastigotes are the glycoconjugates lipophosphoglycan and a glycoprotein of approximately 63 kDa. These major surface molecules vary structurally between Leishmania species and throughout the life-cycle of the parasite. In addition to these major glycoconjugates, Leishmania produce a number of less abundant surface molecules, including a family of glycosylinositol phospholipids, the Promastigote Surface Antigen-2 complex of glycoproteins and a glycoprotein of M r 46000. These molecules share the common feature of attachment to the plasma membrane via glycosylphosphatidylinositol lipid anchors. Leishmania also release molecules from their surface in a species specific manner. In this review we will examine the molecular variation of these molecules and their biological importance. We will also discuss the potential of these molecules as targets for chemotherapy and as candidate vaccines.

Characterization of a surface metalloprotease from Herpetomonas samuelpessoai and comparison with Leishmania major promastigote surface protease

The monogenetic kinetoplastid protozoan parasite Herpetomonas samuelpessoai expresses a surface-exposed metalloprotease [1]. Comparable to the Leishmania promastigote surface protease, or PSP, the protease of Herpetomonas is active at the surface of fixed and live organisms, and both enzymes display an identical cleavage specificity toward a nonapeptide substrate. The protease was enriched 440 times by partition into Triton X-114 followed by 2 steps of anion exchange chromatography. The 56-kDa enzyme is inhibited by the metal chelator 1,10-phenanthroline and is susceptible to cleavage by glycosyl-phosphatidylinositol phospholipase C (GPI-PLC). The conservation of an identical surface protease activity in these monogenetic and digenetic trypanosomatids suggests that the enzyme has a physiological function in the promastigote (insect) stage of these parasites.

Pure protein from Leishmania donovani protects mice against both cutaneous and visceral leishmaniasis.

A protein purified from Leishmania donovani promastigotes, dp72, was shown to partially protect BALB/c mice against a challenge by this parasite. Immunized mice infected i.v. with 10(7) L. donovani promastigotes showed a 0, 60, and 78% reduction in liver parasite burden compared with the control mice at each time point examined after challenge, 1, 20, and 108 days, respectively. Western blotting demonstrated that the sera from the immune mice, which reacted specifically with dp72 in lysates of L. donovani, cross-reacted with one major band in total homogenates of Leishmania major and Leptomonas collosoma. Lymphocyte proliferation to crude and pure parasite Ag was also examined in mice immunized with dp72. Strong proliferation was found at low concentrations of crude L. donovani Ag (0.5 micrograms/ml) and with pure dp72. Proliferation at higher concentrations to crude L. major and L. collosoma Ag was observed. Little or no reaction (stimulation index < 1.0) was seen with other pure leishmanial Ag, including gp70-2, the promastigote surface protease, and lipophosphoglycan. Depletion in vitro of the CD4+ T cell subset from immune spleen cells abolished proliferation to dp72, whereas depletion of CD8+ T cells enhanced proliferation to the pure Ag. Experiments in vivo showed that immunized mice treated with antibodies to either CD4+, CD8+, both T cell subsets, or to IFN-gamma had larger LPB (178, 173, 176, and 130%, respectively) after challenge with L. donovani than nonimmunized controls. Mice immunized with dp72 but treated with either PBS or mouse Ig showed reduced LPB, 81 and 61%, respectively, compared with the nonimmunized animals. BALB/c mice immunized with dp72 were also protected against L. major which causes cutaneous leishmaniasis. Immunized mice infected with either 10(4) or 10(6) promastigotes did not develop lesions. Limiting dilution assays confirmed the protection.

Leishmania donovani surface glycoconjugate GP36 is the major immunogen component of the Fucose-Mannose Ligand (FML)

Leishmania donovani promastigote glycoconjugate ligands, studied in our laboratory, that interact with the internalization receptors on BALB/c macrophages: the ‘fucose mannose ligand’ (FML), the ‘phosphate mannogalactan ligand’ (PMGL), and the ‘lipopeptidephosphoglycan’ (LPPD), interfered also with interaction between amastigotes and host cells in vitro. Among the three compounds studied, the FML was shown to be the most potent inhibitor of both promastigote and amastigote internalization, and to be present on parasite surface during the vertebrate-host cycle. The FML, but not the other two glycoconjugates, is a potent immunogen in rabbits (ELISA, agglutination and immuno-blots). Rabbit hyperimmune sera recognized essentially the 36 kDa band of FML. Mouse monoclonal antibodies against FML recognized either the 36 kDa or the 55 kDa band. No cross-reactivity between these two FML components was detected. No antigenic similarity could be detected between the 36 and 55 kDa bands of FML and the ‘GP63’ (promastigote surface proteinase) major surface leishmanial antigen. The 36 kDa-glycoprotein was identified as the major FML antigenic fraction and designated ‘GP36’. The integrity of the glycidic moiety was necessary for its antigenicity. This L. donovani surface glycoprotein is apparently one of the major molecules involved in interactions between the parasite and the vertebrate host.

A Fluorescent Peptide Substrate for the Surface Metalloprotease of Leishmania

A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry29, 10113–10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 × 106M−1sec−1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.

Membrane insertion and antibody recognition of a glycosylphosphatidylinositol-anchored protein: an optical study.

The kinetics of binding of a glycolipid-anchored protein (the promastigote surface protease, PSP) to planar lecithin bilayers is studied by an integrated optics technique, in which the bilayer membrane is supported on an optical wave guide and the phase velocities of guided light modes in the wave guide are measured. From these velocities, the optical parameters of the membrane and PSP layers deposited on the waveguide are determined, yielding in particular the mass of PSP bound to the membrane, which is followed in real time. From a comparison of the binding rates of PSP and PSP from which the lipid moiety has been removed, it is shown that the lipid moiety plays a key role in anchoring the protein to the membrane. Specific and nonspecific binding of antibodies to membrane-anchored PSP is also investigated. As little as a fifth of a monolayer of PSP is sufficient to suppress the appreciable nonspecific binding of antibodies to the membrane.

Structural characterization of a novel class of glycophosphosphingolipids from the protozoan Leptomonas samueli.

Aqueous phenol extraction of the lower trypanosomatid Leptomonas samueli released into the aqueous layer a chloroform/methanol/water-soluble glycophosphosphingolipid fraction. Alkaline degradation and purification by gel filtration chromatography resulted in a tetrasaccharide (phosphatidylinositol (PI)-oligosaccharide A), and a pentasaccharide (PI-oligosaccharide B), each containing 2 mol of 2-aminoethylphosphonate and 1 mol of phosphate. Nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectrometry suggested that the structure of PI-oligosaccharide A is [formula: see text] and that of PI-oligosaccharide B is as shown. [formula: see text] Both compounds contain an inositol unit linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion are stearic acid, lignoceric acid, and C20-phytosphingosine. These novel glycolipids fall within the glycosylated phosphatidylinositol (GPI) family, since they contain the core structure Man alpha (1-->4)GlcNH2 alpha (1-->6)myo-inositol-1-PO4, which is also found in the glycoinositolphospholipids and lipophosphoglycan of Leishmania spp., the L. major promastigote surface protease, the glycosylphosphatidylinositol anchor of Trypanosoma brucei variant surface glycoprotein, and the lipopeptidophosphoglycan of Trypanosoma cruzi. The glycophosphosphingolipids of Leptomonas have features in common with the glycolipids of both Leishmania and T. cruzi, resembling the former by the alpha (1-->3) linkage of mannose to the GPI core, while the 2-aminoethylphosphonate substituent on O-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Thus these data lend some support to the hypothesis that both T. cruzi and Leishmania evolved from a Leptomonas-like ancestor.

Leishmania major: Differential regulation of the surface metalloprotease in amastigote and promastigote stages

During its life cycle, the protozoan parasite Leishmania major alternates from an intracelluar amastigote form in the mammalian host to a flagellated promastigote form in the insect vector. The expression of the surface metalloprotease (PSP) during differentiation in vitro was investigated by Western and Northern blots, by immunoprecipitation of cells metabolically labeled with [ 35S]methionine or labeled at the surface with radioactive iodine, and by quantification of the proteolytic activity in substrate-containing polyacrylamide gels. We report that the surface metalloprotease is down-regulated at both the mRNA and the protein level in amastigotes, where it represents less than 1% of the equivalent proteolytic activity detected in promastigotes. A significant amount of mRNA is detected 4 hr after the onset of differentiation. The expression of the protease begins at that time and reaches steady state 8 hr later. The synthesis of PSP precedes the complete morphological differentiation to the promastigote stage and the appearance of the lipophosphoglycan, another major promastigote surface component. In contrast to PSP, a family of mercaptoethanol-activated proteases present in the amastigote exists only at a reduced level in the promastigote. The confinement of the surface metalloprotease to the insect stage of the parasite suggests that it has no physiological function in the parasitism maintenance of mammalian host macrophages.

Heparin enhances the interaction of infective Leishmania donovani promastigotes with mouse peritoneal macrophages. A fluorescence flow cytometric analysis.

Human visceral leishmaniasis results from the infection of macrophages by the protozoan parasite Leishmania donovani. Both forms of the parasite, the extracellular promastigote and the obligate intracellular amastigote, require cell surface molecules to ensure their recognition and uptake by the host cell, the macrophage. We have proposed previously that the heparin-binding protein on the surface of promastigotes is an adhesion molecule. The present report provides experimental evidence to support this hypothesis. Fluorescence flow cytometry using FITC-heparin was employed to study the heparin-binding protein of L. donovani promastigotes and amastigotes. We demonstrate the presence of the heparin-binding protein on the surface of amastigotes and document the heparin specificity of the binding protein for both forms of the parasite. Two-color fluorescence analysis was performed to compare R-PNA reactivity and FITC-heparin binding during the parasite's 7-day growth curve. Using this strategy we show that the expression of heparin binding activity coincides with the differentiation of the noninfective promastigote into the infective metacyclic from of the parasite. Macrophages that were challenged for 30 min with heparin-treated, FITC-labeled parasites became 2.82-fold more fluorescent than their counterparts which were exposed to non-heparin-treated FITC-labeled promastigotes. Finally, using Kolmogorov-Smirnov analysis we show that the adhesion of promastigotes to mouse peritoneal macrophages is significantly enhanced in the presence of 3.3 microM heparin. The experiments described in the present report provide evidence for the hypothesis that L. donovani's heparin-binding protein is a virulence factor that functions as an adhesion molecule in the parasite-macrophage interaction.