SCID mice reconstituted with Oct-2-deficient lymphocytes can cure Leishmania major infection and generate normal antigen-specific T cells.
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Animals↗
Biomedical subjects
E Handman
Publications and source records attributed to E Handman.
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) 80000 polypeptide appearing first, followed by the M(r) 96000 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.
Amidohydrolases↗
Lipophosphoglycan blocks attachment of Leishmania major amastigotes to macrophages.
Promastigotes of the intracellular protozoan parasite Leishmania major invade mononuclear phagocytes by a direct interaction between the cell surface lipophosphoglycan found on all Leishmania species and macrophage receptors. This interaction is mediated by phosphoglycan repeats containing oligomers of beta (1-3)Gal residues specific to L. major. We show here that although amastigotes also use lipophosphoglycan to bind to both primary macrophages and a cell line, this interaction is independent of the beta (1-3)Gal residues employed by promastigotes. Binding of amastigotes to macrophages could be blocked by intact lipophosphoglycan from L. major amastigotes as well as by lipophosphoglycan from promastigotes of several other Leishmania species, suggesting involvement of a conserved domain. Binding of amastigotes to macrophages could be blocked significantly by the monoclonal antibody WIC 108.3, directed to the lipophosphoglycan backbone. The glycan core of lipophosphoglycan could also inhibit attachment of amastigotes, but to a considerably lesser extent. The glycan core structure is also present in the type 2 glycoinositolphospholipids which are expressed on the surface of amastigotes at 100-fold-higher levels than lipophosphoglycan. However, their inhibitory effect could not be increased even when they were used at a 300-fold-higher concentration than lipophosphoglycan, indicating that lipophosphoglycan is the major macrophage-binding molecule on amastigotes of L. major. In the presence of complement, the attachment of amastigotes to macrophages was not altered, suggesting that lipophosphoglycan interacts directly with macrophage receptors.
Animals↗
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.
Animals↗
Anti-galactosyl antibodies that react with unmodified agarose: a potential source of artifacts in immunoaffinity chromatography.
Rabbits were immunized with glycolipid and membrane glycoprotein extracts of the promastigotes of Leishmania major partially purified by two-phase extraction with Triton X-114. The resulting antibodies were affinity purified on agarose immunoadsorbents to which a recombinant DNA-produced L. major polypeptide had been coupled. In addition to the expected reaction with the polypeptide, it was found that the affinity-purified antibodies reacted strongly with lipophosphoglycan of L. major amastigotes and to a lesser extent with the lipophosphoglycan of promastigotes. Antibodies with this reactivity could be affinity purified on unmodified agarose and were probably directed against beta-1,3-galactosyl determinants shared between the agarose matrix and Leishmania. In this and other special situations the properties of the support matrix can be exploited to obtain antibodies of defined specificity which may be useful probes for the identification and characterization of carbohydrate structures. This work also points to a potential source of artifacts in affinity chromatography, in which the bead matrix is usually considered as inert. Carbohydrate antigens are ubiquitous, particularly in micro-organisms, and the presence of such antibodies may be more common than previously recognized.
Animals↗
Cloning and sequence of a Leishmania major homologue to the fibrillarin gene.
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Amino Acid Sequence↗
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 supernatants. 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 showed they are similar but distinct, consistent with being members of a 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.
Amino Acid Sequence↗
Epitope mapping of monoclonal antibodies directed against lipophosphoglycan of Leishmania major promastigotes.
Monoclonal antibodies (MAbs) were generated against Leishmania major promastigote lipophosphoglycan (LPG) to use as tools in defining functional epitopes of this major cell surface glycoconjugate. Epitope mapping of four MAbs, designated 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2, revealed that the phosphorylated oligosaccharide repeat unit PO4-6[Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, P3, is a highly immunogenic epitope which has previously been demonstrated, by chemical analyses, to be a repeat unit specific to L. major. Two antibodies, 4A2-A2 and 5E10-F2, also recognised the repeat unit PO4-6[Ara(beta 1-2)Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, 4Pa, with less affinity than P3, while 2G11-A3 recognised P4a with greater affinity than for P3. The L. major metacyclic-specific antibody 3F12 only recognised repeat units terminating with arabinose residues. In particular, 3F12 recognised P4a, which is upregulated in metacyclic LPG compared to the procyclic form of the molecule. The oligosaccharides P3, P4a and P5a are specific to L. major LPG. The epitopes of 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2 were found on the cell surface and in the flagellar pocket of both procyclic and metacyclic V121 promastigotes, but were only detected at very low levels on amastigotes. The repeat unit P3 is able to inhibit attachment of procyclic promastigotes to the midgut of the sandfly vector, but neither Fab fragments of the four antibodies nor purified P3 could inhibit attachment of metacyclic promastigotes to the macrophage cell line J774. It was also shown that human sera from patients with cutaneous leishmaniasis recognised purified P3. The data suggests that while P3 is an immunogen in the natural course of infection of the human host, P3 plays no role in attachment and internalisation of promastigotes into the macrophages of the mammalian host.
Animals↗
Ricin-resistant mutants of Leishmania major which express modified lipophosphoglycan remain infective for mice.
Glycosylation variants of the virulent Leishmania major clone V121 were generated by mutagenesis with N-methyl-N-nitroso-N-nitroguanidine and selected using the galactose-specific lectin Ricinus communis II (RCA II). Three mutants, 4B9, 1D1 and 1C12, which failed to bind RCA II, were found to have an altered expression of lipophosphoglycan (LPG), a molecule implicated in the attachment to host macrophages and survival within the phagolysosome. There were differences in the antigenicity, molecular weight and localization of LPG from mutant parasites as compared to V121. Expression of gp63, a surface molecule also implicated in attachment to macrophages, was unaltered. All 3 mutants caused disease when injected into genetically susceptible BALB/c mice but lesions developed at a much slower rate than those caused by the virulent V121 clone. This slow rate of lesion development did not correlate with promastigotes' ability to invade macrophages in vitro. Karyotype analysis showed that there was a reduction in the size of chromosome band number 2 in all 3 mutants. The differences in LPG and chromosome band 2 were retained by mutant clones following passage through mice, suggesting that these phenotypes are stable. Although the mutant parasites were infective and caused lesions, the changed structure of the LPG appeared to influence the virulence of the parasites.
Agglutination↗
Biosynthesis of lipophosphoglycan from Leishmania major: characterization of (beta 1-3)-galactosyltransferase(s).
Lipophosphoglycan (LPG) is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved GPI anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6-Gal(beta 1-4)Man(alpha 1-) backbone variously substituted with galactose, glucose and arabinose residues in L.major and capped with a neutral oligosaccharide. Using a microsomal membrane preparation from L.major, we have been able to demonstrate that galactose from UDP-[14C]galactose can be transferred to an endogenous acceptor, characterized as LPG. An in vitro assay was established, based on anion-exchange HPLC, that concurrently identifies and quantitates the products of the galactosyltransferases. We show that the products formed are [14C]galactose-labelled P3 (PO4-6-[Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-), P4b (PO4-6-[Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-) and P5b(PO4-6-[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1- 4)Man(alpha 1-). These are major galactosylated repeating units of the backbone of L.major LPG. The same products are also formed when LPG from L.donovani, which contains an unbranched backbone of P2 repeats, is used as an exogenous acceptor with L.major microsomal membranes and UDP-[14C]galactose. In addition, no formation of radioactive backbone repeats (P2) was detected in membrane incubations containing UDP-[14C]galactose with or without added unlabelled GDP-mannose, indicating that the addition of the (beta 1-3)-linked galactose branches is independent of the synthesis of the repeating disaccharide (P2) backbone. Preliminary kinetic analyses suggest that the addition of multiple (beta 1-3)-linked galactose residues may be catalysed by more than one (beta 1-3) galactosyltransferase. The (beta 1-3)galactosyltransferase(s) activity was not detected in microsomal membrane preparations from promastigotes of L.donovani.
Animals↗
Intravenous injection of irradiated Leishmania major into susceptible BALB/c mice: immunization or protective tolerance.
It is well established that BALB/c mice can be protected from fatal infection with Leishmania major by prophylactic intravenous (i.v.) immunization with irradiated parasites. Protection is critically dependent on the route of injection with i.v. injection being protective and subcutaneous injection not protective. We used this BALB/c-L. major model system to investigate this phenomenon. We analyzed quantitatively the parasite-specific, CD4+ T cell mediated immune responses by limiting dilution. Subcutaneous vaccination resulted in priming of CD4+ precursor T cells, whereas i.v. vaccination was ineffectual. Moreover, i.v. injection prevented the increase in the number of specific precursor cells induced by infection of normal mice during the first weeks post-challenge with virulent parasites. We show here that this was not due to the elimination of the virulent challenge parasites as a result of immunity nor to inefficient antigen presentation of the irradiated organisms after i.v. injection. The data presented here suggest that i.v. injection results in tolerization rather than immunization. Tolerization as a mechanism of host protection is consistent with earlier observations that transient immunosuppression results in cure of L. major infection in BALB/c mice. Transfer of antigen presenting cells (APC) isolated from spleens of mice injected previously with irradiated parasites mimicked to some extent the effect of i.v. immunization with irradiated parasites. The possible involvement of these APC in decreasing the parasite-specific T cell response is discussed.
Animals↗
The structure of Leishmania major amastigote lipophosphoglycan.
Intracellular amastigotes of Leishmania major produce 6 x 10(4) copies/cell of a lipophosphoglycan (LPG) that is structurally distinct from the LPG produced by the extracellular promastigote form of L. major, Leishmania donovani, and Leishmania mexicana (reviewed by McConville, M. J. (1991) Cell Biol. Int. Rep. 15, 779-798). L. major amastigote LPG is composed of a lysoalkyl phosphatidylinositol lipid anchor that links via a diphosphorylated hexasaccharide core to a phosphoglycan (6-100 kDa). The structures of the anchor, the core, and the phosphoglycan were determined by monosaccharide and linkage analysis, fast atom bombardment-mass spectrometry, one-dimensional 1H NMR spectroscopy, and exoglycosidase microsequencing. The lipid anchor contains predominantly 1-O-alkylglycerols with 24:0 and 22:0 alkyl chains. The lipids are linked via a glycerol-myo-inositol-PO4 to a core glycan with the structure -PO4-6)Gal(alpha 1-)Gal(alpha 1-) Galf(beta 1-)[Glc(alpha 1-PO4-)]Man(alpha 1-)Man(alpha 1-)GlcN(alpha 1-). The chromatographic characteristics of the core glycan suggest that the saccharide components are linked similarly in amastigote and promastigote LPG. The phosphoglycan attached to the core consists of -PO4-6)Gal(beta 1-4)Man(alpha 1- repeats units which are either unsubstituted (70%) or substituted (30%) at the 3-position of the Gal residues with oligosaccharide side chains containing primarily Gal and some Glc. Thirteen different types of side chains were identified with the structures [Gal(beta 1-3)]x, where x = 1-11, or Glc(1-3)Glc(1-3), or Glc(1-3)Gal(beta 1-3), where glucose is probably in the beta-configuration. All monosaccharides in the phosphoglycan domain are in the pyranose configuration. The average number of repeat units per molecule is 36. The nonreducing terminus of the phosphoglycan chains probably terminates predominantly in the neutral disaccharide Gal(beta 1-4)Man(alpha 1-. Comparison of the structure of L. major amastigote LPG to L. major promastigote procyclic and metacyclic LPG forms (McConville, M. J., Turco, S. J., Furguson, M. A. J., and Sacks, D. L. (1992) Embo J. 11, 3593-3600) indicates that this molecule is developmentally modified throughout the different stages of the parasites' life cycle.
Animals↗
Identification of truncated forms of lipophosphoglycan in mutant cloned lines of Leishmania major that are deficient in mature lipophosphoglycan.
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Animals↗
Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein.
Antibodies against Leishmania major wheat germ agglutinin-binding glycoproteins were used to select from a genomic lambda gt11 expression library a clone coding for a L. major glycoprotein. The partial DNA sequence indicated the presence of a mosaic of repetitive sequences. Southern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to chromosome band 18. Northern blot analysis of L. major mRNA detected a major transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies affinity-purified on the fusion protein identified a complex of two water-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polypeptides in other Leishmania species, in Crithidia lucilliae and very weakly in Leptomonas. The apparent molecular weight of these polypeptides, while conserved within each species, varied between species. A peptide map of the two polypeptides from L. major generated an identical pattern suggesting a close relatedness at the protein level. This protein complex was not hydrolysed by N-glycanase and was not affected by tunicamycin, but treatment with anhydrous hydrogen fluoride suggested that it is O-glycosylated. The glycan moiety appears to be N-acetylglucosamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was capable of adding [3H]galactose to it. This was susceptible to beta elimination and beta-galactosidase treatment. Taken together, the data indicates that gp96/92 belongs to the newly described class of cytoplasmic and nuclear glycoproteins containing O-linked N-acetylglucosamine.
Acetylglucosamine↗
Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major.
This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major. The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L. major cloned line V121. The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced. At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT. The LmYPT gene was present as a single copy gene in both the L. major and L. donovani genomes. Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L. major isolate L119. The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite. Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein. Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.
Amino Acid Sequence↗
Resistance of BALB/c mice to Leishmania major infection is associated with a decrease in the precursor frequency of antigen-specific CD4+ cells secreting interleukin-4.
BALB/c mice are highly susceptible to infection with the protozoan parasite Leishmania major and develop a chronic fatal disease. They can, however, be manipulated to resist disease and this has been shown to correlate with increased expression of IFN-gamma mRNA and the absence of IL-4 mRNA in the draining lymph nodes and spleens of these animals. Here we show that anti-IL-4 or anti-CD4 treatment of BALB/c mice resulted in a reduction in the size of the lesion and in the number of parasites in the draining lymph nodes compared with untreated mice. The precursor frequency of CD4+ T cells proliferating in response to Leishmania antigens in vitro in the treated animals was not significantly different from untreated animals. Analysis of the lymphokines secreted by the clonal progeny of these cells showed that the precursor frequency of IL-4 secreting clones was at least 10-fold lower in animals treated with either mAb. However, there was no reciprocal increase in the precursor frequencies of IFN-gamma secreting clones. Comparisons of the total number of precursors of specific CD4+ cells secreting IFN-gamma showed that anti-CD4-treated animals, which are resistant to disease, had considerably fewer for the first 6 weeks than untreated mice with chronic disease. Protection of BALB/c mice was therefore associated with a reduction in the numbers of precursors of cells secreting IL-4 without a concomitant increase in the number of precursors of IFN-gamma secreting cells.
Animals↗
Persistence of virulent Leishmania major in murine cutaneous leishmaniasis: a possible hazard for the host.
The persistence of Leishmania major parasites in mice resistant to infection was investigated by the polymerase chain reaction and in vitro culture methods. Parasite-specific DNA was detected in the lymph nodes, spleens, bone marrow, and livers of C57BL/6 mice 1 year after their recovery from infection. Live parasites were also recovered from these tissues (except liver tissues) and were used to establish in vitro isolates. Pulsed-field gel electrophoresis, Southern blotting, and Western blot (immunoblot) analyses showed that these isolates retained the karyotype and the phenotype of the original inoculum, including the levels of expression of gp63 and lipophosphoglycan, the two major surface molecules of Leishmania species. More importantly, these isolates were virulent and induced fatal disease when injected into susceptible BALB/c mice. Persistence was shown to be a more general phenomenon, since several different strains of mice which were resistant to L. major infection also harbored persistent parasites. The implications for the etiology of human leishmaniasis in immunocompromised individuals such as AIDS patients are discussed.
Animals↗
Interleukin-4 but not gamma interferon production correlates with the severity of murine cutaneous leishmaniasis.
For murine cutaneous leishmaniasis, data to date suggest a correlation between the presence of gamma interferon (IFN-gamma) and resistance in C57BL/6 mice and the presence of interleukin-4 (IL-4) and disease in BALB/c mice. In this study, 13 inbred strains of mice covering the range of susceptibility to disease were infected with Leishmania major to determine whether the subsequent expression of IFN-gamma or IL-4 is a reliable indicator of cure or progressive disease. The presence of IL-4 and IFN-gamma mRNAs in the draining lymph nodes was examined 9 weeks after infection, when differences in disease severity became obvious. There were large differences in the levels of IL-4 mRNA among the different strains, whereas IFN-gamma mRNA was detected at similar levels in all strains. The levels of IL-4 mRNA correlated with lesion score, with susceptible and intermediate strains containing up to 100-fold more than any of the resistant strains. Differences in the levels of IFN-gamma mRNA were within only a fourfold range, with significant overlap among susceptible, intermediate, and resistant strains. Similarly, the levels of IFN-gamma secreted in vitro by lymph node cells from infected mice in response to L. major antigens were within a 10-fold range for most strains, and there was no correlation with lesion score. Analysis of Leishmania-specific antibody levels revealed a correlation between immunoglobulin G1 (IgG1) titers and lesion score, consistent with the role of IL-4 as a switch factor for IgG1. In contrast, there was no correlation between IgG2a titers and lesion score, supporting the notion that IFN-gamma synthesis (which promotes IgG2a production) is not correlated with disease state. These data suggest that along the spectrum of murine cutaneous leishmaniasis, IL-4 is a reliable indicator of disease, but IFN-gamma is not prognostic for resistance.
Animals↗