Regulation of genes encoding the major surface protease of Leishmania chagasi via mRNA stability

The intercoding regions between many Leishmania sp. genes regulate their mRNA expression. The MSPL mRNA, encoding a subclass of the major surface protease (MSP) of Leishmania chagasi, increases in abundance, when protein synthesis is arrested, while alpha-tubulin (alpha-TUB) mRNA and most other mRNAs do not. We found that the intercoding region between MSPL-coding regions, when cloned downstream of the beta-galactosidase reporter gene (beta-GAL), caused beta-GAL mRNA to increase 8- to 10-fold after inhibiting protein synthesis with cycloheximide. Stable L. chagasi transfectants containing hybrid MSPL/alpha-TUB intercoding regions cloned downstream of beta-GAL were made. The alpha-TUB intercoding region induced high-level baseline beta-GAL mRNA that increased only 1.3-fold after incubation with cycloheximide. In contrast, the MSPL intercoding region, as well as constructs containing nucleotides 303-505 from the MSPL 3'UTR, caused steady-state beta-GAL mRNA levels in the absence of cycloheximide that were approximately 10% of alpha-TUB constructs. These levels increased between 4.4- and 13.2-fold after cycloheximide was added. Constructs containing half of this region (303-394 or 395-505) produced intermediate levels of beta-GAL mRNA and intermediate levels of cycloheximide induction. The kinetics of cycloheximide induction of beta-GAL mRNA was similar with region 303-505 constructs as with constructs bearing the entire endogenous MSPL intercoding region. Furthermore, region 303-505 increased reporter mRNA abundance after cycloheximide by increasing mRNA half-life. Hence, we have identified a 202-nucleotide region within the MSPL 3'UTR that is in part responsible for cycloheximide induction. We hypothesize that this region may interact with labile regulatory protein factor(s).     

Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis

In this report we describe the construction and analysis of a genomic library of Leishmania donovani gene segments in the bacteriophage vector lambda gt11. This cloning vector permits the expression of parasite polypeptides as fusion products with Escherichia coli beta-galactosidase. A group of 90 clones which express L. donovani antigens have been isolated from this library using various polyvalent antisera. Many of these clones appear to encode parasite membrane antigens some of which are recognized by sera from patients with visceral leishmaniasis (kala azar). Some clones reacted with specific subsets of kala azar sera while others reacted with all kala azar sera tested. In addition, some of the clones appear to encode conserved antigens that are recognized by sera from mice immunized with L. major. Preliminary characterization of five clones indicated that each one contains a distinct genetic element and that at least four encode different fusion peptides.     

Cloning of a gene encoding the immunodominant surface antigen of Leishmania donovani promastigotes

This study describes the characterisation of externally oriented surface peptides of both morphological forms of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar). Using 125I surface labelling techniques and peptide extraction in the detergents Triton X-100 and Triton X-114, a major iodinable promastigote peptide at 63 kDa or 65 kDa (depending on detergent used) was identified. This peptide was demonstrated to be the immunodominant membrane peptide of L. donovani and was strongly recognised by human sera from parasitologically confirmed cases of kala-azar. This peptide was not demonstrated on the surface of tissue amastigotes, although in vitro translations of poly(A+) RNA from both promastigotes and amastigotes demonstrated that both forms possessed mRNA that directs the synthesis of a 63 kDa peptide. It is suggested therefore that in amastigotes this peptide may be a processed antigen. We also report the isolation of a recombinant cDNA clone in the bacteriophage vector lambda gt10 which encodes a 63 kDa polypeptide that is recognised by human kala-azar sera. It is proposed that this surface peptide could be used in a specific immunodiagnostic test for leishmaniasis.     

Antigenic specificity of the 72-kilodalton major surface glycoprotein of Leishmania braziliensis braziliensis.

We examined the expression and the antigenicity of the major surface polypeptides of Leishmania braziliensis braziliensis and Leishmania donovani chagasi, parasites which commonly coexist in the same endemic areas of Bolivia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles from surface-iodinated promastigotes showed the presence of a unique iodinatable polypeptide of 72 kDa on the L. b. braziliensis surface and of two major components of 65 and 50 kDa exposed at the surface of L. d. chagasi. Comparison of the peptide digestion profiles of the major iodinated polypeptides of both strains showed no similarity between the maps of the 72- and the 65-kDa polypeptides of L. b. braziliensis and L. d. chagasi, respectively. Immunoprecipitation of surface-labeled L. b. braziliensis Nonidet P-40 extracts with 35 serum specimens obtained from Bolivian patients with cutaneous and mucocutaneous leishmaniasis showed that all serum specimens recognized predominantly the 72-kDa antigen and high-molecular-mass proteins in some cases. The recognition patterns were independent of the geographical origin of the patient, the type of lesion, and the serum antibody titer. Serum specimens from children with visceral leishmaniasis did not precipitate the L. b. braziliensis 72-kDa antigen. Hamster hyperimmune serum against L. b. braziliensis also recognized the 72-kDa surface antigen. However, this recognition was inhibited in the presence of the homologous nonlabeled antigen but not in the presence of heterologous (L. d. chagasi and Trypanosoma cruzi) antigens. The specific recognition of 72-kDa surface antigen in both natural and experimental L. b. braziliensis infections suggests that this antigen could be a good candidate for use in the differential immunodiagnosis and prognosis of the disease.     

Molecular characterization of two genes encoding members of the glucose transporter superfamily in the parasitic protozoan Leishmania donovani.

The polymerase chain reaction was used to clone two genes from Leishmania donovani, each of which encodes a member of a superfamily of membrane transporters which include the mammalian facilitative glucose transporters. One of these transporters, designated D2, is similar in sequence and overall structural features to a previously cloned Leishmania transporter Pro-1. Both D2 and Pro-1 are developmentally regulated genes which are expressed primarily in the insect stage of the parasite life cycle. In contrast, the second novel transporter, D1, is structurally quite different from either D2 or Pro-1, and its expression is not regulated during the parasite life cycle. All three genes are located on different chromosomes in L. donovani.     

Subspecies-specific surface antigens of promastigotes of the Leishmania donovani complex.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of proteins and externally exposed labeled surface constituents were analyzed in promastigotes of three etiological agents of kala azar (Leishmania donovani, HS70 strain from India; L. chagasi, Imperatriz strain from Brazil; L. infantum, ITMPA K263 strain from Morocco and MO strain from France). Coomassie blue-stained gels showed similar protein patterns for L. donovani and L. chagasi and a more distinct one for L. infantum. Surface radioiodination with two different methods, lactoperoxidase and IODO-GEN, gave identical autoradiographic patterns for each parasite. Four major labeled proteins with apparent Mr values of 65,000, 60,000, 50,000, and 26,000 were detected in both L. chagasi and L. donovani. However, the radioiodinated polypeptide pattern of L. infantum only showed two major bands with an apparent Mr of 62,000 and a doublet of 26,000 to 23,000. Immunoprecipitation of detergent extracts of labeled promastigote subspecies with immune sera from rabbits immunized with either L. chagasi or L. infantum and from patients and mice infected with these two parasites, as well as with a monoclonal antibody against the surface of L. donovani promastigotes, demonstrated that the surface antigenic expression of L. infantum is different from that noticed in the two other subspecies, which are similar. Immunofluorescence experiments with some of these antibodies confirmed these results. The present findings should be considered in taxonomic and immunological studies in visceral leishmaniasis.     

DNA synthesis in promastigotes of Leishmania major and L. donovani

The requirement of protozoan parasites for pre-formed purines affords the opportunity for quantitation of nucleic acid synthesis from incorporation of radioactively labeled purines into DNA and RNA. We have developed rapid and simple assays to quantitate DNA and RNA synthesis in promastigotes of Leishmania major and L. donovani from the incorporation of [3H]hypoxanthine. DNA but not RNA synthesis in L. major or L. donovani promastigotes was inhibited by aphidicolin (50% inhibition by 0.2-0.3 microM) and by hydroxyurea (50% inhibition by 0.3-0.5 mM). The inhibition of DNA synthesis by aphidicolin or hydroxyurea was reversible when the inhibitor was removed 2, 4 or 24 h after its addition. Several well-characterized agents that inhibit DNA synthesis in mammalian cells, 1-beta-D-arabinofuranosylcytosine (araC), 9-beta-D-arabinofuranosyladenine (araA), phosphonoacetic acid, novobiocin and N2-(p-n-butylphenyl)guanine (BuPG), failed to inhibit DNA synthesis in promastigotes of L. major even when used at very high concentrations, demonstrating differences between DNA replication components of parasite and host.     

Comparison of the effects of Leishmania major or Leishmania donovani infection on macrophage gene expression

The intracellular parasite Leishmania causes a wide spectrum of human disease, ranging from self-resolving cutaneous lesions to fatal visceral disease, depending on the species of Leishmania involved. The mechanisms by which different Leishmania species cause different pathologies are largely unknown. We have addressed this question by comparing the gene expression profiles of bone marrow-derived macrophages infected with either Leishmania donovani or L. major promastigotes. We found that the two species had very similar effects on macrophage gene expression. Both species caused a small (<2.5-fold) but statistically significant repression of several hundred genes. In addition, both species strongly induced and repressed about 60 genes. Comparing the effects of the two species showed that only 26 genes were regulated differently by L. major as opposed to L. donovani, including those for metallothioneins 1 and 2, HSP70 and -72, CCL4, Gadd45β, Dsp1, matrix metalloprotease 13, T-cell death-associated gene 51 (Tdag51), RhoB, spermine/spermidine N1-acyl transferase 1 (SSAT), and Cox2. L. donovani-infected macrophages were also found to express higher levels of Cox2 protein and prostaglandin E synthase mRNA than L. major-infected macrophages. While both species have previously been shown to trigger prostaglandin E synthesis by bystander cells, this study suggests that infected macrophages themselves express prostaglandin E-synthesizing genes only in response to L. donovani.     

Protection against leishmaniasis by injection of DNA encoding a major surface glycoprotein, gp63, of L. major.

cDNA encoding the highly conserved major surface glycoprotein, gp63, of Leishmania major was cloned, together with a signal sequence, into an eukaryotic expression vector, pCDNAI, which carries the human cytomegalovirus (CMV) promoter. This construct, pCMV/glycoprotein 63 (gp63), when injected into the skeletal muscle of BALB/c mice expressed sustained levels of gp63 in the muscle tissue for at least 40 days. Spleen and lymph node cells from the immunized mice produced significant amounts of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) but no detectable IL-4 when cultured with L. major antigens in vitro. The immunized mice also developed significant resistance against L. major infection compared to control mice injected with the empty plasmid. These results suggest that nucleic acid vaccine is effective against parasite infections.     

Identification of major antigens of Leishmania donovani using kala azar sera

The study was conducted with the prime objective of isolating an antigen from the crude preparation of whole promastigotes (Leishmania donovani) with a view to future exploitation in serodiagnosis and production of monoclonal antibodies. Soluble antigen, prepared from promastigotes isolated from actively growing cultures, was fractionated by gel filtration chromatography over a column of Sephadex G200. Three peaks of proteins could be recovered. The antigenic reactivity of different fractions was checked against sera of kala azar patients by immunoelectrophoresis (IEP), rocket immunoelectrophoresis (RIEP) and enzyme-linked immunosorbent assay (ELISA). The first peak was found to be highly reactive in comparison with other peaks. SDS-PAGE and western blot analysis of this antigen revealed a major antigen of promastigotes in the vicinity of 65 to 66 kDa, while a weakly reactive triplet could also be detected in the low molecular weight region.Abbreviations IEP Immunoelectrophoresis - RIEP Rocket Immunoelectrophoresis - ELISA Enzyme-Linked Immunosorbent Assay - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - BHI Brain Heart Infusion - HBSS Hanks' Balanced Salt Solution - NCP Nitrocellulose Paper - DAB Diamino Benzidine Tetrahydrochloride - kDa Kilo Daltons     

Oxidant-mediated damage of Leishmania donovani promastigotes.

Dissemination of Leishmania within the host is related to parasites undergoing unchecked proliferation. We therefore studied the effects of oxidant generating systems on promastigote multiplication by (i) direct determinations of organism proliferation and (ii) the incorporation of [3H]uracil into promastigote nucleoprotein. These two parameters correlated closely as measures of organism replication as demonstrated by parallel suppression of them by the protein synthesis inhibitors puromycin and cycloheximide and the nucleic acid synthesis inhibitors actinomycin D and mitomycin C. Promastigotes showed dose-related susceptibility to reagent and generated hydrogen peroxide (H2O2) as reflected in quantitatively similar decreases in multiplication and [3H]uracil incorporation. These effects were specific for H2O2 as catalase abrogated the dimunition in multiplication. The generation of superoxide anion by acetaldehyde-xanthine oxidase (10 mU/ml) did not alter promastigote replication or nucleoprotein synthesis. These results indicate that Leishmania donovani promastigotes are damaged by H2O2 and that the incorporation of [3H]uracil into promastigote nucleoprotein may be useful for studying the interaction of this parasite with host effector cells.     

Identification and characterization of host-protective T-cell epitopes of a major surface glycoprotein (gp63) from Leishmania major

By using a series of overlapping synthetic peptides that cover more than 75% of the amino acid sequence of the major surface glycoprotein (gp63) from Leishmania major, 11 T-cell epitopes in CBA and BALB/c mice have been identified. Six of the peptides were recognized by T cells of CBA mice recovered from L. major infection, while one was recognized by the T cells from BALB/c mice recovered from the infection following sublethal doses of gamma-irradiation. Lymph node cells from mice immunized with the peptides also responded to a number of the same peptides (seven in CBA and one in BALB/c). Peptide p10-28 induced proliferative T-cell responses in both CBA and BALB/c mice. Five of the peptides (p10-28, p22-40, p289-309, p459-471 and p467-482) induced vigorous T-cell response in CBA mice but were not recognized by T cells from recovered mice. Four other peptides (p321-336, p364-476, p372-385 and p378-396) were recognized by T cells from recovered CBA mice but could not induce a T-cell response in normal CBA mice. Three peptides (p146-171, p289-309 and p395-414) were both able to induce a T-cell response and were recognized by T cells from recovered mice. However, only two peptides (p146-171 and p467-482) were able to activate T cells, which also recognized epitopes expressed by antigen-presenting cells infected with promastigotes. T cells induced by p146-171 and p467-171 or a mixture of these two peptides were mainly CD4+ and produced interleukin (IL-2) and interferon-gamma (IFN-gamma) but not IL-4 upon antigen stimulation in vitro. These two peptides also induced a classical delayed type hypersensitivity (DTH) response in CBA mice. Furthermore, CBA mice immunized with a mixture of the two peptides in Coryne parvum or entrapped in liposomes induced significant resistance against L. major infection. The implications of these results in terms of a synthetic vaccine against leishmaniasis and the mechanism of the induction of Th1 and Th2 cells are discussed.     

Molecular characterization of clustered variants of genes encoding major surface antigens of human Pneumocystis carinii.

A 13-kb genomic fragment from human Pneumocystis carinii was cloned as repetitive DNA. The fragment contains a cluster of three related genes, each 3 kb in size, and the 5' end of a fourth gene. The predicted polypeptide of the first gene in the cluster comprises 1,030 amino acid residues with a total molecular mass of 116 kDa. The gene's predicted amino acid sequence bears 32% identity to predicted sequences of recently described gene fragments of ferret P. carinii, which encode an immunodominant surface glycoprotein (gpA) (P. J. Haidaris, T. W. Wright, F. Gigliotti, and C. G. Haidaris, J. Infect. Dis. 166:1113-1123, 1992), and 36% identity to the predicted sequence of a rat P. carinii major surface glycoprotein gene (msg) (J. A. Kovacs, F. Powell, J. C. Edman, B. Lundgren, A. Martinez, B. Drew, and C. W. Angus, J. Biol. Chem. 268:6034-6040). DNA hybridization showed that sequences related to the cloned msg genes reside on at least 12 chromosomes of human P. carinii at various degrees of multiplicity and/or homology. Affinity-purified antibodies with specificity to a fusion protein made from the human P. carinii msgI gene recognized two bands on a Western immunoblot containing total human P. carinii protein; they also recognized fusion proteins derived from the other two genes of the cluster. Monoclonal antibodies with reactivity to Msg of human P. carinii recognized fusion proteins produced from two msg genes. Fusion proteins were also recognized by sera from healthy humans and from patients. The msg genes are candidates for the development of immunotherapy and subunit vaccines for the treatment and prevention of P. carinii pneumonia.     

Characterization of RNA from Leishmania tropica and Leishmania d.donovani promastigotes

RNA has been prepared from promastigotes of Leishmania tropica and Leishmania d.donovani using three different methods. Extraction by hot phenol/isothiocyanate gave the best quantitative and qualitative results. The analysis of total RNA on methyl mercuric agarose gels shows that the large rRNA species is nicked: it is composed of a 630 and a 560 kDa molecule. The small rRNA species has a molecular weight of 800,000. Poly(A+) RNA can be translated in a rabbit reticulocyte lysate system. The newly synthesized products comprise high molecular weight proteins and show different patterns using RNA from L. tropica or from L. d. donovani promastigotes.