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1.
Vaccine ; 25(8): 1534-43, 2007 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-17178178

RESUMO

The P-8 proteoglycolipid complex (P-8 PGLC), an amastigote antigen of Leishmania pifanoi, has been demonstrated to induce protection in mouse models, as well as to induce Tc1/Th1-like cellular responses in American cutaneous leishmaniasis patients. Because the immunization with P-8 PGLC in the murine model does not appear to be genetically restricted, we have studied the reactivity of the P-8 PGLC in Leishmania infantum infected dogs. In this study, it is shown that PBMC from experimentally infected dogs (asymptomatic, oligosymptomatic) significantly proliferated in response to soluble leishmanial antigen (SLA) or the P-8 PGLC. Further, quantification of the gene expression induced by the stimulation with P-8 in asymptomatically infected dogs showed an up-regulation of IFN-gamma and TNF-alpha, which were three to 4-fold higher than that induced by soluble Leishmania antigen (SLA). While no measurable induction of IL-10 was observed, low levels of IL-4 mRNA were observed in response to both P-8 and SLA antigens. Thus, our studies establish that P-8 is recognized by infected canines and elicits a potentially curative/protective Th1-like immune response. The identification of Leishmania antigens that elicit appropriate immune responses across different host species (humans, canine) and disease manifestations (cutaneous or visceral) could be an advantage in generating a general vaccine for leishmaniasis.


Assuntos
Antígenos de Protozoários/imunologia , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Vacinas Protozoárias/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Citocinas/biossíntese , Citocinas/genética , Citocinas/imunologia , Cães , Feminino , Interferon gama/imunologia , Leishmania infantum/parasitologia , Leishmaniose Visceral/parasitologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Th1/imunologia , Fator de Necrose Tumoral alfa/imunologia
2.
Scand J Immunol ; 59(3): 294-304, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15030581

RESUMO

Innate mechanisms involving natural killer cells have been implied to play an important role in immunity against Leishmania infection. Previous studies have evaluated responses to three purified amastigote antigens, P-2, P-4 and P-8, of Leishmania pifanoi. The P-4 and P-8 antigens have been demonstrated to induce protection in mouse models, as well as to induce cellular responses in American cutaneous leishmaniasis patients. Cells from Leishmania aethiopica-infected leishmaniasis patients preferentially responded to P-8 and, to a lesser extent, to the cysteine proteinase, P-2. In this study, it is shown that cells from healthy donors, including cells from truly naïve donors (cord blood), could be stimulated to proliferation and cytokine production by P-2. The main proliferating cell types in healthy adult donors were CD16/56(+) and the CD8(+) cells. Blocking of major histocompatibility complex (MHC) class II with alpha-MHC class II antibodies markedly inhibited proliferation and interferon-gamma (IFN-gamma) production, whereas interleukin-10 production was not affected. Experimental evidence indicates that CD4(+) cells were not necessary for the proliferative and IFN-gamma responses; however, an adherent cell population was required. Furthermore, CD16/56(+) cells expressing MHC class II were expanded following P-2 stimulation. The responses to P-2 show a striking similarity to responses induced by the vaccine candidate Leishmania homologue of receptors for activated C-kinase (LACK) in healthy donors. The responses described here may not be desirable when aiming at inducing protective immune responses with a vaccine, and the implications of these results for the development of vaccines against leishmaniasis are discussed.


Assuntos
Antígenos de Protozoários/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Células Matadoras Naturais/imunologia , Leishmania/imunologia , Leishmaniose/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Sangue Fetal/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/imunologia , Leishmaniose/prevenção & controle , Ativação Linfocitária/imunologia , Vacinas Protozoárias/imunologia , RNA Mensageiro/química , RNA Mensageiro/genética , Receptores de IgG/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Clin Exp Immunol ; 132(2): 316-22, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12699423

RESUMO

Lymphoproliferative responses to three affinity chromatography purified amastigote antigens of Leishmania pifanoi, P-2, P-4 and P-8, were evaluated in peripheral blood mononuclear cells (PBMC) from patients with Ethiopian cutaneous leishmaniasis. Antigen-stimulated cells were analysed for the percentage of CD4+, CD8+ and CD16/56+ cells and the expressed levels of gamma interferon (IFNgamma) and interleukin (IL)-10 were determined in culture supernatants. The amastigote antigens induced cellular responses in leishmaniasis patients with heterologous Leishmania parasite infection. These responses were compared to those of freeze-thawed L. aethiopica promastigote antigen stimulation. The membrane protein (P-8), and to a lesser extent the megasomal/cytoplasmic cysteine proteinase(P-2), induced proliferation with high levels of IFNgamma and IL-10 production in cells from patients with active L. aethiopica lesions. CD16/56+ NK cells were the main cell types induced to proliferate in response to P-8 and P-2 stimulation, followed by CD8+ cell populations. P-4 had no such effect. This contrasts from previous studies of New World human leishmaniasis where P-4 and P-8 were stimulatory. The success of a particular molecule in the induction of a response with a protective phenotype may be dependent on the infecting Leishmania spp. To our knowledge, there are no studies that directly compare the New versus Old World cutaneous leishmaniasis in respect of NK cell and IL-10 responses. Our studies indicate that some leishmanial molecules are recognized across the species, while others are apparently more species specific.


Assuntos
Antígenos de Protozoários/imunologia , Leishmania/imunologia , Leishmaniose Cutânea/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Animais , Linfócitos T CD8-Positivos/imunologia , Divisão Celular , Células Cultivadas , Etiópia , Feminino , Humanos , Imunofenotipagem , Interferon gama/metabolismo , Interleucina-10/metabolismo , Células Matadoras Naturais/imunologia , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Contagem de Linfócitos , Masculino
4.
Infect Immun ; 69(11): 6776-84, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11598050

RESUMO

The Leishmania pifanoi amastigote antigen P-8 has been previously shown to induce protective immunity in a murine model of cutaneous leishmaniasis (L. Soong, S. M. Duboise, P. Kima, and D. McMahon-Pratt, Infect. Immun. 63:3559-3566, 1995). As this antigen is of interest for further vaccine studies, the biochemical characterization of P-8 was undertaken. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot analysis, and gel filtration chromatography revealed that P-8 antigen consisted of two proteoglycolipid complexes. The P-8 epitope is associated with the L. pifanoi amastigote-specific glycolipid components found in the two complexes. The P-8 complex 1 (P-8c1) consists of a 56-kDa serine metalloproteinase, apolipoprotein E (derived from fetal bovine serum), and amastigote-specific glycolipids. The P-8 complex 2 (P-8c2) consists of a 31-kDa cysteine proteinase associated with amastigote glycolipids. Biochemical analyses suggest that the P-8 antigenic glycolipids may be distinct from previously described Leishmania glycolipids (glycosylinositol-phospholipids and sphingoglycolipids). Protective immunity studies revealed that P-8c1 (serine metalloproteinase-glycolipid complex) confers comparable protection against infection as immunopurified P-8. The isolated P-8c2 (cysteine proteinase-glycolipid complex) does not provide significant protection, nor does stimulation with P-8c2 result in significant T-cell activation in P-8- or P-8c2-vaccinated mice. Consequently, the P-8c1 complex appears to be the immunodominant component of P-8.


Assuntos
Antígenos de Protozoários/análise , Leishmania/imunologia , Proteínas de Membrana/análise , Proteínas de Protozoários/análise , Animais , Antígenos de Protozoários/imunologia , Feminino , Glicolipídeos/análise , Glicolipídeos/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteolipídeos/análise , Proteolipídeos/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia
5.
Int J Parasitol ; 31(5-6): 536-43, 2001 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11334939

RESUMO

Pathogenic trypanosomatids cause a plethora of diseases marked by the lack of efficient vaccines and therapies. As a consequence, studies are being conducted that are geared towards the understanding of basic mechanisms and various biological aspects of these parasites that might be used as targets for new developments in these areas. One such aspect is the understanding of specific cellular trafficking mechanisms that might be attacked with the intention of disease control. In this paper, we give an overview of our current knowledge of cellular targeting mechanisms in trypanosomatids, with special emphasis on our data related to lysosomal targeting of cysteine proteinases in Leishmania.


Assuntos
Antiprotozoários/farmacologia , Leishmania/metabolismo , Trypanosoma/metabolismo , Animais , Antiprotozoários/farmacocinética , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmaniose/tratamento farmacológico , Proteínas Luminescentes/genética , Proteínas Luminescentes/farmacocinética , Lisossomos/enzimologia , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/farmacocinética , Trypanosoma/efeitos dos fármacos , Trypanosoma/genética , Tripanossomíase Africana/tratamento farmacológico
6.
Parasitol Int ; 49(4): 301-7, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11077264

RESUMO

We have previously identified and characterized two amastigote-specific cysteine proteinases of Leishmania pifanoi. The slightly different isoforms of the more abundant proteinase are coded by a gene family of approximately 20 gene copies, that contain a C-terminal extension characteristic of cysteine proteinases of trypanosomatids. In this gene family, we have detected a copy that codes for a truncated form of this proteinase, lacking the C-terminal extension. Interestingly, when the deletion of a nucleotide that creates a stop codon causing this truncation is disregarded, the translated sequence gives rise to a divergent C-terminal extension that has many conserved amino acids when compared to Leishmania and Trypanosome, suggesting that a recent mutation led to the truncation.


Assuntos
Cisteína Endopeptidases/genética , Leishmania/enzimologia , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Dosagem de Genes , Leishmania/genética , Dados de Sequência Molecular , Análise de Sequência de DNA
8.
J Immunol ; 159(4): 1828-34, 1997 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-9257846

RESUMO

CD8+ T cells play a protective role in immunity to cutaneous leishmaniasis. However, it has been unclear how these cells execute this function, since results from several investigations attempting to demonstrate recognition of Leishmania-infected macrophages by CD8+ T cells have been contradictory. In this study, we report the generation of CD8+ T cell lines specific for GP46/M-2, a leishmanial Ag, previously shown to protectively immunize mice against a Leishmania amazonensis challenge. Using T cell cytolysis and IFN-gamma production to assess CD8+ T cell activation, we show that in addition to recognizing mammalian cells transfected with GP46/M-2, these CD8+ T cell lines also recognize macrophages infected with Leishmania amazonensis. MHC class I presentation of GP46/M-2 by infected macrophages can be blocked by treatment with brefeldin A and also by inhibitors of the cytosolic multicatalytic proteasome, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal and N-acetyl-L-leucinyl-L-leucinylmethional. These results suggest that this leishmanial Ag is processed in the macrophage cytoplasm and is presented to CD8+ T cells via the classical pathway of MHC class I presentation. The relevance of these findings as they impact on our understanding of the biology of the parasite within the macrophage is discussed.


Assuntos
Apresentação de Antígeno , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Leishmania/imunologia , Macrófagos/imunologia , Animais , Citocinas/biossíntese , Feminino , Ativação Linfocitária , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos CBA
9.
Eur J Immunol ; 26(12): 3163-9, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8977318

RESUMO

CD4+ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the I4.07 macrophage cell line (H-2k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4+ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4+ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4+ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.


Assuntos
Apresentação de Antígeno , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Leishmania/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Animais , Feminino , Leishmania/crescimento & desenvolvimento , Leishmania major/crescimento & desenvolvimento , Leishmania major/imunologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/imunologia , Camundongos , Camundongos Endogâmicos CBA
10.
Exp Parasitol ; 84(2): 144-55, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8932764

RESUMO

Patients suffering from American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) or by three proteins (A-2/P-2, P-4, and P-8) derived from Leishmania pifanoi amastigotes were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma), interleukin 2 (IL-2) and interleukin 4 (IL-4) produced were also determined. Results show two different patterns of Lb-induced T cell responses: (a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma, IL-2, and IL-4) during the active disease, (b) similar proportions of responding CD4+ and CD8+ cells and type 1 cytokine production (presence of IFN-gamma and IL-2 and very low IL-4) at the end of therapy (healed lesions). Thus, this last pattern is probably associated with a beneficial T cell response. The A-2/P-2 amastigote cysteine proteinase provided only marginal (s.i. approximately or = 2.5) T cell stimulation in 25% of patients studied; in contrast, the L. pifanoi P-4 and P-8 amastigote antigens induced significant stimulation (s.i. approximately or = 5) in approximately 50% of the patients. In comparison to Lb-stimulated cultures, lower proliferative responses of T lymphocytes to P-4 or P-8 were observed. However, the P-4- or P-8-stimulated cultures had similar percentages of reactive CD4+ and CD8+ cells, as well as type 1 cytokines (presence of IFN-gamma and IL-2, and low levels or absence of IL-4) in the supernatants both before and at the end of therapy. The consistent induction of apparently beneficial T cell responses by the P-4 and P-8 amastigote glycoproteins points to the possibility that these molecules be considered as candidates for future defined vaccines against leishmaniasis.


Assuntos
Antígenos de Protozoários/imunologia , Citocinas/biossíntese , Leishmania braziliensis/imunologia , Leishmania/imunologia , Leishmaniose Cutânea/imunologia , Linfócitos T/imunologia , Animais , Antimônio/uso terapêutico , Antiprotozoários/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Feminino , Humanos , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Leishmaniose Cutânea/tratamento farmacológico , Ativação Linfocitária , Masculino , Meglumina/uso terapêutico , Antimoniato de Meglumina , Compostos Organometálicos/uso terapêutico
11.
Immunity ; 4(3): 263-73, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8624816

RESUMO

To study the role of CD40 ligand (CD40L) in the host immune responses against intracellular pathogens, we infected CD40L knockout (CD40L-/-) mice with Leishmania amazonensis. Although wild-type mice were susceptible to infection and developed progressive ulcerative lesions, tissue parasite burdens in CD40L-/- mice were significantly higher. This heightened susceptibility to infection was associated with an impaired T cell and macrophage activation and altered inflammatory response, as reflected by low levels of IFN gamma, lymphotoxin-tumor necrosis factor (LT-TNF), and nitric oxide (NO) production. Furthermore, CD40L-/- mice failed to generate a protective immune response after immunization. These results indicate an essential role of cognate CD40-CD40L interactions in the generation of cellular immune responses against an intracellular parasite.


Assuntos
Antígenos CD40/metabolismo , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Ligante de CD40 , Suscetibilidade a Doenças , Interações Hospedeiro-Parasita , Leishmaniose Cutânea/patologia , Ligantes , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Mutantes , Óxido Nítrico/biossíntese
12.
Exp Parasitol ; 82(1): 34-44, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8617329

RESUMO

As a means of assessing the usefulness of the Rhesus macaque (Macaca mulatta) as a nonhuman primate model for studying cutaneous leishmaniasis, monkeys were infected with Leishmania amazonensis. Variation in the level of susceptibility was found; however, animals inoculated with 10(8) promastigotes provided consistent results as indicated by an earlier onset and/or larger size of lesions. Three monkeys, which had recovered from skin lesions, were challenge-infected using the same parasite strain/dose; although these animals remained susceptible to homologous infection, lesion size was smaller and healed faster than in the initial infection. The immunologic features during infection were assessed. Levels of IgM and IgG antibodies to promastigote antigens rose during active infection and then declined; immunoblot analyses indicated that numerous leishmanial antigens (predominately >30 kDa) were recognized. Delayed type hypersensitivity (DTH) responses and proliferative responses (PBL) developed during active infection and/or rechallenge. Circulating peripheral T cell subpopulations varied throughout the course of infection. Initially (6-8 weeks p.i.), CD4+ T cells appear to predominate; subsequently (15-21 weeks p.i.), an increase in CD8+ T cells was observed. Pathologic analyses indicated that lesions contained amastigotes with a mononuclear infiltrate of macrophages, lymphocytes, and plasma cells, and formation of tuberculoid-type granulomas. As the progression and resolution of leishmanial infection in rhesus macaques are very similar to those observed in humans, this primate model could be employed for elucidating the mechanisms of protective immunity in cutaneous leishmaniasis.


Assuntos
Modelos Animais de Doenças , Leishmaniose Cutânea/veterinária , Macaca mulatta/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Western Blotting , Suscetibilidade a Doenças , Feminino , Imunidade Celular , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Ativação Linfocitária , Masculino , Pele/patologia
13.
J Exp Med ; 182(5): 1423-33, 1995 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-7595213

RESUMO

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.


Assuntos
Regulação da Expressão Gênica , Genes de Protozoários , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Leishmania donovani/genética , Macrófagos/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/genética , Feminino , Antígenos de Histocompatibilidade Classe II/biossíntese , Leishmania/classificação , Leishmania/genética , Leishmania/imunologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Fases de Leitura Aberta , Fragmentos de Peptídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas de Protozoários/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
14.
Mol Biochem Parasitol ; 68(1): 119-32, 1994 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7891736

RESUMO

Biosynthesis, enzymatic processing, and immunocytochemical localization of an abundant developmentally regulated cysteine proteinase of Leishmania pifanoi, Lpcys2, were investigated employing axenic cultured amastigotes and monoclonal antibodies specifically recognizing either the mature proteinase or the carboxy-terminal extension domain. Pulse labeling and protein sequence data indicated that a 45-kDa precursor is processed to a 40-kDa intermediate, which is further cleaved to generate the 27-kDa mature enzyme and a 15-kDa COOH-terminal domain. Evidence indicates that proteolytic activity is associated with the intermediate form as well as the mature proteinase. Treatment with selected cysteine but not aspartic acid proteinase inhibitors arrested proteolytic processing of Lpcys2 in vivo and inhibited parasite cell division. Electron microscopic immunolocalization of both catalytic and COOH-terminal domains in L. pifanoi and Leishmania amazonensis amastigotes showed intense labeling of megasomes, indicating that cleavage of the COOH-terminal domain probably occurs in the megasome. A low level of the mature proteinase was also associated with the flagellar pocket and plasma membrane; consistent with this observation, low level secretion of Lpcys2 into the culture medium was detected. Lpcys1, a related, less abundant amastigote-specific cysteine proteinase lacking a comparable COOH-terminal domain, was localized to the flagellar pocket and megasomes. Consequently, enzyme sorting to megasomes does not appear to depend upon the COOH-terminal domain; hence this region of Lpcys2 may not be essential for its intracellular targeting.


Assuntos
Cisteína Endopeptidases/biossíntese , Leishmania/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Genes de Protozoários , Imuno-Histoquímica , Leishmania/genética , Leishmania/crescimento & desenvolvimento , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional
15.
Mol Biochem Parasitol ; 57(1): 101-15, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8426606

RESUMO

A developmentally regulated cysteine proteinase associated with an unique lysosomal organelle, the megasome, has been described for the intracellular amastigotes of the Leishmania mexicana complex; this proteinase appears to be important in the survival of the parasite. Degenerate primers encoding the active sites residues have been used to amplify cysteine proteinase cDNA sequences from axenically cultured amastigotes of Leishmania pifanoi, a member of the L. mexicana complex. Based on sequence data, two distinct genes (Lpcys1 and Lpcys2) were identified. Although both genes are preferentially transcribed in the amastigote stage, each is distinct in genomic arrangement and chromosome location, with Lpcys2 showing evidence for the presence of 8-20 tandemly arrayed copies and mRNA levels 10-fold higher than Lpcys1. Related forms of the Lpcys1 and Lpcys2 genes exist in other species of the genus Leishmania, including Leishmania braziliensis, Leishmania major and Leishmania donovani. The protein sequence of an abundant immunoaffinity purified amastigote cysteine proteinase (A-2) is identical to that predicted for the product of Lpcys2; immunofluorescence studies show an intracellular pattern/distribution for the A-2 proteinase consistent with a putative megasomal association. The DNA sequence of a genomic copy of Lpcys2 predicts a C-terminal extension for the proteinase; comparative sequence analyses of the C-terminal extensions found for Trypanosoma cruzi and Trypanosoma brucei reveal the selective conservation of cysteine, as well as proline and glycine residues, suggesting that conservation of folding and secondary structure may be required for biological function.


Assuntos
Cisteína Endopeptidases/genética , Genes de Protozoários , Leishmania/enzimologia , Leishmania/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cisteína Endopeptidases/metabolismo , DNA de Protozoário/genética , Leishmania/crescimento & desenvolvimento , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA de Protozoário/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
16.
Mol Biochem Parasitol ; 49(1): 111-8, 1991 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1775152

RESUMO

Inability to culture the disease-producing amastigote form of Leishmania has greatly hampered its study. We have biochemically characterized an axenically cultured amastigote-like form of Leishmania pifanoi. This form closely resembles amastigotes in proteinase, ribonuclease, adenine deaminase and peroxidase activity. It also exhibits comparable rates of growth, transformation, synthesis of DNA, RNA and protein, and metabolism of glucose and linoleic acid. It is distinct from promastigotes in these characteristics. The expression of the genes for beta-tubulin and the P100/11E reductase is developmentally regulated in this axenic form as in amastigotes. These results, combined with previous demonstrations of amastigote morphology and antigenicity in the culture form, confirm that Leishmania amastigotes have been successfully propagated in axenic media. This strain should serve as an excellent model for the study of amastigote biochemistry, pharmacology and immunology, and the molecular genetics of the transformation between amastigote and promastigote forms.


Assuntos
Leishmania/metabolismo , Adenosina Desaminase/metabolismo , Animais , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Leishmania/genética , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Ácido Linoleico , Ácidos Linoleicos/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo
17.
Mol Biochem Parasitol ; 47(2): 235-43, 1991 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1719417

RESUMO

Biochemical features of the immunologically protective, membrane glycoprotein GP46/M-2 of Leishmania amazonensis have been investigated. The protein appears to have a single carbohydrate side chain of approximately 3 kDa, representing 7% of the mass of the mature GP46/M-2 protein. Experiments removing this carbohydrate side chain from GP46/M-2 indicate that the carbohydrate is not involved in the epitope recognized by the monoclonal antibody, M-2. As this monoclonal antibody recognizes a species-specific epitope, these data suggest that this determinant is defined by the polypeptide portion of the molecule. Studies employing the VSG-lipase as well as anti-CRD antibody clearly indicate that the molecule is anchored to the surface membrane of the promastigote via a phosphatidylinositol-linked lipid anchor. Neither the carbohydrate side chain nor the lipid anchor appear to be responsible for the apparent refractoriness of this protein to protease digestion, suggesting that properties of the polypeptide itself may be responsible. These data are discussed in terms of recent DNA-derived protein sequence of the GP46/M-2.


Assuntos
Leishmania mexicana/química , Glicoproteínas de Membrana/química , Proteínas de Protozoários/química , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/química , Endopeptidases , Epitopos/química , Imunoquímica , Leishmania mexicana/imunologia , Lipase , Glicoproteínas de Membrana/imunologia , Estrutura Molecular , Proteínas de Protozoários/imunologia
18.
Am J Trop Med Hyg ; 44(6): 662-75, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1677544

RESUMO

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).


Assuntos
Leishmania/classificação , Leishmaniose/parasitologia , Psychodidae/parasitologia , Bichos-Preguiça/parasitologia , Adulto , Animais , Anticorpos Monoclonais/imunologia , Colômbia , DNA Circular/análise , DNA de Cinetoplasto , DNA de Protozoário/análise , Feminino , Humanos , Isoenzimas/análise , Leishmania/citologia , Leishmania/isolamento & purificação , Leishmania/patogenicidade , Leishmaniose/veterinária , Macrófagos/parasitologia , Masculino , Panamá , Phlebotomus/parasitologia , Polimorfismo de Fragmento de Restrição
19.
Proc Natl Acad Sci U S A ; 87(21): 8393-7, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2236047

RESUMO

Immunization of mice with the GP46/M-2 membrane glycoprotein has been demonstrated to elicit protection against infection with the parasitic protozoan Leishmania amazonensis. As this molecule is important for future vaccine studies of leishmaniasis, the gene encoding the GP46/M-2 surface membrane glycoprotein of Leishmania amazonensis has been cloned and sequenced. The protein sequence derived from the DNA sequence data is consistent with the known biochemical and immunochemical properties of the protein and indicates a number of structural areas of interest. A repetitive sequence (24 amino acids repeated four times) occurs within the amino-terminal portion of the molecule and constitutes approximately 22% of the total mature protein. The protease-resistant immunodominant carboxyl-terminal domain of the protein comprises approximately half of the molecule and consists of proline-rich and cysteine-rich areas of sequence; the distribution of cysteine residues is suggestive of metal binding motifs. The sequence predicts a hydrophobic leader peptide, and a putative attachment site for a glycosyl-phosphatidylinositol anchor is indicated at the carboxyl terminus, consistent with the membrane location of the protein. Southern blot analyses also indicate the presence of a GP46/M-2 gene family.


Assuntos
Leishmania/genética , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , DNA/genética , Biblioteca Gênica , Leishmania/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição
20.
J Protozool ; 36(5): 502-10, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2810145

RESUMO

Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32 degrees C for L. panamensis (WR442; L. braziliensis panamensis) and at 28 degrees C for L. braziliensis (M5052; L. braziliensis braziliensis). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.


Assuntos
Leishmania braziliensis/crescimento & desenvolvimento , Leishmania/crescimento & desenvolvimento , Macrófagos/parasitologia , Animais , Linhagem Celular , Meios de Cultura , Leishmania braziliensis/ultraestrutura , Microscopia Eletrônica , Temperatura
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