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1.
Mol Cell Biochem ; 404(1-2): 53-77, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25763714

RESUMEN

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts.


Asunto(s)
Leishmania donovani/enzimología , Leishmaniasis Visceral/parasitología , beta-Fructofuranosidasa/genética , Secuencia de Aminoácidos , Regulación Enzimológica de la Expresión Génica , Humanos , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/patogenicidad , Leishmaniasis Visceral/enzimología , Leishmaniasis Visceral/genética , Macrófagos/enzimología , Macrófagos/parasitología , Datos de Secuencia Molecular , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/parasitología , beta-Fructofuranosidasa/biosíntesis
2.
Mol Cell Biochem ; 361(1-2): 169-79, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22020747

RESUMEN

Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.


Asunto(s)
Antígenos de Protozoos/sangre , Esterasas/sangre , Leishmania donovani/enzimología , Leishmaniasis Visceral/parasitología , África Oriental , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Southern Blotting , Brasil , Línea Celular , Cromatografía de Afinidad , Perros , Esterasas/inmunología , Esterasas/metabolismo , Humanos , Inmunoprecipitación , India , Leishmania donovani/genética , Leishmania donovani/inmunología , Leishmania donovani/aislamiento & purificación , Leishmania infantum/enzimología , Leishmania infantum/genética , Leishmaniasis Visceral/inmunología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido
3.
Mol Cell Biochem ; 341(1-2): 17-31, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20349119

RESUMEN

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a approximately 33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.


Asunto(s)
Leishmania donovani/enzimología , Lipasa , Animales , Genes Protozoarios , Humanos , Estadios del Ciclo de Vida , Lipasa/química , Lipasa/genética , Lipasa/fisiología , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Transfección
4.
Cell Microbiol ; 10(6): 1363-72, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18284631

RESUMEN

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis. Compared with control plasmid transfectants, the overexpression of chitinase was found to increase the average number of parasites per sand fly and accelerate the escape of parasites from the peritrophic matrix-enclosed blood meal as revealed by earlier arrival at the stomodeal valve. Such flies also exhibited increased damage to the structure of the stomodeal valve, which may facilitate transmission by regurgitation. When exposed individually to BALB/c mice, those flies with chitinase-overexpressing parasites spent on average 2.4-2.5 times longer in contact with their host during feeding, compared with flies with control infections. Furthermore, the lesions that resulted from these single fly bite infections were both significantly larger and with higher final parasite burdens than controls. These data show that chitinase is a multifunctional virulence factor for L. mexicana which assists its survival in Lu. longipalpis. Specifically, this enzyme enables the parasites to colonize the anterior midgut of the sand fly more quickly, modify the sand fly stomodeal valve and affect its blood feeding, all of which combine to enhance transmission.


Asunto(s)
Quitinasas/fisiología , Insectos Vectores/parasitología , Leishmania mexicana/fisiología , Leishmaniasis Cutánea/parasitología , Psychodidae/parasitología , Animales , Interacciones Huésped-Parásitos , Insectos Vectores/patogenicidad , Leishmania mexicana/patogenicidad , Ratones , Ratones Endogámicos BALB C , Virulencia , Factores de Virulencia/fisiología
5.
J Mol Biol ; 373(2): 296-307, 2007 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-17850817

RESUMEN

Here, we show that Crithidia luciliae, a primitive trypanosomatid, purine auxotroph, up-expressed its unique, bi-functional, surface membrane 3'-nucleotidase/nuclease (Cl 3'NT/NU) activity by approximately 1000-fold in response to purine starvation. A second surface membrane phospho-monoesterase, i.e. a tartrate-resistant acid phosphatase (Cl MAcP) was also found to be up-expressed in such purine-starved cells. Here, we used homologous episomal-expression of an antisense construct of the Cl3'NT/NU to dissect the functional expression of these two surface membrane enzymes. In antisense transfected cells, a large excess of the antisense transcript was produced and no trace of any endogenous Cl3'NT/NU sense message was detected. Further, the purine-starvation hyper-induced levels of 3'NT/NU enzyme activity were completely abrogated in these transfected cells versus controls. Moreover, such antisense transcription completely abolished the ability of these transfectants to grow in poly(A)-containing medium demonstrating the essential nature of the 3'NT/NU for the growth/survival of this parasite. In contrast, antisense transcription had no apparent deleterious effects on either endogenous or purine-starvation-induced levels of MAcP enzyme activity, its steady-state mRNA levels, or the constitutive expression of house-keeping genes (e.g. Cl alpha-tubulin) in these transfectants. Cumulatively, results of our antisense experiments demonstrated that the functional nuclease activity of the surface membrane Cl 3'NT/NU was, in fact, critical/essential for the growth and development of these primitive parasites.


Asunto(s)
Crithidia/enzimología , Nucleotidasas/genética , ARN sin Sentido/genética , ARN Mensajero/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Membrana Celular/metabolismo , Crithidia/metabolismo , Inducción Enzimática , Nucleotidasas/antagonistas & inhibidores , Nucleotidasas/metabolismo , Plásmidos/genética , ARN Mensajero/genética , ARN Protozoario/genética , ARN Protozoario/metabolismo , Transcripción Genética , Transfección
6.
Int J Parasitol ; 34(2): 205-17, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15037106

RESUMEN

In this report, we describe an in vitro culture system for the generation and propagation of axenic amastigotes from the well characterised 1S-CL2D line of Leishmania donovani. Fine structure analyses of these in vitro-grown amastigotes demonstrated that they possessed morphological features characteristic of L. donovani tissue-derived amastigotes. Further, these axenic amastigotes (LdAxAm) were shown to synthesise and release a secretory acid phosphatase isoform similar to that produced by intracellular amastigotes. Such LdAxAm also expressed surface membrane 3'-nucleotidase enzyme activity similar to that of tissue-derived amastigotes. Moreover, LdAxAm, in contrast to promastigotes, expressed significant levels of the amastigote-specific A2 proteins. In addition, LdAxAm, derived from long term cultures of Ld 1S-CL2D promastigotes, had significant infectivity for both human macrophages in vitro and for hamsters in vivo. Thus, the in vitro culture system described herein provides a useful tool for the generation of large quantities of uniform populations of axenic amastigotes of the L. donovani 1S-CL2D line. The availability of such material should greatly facilitate studies concerning the cell and molecular biology of this parasite developmental stage.


Asunto(s)
Leishmania donovani/fisiología , Parasitología/métodos , Fosfatasa Ácida/metabolismo , Animales , Cricetinae , Interacciones Huésped-Parásitos , Humanos , Leishmania donovani/ultraestructura , Macrófagos/parasitología , Microscopía Electrónica de Rastreo , Virulencia
7.
Vaccine ; 29(34): 5657-65, 2011 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-21704111

RESUMEN

Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified.


Asunto(s)
Escherichia coli/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza , Vacunas contra la Influenza/inmunología , Proteínas Recombinantes , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunación
8.
J Biol Chem ; 282(13): 10079-10095, 2007 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-17276983

RESUMEN

The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.


Asunto(s)
Desoxirribonucleasas/química , Desoxirribonucleasas/fisiología , Leishmania donovani/enzimología , Ribonucleasas/química , Ribonucleasas/fisiología , Secuencia de Aminoácidos , Animales , Desoxirribonucleasas/clasificación , Desoxirribonucleasas/metabolismo , Humanos , Datos de Secuencia Molecular , Ribonucleasas/clasificación , Ribonucleasas/metabolismo
9.
J Biol Chem ; 282(43): 31789-802, 2007 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-17766245

RESUMEN

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.


Asunto(s)
Entamoeba histolytica/enzimología , Ribonucleasas/química , Ribonucleasas/fisiología , Secuencia de Aminoácidos , Animales , Catálisis , Sistema Libre de Células/enzimología , Cisteína/química , Disulfuros/química , Entamoeba histolytica/patogenicidad , Epítopos , Genes Protozoarios , Histidina/química , Humanos , Hidrólisis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Pruebas de Precipitina , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Ribonucleasas/clasificación , Ribonucleasas/genética , Ribonucleasas/metabolismo
10.
J Biol Chem ; 280(5): 3847-61, 2005 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-15561707

RESUMEN

Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an approximately 50 kDa protein, with well conserved substrate binding and catalytic domains characteristic of members of the chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release approximately >2-4-fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal expression system was devised and used to express an epitope-tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by reverse transcription-PCR, Western blots, and indirect immunofluorescence analyses. Further, results of coupled immunoprecipitation/enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in the mammalian phase of this important human pathogen.


Asunto(s)
Quitinasas/genética , Quitinasas/metabolismo , Leishmania mexicana/enzimología , Leishmania mexicana/genética , Leishmaniasis Cutánea/parasitología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Quitinasas/química , Humanos , Leishmania mexicana/patogenicidad , Macrófagos/parasitología , Ratones , Datos de Secuencia Molecular , Sitios de Empalme de ARN/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Virulencia
11.
J Eukaryot Microbiol ; 51(1): 108-12, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15068272

RESUMEN

Promastigotes of all pathogenic Leishmania species secrete acid phosphatase (SAcP) activity during their growth in vitro. It has been suggested that this enzyme may play a role in the survival of the parasite within its sandfly-vector host. To carry out such functions, SAcP would have to be relatively resistant to endogenous sandfly gut-proteases. Therefore, the current study was undertaken to ascertain whether L. donovani SAcP activity was affected by treatment with various proteases. Native L. donovani SAcP was treated with a variety of serine-, thiol-, metallo- and mixed-proteases and subsequently assayed for enzymatic activity. Of the eleven proteases tested, only bromelain and subtilisin treatments caused a pronounced reduction in SAcP activity. Treatment of SAcP with seven out of the remaining nine proteases, resulted in an overall enhancement in SAcP enzymatic activity ranging from approximately 10% (e.g. with trypsin) to > or = 90% (e.g. with ficin). The resistance of the Leishmania SAcP to various proteases may prolong its functional life within the sandfly gut and help to facilitate parasite infection in this host.


Asunto(s)
Leishmania donovani/enzimología , Leishmania donovani/patogenicidad , Animales , Endopeptidasas/metabolismo , Humanos , Hidrolasas/aislamiento & purificación , Hidrolasas/metabolismo , Cinética , Especificidad por Sustrato
12.
J Biol Chem ; 277(20): 17994-8001, 2002 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-11882658

RESUMEN

The primitive trypanosomatid pathogen of humans, Leishmania donovani, constitutively expresses a unique externally oriented, tartrate-resistant, acid phosphatase on its surface membrane. This is of interest because these organisms are obligate intracellular protozoan parasites that reside and multiply within the hydrolytic milieu of mammalian macrophage phago-lysosomes. Here we report the identification of the gene encoding this novel L. donovani enzyme. In addition, we characterized its structure, demonstrated its constitutive expression in both parasite developmental forms, and determined the cell surface membrane localization of its translated protein product. Further, we used a variety of green fluorescent protein chimeric constructs as reporters in a homologous leishmanial expression system to dissect the functional domains of this unique, tartrate-resistant, surface membrane enzyme.


Asunto(s)
Fosfatasa Ácida/química , Leishmania donovani/enzimología , Tartratos/farmacología , Fosfatasa Ácida/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Southern Blotting , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Conformación Proteica , Proteínas Recombinantes de Fusión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
13.
Mol Cell Biochem ; 245(1-2): 31-41, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12708742

RESUMEN

Recently, we identified and characterized the genes encoding several distinct members of the histidine-acid phosphatase enzyme family from Leishmania donovani, a primitive protozoan pathogen of humans. These included genes encoding the heavily phosphorylated/glycosylated, tartrate-sensitive, secretory acid phosphatases (Ld SAcP-1 and Ld SAcP-2) and the unique, tartrate-resistant, externally-oriented, surface membrane-bound acid phosphatase (Ld MAcP) of this parasite. It had been previously suggested that these enzymes may play essential roles in the growth, development and survival of this organism. In this report, to further examine this hypothesis, we assessed whether members of the L. donovani histidine-acid phosphatase enzyme family were conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Such phylogenetic conservation would clearly indicate an evolutionary selection for this family of enzymes and strongly suggest and support an important functional role for acid phosphatases to the survival of these parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that homologs of both the Ld SAcPs and Ld MAcP were present in each of the visceral and cutaneous Leishmania species examined (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of enzyme assays showed that all of these organisms expressed both tartrate-sensitive and tartrate-resistant acid phosphatase activities. In addition, homologs of both the Ld SAcPs and Ld MAcP genes and their corresponding enzyme activities were also identified in two Crithidia species (C. fasciculata and C. luciliae) and in Leptomonas seymouri. In contrast, Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens had only very-low levels of such enzyme activities. Cumulatively, results of this study showed that homologs of the Ld SAcPs and Ld MAcP are conserved amongst all pathogenic Leishmania sps. suggesting that they may play significant functional roles in the growth, development and survival of all members of this important group of human pathogens.


Asunto(s)
Fosfatasa Ácida/química , Células Eucariotas/química , Histidina , Isoenzimas/química , Leishmania donovani/enzimología , Trypanosoma/química , Fosfatasa Ácida/metabolismo , Animales , Biomarcadores de Tumor , Cromosomas , ADN Protozoario/análisis , Humanos , Isoenzimas/metabolismo , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmania infantum/enzimología , Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmania major/enzimología , Leishmania major/genética , Leishmania major/metabolismo , Leishmania mexicana/enzimología , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Leishmania tropica/enzimología , Leishmania tropica/genética , Leishmania tropica/metabolismo , Fosfatasa Ácida Tartratorresistente , Trypanosoma/genética , Trypanosoma/metabolismo
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