RESUMO
The saprophytic nematode Halicephalobus is a rare cause of fatal human meningo-encephalitis, and West Nile virus is neurotropic flavivirus implicated in a variety of clinical neurologic syndromes. Here we report a case of rapidly progressive CNS encephalopathy and death. Serologic, immuno-histochemical, histopathologic and nucleic acid studies demonstrate the presence of active Halicephalobus and West Nile virus in the CNS tissue. This is the first reported case of co-infection with these neurotropic pathogens.
Assuntos
Helmintíase do Sistema Nervoso Central/patologia , Nematoides/classificação , Infecções por Nematoides/parasitologia , Febre do Nilo Ocidental/patologia , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Helmintíase do Sistema Nervoso Central/complicações , Coinfecção , Evolução Fatal , Feminino , Humanos , Pessoa de Meia-Idade , Infecções por Nematoides/complicações , Infecções por Nematoides/patologia , Febre do Nilo Ocidental/complicaçõesRESUMO
Leishmaniasis is a global term for cutaneous and visceral anthroponotic and zoonotic diseases caused by the vector-borne parasites of the genus Leishmania. These diseases afflict at least 2 million people each year with more than 350 million at risk in 98 countries worldwide. These are diseases mostly of the impoverished making prevention, diagnosis and treatment difficult. Therapy of leishmaniasis ranges from local treatment of cutaneous lesions to systemic, often toxic, therapy for disseminated cutaneous, mucocutaneous and deadly visceral disease. This review is a summary of the clinical syndromes caused by Leishmania and treatment regimens currently used for various forms of leishmaniasis.
Assuntos
Leishmaniose/diagnóstico , Leishmaniose/terapia , Antiprotozoários/uso terapêutico , Humanos , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/terapia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/terapia , Estágios do Ciclo de Vida , SíndromeRESUMO
The most abundant protein on the surface of the promastigote form of the protozoan parasites Leishmania spp. is a 63-kDa molecule, designated gp63 or leishmanolysin. Because gp63 has been shown to possess fibronectin-like properties, we examined the interaction of gp63 with the cellular receptors for fibronectin. We measured the direct binding of Leishmania to human macrophages or to transfected mammalian cells expressing human fibronectin receptors. Leishmania expressing gp63 exhibited modest but reproducible adhesion to human macrophages and to transfected CHO cells expressing alpha4/beta1 fibronectin receptors. In both cases, this interaction depended on gp63 but occurred independently of the SRYD sequence of gp63, because parasites expressing gp63 with a mutated SRYD sequence bound to macrophages and alpha4/beta1 receptor-expressing cells as well as did wild-type parasites. The contribution of gp63 to parasite adhesion was more pronounced when the assays were performed in the presence of complement, suggesting that the receptors for complement and fibronectin may cooperate to mediate the efficient adhesion of parasites to macrophages. The interaction of gp63 with fibronectin receptors may also play an important role in parasite internalization by macrophages. Erythrocytes to which gp63 was cross-linked were efficiently phagocytized by macrophages, whereas control erythrocytes opsonized with complement alone bound to macrophages but remained peripherally attached to the outside of the cell. Similarly, parasites expressing wild-type gp63 were rapidly and efficiently phagocytized by resting macrophages, whereas parasites lacking gp63 were internalized more slowly. This rapid internalization of gp63-expressing parasites was dependent on the beta1 integrins, because pretreatment of macrophages with monoclonal antibodies to the beta1 integrins decreased the internalization of gp63-expressing parasites. These observations indicate that complement receptors are the primary mediators of parasite adhesion; however, maximal parasite adhesion and internalization may require the participation of the beta1 integrins, which recognize fibronectin-like molecules such as gp63 on the surface of the parasite.
Assuntos
Integrinas/metabolismo , Leishmania/metabolismo , Macrófagos/metabolismo , Metaloendopeptidases/metabolismo , Proteínas de Protozoários/metabolismo , Receptores de Fibronectina/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Animais , Células CHO , Células Cultivadas , Complemento C3/metabolismo , Cricetinae , Humanos , Integrina alfa4beta1 , Integrinas/genética , Leishmania/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Monócitos/metabolismo , Fagocitose/imunologia , Receptores de Fibronectina/genética , Receptores de Retorno de Linfócitos/genética , Ovinos , TransfecçãoRESUMO
Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Macrófagos/parasitologia , Animais , Cricetinae , Glicolipídeos/análise , Glicolipídeos/isolamento & purificação , Glicosilfosfatidilinositóis/química , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Leishmania mexicana/patogenicidade , Mesocricetus , Camundongos , Polissacarídeos/análise , Transfecção , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , VirulênciaRESUMO
Specific receptors may be involved in the process of attachment of Leishmania donovani promastigotes to macrophage surfaces and their subsequent internalization. Two virulent strains of Indian L. donovani (AG83 and GE-I) were found to enter into macrophages much faster than the avirulent ones (UR6). These virulent promastigotes express surface glycoprotein (gp63) and lipophosphoglycan (LPG) to a greater extent than avirulent strains. We examined their interaction with macrophages as a function of time by preblocking the macrophage receptors with the exogenous addition of gp33 or LPG. In experiments where gp63 was used as the blocking agent, the entry of one virulent strain (GE-I) was affected. In other experiments where LPG was used, the entry of another virulent strain (AG83) was affected. Entry of the avirulent strain (UR6) was unaffected by either of these treatments. Exposed LPG or gp63 on the surface of promastigotes thus appear to expedite their recognition and entry into the host cell. To assess the role of gp63 further in the entry of Leishmania into the macrophages, an avirulent UR6 strain was transfected with the gp63 gene cloned from L. amazonensis. The transfected UR6 as expected expressed more GP63 at a faster rate and entered into the macrophages like the virulent strain when compared to the nontransfected UR6 or UR6 transfected with vector alone. Thus, the expression of the gp63 gene is involved in the recognition and intracellular entry of visceral Leishmania into the macrophages in addition to the cutaneous species demonstrated previously.
Assuntos
Glicoesfingolipídeos/fisiologia , Leishmania donovani/patogenicidade , Macrófagos Peritoneais/parasitologia , Metaloendopeptidases/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Membrana Celular/química , Regulação da Expressão Gênica , Cinética , Leishmania donovani/genética , Leishmania donovani/ultraestrutura , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos BALB C , Transfecção , Virulência , ViscosidadeRESUMO
Leishmanolysin (EC 3.4.24.36) (gp63) is a HEXXH metalloprotease, encoded by multicopied genes in Leishmania and implicated in the infectivity of these parasitic protozoa. We examined posttranslational regulation of gp63 expression by site-specific mutagenesis of the predicted catalytic/zinc-binding sites in the H264EXXH motif, the potential sites of N-glycosylation and glycosyl phosphatidylinositol addition. Mutant and wild-type genes were cloned into a Leishmania-specific vector for transfecting a deficient variant, which produced gp63 approximately 20-fold less than wild-type cells. The selective conditions chosen fully restored this deficiency in transfectants with the wild-type gene. Under these conditions, all transfectants were found comparable in both the plasmid copy number per cell and elevation of gp63 transcripts. Mutant and wild-type products in the transfectants were then compared quantitatively and qualitatively by specific immunologic and protease assays. The results indicate the following. 1) Glu-265 in the HEXXH motif is indispensable for the catalytic activity of gp63. The propeptide of the inactive mutant products was cleaved, suggestive of a non-intramolecular event. 2) Substitution of either His residue in HEXXH leads to apparent intracellular degradation of the mutant products, pointing to a role for zinc binding in in vivo stability of gp63. 3) The three potential sites of N-glycosylation at Asn-300, Asn-407, and Asn-534 are all utilized and contribute to intracellular stability of gp63. 4) Substitution of Asn-577 causes release of all mutant products, indicative of its specificity as a glycosyl phosphatidylinositol addition site for membrane anchoring of gp63. It is suggested that expression of gp63 as a functional protease is regulated by these posttranslational modification pathways.
Assuntos
Leishmania/enzimologia , Metaloendopeptidases/metabolismo , Animais , Sequência de Bases , Compartimento Celular , Expressão Gênica , Glicosilação , Glicosilfosfatidilinositóis , Histidina/química , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Zinco/químicaRESUMO
The Leishmania surface protease gp63 has been identified as a parasite virulence factor. To better define the role of gp63 in Leishmania infectivity, the interaction of recombinant gp63 with complement and complement receptors was examined. On Leishmania, gp63 was not necessary for complement fixation. Complement activation occurred on transfected organisms expressing varying amounts of gp63 and on organisms expressing a mutant form of gp63 devoid of proteolytic activity. However, organisms expressing wild-type gp63 on their surface fixed only small amounts of the terminal complement components and were dramatically more resistant to lysis by complement than were those lacking functional gp63. Organisms expressing wild-type gp63 more rapidly converted C3b on their surface to a form that exhibited the neoantigen of iC3b and interacted avidly with cells expressing Mac-1, the receptor for iC3b. Complement fixation by transfected mammalian cells expressing recombinant Leishmania gp63 on their surface was also examined. The presence of Leishmania gp63 on the surface of these cells converted them into efficient activators of complement. Cells expressing gp63 on their surface fixed complement and bound avidly to the human complement receptors. The proteolytic activity of this molecule was not necessary for complement activation or adhesion to complement receptors. Thus, gp63 may contribute to parasite virulence by exerting a novel type of control over complement fixation. Organisms expressing gp63 can exploit the opsonic properties of complement while avoiding its lytic effects.
Assuntos
Antígenos de Protozoários/fisiologia , Leishmania/enzimologia , Glicoproteínas de Membrana/fisiologia , Metaloendopeptidases/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Células CHO , Adesão Celular , Células Cultivadas , Ativação do Complemento , Cricetinae , Técnicas de Transferência de Genes , Humanos , Macrófagos/fisiologia , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genéticaRESUMO
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Leishmania major/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Sequência de Carboidratos , Glicoesfingolipídeos/metabolismo , Glicosilfosfatidilinositol Diacilglicerol-Liase , Membranas Intracelulares/enzimologia , Leishmania major/genética , Leishmania major/crescimento & desenvolvimento , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Fenótipo , Fosfatidilinositol Diacilglicerol-Liase , Diester Fosfórico Hidrolases/genética , TransfecçãoRESUMO
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoesfingolipídeos/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Leishmania major/metabolismo , Metaloendopeptidases/metabolismo , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/metabolismo , Fosfolipases Tipo C/biossíntese , Animais , Compartimento Celular , Citoplasma/metabolismo , Glicosilfosfatidilinositóis/química , Hemoglobinúria Paroxística/metabolismo , Leishmania major/genética , Mamíferos , Linfócitos T/metabolismoRESUMO
The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots, gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man (1Ó6)Man(1Ó4)GlcN(1Ó6)-myo-inositol-1-phospholipid, is cystolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma
Assuntos
Humanos , Retículo Endoplasmático , Fosfatidilinositóis/biossíntese , Glicolipídeos/biossíntese , Hemoglobinúria Paroxística/metabolismo , Leishmania tropica , Trypanosoma brucei brucei , Fosfolipases Tipo C , Linhagem Celular , Fosfatidilinositóis/metabolismo , Glicolipídeos/metabolismo , Mamíferos , Glicoproteínas Variantes de Superfície de TrypanosomaRESUMO
Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.
Assuntos
Linhagem Celular/microbiologia , Simplexvirus/isolamento & purificação , Proteínas Virais/metabolismo , Animais , Membrana Celular/metabolismo , Cães , Eletroforese em Gel de Poliacrilamida , Rim/citologia , Rim/microbiologia , Rim/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Células Vero/ultraestrutura , Proteínas do Envelope Viral/metabolismoRESUMO
An immunostimulating complex (iscom) containing the envelope proteins of pseudorabies virus (PRV) was prepared and its efficacy was evaluated in two experiments on sheep. In the first experiment, sheep were intramuscularly (i.m.) or intradermally (i.d.) vaccinated with PRV iscom doses varying between 1 and 81 micrograms. The vaccination was repeated on Day 21 and the animals were exposed to challenge infection by subcutaneous inoculation of 1000 TCID50 of the virulent Phylaxia strain on Day 35 after first vaccination. In the second experiment, sheep were i.m. vaccinated with single doses of iscom varying between 1 and 27 micrograms and challenge-infected on Day 14. It was found that: (1) the i.d. administration of PRV iscom has no advantage over i.m. administration (2); a single dose of greater than or equal to 3 micrograms of PRV iscom provided protection against the disease. In immunoblots, viral proteins of molecular masses 120, 109, 55, 53 and 32 kDa were detected with the sera obtained from iscom-vaccinated and subsequently challenge-infected sheep, but not with sera from sheep which were iscom-vaccinated only. The above findings indicated that: (1) by using iscom technology, potent subunit vaccines can be prepared to prevent Aujeszky's disease; (2) the selective incorporation of viral envelope proteins into iscoms gives the opportunity to discriminate between iscom-vaccinated and naturally infected animals.