RESUMEN
Intracellular parasites of the phylum Apicomplexa contain specialized rhoptry secretory organelles that have a crucial function in host-cell invasion and establishment of the parasitophorous vacuole. Here we show that localization of the Toxoplasma gondii rhoptry protein ROP2 is dependent on a YEQL sequence in the cytoplasmic tail that binds to micro-chain subunits of T. gondii and mammalian adaptors, and conforms to the YXXstraight phi mammalian sorting motif. Chimaeric reporters, containing the transmembrane domains and cytoplasmic tails of the low-density lipoprotein receptor and of Lamp-1, are sorted to the Golgi or the trans-Golgi network (TGN), and partially to apical microneme organelles of the parasite, respectively. Targeting of these reporters is mediated by YXXstraight phi- and NPXY-type signals. This is the first demonstration of tyrosine-dependent sorting in protozoan parasites, indicating that T. gondii proteins may be targeted to, and involved in biogenesis of, morphologically unique organelles through the use of evolutionarily conserved signals and machinery.
Asunto(s)
Secuencia Conservada/fisiología , Evolución Molecular , Orgánulos/metabolismo , Señales de Clasificación de Proteína/fisiología , Toxoplasma/citología , Toxoplasma/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Transporte Biológico , Clatrina/metabolismo , Secuencia Conservada/genética , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/metabolismo , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Orgánulos/ultraestructura , Unión Proteica , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Receptores de LDL/química , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Toxoplasma/genética , Toxoplasma/ultraestructura , Técnicas del Sistema de Dos Híbridos , Tirosina/genética , Tirosina/metabolismoRESUMEN
The binding of complement by two developmentally distinct stages of Leishmania major has been studied. Noninfective log phase growth (LOG) promastigotes (serum sensitive) activate complement with deposition of covalently bound C3b onto the surface of the parasite. Infective, peanut agglutinin (PNA-) metacyclic stage promastigotes (serum resistant) also bear mainly C3b after incubation in serum, but a major portion of deposited C3 is present as a 110 X 10(3) mol wt C3 fragment. Whereas deposition of C3b on LOG promastigotes is mediated through the alternative pathway. PNA- parasites are unable to activate the alternative pathway in nonimmune serum. C3 is released from the parasite surface by proteolytic cleavage, at a rate which is nearly threefold greater for LOG than for PNA- promastigotes. Immunoprecipitation experiments show that the developmentally regulated lipophosphoglycan is a major C3 acceptor on both LOG and PNA- parasites. These experiments, which are the first to compare the form and processing of complement on infective and noninfective promastigotes of Leishmania, provide a framework for further definition of the differential C3 receptor-dependent uptake and survival of these parasites within mononuclear phagocytes.
Asunto(s)
Antígenos de Superficie/metabolismo , Complemento C3/metabolismo , Glicoesfingolípidos/metabolismo , Leishmania tropica/metabolismo , Receptores de Complemento/metabolismo , Animales , Vía Alternativa del Complemento , Leishmania tropica/crecimiento & desarrollo , Leishmania tropica/inmunología , Antígeno de Macrófago-1 , Receptores Mitogénicos/análisisRESUMEN
The mechanism was sought by which bactericidal IgG for E. coli 0111 (strain 12015) increases the bactericidal efficiency of C5b-9. IgG did not affect the distribution of C3 deposition on the O-Ag capsule and the outer membrane of 12015, suggesting that bactericidal IgG was not directing complement activation to different sites on the bacterial surface. However, one-fifth of the C3 that was deposited in the presence of IgG attached covalently to the antibody molecule. Covalent complexes between purified C3b and IgG were prepared in order to study the role of C3b-IgG in the bactericidal reaction. 8-10-fold less C3b-IgG than IgG was necessary to sensitize 12015 for serum killing. When aggregates were eliminated from the C3b-IgG and IgG preparations by sucrose density gradient ultracentrifugation, C3b-IgG remained three- to fourfold more effective than IgG on a molecule-for-molecule bound basis in mediating the serum bactericidal reaction. These results suggest that formation of C3b-IgG during the serum bactericidal reaction is critical for killing, and have important implications for the development of effective bactericidal vaccines.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Actividad Bactericida de la Sangre , Complemento C3b/inmunología , Escherichia coli/inmunología , Inmunoglobulina G/inmunología , Centrifugación por Gradiente de Densidad , Complemento C3b/metabolismo , Vía Alternativa del Complemento , Humanos , Inmunoglobulina G/metabolismoRESUMEN
Trypanosoma cruzi enters host cells via formation of an acidic vacuole which is subsequently disrupted, allowing the parasite access to the cytoplasm. We show that in an acid environment, release of the parasite surface neuraminidase is enhanced, and this release is likely mediated by a phosphatidylinositol-specific phospholipase C (PIPLC), since antibodies to a carbohydrate epitope (CRD) revealed in glycosylphosphatidylinositol (GPI)-anchored proteins after PIPLC cleavage remove the great majority of the soluble neuraminidase activity from culture supernatants. The neuraminidase is active at acidic pH, and is capable of desialylating known vacuolar constituents, i.e., lysosomal membrane glycoproteins. Parasite escape into the cytoplasm is significantly facilitated in terminal sialylation-defective mutant Lec 2 cells, and enzymatically desialylated membranes are more susceptible to lysis by a parasite hemolysin previously implicated in vacuole membrane rupture. These findings provide evidence that terminal sialylation on carbohydrate moieties contributes to maintaining lysosomal membrane integrity, and indicate a role for a protozoan-derived neuraminidase in facilitating parasite entry into host cells. These observations raise the possibility that other microbial neuraminidases may serve a similar function in acidic intracellular compartments.
Asunto(s)
Antígenos CD , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neuraminidasa/metabolismo , Ácidos Siálicos/metabolismo , Trypanosoma cruzi/enzimología , Animales , Células CHO , Línea Celular , Cricetinae , Citoplasma/parasitología , Técnica del Anticuerpo Fluorescente , Concentración de Iones de Hidrógeno , Proteínas de Membrana de los Lisosomas , Lisosomas/ultraestructura , Microscopía Electrónica , Ácido N-Acetilneuramínico , Especificidad por Sustrato , Trypanosoma cruzi/patogenicidadRESUMEN
The mechanism of resistance of gram-negative bacteria to killing by complement was investigated. Complement consumption and uptake of purified, radiolabeled complement components on bacteria was studied using a serum- sensitive and a serum-resistant strain of Salmonella minnesota. Twice as many molecules of (125)I C3 were bound per colony-forming unit (CFU) of the smooth, serum-resistant S. minnesota S218 as were bound per CFU of the rough, serum-sensitive S. minnesota Re595 in 10 percent pooled normal human serum (PNHS), although 75-80 percent of C3 was consumed by both organisms. Hemolytic titrations documented total consumption of C9 by 5 min and more than 95 percent consumption of C5 and C7 by 15 min in the reaction with S218 with 10 percent PNHS. In contrast, negligible C5 depletion, 10 percent C7 consumption, and only a 26 percent decrease in C9 titer occurred with the serum-sensitive Re595. Binding of (125)I C5, (125)I C7, and (125)I C9 to S218 and Re595 was measured in 10 percent PNHS. A total of 6,600 molecules C5/CFU, 5,200 molecules C7/CFU, and 3,100 molecules C9/CFU bound to S218 after 5-10 min of incubation at 37 degrees C, but 50-70 percent of the C5, C7, and C9 bound to S218 was released from the organism during incubation at 37 degrees C for 60 min. Binding of 2,000 molecules C5/CFU, 1,900 molecules C7/CFU, and 9,000 molecules C9/CFU to Re595 was achieved by 20 min and was stable. The ratio of bound C9 molecules to bound C7 molecules, measured using (131)I C9 and (125)I C7, was constant for both organisms after 15 min and was 4.3:1 on Re595 and 0.65:1 on S218 in 10 percent PNHS. With addition of increasing amounts of purified, unlabeled (29 to 10 percent PNHS, there was no change in the C9:C7 ratio on Re595. However, with S218 there was a linear increase of the C9:C7 ratio, which approached the ratio on Re595. There was no (14)C release from S218 incubated in PNHS, nor was there evidence by electron microscopy of outer membrane damage to S218. Therefore, S. minnesota S218 is resistant to killing by PNHS, despite the fact that the organism consumes terminal complement components efficiently and that terminal components are deposited on the surface in significant amounts. The C5b-9 complex is released from the surface of S218 without causing lethal outer membrane damage.
Asunto(s)
Actividad Bactericida de la Sangre , Proteínas del Sistema Complemento/metabolismo , Salmonella/inmunología , Adsorción , Animales , Complemento C3/metabolismo , Complemento C5/metabolismo , Complemento C7/metabolismo , Complemento C9/metabolismo , Ácido Egtácico/farmacología , Humanos , Conejos , Salmonella/metabolismo , Salmonella/ultraestructuraRESUMEN
The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.
Asunto(s)
Actividad Bactericida de la Sangre , Receptores de Complemento , Salmonella/metabolismo , Centrifugación por Gradiente de Densidad , Complemento C5/metabolismo , Complemento C6/metabolismo , Complemento C7/metabolismo , Complemento C8/metabolismo , Complemento C9/metabolismo , Ácido Edético/farmacología , Humanos , Octoxinol , Polietilenglicoles/farmacología , Salmonella/inmunología , Tripsina/farmacologíaRESUMEN
Toxoplasma gondii uses a highly coordinated arsenal of three structurally and biochemically distinct secretory granules to invade and develop in a wide range of host cells. Proteins of these secretory granules are sorted to strategic subcellular locations using distinctive sorting signals and are then triggered differentially for exocytosis. These secreted proteins are subsequently targeted and inserted into membrane domains.
Asunto(s)
Interacciones Huésped-Parásitos , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Animales , Apicomplexa , Gránulos Citoplasmáticos , Humanos , Señales de Clasificación de Proteína , Toxoplasma/metabolismo , Toxoplasmosis/metabolismoRESUMEN
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.
Asunto(s)
Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/fisiología , Toxoplasma/química , Vacuolas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células Cultivadas , Cricetinae , Citoplasma/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Microsomas Hepáticos/metabolismo , Mitocondrias/metabolismo , Mitocondrias Hepáticas/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Matriz Nuclear/metabolismo , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Fracciones Subcelulares , Transfección , Tripsina/farmacologíaRESUMEN
The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.
Asunto(s)
Colesterol/metabolismo , Endocitosis , Receptores de LDL/metabolismo , Toxoplasma/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Colesterol/biosíntesis , Colesterol/farmacología , Cricetinae , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Endocitosis/efectos de los fármacos , Farnesil Difosfato Farnesil Transferasa/genética , Farnesil Difosfato Farnesil Transferasa/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/fisiología , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ácido Mevalónico/metabolismo , Modelos Biológicos , Mutación/genética , Receptores de LDL/antagonistas & inhibidores , Reproducción/efectos de los fármacos , Toxoplasma/citología , Toxoplasma/efectos de los fármacos , Toxoplasma/crecimiento & desarrollo , Vacuolas/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , Neutrófilos/fisiología , Proteínas Opsoninas , Fagocitosis , Fagosomas/metabolismo , Salmonella typhimurium , Animales , Antígenos CD/análisis , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Humanos , Inmunoglobulina G , Lactoferrina/análisis , Ligandos , Microscopía Electrónica , Peso Molecular , Neutrófilos/ultraestructura , Fagosomas/ultraestructura , Conejos/inmunología , Receptores de Complemento/análisis , Receptores de Complemento/metabolismo , Receptores Fc/análisis , Receptores Fc/metabolismo , Receptores de IgG , Coloración y Etiquetado , Transcobalaminas/análisisRESUMEN
The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM-staining, identified with fraction-specific antibodies, cofractionated with known rhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, was cloned and sequenced, predicting and integral membrane protein. Antibodies specific for ROP 2 reacted with the intact PVM. These results provide the first direct evidence that rhoptry contents participate in the formation of the PVM of T. gondii and suggest a possible role of ROP 2 in parasite-host cell interactions.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Orgánulos/metabolismo , Proteínas Protozoarias/biosíntesis , Toxoplasma/fisiología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/biosíntesis , Secuencia de Bases , Células CHO , Membrana Celular/metabolismo , Células Cultivadas , Clonación Molecular , Cricetinae , Fibroblastos/parasitología , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Orgánulos/ultraestructura , Proteínas Protozoarias/análisis , Proteínas Protozoarias/aislamiento & purificación , Piel/parasitología , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli beta-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline phosphatase fused to the vesicular stomatitis virus G protein transmembrane domain and cytoplasmic tail localized primarily to the Golgi, although staining of dense granules and the intravacuolar network was also detected; truncating the cytoplasmic tail decreased Golgi staining and increased delivery to dense granules but blocked delivery to the intravacuolar network. Targeting of secreted proteins to T. gondii dense granules and the plasma membrane uses general mechanisms identified in higher eukaryotic cells but is simplified and exaggerated in scope, while targeting of secreted proteins beyond the boundaries of the parasite involves unusual sorting events.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Glicoproteínas de Membrana , Toxoplasma/metabolismo , beta-Lactamasas/metabolismo , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Sitios de Unión , Brefeldino A , Membrana Celular/metabolismo , Chlorocebus aethiops , Ciclopentanos/farmacología , Citoplasma/metabolismo , Escherichia coli/enzimología , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/metabolismo , Aparato de Golgi/metabolismo , Macrólidos , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Fracciones Subcelulares , Vacuolas , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , beta-Lactamasas/genéticaRESUMEN
After actively entering its host cells, the protozoan parasite Toxoplasma gondii resides in an intracellular vacuole that is completely unable to fuse with other endocytic or biosynthetic organelles. The fusion blocking requires entry of viable organisms but is irreversible: fusion competence of the vacuole is not restored if the parasite is killed after entry. The fusion block can be overcome, however, by altering the parasite's route of entry. Thus, phagocytosis of viable antibody-coated T. gondii by Chinese hamster ovary cells transfected with macrophage-lymphocyte Fc receptors results in the formation of vacuoles that are capable of both fusion and acidification. Phagocytosis and fusion appear to involve a domain of the Fc receptor cytoplasmic tail distinct from that required for localization at clathrin-coated pits. These results suggest that the mechanism of fusion inhibition is likely to reflect a modification of the vacuole membrane at the time of its formation, as opposed to the secretion of a soluble inhibitor by the parasite.
Asunto(s)
Receptores Fc/fisiología , Toxoplasma/fisiología , Transfección , Vacuolas/parasitología , Animales , Línea Celular , Fibroblastos/parasitología , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Técnica del Anticuerpo Fluorescente , Lisosomas/fisiología , Lisosomas/ultraestructura , Macrófagos/inmunología , Fusión de Membrana , Ratones , Ratones Endogámicos BALB C , Fagocitosis , Receptores Fc/genética , Toxoplasma/crecimiento & desarrollo , Vacuolas/fisiología , Vacuolas/ultraestructuraRESUMEN
Lipid X, a precursor of lipid A (the toxic moiety of endotoxin), has been shown to protect animals from the lethal effects of endotoxin challenge. We investigated the mechanism of action of lipid X and 3-aza-lipid X, a diamino-analogue, in vitro, using the ability of lipopolysaccharide (LPS) to prime neutrophils for an enhanced release of toxic oxygen radicals. Lipid X and 3-aza-lipid X inhibited LPS-induced neutrophil priming in a concentration-dependent manner. At high concentrations, 3-aza-lipid X was a partial agonist of priming. Lipid X was found to inhibit LPS-induced priming by directly interacting with the neutrophil in contrast to polymyxin B, which neutralized LPS by binding to it. Increasing concentrations of lipid X shifted the LPS dose response curve of neutrophils rightward but did not prevent maximum priming at higher LPS concentrations, a finding consistent with competitive inhibition. These results suggest that lipid X, a compound structurally related to lipid A, may block neutrophil priming by competing with LPS for cellular binding sites. Lipid X appears to have a novel mechanism of inhibiting LPS effect and may have efficacy in the treatment of gram-negative sepsis.
Asunto(s)
Endotoxinas/farmacología , Glucolípidos/farmacología , Neutrófilos/efectos de los fármacos , Salmonella , Relación Dosis-Respuesta a Droga , Endotoxinas/antagonistas & inhibidores , Humanos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Concentración OsmolarRESUMEN
We examined the fate of desialated autologous erythrocytes injected intravenously into guinea pigs (GP). Desialated GP erythrocytes (E) were lysed directly or cleared by the reticuloendothelial system in normal GP (NIH-GP) and cleared by the reticuloendothelial system in GP genetically deficient in the classical complement pathway component C4 (C4D-GP), which activate complement only via the alternative pathway. Desialated E were also cleared in cobra venom factor-treated GP (CVF-GP), which had less than 1% of normal C3 levels, but were not cleared at all in C4D-CVF-GP. Preinjection of asialoorosomucoid (ASOR) and ovalbumin (OVA) had no effect on the rate of E clearance. These in vivo studies indicated that complement activation is essential for clearance of desialated E and that clearance is unaffected by blockade of galactose or mannose receptors. Inhibition of complement-mediated clearance required blockade of both classical and alternative complement pathways. In vitro studies showed that lysis of desialated E could occur in NIH-GP serum (GPS) but not in C4D-GPS. Surprisingly, CVF-GPS also caused lysis of desialated E. Lysis was dependent on both natural antibody to desialated E and classical pathway activation; natural antibody was of both the IgG and IgM classes. C3 uptake studies demonstrated that almost 10 times as many C3 molecules/E were deposited by NIH-GPS as by C4D-GPS or CVF-GPS onto desialated E. Approximately equal numbers of C3 molecules were deposited by CVF-GPS, which did lyse desialated E, and by C4D-GPS, which did not. We suggest that the molecular mechanism of in vivo clearance and in vitro lysis of desialated E by CVF-GP is via classical pathway deposition of C3b into sites on the erythrocyte surface protected from inactivation by H (beta 1H) and I (C4b/3b inactivator). Deposition of C3b into these sites by alternative pathway activation is sufficient to cause clearance but not lysis of desialated E. CVF-GPS may not represent an adequate reagent for testing the complement dependence of various biologic phenomena, particularly if the question involves surfaces that can provide protected sites for C3b molecules.
Asunto(s)
Activación de Complemento , Vía Alternativa del Complemento , Vía Clásica del Complemento , Eritrocitos/inmunología , Ácidos Siálicos/sangre , Animales , Complemento C3/deficiencia , Complemento C3b/metabolismo , Complemento C4/deficiencia , Venenos Elapídicos/farmacología , Femenino , Cobayas , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Cinética , NeuraminidasaRESUMEN
Campylobacter fetus ssp. fetus strains causing systemic infections in humans are highly resistant to normal and immune serum, which is due to the presence of high molecular weight (100,000, 127,000, or 149,000) surface (S-layer) proteins. Using serum-resistant parental strains (82-40 LP and 23D) containing the 100,000-mol wt protein and serum-sensitive mutants (82-40 HP and 23B) differing only in that they lack the 100,000-mol wt protein capsule, we examined complement binding and activation, and opsono-phagocytosis by polymorphonuclear leukocytes. C3 consumption was similar for all four strains but C3 was not efficiently bound to 82-40 LP or 23D even in the presence of immune serum, and the small amount of C3 bound was predominently the hemolytically inactive iC3b fragment. Consumption and binding of C5 and C9 was significantly greater for the unencapsulated than the encapsulated strains. Opsonization of 82-40 HP with heat-inactivated normal human serum caused greater than 99% killing by human PMN. Similar opsonization of 82-40 LP showed no kill, but use of immune serum restored killing. Findings in a PMN chemiluminescence assay showed parallel results. Association of 32P-labeled 82-40 HP with PMN in the presence of HINHS was 19-fold that for the 82-40 LP, and electron microscopy illustrated that the difference was in uptake rather than in binding. These results indicate that presence of the 100,000-mol wt protein capsule on the surface of C. fetus leads to impaired C3b binding, thus explaining serum resistance and defective opsonization in NHS, mechanisms that explain the capacity of this enteric organism to cause systemic infections.
Asunto(s)
Infecciones por Campylobacter/etiología , Campylobacter fetus/inmunología , Complemento C3/metabolismo , Neutrófilos/inmunología , Actividad Bactericida de la Sangre , Infecciones por Campylobacter/inmunología , Campylobacter fetus/patogenicidad , Campylobacter fetus/ultraestructura , Complemento C3/análisis , Complemento C3/inmunología , Complemento C5/inmunología , Complemento C5/metabolismo , Complemento C9/inmunología , Complemento C9/metabolismo , Humanos , Microscopía Electrónica , Proteínas Opsoninas , Fagocitosis , VirulenciaRESUMEN
Internalization and infectivity of Trypanosoma cruzi trypomastigotes by macrophages is enhanced by prior treatment of parasites with normal human serum. Heating serum or removing C1q from serum abrogates the enhancement, but augmentation of attachment and infectivity is restored by addition of purified C1q to either serum source. Although both noninfective epimastigotes (Epi) and vertebrate-stage tissue culture trypomastigotes (TCT) bind C1q in saturable fashion at 4 degrees C, internalization by monocytes and macrophages of TCT but not Epi-bearing C1q is enhanced in comparison to untreated parasites. Adherence of human monocytes and macrophages to surfaces coated with C1q also induces a marked enhancement of the internalization of native TCT. C1q enhances attachment of both Epi and TCT to human foreskin fibroblasts, but only when C1q is on the parasite and not when the fibroblasts are plated on C1q-coated surfaces. Only TCT coated with C1q show enhanced invasion into fibroblasts. Although trypomastigotes produce an inhibitor of the complement cascade which limits C3 deposition during incubation in normal human serum, C1q binds to the parasite and enhances entry of trypomastigotes into target cells.
Asunto(s)
Complemento C1q/fisiología , Macrófagos/parasitología , Trypanosoma cruzi/fisiología , Animales , Sangre , Activación de Complemento , Complemento C1q/inmunología , Fibroblastos/inmunología , Fibroblastos/parasitología , Calor , Humanos , Macrófagos/inmunología , Monocitos/parasitología , Trypanosoma cruzi/inmunologíaRESUMEN
Blocking immunoglobulin G (IgG) inhibits complement-mediated killing of serum-resistant Neisseria gonorrhoeae (GC) in immune human serum. We examined the mechanism of action of blocking IgG. Presensitization of GC with increasing concentrations of blocking IgG or F(ab')2 before incubation with bactericidal antibody and absorbed pooled normal human serum increased consumption and deposition of the third component of human complement (C3) and the ninth component of human complement (C9) but inhibited killing in dose-related fashion. We next showed that blocking IgG or F(ab')2 partially inhibited binding of bactericidal IgG to GC. Also, binding of a monoclonal antibody recognizing GC outer membrane protein PIII was almost completely inhibited by blocking F(ab')2, confirming other work (Rice, P. A., M. R. Tam, and M. S. Blake, manuscript submitted for publication) showing that PIII is a target for blocking antibody. Studies of the C3 deposition site showed that one quarter of the C3 deposited on GC in the presence of blocking IgG bound covalently to the antibody molecule. Finally, 125I-GC constituents with covalently bound C3 were affinity purified on Sepharose bearing antibodies to C3 and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. C3 deposition on a 40,000-mol wt surface protein was enhanced six- to ninefold by blocking IgG, which indicates that the site of complement deposition was altered by blocking antibody. These studies show that blocking IgG competes with bactericidal antibody for binding to GC, but enhances rather than blocks complement activation, and leads to complement deposition at new sites that do not result in serum killing.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Complemento C3/inmunología , Neisseria gonorrhoeae/inmunología , Complejo Antígeno-Anticuerpo , Sitios de Unión , Unión Competitiva , Activación de Complemento , Complemento C9/inmunología , Citotoxicidad Inmunológica , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunologíaRESUMEN
Strains of Neisseria gonorrhoeae were used to evaluate bactericidal and opsonic properties of McAb 10 directed against the Neisserial outer membrane antigen, H.8. Gonococci were either serum resistant in the absence but serum sensitive in the presence, of McAb 10, or serum sensitive or serum resistant regardless of the presence of McAb 10. Strain JS3, which fell in the former category, was used in subsequent studies. C1 zymogen formed by reassociation of isolated C1 subunits was not directly activated by JS3 in the presence or absence of C1-inhibitor. JS3 thus was unable to directly activate the classical pathway independently of antibody. When purified classical pathway components were used to deposit C3 on JS3 in the absence of serum regulatory proteins or antibodies, added C1-inhibitor reduced C3 binding to background levels. When McAb 10 was present, C3 binding was unaffected by C1-inhibitor. Covalently bound, large molecular weight C3 alpha-chain-gonococcal complexes were disbanded by methylamine release of ester linkages. Released 125I-C3 migrated as C3b without degradation by gonococcal proteases. Purified classical components alone or McAb 10 alone facilitated JS3 killing by neutrophils; when combined, the two provided maximal killing. Levels of McAb 10 that only slightly increase C3 deposition on JS3 are bactericidal in serum and maximally opsonic in combination with purified classical pathway components.
Asunto(s)
Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Inactivadoras del Complemento 1/inmunología , Neisseria gonorrhoeae/inmunología , Actividad Bactericida de la Sangre , Complemento C1/inmunología , Vía Clásica del Complemento , Humanos , Peso Molecular , Neutrófilos/inmunología , FagocitosisRESUMEN
The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains, a cysteine-rich NH2-terminal domain that stabilizes the collagen alpha helix of the second domain and a third COOH-terminal carbohydrate recognition domain. Previous studies have shown that both native and recombinant human MBP bind to wild-type virulent Salmonella montevideo that expresses a mannose-rich lipopolysaccharide. Interaction with MBP results in opsonization and killing by phagocytes. In this report we show that low concentration of MBP (less than 10 micrograms/ml) markedly enhance complement deposition via the alternative complement pathway on S. montevideo. Despite structural similarities between MBP and the C1q subcomponent of the first complement component, MBP did not restore classical pathway activity to C1q-deficient serum, nor did it activate C1s when added to a mixture of C1r and C1s. In the presence of MBP the C3 bound to S. montevideo during incubation in serum was in the form of C3b and iC3b at a ratio of 1:2. Presensitization of S. montevideo with MBP rendered this normally serum resistant organism susceptible to complement-mediated killing. These results emphasize that MBP and complement cooperate in first line defense of the nonimmune host.