RESUMEN
OBJECTIVE: To evaluate the effect of pregnancy loss (PL) on MS disease activity. METHODS: Eleven women with first-trimester PLs were identified through a reproductive questionnaire. MS activity (MRI lesions and/or clinical relapses) was compared for the 12â¯months before conception and after PL. RESULTS: There was MS activity in 7/11 participants after, compared with 3/11 before PL (McNemar's test, pâ¯=â¯.29), including MRI activity in 7/11 after, compared with 2/11 before PL (McNemar's test, pâ¯=â¯.13). CONCLUSION: Larger studies are needed to confirm this observed trend of increased MS activity following PL.
Asunto(s)
Aborto Espontáneo , Encéfalo/diagnóstico por imagen , Esclerosis Múltiple/diagnóstico , Aborto Terapéutico , Adulto , Progresión de la Enfermedad , Femenino , Humanos , Imagen por Resonancia Magnética , Esclerosis Múltiple/diagnóstico por imagen , Embarazo , Primer Trimestre del Embarazo , Recurrencia , Índice de Severidad de la Enfermedad , Encuestas y CuestionariosRESUMEN
Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.
Asunto(s)
Malaria/parasitología , Plasmodium/fisiología , Transducción de Señal/fisiología , Animales , Hepatocitos/parasitología , Humanos , Estadios del Ciclo de Vida , Malaria/fisiopatología , Plasmodium berghei/genética , Plasmodium berghei/fisiología , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Toxoplasma/genética , Toxoplasma/fisiologíaRESUMEN
The receptor-binding domain of Plasmodium vivax Duffy-binding protein, region II (PvRII), is an attractive candidate for a vaccine against P. vivax malaria. Here, we have studied the safety and immunogenicity of recombinant PvRII in Macaca mulatta (rhesus monkeys). Recombinant PvRII with a C-terminal 6-histidine tag was expressed in E. coli, recovered from inclusion bodies, refolded into its functional conformation, purified to homogeneity and formulated with three adjuvants, namely, Alhydrogel, Montanide ISA 720 and the GSK proprietary Adjuvant System AS02A for use in immunogenicity studies. All the PvRII vaccine formulations tested were safe and highly immunogenic. The overall magnitude of the antibody response was significantly higher for both Montanide ISA 720 and AS02A formulations in comparison with Alhydrogel. Furthermore, there was a significant correlation between antibody recognition titers by ELISA and binding inhibition titers in in vitro binding assays. The PvRII vaccine formulations also induced IFN-gamma recall responses that were identified using ex vivo ELISPOT assays. These results provide support for further clinical development of a vaccine for P. vivax malaria based on recombinant PvRII.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria/veterinaria , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/sangre , Ensayo de Inmunoadsorción Enzimática , Interferón gamma/metabolismo , Linfocitos/inmunología , Macaca mulatta , Malaria/prevención & control , Manitol/administración & dosificación , Manitol/análogos & derivados , Ácidos Oléicos/administración & dosificación , Unión Proteica , Vacunas Sintéticas/inmunologíaRESUMEN
The blood stage of the malaria parasite's life cycle is responsible for all the clinical symptoms of malaria. The development of clinical disease is dependent on the interplay of the infecting parasite with the immune status and genetic background of the host. Following repeated exposure to malaria parasites, individuals residing in endemic areas develop immunity. Naturally acquired immunity provides protection against clinical disease, especially severe malaria and death from malaria, although sterilizing immunity is never achieved. Given the absence of antigen processing in erythrocytes, immunity to blood stage malaria parasites is primarily conferred by humoral immune responses. Cellular and innate immune responses play a role in controlling parasite growth but may also contribute to malaria pathology. Here, we analyze the natural humoral immune responses acquired by individuals residing in P. falciparum endemic areas and review their role in providing protection against malaria. In addition, we review the dual potential of cellular and innate immune responses to control parasite multiplication and promote pathology.
Asunto(s)
Eritrocitos/parasitología , Interacciones Huésped-Parásitos/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/fisiología , Animales , Humanos , Inmunidad Celular , Inmunidad Innata , Inmunidad Materno-Adquirida , Malaria Falciparum/sangre , Parasitemia/inmunología , Parasitemia/parasitologíaRESUMEN
Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Sistema del Grupo Sanguíneo Duffy/metabolismo , Plasmodium vivax/metabolismo , Proteínas Protozoarias , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/aislamiento & purificación , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Dicroismo Circular , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/metabolismo , Eritrocitos/parasitología , Escherichia coli , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Conformación Proteica , Pliegue de Proteína , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The invasion of erythrocytes by malaria parasites is a multi-step process that requires a series of highly specific molecular interactions. Here, the authors review what has been learned about receptor-ligand interactions that mediate erythrocyte invasion. Parasite proteins involved in these interactions are promising candidates for malaria vaccines. Clear understanding of these interactions is important for the rational design of vaccines that attempt to inhibit invasion and prevent malaria.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/metabolismo , Eritrocitos/parasitología , Proteínas Protozoarias , Receptores de Superficie Celular/metabolismo , Animales , Sitios de Unión , Eritrocitos/metabolismo , Humanos , Malaria/parasitología , Proteína 1 de Superficie de Merozoito/metabolismo , Plasmodium/crecimiento & desarrolloRESUMEN
The complex life cycle of the malaria parasite includes three specialized invasive stages, distinct both in terms of their cellular architecture and in their choice of target host cell. Despite the dissimilarities between these forms, there are clear parallels in the manner by which they enter their respective host cells. Advances in the area of erythrocyte invasion by the malaria merozoite, outlined here by Chetan Chitnis and Mike Blackman and discussed at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000, will undoubtedly impact on our understanding of mechanisms of cell entry by the other invasive forms. Similarly, recent progress in dissecting the functional role of surface proteins expressed by sporozoite and ookinete stages has provided fascinating insights into general aspects of invasion by all invasive stages of apicomplexan parasites.
Asunto(s)
Eritrocitos/parasitología , Malaria/parasitología , Plasmodium falciparum/patogenicidad , Plasmodium/genética , Plasmodium/patogenicidad , Animales , Humanos , Malaria Falciparum/parasitología , Plasmodium/crecimiento & desarrollo , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , VirulenciaRESUMEN
Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas Plasmodium vivax and Plasmodium knowlesi use the Duffy blood group antigen, Plasmodium falciparum uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes. P. knowlesi uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi beta and gamma proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteine-rich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within P. vivax and P. knowlesi beta region II. Chimeric domains containing P. vivax region II sequences fused to P. knowlesi beta region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of P. vivax region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of P. knowlesi beta region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/genética , Sistema del Grupo Sanguíneo Duffy , Eritrocitos/metabolismo , Plasmodium knowlesi/genética , Plasmodium vivax/genética , Proteínas Protozoarias , Receptores de Superficie Celular/genética , Animales , Sitios de Unión , Células COS , Proteínas Portadoras/metabolismo , Mapeo Cromosómico , Humanos , Plasmodium knowlesi/metabolismo , Plasmodium vivax/metabolismo , Unión Proteica , Receptores de Superficie Celular/metabolismo , SolubilidadRESUMEN
Erythrocyte invasion by malaria parasites is mediated by specific molecular interactions. Sialic acid residues of glycophorin A are used as invasion receptors by Plasmodium falciparum. In vitro invasion studies have demonstrated that some cloned P. falciparum lines can use alternate receptors independent of sialic acid residues of glycophorin A. It is not known if invasion by alternate pathways occurs commonly in the field. In this study, we used in vitro growth assays and erythrocyte invasion assays to determine the invasion phenotypes of 15 P. falciparum field isolates. Of the 15 field isolates tested, 5 multiply in both neuraminidase and trypsin-treated erythrocytes, 3 multiply in neuraminidase-treated but not trypsin-treated erythrocytes, and 4 multiply in trypsin-treated but not neuraminidase-treated erythrocytes; 12 of the 15 field isolates tested use alternate invasion pathways that are not dependent on sialic acid residues of glycophorin A. Alternate invasion pathways are thus commonly used by P. falciparum field isolates. Typing based on two polymorphic markers, MSP-1 and MSP-2, and two microsatellite markers suggests that only 1 of the 15 field isolates tested contains multiple parasite genotypes. Individual P. falciparum lines can thus use multiple invasion pathways in the field. These observations have important implications for malaria vaccine development efforts based on EBA-175, the P. falciparum protein that binds sialic acid residues of glycophorin A during invasion. It may be necessary to target parasite ligands responsible for the alternate invasion pathways in addition to EBA-175 to effectively block erythrocyte invasion by P. falciparum.
Asunto(s)
Eritrocitos/parasitología , Glicoforinas/fisiología , Ácido N-Acetilneuramínico/fisiología , Plasmodium falciparum/fisiología , Animales , Genotipo , Humanos , Neuraminidasa/farmacología , Reacción en Cadena de la Polimerasa , Tripsina/farmacologíaAsunto(s)
Antígenos de Protozoos , Proteínas Portadoras/genética , Genes Protozoarios/genética , Plasmodium cynomolgi/genética , Receptores de Superficie Celular/genética , Animales , Clonación Molecular , Dosificación de Gen , Humanos , Intrones/genética , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Plasmodium vivax and the related simian malarial parasite P. knowlesi use the Duffy blood group antigen as a receptor to invade human erythrocytes and region II of the parasite ligands for binding to this erythrocyte receptor. Here, we identify the peptide within the Duffy blood group antigen of human and rhesus erythrocytes to which the P. vivax and P. knowlesi ligands bind. Peptides from the NH2-terminal extracellular region of the Duffy antigen were tested for their ability to block the binding of erythrocytes to transfected Cos cells expressing on their surface region II of the Duffy-binding ligands. The binding site on the human Duffy antigen used by both the P. vivax and P. knowlesi ligands maps to a 35-amino acid region. A 34-amino acid peptide from the equivalent region of the rhesus Duffy antigen blocked the binding of P. vivax to human erythrocytes, although the P. vivax ligand expressed on Cos cells does not bind rhesus erythrocytes. The binding of the rhesus peptide, but not the rhesus erythrocyte, to the P. vivax ligand was explained by interference of carbohydrate with the binding process. Rhesus erythrocytes, treated with N-glycanase, bound specifically to P. vivax region II. Thus, the interaction of P. vivax ligand with human and rhesus erythrocytes appears to be mediated by a peptide-peptide interaction. Glycosylation of the rhesus Duffy antigen appears to block binding of the P. vivax ligand to rhesus erythrocytes.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/inmunología , Quimiocinas CXC , Sistema del Grupo Sanguíneo Duffy/inmunología , Epítopos , Eritrocitos/inmunología , Péptidos y Proteínas de Señalización Intercelular , Plasmodium/inmunología , Proteínas Protozoarias , Receptores de Superficie Celular/inmunología , Amidohidrolasas/farmacología , Secuencia de Aminoácidos , Animales , Unión Competitiva , Proteínas Portadoras/metabolismo , Quimiocina CXCL1 , Factores Quimiotácticos/farmacología , Eritrocitos/parasitología , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Humanos , Macaca mulatta , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Plasmodium knowlesi/inmunología , Plasmodium vivax/inmunología , Unión Proteica/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Especificidad de la EspecieRESUMEN
Plasmodium falciparum expresses on the host erythrocyte surface clonally variant antigens and ligands that mediate adherence to endothelial receptors. Both are central to pathogenesis, since they allow chronicity of infection and lead to concentration of infected erythrocytes in cerebral vessels. Here we show that expression of variant antigenic determinants is correlated with expression of individual members of a large, multigene family named var. Each var gene contains copies of a motif that has been previously shown to bind diverse host receptors; expression of a specific var gene correlated with binding to ICAM-1. Thus, our findings are consistent with the involvement of var genes in antigenic variation and binding to endothelium.
Asunto(s)
Variación Antigénica/genética , Antígenos de Protozoos/genética , Membrana Eritrocítica/parasitología , Regulación de la Expresión Génica/genética , Genes Protozoarios/genética , Plasmodium falciparum/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/inmunología , Secuencia de Bases , Moléculas de Adhesión Celular/metabolismo , Clonación Molecular , Cartilla de ADN , Reordenamiento Génico/genética , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes/genética , Plasmodium falciparum/inmunología , ARN Mensajero/análisis , ARN Protozoario/análisis , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB. Mutant chemokines like E6A could prove to be useful therapeutically for the design of receptor blocking drugs that inhibit erythrocyte invasion by Plasmodium vivax malaria.
Asunto(s)
Alanina , Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Eritrocitos/parasitología , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Neutrófilos/fisiología , Plasmodium knowlesi/patogenicidad , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Quimiocina CXCL1 , Factores Quimiotácticos/química , Clonación Molecular , Secuencia Conservada , Sistema del Grupo Sanguíneo Duffy/metabolismo , Eritrocitos/efectos de los fármacos , Escherichia coli , Sustancias de Crecimiento/química , Humanos , Riñón , Cinética , Malaria/sangre , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacología , Activación Neutrófila , Neutrófilos/efectos de los fármacos , Plasmodium knowlesi/efectos de los fármacos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Plasmodium vivax and the related monkey malaria, P. knowlesi, require interaction with the Duffy blood group antigen, a receptor for a family of chemokines that includes interleukin 8, to invade human erythrocytes. One P. vivax and three P. knowlesi proteins that serve as erythrocyte binding ligands in such interactions share sequence homology. Expression of different regions of the P. vivax protein in COS7 cells identified a cysteine-rich domain that bound Duffy blood group-positive but not Duffy blood group-negative human erythrocytes. The homologous domain of the P. knowlesi proteins also bound erythrocytes, but had different specificities. The P. vivax and P. knowlesi binding domains lie in one of two regions of homology with the P. falciparum sialic acid binding protein, another erythrocyte binding ligand, indicating conservation of the domain for erythrocyte binding in evolutionarily distant malaria species. The binding domains of these malaria ligands represent potential vaccine candidates and targets for receptor-blockade therapy.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/metabolismo , Sistema del Grupo Sanguíneo Duffy/metabolismo , Eritrocitos/parasitología , Plasmodium knowlesi/metabolismo , Plasmodium vivax/metabolismo , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/química , Línea Celular , ADN Protozoario , Eritrocitos/inmunología , Técnica del Anticuerpo Fluorescente , Haplorrinos , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Protozoarias/químicaRESUMEN
A 175-kilodalton erythrocyte binding protein, EBA-175, of the parasite Plasmodium falciparum mediates the invasion of erythrocytes. The erythrocyte receptor for EBA-175 is dependent on sialic acid. The domain of EBA-175 that binds erythrocytes was identified as region II with the use of truncated portions of EBA-175 expressed on COS cells. Region II, which contains a cysteine-rich motif, and native EBA-175 bind specifically to glycophorin A, but not to glycophorin B, on the erythrocyte membrane. Erythrocyte recognition of EBA-175 requires both sialic acid and the peptide backbone of glycophorin A. The identification of both the receptor and ligand domains may suggest rational designs for receptor blockade and vaccines.
Asunto(s)
Antígenos de Protozoos , Proteínas Portadoras/metabolismo , Eritrocitos/parasitología , Glicoforinas/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Ácidos Siálicos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/genética , Línea Celular , Eritrocitos/metabolismo , Glicopéptidos/química , Glicopéptidos/metabolismo , Glicoforinas/química , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo. Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AlgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.
Asunto(s)
Alginatos/metabolismo , Carbohidrato Epimerasas/metabolismo , Ácidos Hexurónicos/metabolismo , Polisacáridos Bacterianos/metabolismo , Pseudomonas aeruginosa/enzimología , Ácidos Urónicos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Carbohidrato Epimerasas/genética , Compartimento Celular , Escherichia coli/genética , Prueba de Complementación Genética , Isomerismo , Datos de Secuencia Molecular , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADNRESUMEN
Plasmodium vivax and P. falciparum are the major causes of human malaria, except in sub-Saharan Africa where people lack the Duffy blood group antigen, the erythrocyte receptor for P. vivax. Duffy negative human erythrocytes are resistant to invasion by P. vivax and the related monkey malaria, P. knowlesi. Several lines of evidence in the present study indicate that the Duffy blood group antigen is the erythrocyte receptor for the chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA). First, IL-8 binds minimally to Duffy negative erythrocytes. Second, a monoclonal antibody to the Duffy blood group antigen blocked binding of IL-8 and other chemokines to Duffy positive erythrocytes. Third, both MGSA and IL-8 blocked the binding of the parasite ligand and the invasion of human erythrocytes by P. knowlesi, suggesting the possibility of receptor blockade for anti-malarial therapy.
Asunto(s)
Antígenos de Protozoos , Quimiocinas CXC , Sistema del Grupo Sanguíneo Duffy , Eritrocitos/parasitología , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/metabolismo , Plasmodium knowlesi/metabolismo , Plasmodium vivax/metabolismo , Proteínas Protozoarias , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Quimiocina CCL5 , Quimiocina CXCL1 , Factores Quimiotácticos/metabolismo , Citocinas/metabolismo , Sustancias de Crecimiento/metabolismo , Humanos , Linfocinas/metabolismo , Proteínas Quimioatrayentes de Monocitos , Plasmodium knowlesi/fisiología , Plasmodium vivax/fisiología , Receptores de Interleucina-8ARESUMEN
Strains of Pseudomonas aeruginosa causing pulmonary infections in cystic fibrosis patients have an unusual mucoid phenotype because of production of the capsule-like exopolysaccharide, alginate. Transcriptional activation of algD, the first gene of a large alginate biosynthetic gene cluster, is associated with conversion to the alginate-producing (Alg+) phenotype. In this study, we examined the regulation of alginate genes immediately downstream of algD. Mutants of the Alg+ strain FRD1 were constructed by gene replacement with defined Tn501 (8.2kb) insertions in the alginate biosynthetic gene cluster, resulting in an Alg- phenotype. The Alg+ phenotype of these mutants was restored by integration of narrow-host-range plasmids containing DNA fragments from P. aeruginosa that reconstructed a continuous alginate gene cluster. A broad-host-range plasmid containing the entire alginate gene cluster except for the terminal gene, algA, was unable to complement an alG::Tn501 mutant unless algA was transcribed from a second plasmid. This indicated that any Tn501 insertion in the cluster was polar on downstream alginate genes. Northern blot hybridization experiments also showed that a transposon insertion downstream of algD adversely affected algG and algA transcription. These results provided evidence that the alginate biosynthetic gene cluster has an operonic structure and is cotranscribed from the algD promoter.
Asunto(s)
Alginatos/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Operón , Pseudomonas aeruginosa/genética , Metabolismo de los Hidratos de Carbono , Expresión Génica , Prueba de Complementación Genética , Ácido Glucurónico , Ácidos Hexurónicos , Familia de Multigenes , Mutagénesis Insercional , Pseudomonas aeruginosa/metabolismo , ARN Bacteriano/genética , ARN Mensajero/genéticaRESUMEN
The biochemical mechanism by which alpha-L-guluronate (G) residues are incorporated into alginate by Pseudomonas aeruginosa is not understood. P. aeruginosa first synthesizes GDP-mannuronate, which is used to incorporate beta-D-mannuronate residues into the polymer. It is likely that the conversion of some beta-D-mannuronate residues to G occurs by the action of a C-5 epimerase at either the monomer (e.g., sugar-nucleotide) or the polymer level. This study describes the results of a molecular genetic approach to identify a gene involved in the formation or incorporation of G residues into alginate by P. aeruginosa. Mucoid P. aeruginosa FRD1 was chemically mutagenized, and mutants FRD462 and FRD465, which were incapable of incorporating G residues into alginate, were independently isolated. Assays using a G-specific alginate lyase from Klebsiella aerogenes and 1H-nuclear magnetic resonance analyses showed that G residues were absent in the alginates secreted by these mutants. 1H-nuclear magnetic resonance analyses also showed that alginate from wild-type P. aeruginosa contained no detectable blocks of G. The mutations responsible for defective incorporation of G residues into alginate in the mutants FRD462 and FRD465 were designated algG4 and algG7, respectively. Genetic mapping experiments revealed that algG was closely linked (greater than 90%) to argF, which lies at 34 min on the P. aeruginosa chromosome and is adjacent to a cluster of genes required for alginate biosynthesis. The clone pALG2, which contained 35 kilobases of P. aeruginosa DNA that included the algG and argF wild-type alleles, was identified from a P. aeruginosa gene bank by a screening method that involved gene replacement. A DNA fragment carrying algG was shown to complement algG4 and algG7 in trans. The algG gene was physically mapped on the alginate gene cluster by subcloning and Tn501 mutagenesis.