RESUMEN
Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.
Asunto(s)
Eritroblastos/metabolismo , Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Pez Cebra/metabolismo , Anemia/sangre , Anemia/metabolismo , Animales , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular , Secuencia Conservada , Eritroblastos/citología , Eritroblastos/patología , Regulación de la Expresión Génica , Prueba de Complementación Genética , Hemo/metabolismo , Homeostasis , Humanos , Sobrecarga de Hierro , Proteínas Hierro-Azufre/biosíntesis , Proteínas Hierro-Azufre/genética , Proteínas de Transporte de Membrana/genética , Ratones , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutación/genética , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Madre/citología , Células Madre/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.
Asunto(s)
Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares , Proteínas de Pez Cebra , Pez Cebra/embriología , Animales , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Hematopoyesis/fisiología , Hibridación in Situ , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Elementos Reguladores de la Transcripción/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide variety of infections that are responsible for significant morbidity, mortality and costs to our healthcare system. An efficacious vaccine against ExPEC would be desirable. Previously, we demonstrated that nasal immunization with a genetically engineered strain in which capsule and O-antigen are no longer expressed (CP923) was immunogenic, generated antibodies that bound a subset of heterologous ExPEC strains, and enhanced neutrophil-mediated bactericidal activity against the homologous and a heterologous strain in vitro. In the work reported here we tested the hypothesis that nasal immunization with CP923 conferred protection in a mouse intravenous sepsis model. Nasal immunization with the wild-type strain CP9 conferred protection against challenge with itself and this protection was enhanced when IL-12 was used as an adjuvant. However, when CP923 was used the immunogen, protection was not observed against challenge with CP9. Next, we hypothesized that the observed lack of protection may be due to capsule and the O-antigen moiety of lipopolysaccharide (LPS) impeding antibody binding to non-capsule and O-antigen epitopes. This hypothesis was substantiated by in vitro binding assays, which demonstrated that binding of polyclonal anti-CP923 antisera was decreased when capsule and/or O-antigen were present. Lastly, neutrophil-mediated bactericidal activity against CP923, opsonisized with anti-CP923 antisera, was significantly increased compared to CP9. Taken together, these results demonstrate that the capsule and O-antigen form a biologically significant barrier against antibodies directed against non-capsular and O-antigen epitopes. This defense against the acquired immune response will need to be overcome for the development of a successful vaccine against ExPEC.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Cápsulas Bacterianas/inmunología , Vacunas contra Escherichia coli/inmunología , Antígenos O/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana , Unión Proteica , Sepsis/microbiología , Sepsis/prevención & control , Análisis de SupervivenciaRESUMEN
Infections due to extraintestinal pathogenic E. coli (ExPEC) result in significant morbidity, mortality and increased healthcare costs. An efficacious vaccine against ExPEC would be desirable. In this report, we explore the use of killed-whole E. coli as a vaccine immunogen. Given the diversity of capsule and O-antigens in ExPEC, we have hypothesized that alternative targets are viable vaccine candidates. We have also hypothesized that immunization with a genetically engineered strain that is deficient in the capsule and O-antigen will generate a greater immune response against antigens other than the capsular and O-antigen epitopes than a wild-type strain. Lastly, we hypothesize that mucosal immunization with killed E. coli has the potential to generate a significant immune response. In this study, we demonstrated that nasal immunization with a formalin-killed ExPEC derivative deficient in capsule and O-antigen results in a significantly greater overall humoral response compared to its wild-type derivative (which demonstrates that capsule and/or the O-antigen impede the development of an optimal humoral immune response) and a significantly greater immune response against non-capsular and O-antigen epitopes. These antibodies also bound to a subset of heterologous ExPEC strains and enhanced neutrophil-mediated bactericidal activity against the homologous and a heterologous strain. Taken together, these studies support the concept that formalin-killed genetically engineered ExPEC derivatives are whole cell vaccine candidates to prevent infections due to ExPEC.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli , Ingeniería Genética/métodos , Vacunas de Productos Inactivados , Administración Intranasal , Animales , Cápsulas Bacterianas/genética , Sangre/microbiología , Escherichia coli/genética , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/genética , Vacunas contra Escherichia coli/inmunología , Femenino , Formaldehído/farmacología , Humanos , Inmunización , Masculino , Ratones , Antígenos O/genética , Conejos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/genética , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Vascular endothelial growth factor-receptors (VEGF-Rs) are pivotal regulators of vascular development, but a specific role for these receptors in the formation of heart valves has not been identified. We took advantage of small molecule inhibitors of VEGF-R signaling and showed that blocking VEGF-R signaling with receptor selective tyrosine kinase inhibitors, PTK 787 and AAC 787, from 17-21 hr post-fertilization (hpf) in zebrafish embryos resulted in a functional and structural defect in cardiac valve development. Regurgitation of blood between the two chambers of the heart, as well as a loss of cell-restricted expression of the valve differentiation markers notch 1b and bone morphogenetic protein-4 (bmp-4), was readily apparent in treated embryos. In addition, microangiography revealed a loss of a definitive atrioventricular constriction in treated embryos. Taken together, these data demonstrate a novel function for VEGF-Rs in the endocardial endothelium of the developing cardiac valve.
Asunto(s)
Válvulas Cardíacas/embriología , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Transducción de Señal/fisiología , Pez Cebra/embriología , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/genética , Núcleo Celular/metabolismo , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Válvulas Cardíacas/anomalías , Válvulas Cardíacas/efectos de los fármacos , Válvulas Cardíacas/fisiología , Humanos , Factores de Transcripción NFATC/metabolismo , Ftalazinas/farmacología , Piridinas/farmacología , Receptor Notch1/biosíntesis , Receptor Notch1/genética , Transducción de Señal/efectos de los fármacos , Pez Cebra/fisiología , Proteínas de Pez CebraRESUMEN
The red blood cell membrane skeleton is an elaborate and organized network of structural proteins that interacts with the lipid bilayer and transmembrane proteins to maintain red blood cell morphology, membrane deformability and mechanical stability. A crucial component of red blood cell membrane skeleton is the erythroid specific protein 4.1R, which anchors the spectrin-actin based cytoskeleton to the plasma membrane. Qualitative and quantitative defects in protein 4.1R result in congenital red cell membrane disorders characterized by reduced cellular deformability and abnormal cell morphology. The zebrafish mutants merlot (mot) and chablis (cha) exhibit severe hemolytic anemia characterized by abnormal cell morphology and increased osmotic fragility. The phenotypic analysis of merlot indicates severe hemolysis of mutant red blood cells, consistent with the observed cardiomegaly, splenomegaly, elevated bilirubin levels and erythroid hyperplasia in the kidneys. The result of electron microscopic analysis demonstrates that mot red blood cells have membrane abnormalities and exhibit a severe loss of cortical membrane organization. Using positional cloning techniques and a candidate gene approach, we demonstrate that merlot and chablis are allelic and encode the zebrafish erythroid specific protein 4.1R. We show that mutant cDNAs from both alleles harbor nonsense point mutations, resulting in premature stop codons. This work presents merlot/chablis as the first characterized non-mammalian vertebrate models of hereditary anemia due to a defect in protein 4.1R integrity.