RESUMEN
Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.
Asunto(s)
Actinas/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Proteínas de Escherichia coli/metabolismo , Proteína Oncogénica v-crk/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Electroporación , Escherichia coli Enteropatógena/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Proteínas Oncogénicas/metabolismo , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño , TransfecciónRESUMEN
BACKGROUND: Adenoviruses are common pathogens in vertebrates, including humans. In marine mammals, adenovirus has been associated with fatal hepatitis in sea lions. However, only in rare cases have adenoviruses been detected in cetaceans, where no clear correlation was found between presence of the virus and disease status. CASE PRESENTATION: A novel adenovirus was identified in four captive bottlenose dolphins with self-limiting gastroenteritis. Viral detection and identification were achieved by: PCR-amplification from fecal samples; sequencing of partial adenovirus polymerase (pol) and hexon genes; producing the virus in HeLa cells, with PCR and immunofluorescence detection, and with sequencing of the amplified pol and hexon gene fragments. A causative role of this adenovirus for gastroenteritis was suggested by: 1) we failed to identify other potential etiological agents; 2) the exclusive detection of this novel adenovirus and of seropositivity for canine adenoviruses 1 and 2 in the four sick dolphins, but not in 10 healthy individuals of the same captive population; and 3) the virus disappeared from feces after clinical signs receded. The partial sequences of the amplified fragments of the pol and hexon genes were closest to those of adenoviruses identified in sea lions with fatal adenoviral hepatitis, and to a Genbank-deposited sequence obtained from a harbour porpoise. CONCLUSION: These data suggest that adenovirus can cause self-limiting gastroenteritis in dolphins. This adenoviral infection can be detected by serology and by PCR detection in fecal material. Lack of signs of hepatitis in sick dolphins may reflect restricted tissue tropism or virulence of this adenovirus compared to those of the adenovirus identified in sea lions. Gene sequence-based phylogenetic analysis supports a common origin of adenoviruses that affect sea mammals. Our findings suggest the need for vigilance against adenoviruses in captive and wild dolphin populations.
Asunto(s)
Infecciones por Adenoviridae/veterinaria , Adenoviridae , Delfín Mular/virología , Gastroenteritis/veterinaria , Adenoviridae/genética , Adenoviridae/patogenicidad , Infecciones por Adenoviridae/virología , Animales , Animales de Zoológico/virología , ADN Viral/genética , Gastroenteritis/virología , Genes Virales/genética , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinariaRESUMEN
CLEC12B is a C-type lectin-like receptor expressed on myeloid cells. In this study, we have characterized the porcine homologue of CLEC12B (poCLEC12B). To this end, we have generated constructs encoding a c-myc tagged version of the whole receptor, or its ectodomain fused to the Fc portion of human IgG1, from a cDNA clone obtained from an alveolar macrophage library, and raised monoclonal antibodies (mAb) against this molecule. Using these mAbs, poCLEC12B was found to be expressed on alveolar macrophages and, at lower levels, on blood conventional type 1 dendritic cells (cDC1) and plasmacytoid DCs. No binding was detected on monocytes, monocyte-derived macrophages or monocyte-derived DCs. Engagement of CLEC12B on alveolar macrophages with mAbs had no apparent effect on cytokine production (TNF-α, IL-8) induced by LPS. These results provide the basis for future investigations aimed to assess the role of poCLEC12B in different microbial infections and to evaluate its potential in vaccination strategies targeting DCs.
Asunto(s)
Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Macrófagos Alveolares/inmunología , Receptores Mitogénicos/inmunología , Porcinos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Circulación Sanguínea , Células Cultivadas , Humanos , Interleucina-8/metabolismo , Lectinas Tipo C/genética , Lipopolisacáridos/inmunología , Activación de Macrófagos , Receptores Mitogénicos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CLEC12A has been proposed as a suitable target for delivering antigen to dendritic cells (DCs) to enhance vaccine efficacy both in human and mouse. In this study, we have characterized the porcine homolog of CLEC12A (poCLEC12A). Using new monoclonal antibodies (mAb), raised against its ectodomain, poCLEC12A was found to be expressed on alveolar macrophages, blood conventional type 1 and type 2 DCs and plasmacytoid DCs, but not on monocytes, T cells, B cells or NK cells, in contrast to its human and murine homologs. Western blot analysis showed that in alveolar macrophages this receptor is expressed both as a monomer and a dimer. After binding to DCs, anti- poCLEC12A mAb was efficiently internalized. No significant changes were observed in TNFα or IFNα secretion by plasmacytoid DCs stimulated with either CpGs (ODN2216) or polyinosinic-polycytidylic acid (poly I:C), upon incubation with mAb. These results provide the basis for future investigations aimed to assess the ability of anti-poCLEC12A mAbs to improve vaccine efficacy by targeting antigen to DCs.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Células CHO , Clonación Molecular , Cricetulus , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Terapia Molecular Dirigida , Oligodesoxirribonucleótidos/genética , Poli I-C/inmunología , Proteínas Recombinantes de Fusión/genética , Porcinos , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/genética , TranscriptomaRESUMEN
BACKGROUND: Cortactin activates the actin-related 2/3 (Arp2/3) complex promoting actin polymerization to remodel cell architecture in multiple processes (e.g. cell migration, membrane trafficking, invadopodia formation etc.). Moreover, it was called the Achilles' heel of the actin cytoskeleton because many pathogens hijack signals that converge on this oncogenic scaffolding protein. Cortactin is able to modulate N-WASP activation in vitro in a phosphorylation-dependent fashion. Thus Erk-phosphorylated cortactin is efficient in activating N-WASP through its SH3 domain, while Src-phosphorylated cortactin is not. This could represent a switch on/off mechanism controlling the coordinated action of both nucleator promoting factors (NPFs). Pedestal formation by enteropathogenic Escherichia coli (EPEC) requires N-WASP activation. N-WASP is recruited by the cell adapter Nck which binds a major tyrosine-phosphorylated site of a bacterial injected effector, Tir (translocated intimin receptor). Tir-Nck-N-WASP axis defines the current major pathway to actin polymerization on pedestals. In addition, it was recently reported that EPEC induces tyrosine phosphorylation of cortactin. RESULTS: Here we demonstrate that cortactin phosphorylation is absent on N-WASP deficient cells, but is recovered by re-expression of N-WASP. We used purified recombinant cortactin and Tir proteins to demonstrate a direct interaction of both that promoted Arp2/3 complex-mediated actin polymerization in vitro, independently of cortactin phosphorylation. CONCLUSION: We propose that cortactin binds Tir through its N-terminal part in a tyrosine and serine phosphorylation independent manner while SH3 domain binding and activation of N-WASP is regulated by tyrosine and serine mediated phosphorylation of cortactin. Therefore cortactin could act on Tir-Nck-N-WASP pathway and control a possible cycling activity of N-WASP underlying pedestal formation.
RESUMEN
Diarrheic diseases account for the annual death of approximately 1.9 million children under the age of 5 years, and it is a major cause of work absenteeism in developed countries. As diarrheagenic bacteria, enteropathogenic Escherichia coli (EPEC) attach to cells in the small intestine, causing local disappearance of microvilli and inducing the formation of actin-rich pedestals that disrupt the intestinal barrier and help EPEC adhere to and infect intestinal cells. Antibiotics and other bioactive compounds can often be found by analyzing traditional medicines. Here a crude aqueous extract of Hibiscus sabdariffa, which typically grows in subtropical and tropical areas and is a popular medicinal tisane in many countries, was analyzed for antibacterial activity against EPEC. In standard microdilution assays, the extract showed a minimum inhibitory concentration of 6.5 mg/ml against EPEC growth. Time-kill kinetics assays demonstrated significant 24 h bactericidal activity at 25 mg/ml. The extract is able to impede pedestal induction. Not only did the extract inhibit preformed pedestals but it prevented pedestal induction as well. Remarkably, it also promoted the formation of EPEC filaments, as observed with other antibiotics. Our results in vitro support the potential of Hibiscus sabdariffa as an antimicrobial agent against EPEC.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli Enterotoxigénica/crecimiento & desarrollo , Escherichia coli Enterotoxigénica/ultraestructura , Hibiscus/química , Extractos Vegetales/farmacología , Antibacterianos/química , Extractos Vegetales/químicaRESUMEN
Adenoviruses are common pathogens in vertebrates, infecting a wide range of hosts, but only having rarely been detected and correlated with disease in cetaceans. This article describes the first complete genomic sequence of a cetacean adenovirus, bottlenose dolphin adenovirus 1 (BdAdV-1), detected in captive bottlenose dolphin population (Tursiops truncatus) suffering from self-limiting gastroenteritis. The complete genome sequence of BdAdV-1 was recovered from data generated by high-throughput sequencing and validated by Sanger sequencing. The genome is 34,080bp long and has 220 nucleotides long inverted terminal repeats. A total of 29 coding sequences were identified, 26 of which were functionally annotated. Among the unusual features of this genome is a remarkably long 4380bp E3 ORF1, that displays no sequence homology with the corresponding E3 regions of other adenoviruses. In addition, the fiber protein only has 26% identity with fiber proteins described in other adenoviruses. Three hypothetical proteins were predicted. The phylogenetic analysis indicates that the closest known relative to BdAdV-1 is an adenovirus detected in bottlenose dolphin (KR024710), with an amino acid sequence identity between 36 and 79% depending on the protein. Based on the phylogenic analysis, the BdAdV-1 appears to have co-evolved with its host. The results indicate that BdAdV-1 belongs to the Mastadenovirus genus of the Adenoviridae family, however, it is clearly different from other adenoviruses, especially in the 3'-end of the viral genome. The high degree of sequence divergence suggests that BdAdV-1 should be considered as a novel species in the Mastadenovirus genus. The study also demonstrates the usefulness of high-throughput sequencing to obtain full-length genomes of genetically divergent viruses.
Asunto(s)
Infecciones por Adenoviridae/veterinaria , Delfín Mular/virología , Gastroenteritis/veterinaria , Genoma Viral , Mastadenovirus/genética , Filogenia , Proteínas Virales/genética , Infecciones por Adenoviridae/epidemiología , Infecciones por Adenoviridae/virología , Animales , Coevolución Biológica , ADN Viral/genética , Gastroenteritis/epidemiología , Gastroenteritis/virología , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Mastadenovirus/clasificación , Mastadenovirus/aislamiento & purificación , Sistemas de Lectura Abierta , España/epidemiologíaRESUMEN
Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/metabolismo , Proteínas Oncogénicas/metabolismo , Receptores de Superficie Celular/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Adhesinas Bacterianas/metabolismo , Animales , Adhesión Bacteriana , Fibroblastos/metabolismo , Fibroblastos/microbiología , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Ácidos Hidroxámicos/farmacología , Ratones , Fosforilación , Transporte de ProteínasRESUMEN
BACKGROUND: Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. CONCLUSIONS/SIGNIFICANCE: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.