RESUMEN
Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Enfermedades de los Perros , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Complejo Mycobacterium avium , Infección por Mycobacterium avium-intracellulare , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Perros , Infección por Mycobacterium avium-intracellulare/veterinaria , Infección por Mycobacterium avium-intracellulare/genética , Infección por Mycobacterium avium-intracellulare/microbiología , Complejo Mycobacterium avium/genética , Enfermedades de los Perros/genética , Enfermedades de los Perros/microbiología , Eliminación de Secuencia , Linaje , Femenino , Masculino , Secuenciación Completa del Genoma , Homocigoto , Lectinas Tipo C/genéticaRESUMEN
Stress-mediated programmed cell death (PCD) in bacteria has recently attracted attention, largely because it raises novel possibilities for controlling pathogens. How PCD in bacteria is regulated to avoid population extinction due to transient, moderate stress remains a central question. Here, we report that the YihE protein kinase is a key regulator that protects Escherichia coli from antimicrobial and environmental stressors by antagonizing the MazEF toxin-antitoxin module. YihE was linked to a reactive oxygen species (ROS) cascade, and a deficiency of yihE stimulated stress-induced PCD even after stress dissipated. YihE was partially regulated by the Cpx envelope stress-response system, which, along with MazF toxin and superoxide, has both protective and destructive roles that help bacteria make a live-or-die decision in response to stress. YihE probably acts early in the stress response to limit self-sustaining ROS production and PCD. Inhibition of YihE may provide a way of enhancing antimicrobial lethality and attenuating virulence.
Asunto(s)
Apoptosis , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Antiinfecciosos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Ácido Nalidíxico/farmacología , Proteínas Serina-Treonina Quinasas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Transformation requires specialized proteins to facilitate the binding and uptake of DNA. The genes of the Bacillus subtilis comG operon (comGA-G) are required for transformation and to assemble a structure, the pseudopilus, in the cell envelope. No role for the pseudopilus has been established and the functions of the individual comG genes are unknown. We show that among the comG genes, only comGA is absolutely required for DNA binding to the cell surface. ComEA, an integral membrane DNA-binding protein plays a minor role in the initial binding step, while an unidentified protein which communicates with ComGA must be directly responsible for binding to the cell. We show that the use of resistance to DNase to measure 'DNA uptake' reflects the movement of transforming DNA to a protected state in which it is not irreversibly associated with the protoplast, and presumably resides outside the cell membrane, in the periplasm or associated with the cell wall. We suggest that ComGA is needed for the acquisition of DNase resistance as well as for the binding of DNA to the cell surface. Finally, we show that the pseudopilus is required for DNA uptake and we offer a revised model for the transformation process.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , ADN Bacteriano/metabolismo , Transformación Bacteriana , Secuencia de Aminoácidos , Bacillus subtilis/genética , ADN Bacteriano/genética , Desoxirribonucleasas/metabolismo , Orden Génico , Genes Bacterianos , Datos de Secuencia Molecular , Operón , Unión Proteica , Alineación de SecuenciaRESUMEN
Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.
Asunto(s)
Apoptosis , Bordetella avium/patogenicidad , Óxido Nítrico Sintasa/metabolismo , Tráquea/microbiología , Tráquea/patología , Animales , Anexina A5/análisis , Modelos Animales de Enfermedad , Etiquetado Corte-Fin in Situ , Técnicas de Cultivo de Órganos , PavosRESUMEN
BACKGROUND: The continuing emergence of antimicrobial resistance requires the development of new compounds and/or enhancers of existing compounds. Genes that protect against the lethal effects of antibiotic stress are potential targets of enhancers. To distinguish such genes from those involved in drug uptake and efflux, a new susceptibility screen is required. RESULTS: Transposon (Tn5)-mediated mutagenesis was used to create a library of Escherichia coli mutants that was screened for hypersensitivity to the lethal action of quinolones and counter-screened to have wild-type bacteriostatic susceptibility. Mutants with this novel "hyperlethal" phenotype were found. The phenotype was transferable to other E. coli strains by P1-mediated transduction, and for a subset of the mutants the phenotype was complemented by the corresponding wild-type gene cloned into a plasmid. Thus, the inactivation of these genes was responsible for hyperlethality. Nucleotide sequence analysis identified 14 genes, mostly of unknown function, as potential factors protecting from lethal effects of stress. The 14 mutants were killed more readily than wild-type cells by mitomycin C and hydrogen peroxide; nine were also more readily killed by UV irradiation, and several exhibited increased susceptibility to killing by sodium dodecyl sulfate. No mutant was more readily killed by high temperature. CONCLUSIONS: A new screening strategy identified a diverse set of E. coli genes involved in the response to lethal antimicrobial and environmental stress, with some genes being involved in the response to multiple stressors. The gene set, which differed from sets previously identified with bacteriostatic assays, provides an entry point for obtaining small-molecule enhancers that will affect multiple antimicrobial agents.