RESUMEN
Recently, the application of array-based comparative genomic hybridization (array CGH) has improved rates of detection of chromosomal imbalances in individuals with mental retardation and dysmorphic features. Here, we describe three individuals with learning disability and a heterozygous deletion at chromosome 17q21.3, detected in each case by array CGH. FISH analysis demonstrated that the deletions occurred as de novo events in each individual and were between 500 kb and 650 kb in size. A recently described 900-kb inversion that suppresses recombination between ancestral H1 and H2 haplotypes encompasses the deletion. We show that, in each trio, the parent of origin of the deleted chromosome 17 carries at least one H2 chromosome. This region of 17q21.3 shows complex genomic architecture with well-described low-copy repeats (LCRs). The orientation of LCRs flanking the deleted segment in inversion heterozygotes is likely to facilitate the generation of this microdeletion by means of non-allelic homologous recombination.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 17 , Discapacidades del Desarrollo/genética , Discapacidades para el Aprendizaje/genética , Proteínas tau/genética , Adolescente , Adulto , Preescolar , Inversión Cromosómica , Femenino , Marcadores Genéticos , Haplotipos , Heterocigoto , Humanos , Hibridación Fluorescente in Situ , Masculino , Hibridación de Ácido Nucleico , Mapeo Físico de Cromosoma , Polimorfismo de Nucleótido Simple , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow-derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c-Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.
Asunto(s)
Aconitato Hidratasa/biosíntesis , Proteínas Bacterianas/biosíntesis , Proteína Receptora de AMP Cíclico/fisiología , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/patogenicidad , Transcripción Genética , Aconitato Hidratasa/genética , Secuencia de Aminoácidos , Animales , Fusión Artificial Génica , Proteínas Bacterianas/genética , Recuento de Colonia Microbiana , Proteína Receptora de AMP Cíclico/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Genes Bacterianos/fisiología , Genes Reporteros/fisiología , Operón Lac/fisiología , Pulmón/microbiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Análisis de Secuencia por Matrices de Oligonucleótidos , Secuencias Reguladoras de Ácidos Nucleicos , Bazo/microbiología , Tuberculosis/microbiología , Virulencia/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Forkhead-associated (FHA) domains are modular phosphopeptide recognition motifs with a striking preference for phosphothreonine-containing epitopes. FHA domains have been best characterized in eukaryotic signaling pathways but have been identified in six proteins in Mycobacterium tuberculosis, the causative organism of tuberculosis. One of these, coded by gene Rv1747, is an ABC transporter and the only one to contain two such modules. A deletion mutant of Rv1747 is attenuated in a mouse intravenous injection model of tuberculosis where the bacterial load of the mutant is 10-fold lower than that of the wild type in both lungs and spleen. In addition, growth of the mutant in mouse bone marrow-derived macrophages and dendritic cells is significantly impaired. In contrast, growth of this mutant in vitro was indistinguishable from that of the wild type. The mutant phenotype was lost when the mutation was complemented by the wild-type allele, confirming that it was due to mutation of Rv1747. Using yeast two-hybrid analysis, we have shown that the Rv1747 protein interacts with the serine-threonine protein kinase PknF. This interaction appears to be phospho-dependent since it is abrogated in a kinase-dead mutant and by mutations in the presumed activation loop of PknF and in the first FHA domain of Rv1747. These results demonstrate that the protein coded by Rv1747 is required for normal virulent infection by M. tuberculosis in mice and, since it interacts with a serine-threonine protein kinase in a kinase-dependent manner, indicate that it forms part of an important phospho-dependent signaling pathway.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tuberculosis Pulmonar/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Animales , Sitios de Unión , Factores de Transcripción Forkhead , Pulmón/microbiología , Ratones , Mutación , Mycobacterium tuberculosis/genética , Proteínas Nucleares/química , Organismos Modificados Genéticamente , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Bazo/microbiología , Factores de Tiempo , Factores de Transcripción/química , Tuberculosis Pulmonar/microbiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, is phylogenetically closely related to M. tuberculosis, differing in a few biochemical properties. However, these species have different levels of virulence in different hosts; most notably M. microti shows lower virulence for humans than M. tuberculosis. This report presents genomic comparisons using DNA microarray analysis for an extensive study of the diversity of M. microti strains. Compared to M. tuberculosis H37Rv, 13 deletions were identified in 12 strains of M. microti, including the regions RD1 to RD10, which are also missing in Mycobacterium bovis BCG. In addition, four new deleted regions, named MiD1, RD1beta, MiD2 and MiD3, were identified. DNA sequencing was used to define the extent of most of the deletions in one strain. Although RD1 of M. bovis BCG and M. microti is thought to be crucial for attenuation, in this study, three of the four M. microti strains that were isolated from immunocompetent patients had the RD1 deletion. In fact, only the RD3 deletion was present in all of the strains examined, although deletions RD7, RD8 and MiD1 were found in almost all the M. microti strains. These deletions might therefore have some relation to the different host range of M. microti. It was also noticeable that of the 12 strains studied, only three were identical; these strains were all isolated from immunocompetent humans, suggesting that they could have arisen from a single source. Thus, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion in M. microti.
Asunto(s)
Arvicolinae/microbiología , Variación Genética , Genoma Bacteriano , Mycobacterium/clasificación , Mycobacterium/patogenicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Tuberculosis/microbiología , Animales , Proteínas Bacterianas/genética , Eliminación de Gen , Genómica , Humanos , Mycobacterium/química , Mycobacterium/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , VirulenciaRESUMEN
Mycobacterium tuberculosis, the causative organism of tuberculosis, encounters oxidative stress during phagocytosis by the macrophage and following macrophage activation during an acquired immune response, and also from internally generated sources of radical oxygen intermediates through intermediary metabolism. We have identified the SenX3 protein, a sensor in 1 of the 11 complete pairs of two-component signal transduction systems in M. tuberculosis, as a possible orthologue of the Mak2p protein from the fission yeast Schizosaccharomyces pombe that is known to sense peroxide stress. Moreover, the SenX3-RegX3 two-component system was the top scoring hit in a homology search with the Escherichia coli ArcB-ArcA global control system of aerobic genes. Using structural modelling techniques we have determined that SenX3 contains a PAS-like domain found in a variety of prokaryotic and eukaryotic sensors of oxygen and redox. Mutants with knock-outs of senX3 or of the accompanying transcriptional regulator regX3 were constructed and found to have reduced virulence in a mouse model of tuberculosis infection, the mutant bacteria persisting for up to 4 months post-infection; complemented mutants had regained virulence confirming that it was mutations of this two-component system that were responsible for the avirulent phenotype. This work identifies the PAS domain as a possible drug target for tuberculosis and mutations in the senX3-regX signal transduction system as potentially useful components of live vaccine strains.