RESUMO
The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression.
Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/metabolismo , Ferro/metabolismo , Proteínas Repressoras/metabolismo , Bacillus cereus/genética , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Hemolisinas/genética , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD) is a rare and complex pediatric disorder. Despite increased identification and advancing knowledge of the disease course, the variable onset and timing of phenotypic features in ROHHAD often result in delayed or missed diagnosis, potentially leading to fatal central hypoventilation, cardiorespiratory arrest, and impaired neurocognitive development. The 5-hydroxytryptamine receptor 1A (HTR1A), orthopedia (OTP), and pituitary adenylate cyclase activating polypeptide (PACAP) genes were targeted in the etiology of ROHHAD based on their roles in the embryologic development of the hypothalamus and autonomic nervous system. We hypothesized that variations of HTR1A, OTP, and/or PACAP would be associated with ROHHAD. All coding regions and intron-exon boundaries of the HTR1A, OTP, and PACAP genes, in addition to the promoter region of the HTR1A gene, were analyzed by standard sequencing in 25 ROHHAD cases and 25 matched controls. Thirteen variations, including six protein-changing mutations, were identified. None of these variations were significantly correlated with ROHHAD. This report provides evidence that variation of the HTR1A, OTP, and PACAP genes are not responsible for ROHHAD. These results represent a further step in the investigation of the genetic determinants of ROHHAD.
Assuntos
Doenças do Sistema Nervoso Autônomo/genética , Doenças Hipotalâmicas/genética , Hipoventilação/genética , Obesidade/genética , Feminino , Variação Genética , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Fenótipo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptor 5-HT1A de Serotonina/genética , SíndromeRESUMO
HlyIIR is a negative transcriptional regulator of hemolysin II gene from B. cereus. It binds to a long DNA perfect inverted repeat (44bp) located upstream the hlyII gene. Here we show that HlyIIR is dimeric in solution and in bacterial cells. No protein-protein interactions between dimers and no significant modification of target DNA conformation upon complex formation were observed. Two HlyIIR dimers were found to bind to native operator independently with Kd level in the nanomolar range. The minimal HlyIIR binding site was identified as a half of the long DNA perfect inverted repeat.
Assuntos
Bacillus cereus/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Proteínas Hemolisinas/genética , Regiões Operadoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Sítios de Ligação , Dimerização , Genes Bacterianos , Conformação de Ácido Nucleico , Ligação ProteicaRESUMO
Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.