RESUMO
Copper homeostasis is essential for bacterial pathogen fitness and infection, and has been the focus of a number of recent studies. In Salmonella, envelope protection against copper overload and macrophage survival depends on CueP, a major copper-binding protein in the periplasm. This protein is also required to deliver the metal ion to the Cu/Zn superoxide dismutase SodCII. The Salmonella-specific CueP-coding gene was originally identified as part of the Cue regulon under the transcriptional control of the cytoplasmic copper sensor CueR, but its expression differs from the rest of CueR-regulated genes. Here we show that cueP expression is controlled by the concerted action of CueR, which detects the presence of copper in the cytoplasm, and by CpxR/CpxA, which monitors envelope stress. Copper-activated CueR is necessary for the appropriate spatial arrangement of the -10 and -35 elements of the cueP promoter, and CpxR is essential to recruit the RNA polymerase. The integration of two ancestral sensory systems-CueR, which provides signal specificity, and CpxR/CpxA, which detects stress in the bacterial envelope-restricts the expression of this periplasmic copper resistance protein solely to cells encountering surplus copper that disturbs envelope homeostasis, emulating the role of the CusR/CusS regulatory system present in other enteric bacteria.
Assuntos
Cobre/metabolismo , Regulação Bacteriana da Expressão Gênica , Homeostase , Periplasma/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transdução de Sinais , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Regiões Operadoras Genéticas/genética , Periplasma/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Filogenia , Cianeto de Potássio/farmacologia , Regiões Promotoras Genéticas/genética , Regulon/genética , Salmonella typhimurium/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The aim of this systematic review was to identify and characterize articles in indexed scientific journals with quantitative data surveys on administrative or legal proceedings for access to medicines. The SciELO, LILACS, MEDLINE via PubMed, Embase, and Scopus databases were used. We identified 45 articles, of which 17 were selected. The larger studies, each covering between 2,000 and 2,927 lawsuits, were done in the states of São Paulo, Rio de Janeiro, and Santa Catarina, Brazil. Eleven studies specified the type of legal representation, of which six examined cases with public attorneys and five with private attorneys. Only two studies reported whether the lawsuit was individual or class action, and in both the claims were individual. Since the majority of the medicines requested in the lawsuits were medium to high-cost, the review indicates that lawsuits contributed to the incorporation of these drugs into current pharmaceutical care in Brazil.
El objetivo de esta revisión sistemática fue identificar y caracterizar los artículos disponibles en revistas científicas indexadas en bases de datos electrónicas, que llevaron a cabo un estudio cuantitativo de datos, procedimientos administrativos o judiciales sobre la cuestión del acceso a los medicamentos a través de demandas judiciales. Los estudios fueron localizados en las bases de datos SciELO, LILACS, MEDLINE vía PubMed, Embase, Scopus. Se identificaron 45 artículos, de los cuales se seleccionaron 17. Los estudios que se llevaron a cabo engloban de 2.000 a 2.927 procesos judiciales en São Paulo, Río de Janeiro y Santa Catarina, Brasil. En once estudios se realizaron encuestas a los representantes legales de la acción judicial. En seis estudios predominó la representación pública legal y en cinco abogados privados. Sólo dos estudios examinaron si la acción era individual o colectiva y en los dos hubo prevalencia de acciones individuales. Como la mayoría de los medicamentos estaba involucrada en acciones legales de medio y alto coste, se cree que las demandas han contribuido a la incorporación de fármacos en la política pública actual.
O objetivo desta revisão sistemática foi identificar e caracterizar artigos disponíveis em periódicos científicos indexados em bases eletrônicas, que realizaram levantamento de dados quantitativo, em processos administrativos ou judiciais, sobre a questão do acesso a medicamentos por meio de ações judiciais. Foram usadas as bases de dados SciELO, LILACS, MEDLINE via PubMed, Embase e Scopus. Identificamos 45 artigos, dos quais foram selecionados 17 artigos. Os estudos com faixa de 2.000 a 2.927 processos foram conduzidos em São Paulo, Rio de Janeiro e Santa Catarina, Brasil. Em 11 estudos foram pesquisadas qual a representação jurídica da ação. Em seis estudos predominaram a representação de advogados públicos e em cinco particulares. Somente dois estudos observaram se a ação era coletiva ou individual, sendo que nas duas pesquisas a prevalência era de ações individuais. Como a maioria dos medicamentos envolvidos nas ações é de médio e alto custo, acredita-se que as demandas judiciais tenham contribuído para incorporação de medicamentos nas ações de assistência farmacêutica atuais.
Assuntos
Bacteriófago lambda/genética , DNA Viral/fisiologia , Genes de Troca , Instabilidade Genômica , Sítios de Ligação , DNA Viral/química , Regulação Viral da Expressão Gênica , Lisogenia/genética , Modelos Genéticos , Mutação , Conformação de Ácido Nucleico , Regiões Operadoras Genéticas , Processos EstocásticosRESUMO
BACKGROUND: DNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. RESULTS: Our recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML-/-, Daxx-/-, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. CONCLUSION: Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Linhagem Celular , Herpesvirus Humano 1/genética , Papillomavirus Humano 11/genética , Humanos , Óperon Lac , Regiões Operadoras Genéticas , Ligação Proteica , Vírus 40 dos Símios/genéticaRESUMO
The evolution of bacterial regulatory circuits often involves duplication of genes encoding transcription factors that may suffer both modifications in their detected signals, as well as, rewiring of their target operators. This, and subsequent horizontal gene transfer events contribute to generate a diverse array of regulatory pathways. In Salmonella, two homologous transcription factors CueR and GolS are responsible for Cu and Au sensing and resistance respectively. They share similarities not only in their sequence but also in their target binding sites, although they cluster separately among MerR-monovalent metal sensors. Here, we demonstrate that CueR and GolS can selectively distinguish their target binding sites by recognizing bases at positions 3' and 3 of their cognate operators. Swap of these bases results in switching regulator dependency. The differences in promoter architecture plus the environmentally controlled regulator's cytoplasmic availability warrant intra-regulon regulator-operator selectivity, and the proper response to metal injury. Furthermore, the presence of the distinctive operators' bases is widely extended among the two groups of MerR-monovalent metal sensors, providing evidence of the co-evolution of these factors and their target operators. This approach allows the prediction of regulator's dependency and the identification of transcription modules among groups of homologous transcription factors.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/genética , Íons/metabolismo , Metais/metabolismo , Dados de Sequência Molecular , Regiões Operadoras Genéticas , Ligação Proteica , Salmonella typhimurium/metabolismoRESUMO
The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence mechanisms of this bacterium. In the present study we demonstrated that Rv0586, which encodes a putative GntR-like regulator, is part of the mce2 operon. By using a promoter-lacZ fusion approach and bioinformatics tools, we found that Rv0586 represses the expression of Mce2 proteins and of a putative endonuclease IV, encoded by end (Rv0670) gene. For this reason, we have re-named the repressor protein Mce2R. By gel-shift experiments Mce2R binding was determined to be located within the mce2 promoter region. In addition, two FadR-like operator motifs were identified within the promoter regions of both the mce2 operon and the end gene. These motifs overlap putative -10 and -35 promoter boxes. M. tuberculosis carrying mce2 and end promoter-lacZ fusions were used to infect J774 macrophage-like cells. Expression of beta-galactosidase was induced after phagocytocis, suggesting that some cellular factor could be a key component of the molecular switch regulation expression of the mce2 operon. In conclusion, these results add novel evidence of the complex regulation of mce operon expression.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Proteínas Repressoras/genética , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Humanos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica , Virulência/genéticaRESUMO
The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Regulon/genética , Resposta SOS em Genética/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Caulobacter crescentus/metabolismo , Dano ao DNA , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Técnicas de Amplificação de Ácido Nucleico , Regiões Operadoras Genéticas/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Sítio de Iniciação de Transcrição , Raios UltravioletaRESUMO
The long-chain acyl-CoA synthase (ACS) FadD1 plays an important role in timing the levels of antibiotic production in Streptomyces coelicolor. fadD1 and macs1, encoding a putative medium-chain ACS, are part of a two-gene operon, whose expression is induced during the stationary phase of growth. Here it is reported that transcription of the macs1-fadD1 operon is positively regulated by AcsR, a LuxR-type transcriptional regulator. In an acsR mutant, expression of the macs1-fadD1 genes loses its normal up-regulation and the mutant becomes deficient in antibiotic production, in a clear correlation with the phenotype shown by a fadD1 null mutant. The absence of macs1-fadD1 induction in the acsR mutant was restored by complementation with a wild-type copy of the acsR gene, showing a strict link between AcsR and induction of the macs1-fadD1 operon. Gel mobility shift assays and DNase I footprinting indicated that AcsR binds to specific sequences about +162 nucleotides downstream of the macs1 transcriptional start site. In the putative operator sequence three almost identical direct tandem repeats of seven nucleotides were identified where the central sequence is essential for AcsR recognition and binding. Transcriptional fusions of the divergent pacsR and pmacs1 promoters indicated that AcsR does not regulate its own transcription, and that it binds to the operator region to control exclusively the growth-phase induction of the macs1-fadD1 operon.
Assuntos
Coenzima A Ligases/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Streptomyces coelicolor/genética , Transcrição Gênica , Sequência de Aminoácidos , Antibacterianos/biossíntese , Fusão Gênica Artificial , Sequência de Bases , Catecol 2,3-Dioxigenase/análise , Catecol 2,3-Dioxigenase/genética , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reguladores , Teste de Complementação Genética , Luciferases/análise , Luciferases/genética , Dados de Sequência Molecular , Mutação , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Ligação Proteica , Streptomyces coelicolor/fisiologiaRESUMO
The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.