RESUMO
BACKGROUND: Salmonella Typhimurium is a Gram-negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella-containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin-encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post-transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post-transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S. Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild-type strain, suggesting that ompX mRNA is also regulated at a post-transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2-induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Porinas , Salmonella typhimurium , Animais , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , Porinas/genética , Porinas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.
Assuntos
Animais , Camundongos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Porinas/genética , Porinas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologiaRESUMO
The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Typhi (S. Typhi) can be partially attributed to pseudogenes. Pseudogenes are genomic segments homologous to functional genes that do not encode functional products due to the presence of genetic defects. S. Typhi lacks several protein effectors implicated in invasion or other important processes necessary for full virulence of S. Typhimurium. SopA and SopE2, effectors that have been lost by pseudogenization in S. Typhi, correspond to an ubiquitin ligase involved in cytokine production by infected cells, and to a guanine exchange factor necessary for invasion of epithelial cells, respectively. We hypothesized that sopA and/or sopE pseudogenization contributed to the virulence of S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium sopE2 exhibited a decreased invasion in different epithelial cell lines compared with S. Typhi WT. S. Typhimurium sopA completely abolished the hypo-invasive phenotype observed in S. Typhi expressing S. Typhimurium sopE2, suggesting that functional SopA and SopE2 participate concertedly in the invasion process. Finally, the expression of S. Typhimurium sopA and/or sopE2 in S. Typhi, determined changes in the secretion of IL-8 and IL-18 in infected epithelial cells.
Assuntos
Proteínas de Bactérias/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Salmonella typhi/genética , Salmonella typhi/patogenicidade , Febre Tifoide/microbiologia , Virulência/genética , Proteínas de Bactérias/metabolismo , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica , Genótipo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Mutação , PseudogenesRESUMO
In response to antibiotics, bacteria activate regulatory systems that control the expression of genes that participate in detoxifying these compounds, like multidrug efflux systems. We previously demonstrated that the BaeSR two-component system from Salmonella enterica serovar Typhimurium (S. Typhimurium) participates in the detection of ciprofloxacin, a bactericidal antibiotic, and in the positive regulation of mdtA, an efflux pump implicated in antibiotic resistance. In the present work, we provide further evidence for a role of the S. Typhimurium BaeSR two-component system in response to ciprofloxacin treatment and show that it regulates sodA expression. We demonstrate that, in the absence of BaeSR, the transcript levels of sodA and the activity of its gene product are lower. Using electrophoretic mobility shift assays and transcriptional fusions, we demonstrate that BaeR regulates sodA by a direct interaction with the promoter region.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Ciprofloxacina/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Quinases/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Superóxido Dismutase/biossíntese , Transativadores/metabolismo , Fusão Gênica Artificial , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Quinases/genética , Transativadores/genética , Transcrição GênicaRESUMO
OmpW is a minor porin whose biological function has not been clearly defined. Evidence obtained in our laboratory indicates that in Salmonella enterica serovar Typhimurium the expression of OmpW is activated by SoxS upon exposure to paraquat and it is required for resistance. SoxS belongs to the AraC family of transcriptional regulators, like MarA and Rob. Due to their high structural similarity, the genes under their control have been grouped in the mar/sox/rob regulon, which presents a DNA-binding consensus sequence denominated the marsox box. In this work, we evaluated the role of the transcription factors MarA, SoxS and Rob of S. enterica serovar Typhimurium in regulating ompW expression in response to menadione. We determined the transcript and protein levels of OmpW in different genetic backgrounds; in the wild-type and Δrob strains ompW was upregulated in response to menadione, while in the ΔmarA and ΔsoxS strains the induction was abolished. In a double marA soxS mutant, ompW transcript levels were lowered after exposure to menadione, and only complementation in trans with both genes restored the positive regulation. Using transcriptional fusions and electrophoretic mobility shift assays with mutant versions of the promoter region we demonstrated that two of the predicted sites were functional. Additionally, we demonstrated that MarA increases the affinity of SoxS for the ompW promoter region. In conclusion, our study shows that ompW is upregulated in response to menadione in a cooperative manner by MarA and SoxS through a direct interaction with the promoter region.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Transativadores/metabolismo , Vitamina K 3/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Regiões Promotoras Genéticas , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transativadores/genética , Transativadores/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
To survive, Salmonella enterica serovar Typhimurium (S. Typhimurium) must sense signals found in phagocytic cells and modulate gene expression. In the present work, we evaluated the expression and cross-regulation of the transcription factors MarA, Rob, and SoxS in response to NaOCl. We generated strains ΔsoxS and ΔmarA, which were 20 times more sensitive to NaOCl as compared to the wild-type strain; while Δrob only 5 times. Subsequently, we determined that marA and soxS transcript and protein levels were increased while those of rob decreased in a wild-type strain treated with NaOCl. To assess if changes in S. Typhimurium after exposure to NaOCl were due to a cross-regulation, as in Escherichia coli, we evaluated the expression of marA, soxS, and rob in the different genetic backgrounds. The positive regulation observed in the wild-type strain of marA and soxS was retained in the Δrob strain. As in the wild-type strain, rob was down-regulated in the ΔmarA and ΔsoxS treated with NaOCl; however, this effect was decreased. Since rob was down-regulated by both factors, we generated a ΔmarA ΔsoxS strain finding that the negative regulation was abolished, confirming our hypothesis. Electrophoretic mobility shift assays using MarA and SoxS confirmed an interaction with the promoter of rob.
Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Hipoclorito de Sódio/farmacologia , Fatores de Transcrição/genética , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Mutação , Oxidantes/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Salmonella typhimurium/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Two-component systems are one of the most prevalent mechanisms by which bacteria sense, respond and adapt to changes in their environment. The activation of a sensor histidine kinase leads to autophosphorylation of a conserved histidine residue followed by transfer of the phosphoryl group to a cognate response regulator in an aspartate residue. The search for antibiotics that inhibit molecular targets has led to study prokaryotic two-component systems. In this study, we characterized in vitro and in vivo the BaeSR two-component system from Salmonella Typhimurium and evaluated its role in mdtA regulation in response to ciprofloxacin treatment. We demonstrated in vitro that residue histidine 250 is essential for BaeS autophosphorylation and aspartic acid 61 for BaeR transphosphorylation. By real-time PCR, we showed that mdtA activation in the presence of ciprofloxacin depends on both members of this system and that histidine 250 of BaeS and aspartic acid 61 of BaeR are needed for this. Moreover, the mdtA expression is directly regulated by binding of BaeR at the promoter region, and this interaction is enhanced when the protein is phosphorylated. In agreement, a BaeR mutant unable to phosphorylate at aspartic acid 61 presents a lower affinity with the mdtA promoter.
Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Quinases/metabolismo , Salmonella typhimurium/genética , Transativadores/metabolismo , Ácido Aspártico/metabolismo , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Histidina/metabolismo , Histidina Quinase , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutagênese Sítio-Dirigida , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Quinases/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/fisiologia , Transativadores/genéticaRESUMO
Salmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans. This bacterium is also able to persist in its host, causing a chronic disease by colonizing the spleen, liver and gallbladder, in the last of which the pathogen forms biofilms in order to survive the bile. Several genetic components, including the yihU-yshA genes, have been suggested to be involved in the survival of Salmonella in the gallbladder. In this work we describe how the yihU-yshA gene cluster forms a transcriptional unit regulated positively by the cAMP receptor global regulator CRP (cAMP receptor protein). The results obtained show that two CRP-binding sites on the regulatory region of the yihU-yshA operon are required to promote transcriptional activation. In this work we also demonstrate that the yihU-yshA transcriptional unit is carbon catabolite-repressed in Salmonella, indicating that it forms part of the CRP regulon in enteric bacteria.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidroxibutirato Desidrogenase/metabolismo , Óperon , Salmonella typhi/genética , Salmonella typhi/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Repressão Catabólica , Humanos , Hidroxibutirato Desidrogenase/química , Hidroxibutirato Desidrogenase/genética , Mutagênese Sítio-Dirigida , Salmonella typhi/crescimento & desenvolvimento , Febre Tifoide/microbiologiaRESUMO
OmpW of Salmonella enterica serovar Typhimurium has been described as a minor porin involved in osmoregulation, and is also affected by environmental conditions. Biochemical and genetic evidence from our laboratory indicates that OmpW is involved in efflux of and resistance towards paraquat (PQ), and its expression has been shown to be activated in response to oxidative stress. In this study we have explored ompW expression in response to PQ. Primer extension and transcriptional fusions showed that its expression was induced in the presence of PQ. In silico analyses suggested a putative binding site for the SoxS transcriptional factor at the ompW regulatory region. Electrophoretic mobility shift assays (EMSAs) and footprinting experiments showed that SoxS binds at a region that starts close to -54 and ends at about -197 upstream of the transcription start site. Transcriptional fusions support the relevance of this region in ompW activation. The SoxS site is in the forward orientation and its location suggests that the ompW gene has a class I SoxS-dependent promoter.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Salmonella typhimurium/metabolismo , Transativadores/metabolismo , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Herbicidas/farmacologia , Paraquat/farmacologia , Regiões Promotoras Genéticas , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Análise de Sequência de DNA , Ativação Transcricional/efeitos dos fármacosRESUMO
The ubiE gene of Geobacillus stearothermophilus V, with its own promoter, was cloned and introduced into Escherichia coli. The cloned gene complemented the ubiE gene deficiency of E. coli AN70. In addition, the expression of this gene in E. coli JM109 resulted in the evolution of volatile selenium compounds when these cells were grown in selenite- or selenate-amended media. These compounds were dimethyl selenide and dimethyl diselenide.