RESUMO
Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.
Assuntos
Clostridioides difficile/genética , Clostridioides difficile/fisiologia , Sistemas Toxina-Antitoxina , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Biologia ComputacionalRESUMO
OBJECTIVES: To evaluate the contribution of cysK and cysM to the fluoroquinolone (ofloxacin) antibiotic resistance in Salmonella Typhimurium, and their impact on H2S and cysteine production through targeted mutagenesis. METHODS: Salmonella Typhimurium 14028s and its cysK and cysM mutants were tested for their susceptibility to ofloxacin, as determined by a broth microdilution test (to determine the MIC) and survival curves. H2S levels were measured by the Pb(AC)2 method and cysteine levels were determined using 5,5-dithio-bis-2-nitrobenzoic acid. DNA damage induced by antibiotic treatment was determined by PFGE. Finally, expression of cysK and cysM genes under antibiotic treatment was determined by real-time reverse transcription PCR. RESULTS: As determined by MIC, the ΔcysK strain was more resistant to ofloxacin, a reactive oxygen species (ROS)-producing fluoroquinolone, than the WT and ΔcysM strains, which correlates with survival curves. Moreover, the ΔcysK strain exhibited higher H2S levels and lower cysteine levels than the WT strain. Finally, the ΔcysK strain exhibited lower DNA damage upon challenge with ofloxacin than the WT and ΔcysM strains. These results are in accordance with lower expression of cysK under ofloxacin treatment in the WT strain. CONCLUSIONS: This work demonstrated that cysteine metabolism in Salmonella Typhimurium modulated H2S levels, conferring resistance to second-generation fluoroquinolones.
Assuntos
Antibacterianos/metabolismo , Cisteína Sintase/metabolismo , Cisteína/metabolismo , Farmacorresistência Bacteriana , Fluoroquinolonas/metabolismo , Sulfeto de Hidrogênio/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Antioxidantes/metabolismo , Cisteína Sintase/genética , Fluoroquinolonas/antagonistas & inibidores , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Ofloxacino/antagonistas & inibidores , Ofloxacino/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Salmonella typhimurium/fisiologiaRESUMO
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
The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.