RESUMO
Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.
Assuntos
RNA Nuclear , RNA não Traduzido , Infecções por Salmonella/genética , Sobrevivência Celular , Células HeLa , Humanos , Salmonella enterica/genética , Regulação para CimaRESUMO
The envelope of Escherichia coli contains approximately 100 different species of lipoproteins, most of which are localized to the inner leaflet of the outer membrane. The localization of lipoprotein (Lol) system, consisting of five Lol proteins, is responsible for the trafficking of lipoproteins to the outer membrane. LolCDE binds to lipoproteins destined for the outer membrane and transfers them to the periplasmic chaperone LolA. Although the cryo-EM structures of E. coli LolCDE have been reported, the mechanisms by which outer membrane lipoproteins are transferred to LolA remain elusive. In this study, we investigated the interaction between LolCDE and lipoproteins using site-specific photo-crosslinking. We introduced a photo-crosslinkable amino acid into different locations across the four helices which form the central lipoprotein-binding cavity, and identified domains that crosslink with peptidoglycan-associated lipoprotein (Pal) in vivo. Using one of the derivatives containing the photo-crosslinkable amino acid, we developed an in vitro system to analyze the binding of lipoproteins to LolCDE. Our results indicate that compound 2, a LolCDE inhibitor, does not inhibit the binding of lipoproteins to LolCDE, but rather promotes the dissociation of bound lipoproteins from LolCDE.
Assuntos
Proteínas de Escherichia coli , Proteínas Periplásmicas de Ligação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Aminoácidos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas Periplásmicas de Ligação/metabolismoRESUMO
YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet-undefined function in the oxidative-stress response. YajL protects cells against oxidative-stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. Compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA repair proteins, RNA chaperones, and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative-stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/biossíntese , Estresse Oxidativo/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ferro/metabolismo , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Mutação , Proteínas Oncogênicas/genética , Oxirredução , Transtornos Parkinsonianos/genética , Proteína Desglicase DJ-1RESUMO
The Escherichia coli LolA protein is a lipoprotein-specific chaperone that carries lipoproteins from the inner to the outer membrane. A dominant negative LolA mutant, LolA(I93C/F140C), in which both (93)Ile and (140)Phe are replaced by Cys, binds tightly to the lipoprotein-dedicated ABC transporter LolCDE complex on the inner membrane and therefore inhibits the detachment of outer membrane-specific lipoproteins from the inner membrane. We found that the expression of this mutant strongly induced lolA gene transcription. The depletion of the LolA or LolB protein also triggered lolA gene transcription, indicating that the inhibition of outer membrane lipoprotein transport triggers lolA transcription. To elucidate the mechanism, we isolated mutants that are unable to induce lolA transcription using the lacZ gene fused to the lolA promoter as a reporter and found that the Rcs phosphorelay system directly regulates lolA transcription. An outer membrane lipoprotein, RcsF, was essential for this activation, while the coactivator RcsA was dispensable. Taking the observation that an RcsF mutant localized in the inner membrane constitutively activated the Rcs phosphorelay system into consideration, the results shown here strongly suggest that correct lipoprotein sorting to the outer membrane is monitored by RcsF, which plays a key role in the Rcs stress response system.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Lipoproteínas/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Estresse Fisiológico/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Escherichia coli/genética , Mutação , Proteínas Periplásmicas de Ligação/genética , Fatores de Transcrição/genéticaRESUMO
LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress. The inhibition of growth by I93C/F140C was partly suppressed by overproduction of LolCDE, which is an ATP-binding cassette transporter and mediates the transfer of lipoproteins from the inner membrane to LolA. A substantial portion of the oxidized form, but not the reduced one, of I93C/F140C expressed on LolCDE overproduction was recovered in the membrane fraction, whereas wild-type LolA was localized in the periplasm even when LolCDE was overproduced. Moreover, LolCDE overproduction stabilized I93C/F140C and therefore caused an increase in its level. Taken together, these results indicate that oxidized I93C/F140C stably binds to LolCDE, which causes strong envelope stress.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Estresse Fisiológico/fisiologia , Sequência de Aminoácidos , Membrana Celular , Proliferação de Células , Dissulfetos/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Lipoproteínas/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Ligação Proteica , Conformação Proteica , RegulonRESUMO
Peroxiredoxins are a class of peroxide-scavenging enzymes having a conserved cysteine residue(s) in their active centers. Thiol peroxidase (Tpx) is one of the peroxiredoxins identified in Escherichia coli. Despite the absence of the N-terminal signal sequence for transport across the membrane, it has been characterized as a periplasmic protein. Reanalysis of Tpx localization, using active site cysteine mutants of thioredoxin 1 (Trx1), demonstrated that Tpx forms a mixed-disulfide complex with cytoplasmic Trx1, indicating that Tpx localizes in the cytoplasm.
Assuntos
Fracionamento Celular/métodos , Citoplasma/enzimologia , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas Periplásmicas/metabolismo , Peroxidases/metabolismo , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Peróxido de Hidrogênio , Cinética , Mutação , Pressão Osmótica , Oxirredução , Proteínas Periplásmicas/genética , Peroxidases/genética , Plasmídeos/genética , Esferoplastos , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
BACKGROUND: High-level macrolide-resistant Moraxella catarrhalis strains have been isolated; however, the underlying mechanism has not been well elucidated. We investigated the role of mutations in the 23S rRNA gene and the L4 and L22 ribosomal proteins using spontaneous erythromycin-resistant mutants and transformants. MATERIALS AND METHODS: The erythromycin-susceptible M. catarrhalis ATCC25238 and clinical isolate Mc19 were used as parental strains. To obtain spontaneous erythromycin-resistant mutants, in vitro stepwise selection was performed using brain-heart infusion agar plates containing various concentrations of erythromycin. The role of the mutations identified in the spontaneous mutants was validated using transformation experiments. RESULTS: We obtained two spontaneous mutants with high-level resistance to erythromycin, S25-32-af10 and S19-256-af10, from ATCC25238 and Mc19, respectively. S25-32-af10 exhibited mutations of Q61R in L4 and Insertion98SRADRIS in L22. S19-256-af10 exhibited three C2611T-mutated alleles in the 23S rRNA gene and G65A in L4. Transformants with single mutations identified in S25-32-af10 or S19-256-af10 showed higher erythromycin and azithromycin minimum inhibitory concentrations (MICs) than those of each parental strain. However, transformants with multiple mutations identified in S25-32-af10 or S19-256-af10 showed macrolide MICs similar to those of each parental strain. CONCLUSION: Our results provide the first evidence suggesting that Q61R in L4 and Insertion98SRADRIS in L22 are involved in the synergistic acquisition of high-level resistance to both 14- and 15-member macrolides, and that C2611T in the 23S rRNA gene and G65A in L4 also synergistically contribute toward conferring high-level 14-member macrolide resistance to M. catarrhalis.
RESUMO
We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.