Project description:UnlabelledThe twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and ΔtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ΔtatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.ImportanceIn addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and â??tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the â??tatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. Y. pseudotuberculosis IP32953 was grown at 26°C in LB medium under aeration on a rotary shaker. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and âtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the âtatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. Y. pseudotuberculosis IP32953 was grown at 26°C in LB medium under aeration. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and âtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the âtatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. Y. pseudotuberculosis IP32953 was grown in LB medium supplemented with 2.5 mM CaCl2 under aeration for one hour at 26°C and then shifted to 37°C. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.
Project description:The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and âtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the âtatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. Y. pseudotuberculosis IP32953 was grown in LB medium supplemented with 2.5 mM CaCl2 under aeration for one hour at 26°C and then shifted to 37°C. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.