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The two-component system CpxR/A represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD.

De la Cruz MA, Pérez-Morales D, Palacios IJ, Fernández-Mora M, Calva E, Bustamante VH - Front Microbiol (2015)

Bottom Line: Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively.The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2.Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Centro Médico Nacional Siglo XX1-IMSS México DF, Mexico.

ABSTRACT
Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.

No MeSH data available.


Related in: MedlinePlus

CpxR reduces the stability of HilD. (A) Stability of HilD-Myc was determined in the ΔhilD, ΔhilD ΔcpxA, and ΔhilD Δlon mutants carrying plasmid pBAD-HilD, which were grown in LB medium at 37°C. Expression of HilD-Myc, from the arabinose-inducible promoter of plasmid pBAD-HilD, was induced with 0.05% L-arabinose for 45 min; then, transcription and translation were halted by the addition of a cocktail of antibiotics and glucose, and samples of bacterial cultures were taken at indicated times. HilD-Myc was detected from whole cell lysates of the samples by Western blotting using monoclonal anti-Myc antibodies. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. A representative Western blot of three independent experiments is shown. (B) Densitometric analysis of the HilD-Myc bands from the Western blots is indicated as the relative percentage of HilD-Myc at each time with respect to time 0. Intensity values of HilD-Myc bands were normalized with those respective of DnaK bands. The data are the averages of three independent experiments. Bars represent the standard deviations and t1∕2 indicates the half-life of HilD.
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Figure 6: CpxR reduces the stability of HilD. (A) Stability of HilD-Myc was determined in the ΔhilD, ΔhilD ΔcpxA, and ΔhilD Δlon mutants carrying plasmid pBAD-HilD, which were grown in LB medium at 37°C. Expression of HilD-Myc, from the arabinose-inducible promoter of plasmid pBAD-HilD, was induced with 0.05% L-arabinose for 45 min; then, transcription and translation were halted by the addition of a cocktail of antibiotics and glucose, and samples of bacterial cultures were taken at indicated times. HilD-Myc was detected from whole cell lysates of the samples by Western blotting using monoclonal anti-Myc antibodies. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. A representative Western blot of three independent experiments is shown. (B) Densitometric analysis of the HilD-Myc bands from the Western blots is indicated as the relative percentage of HilD-Myc at each time with respect to time 0. Intensity values of HilD-Myc bands were normalized with those respective of DnaK bands. The data are the averages of three independent experiments. Bars represent the standard deviations and t1∕2 indicates the half-life of HilD.

Mentions: On the basis of our results indicating that repression of the SPI-1 genes by CpxR is mostly lost in the absence of the Lon protease, which degrades HilD, we hypothesized that CpxR should reduce the stability of HilD. To investigate this, we determined the in vivo half-life of HilD in the presence or absence of CpxA or Lon. The cellular levels of Myc-tagged HilD (HilD-Myc) expressed from plasmid pBAD-HilD, under an arabinose-inducible promoter, were monitored in the ΔhilD, ΔhilD ΔcpxA and ΔhilD Δlon mutants, at indicated times after adding a cocktail of transcription and translation inhibitors. As shown in Figure 6A, the levels of HilD-Myc were reduced faster in the ΔhilD ΔcpxA mutant than in the ΔhilD mutant, whereas, as expected, the stability of HilD-Myc was drastically increased in the ΔhilD Δlon mutant. In these assays, the half-life of HilD-Myc in the presence and absence of CpxA was 38 and 20 min, respectively (Figure 6B), supporting the notion that the activation of CpxR by the absence of CpxA decreases the stability of HilD.


The two-component system CpxR/A represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD.

De la Cruz MA, Pérez-Morales D, Palacios IJ, Fernández-Mora M, Calva E, Bustamante VH - Front Microbiol (2015)

CpxR reduces the stability of HilD. (A) Stability of HilD-Myc was determined in the ΔhilD, ΔhilD ΔcpxA, and ΔhilD Δlon mutants carrying plasmid pBAD-HilD, which were grown in LB medium at 37°C. Expression of HilD-Myc, from the arabinose-inducible promoter of plasmid pBAD-HilD, was induced with 0.05% L-arabinose for 45 min; then, transcription and translation were halted by the addition of a cocktail of antibiotics and glucose, and samples of bacterial cultures were taken at indicated times. HilD-Myc was detected from whole cell lysates of the samples by Western blotting using monoclonal anti-Myc antibodies. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. A representative Western blot of three independent experiments is shown. (B) Densitometric analysis of the HilD-Myc bands from the Western blots is indicated as the relative percentage of HilD-Myc at each time with respect to time 0. Intensity values of HilD-Myc bands were normalized with those respective of DnaK bands. The data are the averages of three independent experiments. Bars represent the standard deviations and t1∕2 indicates the half-life of HilD.
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Figure 6: CpxR reduces the stability of HilD. (A) Stability of HilD-Myc was determined in the ΔhilD, ΔhilD ΔcpxA, and ΔhilD Δlon mutants carrying plasmid pBAD-HilD, which were grown in LB medium at 37°C. Expression of HilD-Myc, from the arabinose-inducible promoter of plasmid pBAD-HilD, was induced with 0.05% L-arabinose for 45 min; then, transcription and translation were halted by the addition of a cocktail of antibiotics and glucose, and samples of bacterial cultures were taken at indicated times. HilD-Myc was detected from whole cell lysates of the samples by Western blotting using monoclonal anti-Myc antibodies. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. A representative Western blot of three independent experiments is shown. (B) Densitometric analysis of the HilD-Myc bands from the Western blots is indicated as the relative percentage of HilD-Myc at each time with respect to time 0. Intensity values of HilD-Myc bands were normalized with those respective of DnaK bands. The data are the averages of three independent experiments. Bars represent the standard deviations and t1∕2 indicates the half-life of HilD.
Mentions: On the basis of our results indicating that repression of the SPI-1 genes by CpxR is mostly lost in the absence of the Lon protease, which degrades HilD, we hypothesized that CpxR should reduce the stability of HilD. To investigate this, we determined the in vivo half-life of HilD in the presence or absence of CpxA or Lon. The cellular levels of Myc-tagged HilD (HilD-Myc) expressed from plasmid pBAD-HilD, under an arabinose-inducible promoter, were monitored in the ΔhilD, ΔhilD ΔcpxA and ΔhilD Δlon mutants, at indicated times after adding a cocktail of transcription and translation inhibitors. As shown in Figure 6A, the levels of HilD-Myc were reduced faster in the ΔhilD ΔcpxA mutant than in the ΔhilD mutant, whereas, as expected, the stability of HilD-Myc was drastically increased in the ΔhilD Δlon mutant. In these assays, the half-life of HilD-Myc in the presence and absence of CpxA was 38 and 20 min, respectively (Figure 6B), supporting the notion that the activation of CpxR by the absence of CpxA decreases the stability of HilD.

Bottom Line: Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively.The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2.Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Centro Médico Nacional Siglo XX1-IMSS México DF, Mexico.

ABSTRACT
Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.

No MeSH data available.


Related in: MedlinePlus