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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

RpoH, but not CpxR, induces the transcription of lon, clpX, and clpP. (A) Genetic organization of the lon, clpX, and clpP genes and schematic representation of the cat transcriptional fusions of these genes. Expression of the lon-cat, clpX-cat and clpP-cat transcriptional fusions, carried by plasmids plon-cat, pclpX-cat and pclpP-cat, respectively, was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA mutant (B), as well as in the WT S. Typhimurium strain carrying plasmid pK3-CpxR or pK3-RpoH, which constitutively express CpxR and RpoH, respectively, or carrying the vector pMPM-K3 (C). CAT-specific activity was determined from samples collected of bacterial cultures grown for 5 h in LB medium at 37°C. The data are the averages of three different experiments performed in duplicate. Bars represent the standard deviations. *Expression statistically different with respect to that shown by the same fusion in the WT strain containing the vector.
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Figure 7: RpoH, but not CpxR, induces the transcription of lon, clpX, and clpP. (A) Genetic organization of the lon, clpX, and clpP genes and schematic representation of the cat transcriptional fusions of these genes. Expression of the lon-cat, clpX-cat and clpP-cat transcriptional fusions, carried by plasmids plon-cat, pclpX-cat and pclpP-cat, respectively, was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA mutant (B), as well as in the WT S. Typhimurium strain carrying plasmid pK3-CpxR or pK3-RpoH, which constitutively express CpxR and RpoH, respectively, or carrying the vector pMPM-K3 (C). CAT-specific activity was determined from samples collected of bacterial cultures grown for 5 h in LB medium at 37°C. The data are the averages of three different experiments performed in duplicate. Bars represent the standard deviations. *Expression statistically different with respect to that shown by the same fusion in the WT strain containing the vector.

Mentions: As most response regulators, CpxR directly controls gene expression at transcriptional level (Hunke et al., 2012; Vogt and Raivio, 2012; Raivio, 2014). Thus, we tested if CpxR affects the transcription of lon. In E. coli, lon seems to be transcribed from promoters located upstream of lon, or from those of neighboring genes clpX and clpP (RegulonDB database, www.regulondb.ccg.unam.mx). Therefore, to monitor the promoters expressing lon, we constructed lon-cat, clpX-cat and clpP-cat transcriptional fusions, which contain the full intergenic region upstream of the respective gene (Figure 7A). Each of these fusions showed similar levels of expression in the WT strain and its derivative ΔcpxA mutant (Figure 7B). Furthermore, the expression of the lon-cat, clpX-cat, and clpP-cat fusions was not affected in the WT strain by the overexpression of CpxR; in contrast, their expression was increased by the overproduction of RpoH (Figure 7C). These results indicate that CpxR does not affect the transcription of lon and demonstrate that RpoH positively regulates lon, clpX, and clpP in S. Typhimurium.


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)

RpoH, but not CpxR, induces the transcription of lon, clpX, and clpP. (A) Genetic organization of the lon, clpX, and clpP genes and schematic representation of the cat transcriptional fusions of these genes. Expression of the lon-cat, clpX-cat and clpP-cat transcriptional fusions, carried by plasmids plon-cat, pclpX-cat and pclpP-cat, respectively, was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA mutant (B), as well as in the WT S. Typhimurium strain carrying plasmid pK3-CpxR or pK3-RpoH, which constitutively express CpxR and RpoH, respectively, or carrying the vector pMPM-K3 (C). CAT-specific activity was determined from samples collected of bacterial cultures grown for 5 h in LB medium at 37°C. The data are the averages of three different experiments performed in duplicate. Bars represent the standard deviations. *Expression statistically different with respect to that shown by the same fusion in the WT strain containing the vector.
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Related In: Results  -  Collection

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Figure 7: RpoH, but not CpxR, induces the transcription of lon, clpX, and clpP. (A) Genetic organization of the lon, clpX, and clpP genes and schematic representation of the cat transcriptional fusions of these genes. Expression of the lon-cat, clpX-cat and clpP-cat transcriptional fusions, carried by plasmids plon-cat, pclpX-cat and pclpP-cat, respectively, was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA mutant (B), as well as in the WT S. Typhimurium strain carrying plasmid pK3-CpxR or pK3-RpoH, which constitutively express CpxR and RpoH, respectively, or carrying the vector pMPM-K3 (C). CAT-specific activity was determined from samples collected of bacterial cultures grown for 5 h in LB medium at 37°C. The data are the averages of three different experiments performed in duplicate. Bars represent the standard deviations. *Expression statistically different with respect to that shown by the same fusion in the WT strain containing the vector.
Mentions: As most response regulators, CpxR directly controls gene expression at transcriptional level (Hunke et al., 2012; Vogt and Raivio, 2012; Raivio, 2014). Thus, we tested if CpxR affects the transcription of lon. In E. coli, lon seems to be transcribed from promoters located upstream of lon, or from those of neighboring genes clpX and clpP (RegulonDB database, www.regulondb.ccg.unam.mx). Therefore, to monitor the promoters expressing lon, we constructed lon-cat, clpX-cat and clpP-cat transcriptional fusions, which contain the full intergenic region upstream of the respective gene (Figure 7A). Each of these fusions showed similar levels of expression in the WT strain and its derivative ΔcpxA mutant (Figure 7B). Furthermore, the expression of the lon-cat, clpX-cat, and clpP-cat fusions was not affected in the WT strain by the overexpression of CpxR; in contrast, their expression was increased by the overproduction of RpoH (Figure 7C). These results indicate that CpxR does not affect the transcription of lon and demonstrate that RpoH positively regulates lon, clpX, and clpP in S. Typhimurium.

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