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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

Repression of the SPI-1 genes by CpxR is lost in the absence of the Lon protease. Expression of the SPI-1-encoded InvF-FLAG in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon and ΔcpxA Δlon mutants (A), or in the WT S. Typhimurium strain and its isogenic ΔhilE and Δlon mutants carrying plasmid pCA-CpxR (C), was analyzed by Western blotting using monoclonal anti-FLAG antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown for 5 h in LB medium at 37°C. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. Overexpression (+) of CpxR from the T5-lac promoter of plasmid pCA-CpxR was induced by adding 50 μM IPTG at the beginning of the bacterial cultures. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon, and ΔcpxA Δlon mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent.
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Figure 5: Repression of the SPI-1 genes by CpxR is lost in the absence of the Lon protease. Expression of the SPI-1-encoded InvF-FLAG in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon and ΔcpxA Δlon mutants (A), or in the WT S. Typhimurium strain and its isogenic ΔhilE and Δlon mutants carrying plasmid pCA-CpxR (C), was analyzed by Western blotting using monoclonal anti-FLAG antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown for 5 h in LB medium at 37°C. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. Overexpression (+) of CpxR from the T5-lac promoter of plasmid pCA-CpxR was induced by adding 50 μM IPTG at the beginning of the bacterial cultures. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon, and ΔcpxA Δlon mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent.

Mentions: A previous study shown that the overexpression of the sigma factor RpoH represses the SPI-1 genes through the Lon protease that degrades HilD (Matsui et al., 2008). On the other hand, it was reported that CpxR positively regulates rpoH in E. coli (Zahrl et al., 2006). Interestingly, we observed a very similar effect with the overexpression of CpxR or RpoH on the activity of hilD-cat and hilA-cat fusions (Figures 4A,C,D), which was initially tested as an expression control of the promoter expressing CpxR from plasmid pMPM-K3. Therefore, we thought that CpxR could repress the SPI-1 genes through RpoH and Lon. To investigate this, we sought to determine the effect of CpxR on the SPI-1 genes in the absence of RpoH or Lon. After several attempts, we were unable to delete rpoH in the S. Typhimurium 14028s strain by the λRed recombination method (Datsenko and Wanner, 2000), which could suggest that the absence of RpoH affects Salmonella fitness; although, a S. Typhimurium 14028s ΔrpoH mutant was reported previously (Bang et al., 2005). In contrast, a lon deletion strain was successful; thus, we constructed and analyzed Δlon and ΔcpxA Δlon mutants. Furthermore, since HilE regulates the activity of HilD by protein-protein interaction (Baxter et al., 2003), ΔhilE and ΔcpxA ΔhilE mutants were also constructed and used as controls. Interestingly, the expression of InvF-FLAG, as well as the secretion/expression of the SipA-D proteins, was drastically reduced in the ΔcpxA and ΔcpxA ΔhilE mutants, but not in the ΔhilE, Δlon, and ΔcpxA Δlon mutants (Figures 5A,B). Consistently, the overexpression of CpxR clearly repressed the expression of InvF-FLAG in the WT strain and the ΔhilE mutant, but only slightly in the Δlon mutant (Figure 5C). Therefore, these results show that deletion of lon counteracts repression exerted by CpxR on the SPI-1 genes, which supports that CpxR acts through the Lon protease to repress these genes. Nevertheless, even in the absence of Lon, either the absence of CpxA or the overexpression of CpxR slightly repressed the SPI-1 genes (Figure 5), revealing an additional Lon-independent mechanism for the repression of these genes by CpxR.


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)

Repression of the SPI-1 genes by CpxR is lost in the absence of the Lon protease. Expression of the SPI-1-encoded InvF-FLAG in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon and ΔcpxA Δlon mutants (A), or in the WT S. Typhimurium strain and its isogenic ΔhilE and Δlon mutants carrying plasmid pCA-CpxR (C), was analyzed by Western blotting using monoclonal anti-FLAG antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown for 5 h in LB medium at 37°C. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. Overexpression (+) of CpxR from the T5-lac promoter of plasmid pCA-CpxR was induced by adding 50 μM IPTG at the beginning of the bacterial cultures. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon, and ΔcpxA Δlon mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent.
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Related In: Results  -  Collection

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Figure 5: Repression of the SPI-1 genes by CpxR is lost in the absence of the Lon protease. Expression of the SPI-1-encoded InvF-FLAG in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon and ΔcpxA Δlon mutants (A), or in the WT S. Typhimurium strain and its isogenic ΔhilE and Δlon mutants carrying plasmid pCA-CpxR (C), was analyzed by Western blotting using monoclonal anti-FLAG antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown for 5 h in LB medium at 37°C. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. Overexpression (+) of CpxR from the T5-lac promoter of plasmid pCA-CpxR was induced by adding 50 μM IPTG at the beginning of the bacterial cultures. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain and its isogenic ΔcpxA, ΔhilE, ΔcpxA ΔhilE, Δlon, and ΔcpxA Δlon mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent.
Mentions: A previous study shown that the overexpression of the sigma factor RpoH represses the SPI-1 genes through the Lon protease that degrades HilD (Matsui et al., 2008). On the other hand, it was reported that CpxR positively regulates rpoH in E. coli (Zahrl et al., 2006). Interestingly, we observed a very similar effect with the overexpression of CpxR or RpoH on the activity of hilD-cat and hilA-cat fusions (Figures 4A,C,D), which was initially tested as an expression control of the promoter expressing CpxR from plasmid pMPM-K3. Therefore, we thought that CpxR could repress the SPI-1 genes through RpoH and Lon. To investigate this, we sought to determine the effect of CpxR on the SPI-1 genes in the absence of RpoH or Lon. After several attempts, we were unable to delete rpoH in the S. Typhimurium 14028s strain by the λRed recombination method (Datsenko and Wanner, 2000), which could suggest that the absence of RpoH affects Salmonella fitness; although, a S. Typhimurium 14028s ΔrpoH mutant was reported previously (Bang et al., 2005). In contrast, a lon deletion strain was successful; thus, we constructed and analyzed Δlon and ΔcpxA Δlon mutants. Furthermore, since HilE regulates the activity of HilD by protein-protein interaction (Baxter et al., 2003), ΔhilE and ΔcpxA ΔhilE mutants were also constructed and used as controls. Interestingly, the expression of InvF-FLAG, as well as the secretion/expression of the SipA-D proteins, was drastically reduced in the ΔcpxA and ΔcpxA ΔhilE mutants, but not in the ΔhilE, Δlon, and ΔcpxA Δlon mutants (Figures 5A,B). Consistently, the overexpression of CpxR clearly repressed the expression of InvF-FLAG in the WT strain and the ΔhilE mutant, but only slightly in the Δlon mutant (Figure 5C). Therefore, these results show that deletion of lon counteracts repression exerted by CpxR on the SPI-1 genes, which supports that CpxR acts through the Lon protease to repress these genes. Nevertheless, even in the absence of Lon, either the absence of CpxA or the overexpression of CpxR slightly repressed the SPI-1 genes (Figure 5), revealing an additional Lon-independent mechanism for the repression of these genes by CpxR.

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