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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

The absence of CpxA represses SPI-1 genes by the activation of CpxR via the AckA and Pta enzymes. (A) Expression of the invF-cat and sirA-cat transcriptional fusions, carried by plasmids pinvF-cat and psirA-cat, respectively, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain 14028s and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent. (C) Expression of the SPI-1-encoded HilA-FLAG and InvF-FLAG and the SPI-2-encoded SsrB-FLAG and SseB, in the WT S. Typhimurium strain and its isogenic ΔcpxR, ΔcpxA and ΔcpxRA mutants, carrying the respective chromosomal FLAG-tagged gene, was analyzed by Western blotting using monoclonal anti-FLAG or polyclonal anti-SseB antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown in LB medium at 37°C for 5 or 9 h, for detection of SPI-1- and SPI-2-encoded proteins, respectively. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. (D) Expression of the cpxRA-cat transcriptional fusion, carried by plasmid pcpxRA-cat, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxR, ΔcpxA, ΔcpxRA, and ΔhilD ΔcpxA mutants. CAT-specific activity of cat fusions 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.
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Figure 1: The absence of CpxA represses SPI-1 genes by the activation of CpxR via the AckA and Pta enzymes. (A) Expression of the invF-cat and sirA-cat transcriptional fusions, carried by plasmids pinvF-cat and psirA-cat, respectively, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain 14028s and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent. (C) Expression of the SPI-1-encoded HilA-FLAG and InvF-FLAG and the SPI-2-encoded SsrB-FLAG and SseB, in the WT S. Typhimurium strain and its isogenic ΔcpxR, ΔcpxA and ΔcpxRA mutants, carrying the respective chromosomal FLAG-tagged gene, was analyzed by Western blotting using monoclonal anti-FLAG or polyclonal anti-SseB antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown in LB medium at 37°C for 5 or 9 h, for detection of SPI-1- and SPI-2-encoded proteins, respectively. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. (D) Expression of the cpxRA-cat transcriptional fusion, carried by plasmid pcpxRA-cat, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxR, ΔcpxA, ΔcpxRA, and ΔhilD ΔcpxA mutants. CAT-specific activity of cat fusions 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.

Mentions: Results from chloramphenicol acetyltransferase (CAT) assays were analyzed using One-Way analysis of variance (ANOVA) with the Dunnett multiple comparison test for Figures 1A,D, or t-Test with the Mann–Whitney test for Figures 4A–D, 7B,C. A P-value of < 0.05 was considered significant. This statistical analysis was performed using Prism 5 program version 5.04 (GraphPad Software, San Diego, CA).


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)

The absence of CpxA represses SPI-1 genes by the activation of CpxR via the AckA and Pta enzymes. (A) Expression of the invF-cat and sirA-cat transcriptional fusions, carried by plasmids pinvF-cat and psirA-cat, respectively, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain 14028s and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent. (C) Expression of the SPI-1-encoded HilA-FLAG and InvF-FLAG and the SPI-2-encoded SsrB-FLAG and SseB, in the WT S. Typhimurium strain and its isogenic ΔcpxR, ΔcpxA and ΔcpxRA mutants, carrying the respective chromosomal FLAG-tagged gene, was analyzed by Western blotting using monoclonal anti-FLAG or polyclonal anti-SseB antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown in LB medium at 37°C for 5 or 9 h, for detection of SPI-1- and SPI-2-encoded proteins, respectively. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. (D) Expression of the cpxRA-cat transcriptional fusion, carried by plasmid pcpxRA-cat, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxR, ΔcpxA, ΔcpxRA, and ΔhilD ΔcpxA mutants. CAT-specific activity of cat fusions 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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526804&req=5

Figure 1: The absence of CpxA represses SPI-1 genes by the activation of CpxR via the AckA and Pta enzymes. (A) Expression of the invF-cat and sirA-cat transcriptional fusions, carried by plasmids pinvF-cat and psirA-cat, respectively, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants. (B) Secretion analysis of the SPI-1-encoded proteins SipA, SipB, SipC, and SipD was tested in the WT S. Typhimurium strain 14028s and its isogenic ΔcpxA, ΔcpxR, ΔcpxRA, ΔackA-pta, and ΔcpxA ΔackA-pta mutants grown for 9 h in LB medium at 37°C. FliC is a flagellar protein whose secretion is SPI-1-independent. (C) Expression of the SPI-1-encoded HilA-FLAG and InvF-FLAG and the SPI-2-encoded SsrB-FLAG and SseB, in the WT S. Typhimurium strain and its isogenic ΔcpxR, ΔcpxA and ΔcpxRA mutants, carrying the respective chromosomal FLAG-tagged gene, was analyzed by Western blotting using monoclonal anti-FLAG or polyclonal anti-SseB antibodies. Whole cell lysates were prepared from samples of bacterial cultures grown in LB medium at 37°C for 5 or 9 h, for detection of SPI-1- and SPI-2-encoded proteins, respectively. As a loading control, the expression of DnaK was also determined using monoclonal anti-DnaK antibodies. (D) Expression of the cpxRA-cat transcriptional fusion, carried by plasmid pcpxRA-cat, was tested in the WT S. Typhimurium 14028s strain and its isogenic ΔcpxR, ΔcpxA, ΔcpxRA, and ΔhilD ΔcpxA mutants. CAT-specific activity of cat fusions 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.
Mentions: Results from chloramphenicol acetyltransferase (CAT) assays were analyzed using One-Way analysis of variance (ANOVA) with the Dunnett multiple comparison test for Figures 1A,D, or t-Test with the Mann–Whitney test for Figures 4A–D, 7B,C. A P-value of < 0.05 was considered significant. This statistical analysis was performed using Prism 5 program version 5.04 (GraphPad Software, San Diego, CA).

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