The multifaceted RisA regulon of Bordetella pertussis

Abstract

The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA(-) background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD(60)N and the phosphomimetic RisAD(60)E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation.

Figure 1. Transcriptome comparisons of different strains in modulating and non-modulating conditions.

DNA array gene expression ratios, expressed as log₂ ratios, between the mutant and parental strains in modulating and non-modulating conditions are depicted in the scatter plots, each point of which represents one gene. In non-modulating conditions, the ratio between a non-modulated mutant strain and its parental strain (y axis) is plotted against the ratio between two non-modulated parental strains (x axis). In modulating conditions, the ratio between a modulated mutant strain and its non-modulated parental strain (y axis) is plotted against the ratio between the modulated and the non-modulated parental strain (x axis). For the RisAD⁶⁰E and RisAD⁶⁰N mutations the parental strain background is BP536, while for the other strains the parental strain is BPSM. Coloured circles highlight genes of interest; blue for the vags, orange for the vrgs and red for genes related to the flagellar and chemotaxis operons. 50 mM MgSO₄ was used as the modulating condition.

Figure 2. Heat map of RisA-regulated gene expression in non-modulated BPSM.

Rows correspond to array probes. The names and symbols in the left column correspond to the Tohama I Sanger Centre annotation. BPSMΔRisA vs BPSM corresponds to the ratios between BPSMΔRisA and BPSM used as reference, both cultivated in non-modulating conditions. BPSM Mg vs BPSM corresponds to the ratios between BPSM cultivated in the presence of 50 mM MgSO₄ and non-modulated BPSM. The indicated ratios are the means of all the experiments. Data are centred from the first column between genes less transcribed (top) to more transcribed (bottom) in BPSMΔRisA compared to BPSM. Red, increased transcript abundance; green, decreased transcript abundance; black, no significant change in transcript abundance; the level of transcript abundance is defined by the coloured Log₂ scale shown on the bottom of the figure.

Figure 3. Heat map of RisA-regulated gene expression in modulating conditions.

Rows correspond to array probes. The names and symbols in the left column correspond to the Tohama I Sanger Centre annotation. BPSMΔRisA Mg vs BPSM corresponds to the ratios between BPSMΔRisA cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. BPSM Mg vs BPSM corresponds to the ratios between BPSM cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. The indicated ratios are the means of all the experiments. Red, increased transcript abundance; green, decreased transcript abundance; black, no significant change in transcript abundance; the level of transcript abundance is defined by the coloured Log₂ scale.

Figure 4. Role of RisA phosphorylation in gene expression in modulated or non-modulated B. pertussis.

Rows correspond to array probes. The names and symbols in the left column correspond to the Tohama I Sanger Centre annotation. BPSMΔRisA vs BPSM corresponds to the ratios between BPSMΔRisA and BPSM, both cultivated in non-modulating conditions. BPSMΔRisK vs BPSM corresponds to the ratios between BPSMΔRisK and BPSM, both cultivated in non-modulating conditions. BP536 RisAD⁶⁰E vs BP536 corresponds to the ratios between BP536 RisAD⁶⁰E and BP536, both cultivated in non-modulating conditions. BP536 RisAD⁶⁰N vs BP536 corresponds to the ratios between BP536 RisAD⁶⁰N and BP536, both cultivated in non-modulating conditions. BPSM Mg vs BPSM corresponds to the ratios between BPSM cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. BPSMΔRisA Mg vs BPSM corresponds to the ratios between BPSMΔRisA cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. BPSMΔRisK Mg vs BPSM corresponds to the ratios between BPSMΔRisK cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. BP536 RisAD⁶⁰E Mg vs BP536 corresponds to the ratios between BP536 RisAD⁶⁰E cultivated in the presence of 50 mM MgSO₄ and BP536 cultivated in non-modulating conditions. BP536 RisAD⁶⁰N Mg vs BP536 corresponds to the ratios between BP536 RisAD⁶⁰N cultivated in the presence of 50 mM MgSO₄ and BP536 cultivated in non-modulating conditions. The indicated ratios are the means of all the experiments. Red, increased transcript abundance; green, decreased transcript abundance; black, no significant change in transcript abundance; the level of transcript abundance is defined by the coloured Log₂ scale shown on the right.

Figure 5

(A) Quantitative RT-PCR analysis of genes in D420 and D420 RisAD⁶⁰N. D420 (blue), MgSO₄ modulated D420 (red), D420 RisAD⁶⁰N (green) and MgSO₄ modulated D420 RisAD⁶⁰N (violet). (B) Quantitative RT-PCR analysis of genes in iron depleted conditions. BPSM (blue), MgSO₄ modulated BPSM (red), BPSM RisA (green), MgSO₄ modulated BPSM RisA (violet), iron depleted BPSM (cyan), iron depleted MgSO4 modulated BPSM (orange), iron depleted BPSM RisA (light purple) and iron depleted MgSO4 modulated BPSM RisA (pink). (C) Quantitative RT-PCR analysis of genes in glutamate depleted conditions. BPSM (blue), MgSO₄ modulated BPSM (red), BPSM RisA (green), MgSO₄ modulated BPSM RisA (violet), glutamate depleted BPSM (cyan), glutamate depleted MgSO4 modulated BPSM (orange), glutamate depleted BPSM RisA (light purple) and glutamate depleted MgSO4 modulated BPSM RisA (pink). The values represent the Log₂ of mean expression of each gene from mid exponential cultures, normalized to bp3416 using the 2^ΔΔCt method expressed relative to the expression in D420 for (a) or BPSM for (b,c). The error bars represent the ∆∆Ct S.D. 50 mM MgSO₄ was used as the modulating condition.

Figure 6. BvgR-regulated gene expression in modulated and non-modulated BPSM and BPSMΔRisA.

Rows correspond to array probes. The names and symbols in the left column correspond to the Tohama I Sanger Centre annotation. BPSMΔBvgR vs BPSM corresponds to the ratios between BPSMΔBvgR and BPSM, both cultivated in non-modulating conditions. BPSMΔRisA vs BPSM corresponds to the ratios between BPSMΔRisA and BPSM, both cultivated in non-modulating conditions. BPSM Mg vs BPSM corresponds to the ratios between BPSM cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. BPSMΔBvgR Mg vs BPSM corresponds to the ratios between BPSMΔBvgR cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. BPSMΔRisA Mg vs BPSM corresponds to the ratios between BPSMΔRisA cultivated in the presence of 50 mM MgSO₄ and BPSM cultivated in non-modulating conditions. The indicated ratios are the means of all the experiments. Red, increased transcript abundance; green, decreased transcript abundance; black, no significant change in transcript abundance; the level of transcript abundance is defined by the coloured Log₂ scale shown on the right.

Figure 7. Schematic representation of the RisA regulatory network in B. pertussis.

Clusters shown in this figure are the same as those identified in Fig. 3. In non-modulating conditions phosphorylated BvgA induces the expression of vags (clusters 1 and 2), including bvgR, thereby leading to the degradation of c-di-GMP into GMP. In the absence of c-di-GMP RisK-phosphorylated RisA is able to activate many genes of cluster 3. A subset of cluster 3 genes are also activated by non-phosphorylated RisA in the absence of c-di-GMP. Under modulating conditions, and in the absence of BvgR, binding of c-di-GMP to RisA phosphorylated by RisK leads to the expression of vrgs (cluster 6), but also to the repression of flagellar and chemotaxis genes (cluster 4). Unphosphorylated RisA, in the presence or absence of c-di-GMP, inhibits the expression of other cluster 4 genes, including iron-regulated genes. Phosphorylated c-di-GMP-associated RisA is also able to repress the expression of the vags belonging to cluster 2.