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Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

Dupré E, Herrou J, Lensink MF, Wintjens R, Vagin A, Lebedev A, Crosson S, Villeret V, Locht C, Antoine R, Jacob-Dubuisson F - PLoS Pathog. (2015)

Bottom Line: Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase.The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition.This work lays the bases to understand virulence regulation in Bordetella.

View Article: PubMed Central - PubMed

Affiliation: Center for Infection and Immunity (CIIL), Institut Pasteur de Lille, Lille, France; Center for Infection and Immunity (CIIL), University Lille North of France, Lille, France; UMR 8204, Centre National de la Recherche Scientifique (CNRS), Lille, France; U1019, Institut National de la Santé et de la Recherche Médicale (INSERM), Lille, France.

ABSTRACT
Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

No MeSH data available.


Related in: MedlinePlus

In vivo effects of the substitutions in BvgS.A lacZ reporter gene under the control of the Bvg-regulated ptx promoter was used for determination of BvgS kinase activity in standard or modulated culture conditions. Blue and pink bars indicate kinase activity levels of bacteria producing the indicated BvgS variants and grown without or with 8 mM nicotinate, respectively, with the standard errors of the mean calculated from three distinct experiments. The middle column indicates the interfaces in which the targeted interactions are located, with inter- and intra-protomer interfaces designated ‘inter’ and ‘intra’, respectively. Nd, no β-gal activity detected; a, wild type activity and/or modulation recovered when cells were grown in the presence of TCEP; b, BvgS variants only responsive to high nicotinate concentrations (20 mM). The full set of data is shown in S5 Fig.
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ppat.1004700.g005: In vivo effects of the substitutions in BvgS.A lacZ reporter gene under the control of the Bvg-regulated ptx promoter was used for determination of BvgS kinase activity in standard or modulated culture conditions. Blue and pink bars indicate kinase activity levels of bacteria producing the indicated BvgS variants and grown without or with 8 mM nicotinate, respectively, with the standard errors of the mean calculated from three distinct experiments. The middle column indicates the interfaces in which the targeted interactions are located, with inter- and intra-protomer interfaces designated ‘inter’ and ‘intra’, respectively. Nd, no β-gal activity detected; a, wild type activity and/or modulation recovered when cells were grown in the presence of TCEP; b, BvgS variants only responsive to high nicotinate concentrations (20 mM). The full set of data is shown in S5 Fig.

Mentions: We then asked whether VFT1 closing—as might happen upon binding of a ligand—would affect BvgS activity. We locked the VFT1 domains in closed conformations by generating a disulfide (S-S) bonds across their cavity [33,34]. Two residues located on the edges of the lobes were replaced by Cys to obtain BvgSE113C+N177C (S3 Fig). The corresponding point mutations were inserted into the chromosomal bvg locus by allelic exchange, and we verified the production of the protein and the formation of the S-S bond by immunoblotting (S4 Fig). The in vivo effect of the substitution on BvgS function was then measured by using a reporter system with the lacZ gene under the control of the Bvg-regulated ptx promoter [35]. In vivo formation of the S-S bond in VFT1 abrogates the kinase activity of BvgS (Fig. 5). This phenotype is reverted by the addition of a reducing agent, TCEP, to the growth medium (S5 Fig), which confirms that the S-S bond forms in vivo and shows that the loss of function is related to its presence and not to the Cys substitutions.


Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

Dupré E, Herrou J, Lensink MF, Wintjens R, Vagin A, Lebedev A, Crosson S, Villeret V, Locht C, Antoine R, Jacob-Dubuisson F - PLoS Pathog. (2015)

In vivo effects of the substitutions in BvgS.A lacZ reporter gene under the control of the Bvg-regulated ptx promoter was used for determination of BvgS kinase activity in standard or modulated culture conditions. Blue and pink bars indicate kinase activity levels of bacteria producing the indicated BvgS variants and grown without or with 8 mM nicotinate, respectively, with the standard errors of the mean calculated from three distinct experiments. The middle column indicates the interfaces in which the targeted interactions are located, with inter- and intra-protomer interfaces designated ‘inter’ and ‘intra’, respectively. Nd, no β-gal activity detected; a, wild type activity and/or modulation recovered when cells were grown in the presence of TCEP; b, BvgS variants only responsive to high nicotinate concentrations (20 mM). The full set of data is shown in S5 Fig.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352136&req=5

ppat.1004700.g005: In vivo effects of the substitutions in BvgS.A lacZ reporter gene under the control of the Bvg-regulated ptx promoter was used for determination of BvgS kinase activity in standard or modulated culture conditions. Blue and pink bars indicate kinase activity levels of bacteria producing the indicated BvgS variants and grown without or with 8 mM nicotinate, respectively, with the standard errors of the mean calculated from three distinct experiments. The middle column indicates the interfaces in which the targeted interactions are located, with inter- and intra-protomer interfaces designated ‘inter’ and ‘intra’, respectively. Nd, no β-gal activity detected; a, wild type activity and/or modulation recovered when cells were grown in the presence of TCEP; b, BvgS variants only responsive to high nicotinate concentrations (20 mM). The full set of data is shown in S5 Fig.
Mentions: We then asked whether VFT1 closing—as might happen upon binding of a ligand—would affect BvgS activity. We locked the VFT1 domains in closed conformations by generating a disulfide (S-S) bonds across their cavity [33,34]. Two residues located on the edges of the lobes were replaced by Cys to obtain BvgSE113C+N177C (S3 Fig). The corresponding point mutations were inserted into the chromosomal bvg locus by allelic exchange, and we verified the production of the protein and the formation of the S-S bond by immunoblotting (S4 Fig). The in vivo effect of the substitution on BvgS function was then measured by using a reporter system with the lacZ gene under the control of the Bvg-regulated ptx promoter [35]. In vivo formation of the S-S bond in VFT1 abrogates the kinase activity of BvgS (Fig. 5). This phenotype is reverted by the addition of a reducing agent, TCEP, to the growth medium (S5 Fig), which confirms that the S-S bond forms in vivo and shows that the loss of function is related to its presence and not to the Cys substitutions.

Bottom Line: Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase.The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition.This work lays the bases to understand virulence regulation in Bordetella.

View Article: PubMed Central - PubMed

Affiliation: Center for Infection and Immunity (CIIL), Institut Pasteur de Lille, Lille, France; Center for Infection and Immunity (CIIL), University Lille North of France, Lille, France; UMR 8204, Centre National de la Recherche Scientifique (CNRS), Lille, France; U1019, Institut National de la Santé et de la Recherche Médicale (INSERM), Lille, France.

ABSTRACT
Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

No MeSH data available.


Related in: MedlinePlus