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The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways.

Malone JG, Jaeger T, Manfredi P, Dötsch A, Blanka A, Bos R, Cornelis GR, Häussler S, Jenal U - PLoS Pathog. (2012)

Bottom Line: The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN.The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections.These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum of the University of Basel, Basel, Switzerland.

ABSTRACT
The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

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Interactions between the YfiBNR proteins.A) A model of yfiBNR interaction. YfiN is a membrane-localized DGC controlled by YfiR. YfiB, the outer-membrane bound Pal-like protein, activates YfiN by sequestering YfiR. B) Co-immunoprecipitation of YfiN with flag-tagged YfiN and YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum shows YfiN (lower band) co-precipitating with YfiN-flag (upper band) or YfiR-flag. C) Membrane localization of YfiB and YfiR. Immunoblots of fractionated membrane samples with anti-YfiB (upper panel) and M2 antisera (lower 2 panels). The left two panels show membrane fractions for PA01 yfiR-M2, the right panel for ΔyfiBN yfiR-M2. IM1/2: inner membrane fractions, OM1/2: outer membrane fractions.
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ppat-1002760-g001: Interactions between the YfiBNR proteins.A) A model of yfiBNR interaction. YfiN is a membrane-localized DGC controlled by YfiR. YfiB, the outer-membrane bound Pal-like protein, activates YfiN by sequestering YfiR. B) Co-immunoprecipitation of YfiN with flag-tagged YfiN and YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum shows YfiN (lower band) co-precipitating with YfiN-flag (upper band) or YfiR-flag. C) Membrane localization of YfiB and YfiR. Immunoblots of fractionated membrane samples with anti-YfiB (upper panel) and M2 antisera (lower 2 panels). The left two panels show membrane fractions for PA01 yfiR-M2, the right panel for ΔyfiBN yfiR-M2. IM1/2: inner membrane fractions, OM1/2: outer membrane fractions.

Mentions: YfiBNR [11], [33] (also called AwsXRO [34], [35], [36], TpbB [37]) is a tripartite signaling system that modulates intracellular c-di-GMP levels in response to signals received in the periplasm [11]. The effector of the Yfi system, YfiN, is a membrane integral diguanylate cyclase consisting of a periplasmic PAS domain and cytoplasmic HAMP and catalytic GGDEF domains (Figure 1A). YfiN activity is repressed by the soluble periplasmic protein YfiR and stimulated by the outer membrane lipoprotein YfiB [11]. C-di-GMP produced by YfiN stimulates the production of the Pel and Psl exopolysaccharides, thereby promoting surface attachment in wild-type P. aeruginosa and generating an SCV phenotype when YfiN is activated or YfiR repression is released [11]. Homologs of the YfiBNR system are widespread, and have been shown to function similarly in Escherichia coli, Klebsiella pneumonia and Pseudomonas fluorescens SBW25, where they affect biofilm formation through cellulose production [33], [34] or Type 3 fimbriae expression [38].


The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways.

Malone JG, Jaeger T, Manfredi P, Dötsch A, Blanka A, Bos R, Cornelis GR, Häussler S, Jenal U - PLoS Pathog. (2012)

Interactions between the YfiBNR proteins.A) A model of yfiBNR interaction. YfiN is a membrane-localized DGC controlled by YfiR. YfiB, the outer-membrane bound Pal-like protein, activates YfiN by sequestering YfiR. B) Co-immunoprecipitation of YfiN with flag-tagged YfiN and YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum shows YfiN (lower band) co-precipitating with YfiN-flag (upper band) or YfiR-flag. C) Membrane localization of YfiB and YfiR. Immunoblots of fractionated membrane samples with anti-YfiB (upper panel) and M2 antisera (lower 2 panels). The left two panels show membrane fractions for PA01 yfiR-M2, the right panel for ΔyfiBN yfiR-M2. IM1/2: inner membrane fractions, OM1/2: outer membrane fractions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375315&req=5

ppat-1002760-g001: Interactions between the YfiBNR proteins.A) A model of yfiBNR interaction. YfiN is a membrane-localized DGC controlled by YfiR. YfiB, the outer-membrane bound Pal-like protein, activates YfiN by sequestering YfiR. B) Co-immunoprecipitation of YfiN with flag-tagged YfiN and YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum shows YfiN (lower band) co-precipitating with YfiN-flag (upper band) or YfiR-flag. C) Membrane localization of YfiB and YfiR. Immunoblots of fractionated membrane samples with anti-YfiB (upper panel) and M2 antisera (lower 2 panels). The left two panels show membrane fractions for PA01 yfiR-M2, the right panel for ΔyfiBN yfiR-M2. IM1/2: inner membrane fractions, OM1/2: outer membrane fractions.
Mentions: YfiBNR [11], [33] (also called AwsXRO [34], [35], [36], TpbB [37]) is a tripartite signaling system that modulates intracellular c-di-GMP levels in response to signals received in the periplasm [11]. The effector of the Yfi system, YfiN, is a membrane integral diguanylate cyclase consisting of a periplasmic PAS domain and cytoplasmic HAMP and catalytic GGDEF domains (Figure 1A). YfiN activity is repressed by the soluble periplasmic protein YfiR and stimulated by the outer membrane lipoprotein YfiB [11]. C-di-GMP produced by YfiN stimulates the production of the Pel and Psl exopolysaccharides, thereby promoting surface attachment in wild-type P. aeruginosa and generating an SCV phenotype when YfiN is activated or YfiR repression is released [11]. Homologs of the YfiBNR system are widespread, and have been shown to function similarly in Escherichia coli, Klebsiella pneumonia and Pseudomonas fluorescens SBW25, where they affect biofilm formation through cellulose production [33], [34] or Type 3 fimbriae expression [38].

Bottom Line: The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN.The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections.These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum of the University of Basel, Basel, Switzerland.

ABSTRACT
The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

Show MeSH
Related in: MedlinePlus