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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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Clinical YfiBNR mutants.A) Colony morphologies of ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag strains with the point mutants indicated present in either YfiR or YfiN. ‘WT’: both plasmids contain wild-type yfiN/yfiR alleles. All colony morphologies are on LB Congo-red agar. B) Colony morphologies of Clin110 and Clin163 strains. pR indicates that the strain contains pME-araC-yfiR, the asterisk indicates induction. C) Colony morphologies of ΔyfiBNR strains complemented with the yfiBNR operon inserted into the att-Tn7 site. Mutations in the complementing copy of yfiN are indicated in each case. ‘WT’: wild type yfiN. D) Attachment relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (pNpR) of clinical yfiN/yfiR mutants. ‘pR’ and ‘pN’ contain pMR-yfiR-flag or pGm-yfiprom-N only. E) Attachment of Clin110 ± pME-araC-yfiR and Clin163 relative to PA01.
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ppat-1002760-g007: Clinical YfiBNR mutants.A) Colony morphologies of ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag strains with the point mutants indicated present in either YfiR or YfiN. ‘WT’: both plasmids contain wild-type yfiN/yfiR alleles. All colony morphologies are on LB Congo-red agar. B) Colony morphologies of Clin110 and Clin163 strains. pR indicates that the strain contains pME-araC-yfiR, the asterisk indicates induction. C) Colony morphologies of ΔyfiBNR strains complemented with the yfiBNR operon inserted into the att-Tn7 site. Mutations in the complementing copy of yfiN are indicated in each case. ‘WT’: wild type yfiN. D) Attachment relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (pNpR) of clinical yfiN/yfiR mutants. ‘pR’ and ‘pN’ contain pMR-yfiR-flag or pGm-yfiprom-N only. E) Attachment of Clin110 ± pME-araC-yfiR and Clin163 relative to PA01.

Mentions: “In vitro” mutagenesis of the yfiBNR operon has revealed several routes to Yfi-mediated SCV formation. Disruption of yfiR induces a strong SCV phenotype [11], and activating mutations can arise in yfiN or yfiB (Figure 2, 4). To determine whether mutations in the yfi genes were responsible for the SCV phenotypes of clinical P. aeruginosa isolates from CF patients, we sequenced the yfiBNR operon of clinically derived SCVs and analyzed the contribution of candidate SNPs. Firstly, 45 clinical SCV strains were pooled and their genomes sequenced (S. Häussler and A. Dötsch, unpublished data). The yfiBNR operon sequence of each pool (representing a consensus sequence drawn from 15 individual SCV genome sequences) was compared with that of PA01, the positions of SNPs were identified (Table S3), and non-synonymous SNPs reproduced and introduced into the SCV screening strains described above (for details see Materials and Methods). From a total of 11 mutations identified, seven showed no noticeable effect. However, several mutations were identified in yfiN (G173D, D223N, L227M) or yfiR (C26R) that induced an SCV morphology and greatly enhanced attachment in the screening strain (Figure 2C, 7A, D). The amino acid residues altered in YfiN either matched (G173) or were located in the immediate vicinity of positions (D223, L227) identified by in vitro mutagenesis (Figure 2). These data demonstrate that SCVs arise in the airways of CF patients through the selection of clones carrying mutations in yfi genes that lead to de-repression the Yfi pathway.


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)

Clinical YfiBNR mutants.A) Colony morphologies of ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag strains with the point mutants indicated present in either YfiR or YfiN. ‘WT’: both plasmids contain wild-type yfiN/yfiR alleles. All colony morphologies are on LB Congo-red agar. B) Colony morphologies of Clin110 and Clin163 strains. pR indicates that the strain contains pME-araC-yfiR, the asterisk indicates induction. C) Colony morphologies of ΔyfiBNR strains complemented with the yfiBNR operon inserted into the att-Tn7 site. Mutations in the complementing copy of yfiN are indicated in each case. ‘WT’: wild type yfiN. D) Attachment relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (pNpR) of clinical yfiN/yfiR mutants. ‘pR’ and ‘pN’ contain pMR-yfiR-flag or pGm-yfiprom-N only. E) Attachment of Clin110 ± pME-araC-yfiR and Clin163 relative to PA01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375315&req=5

ppat-1002760-g007: Clinical YfiBNR mutants.A) Colony morphologies of ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag strains with the point mutants indicated present in either YfiR or YfiN. ‘WT’: both plasmids contain wild-type yfiN/yfiR alleles. All colony morphologies are on LB Congo-red agar. B) Colony morphologies of Clin110 and Clin163 strains. pR indicates that the strain contains pME-araC-yfiR, the asterisk indicates induction. C) Colony morphologies of ΔyfiBNR strains complemented with the yfiBNR operon inserted into the att-Tn7 site. Mutations in the complementing copy of yfiN are indicated in each case. ‘WT’: wild type yfiN. D) Attachment relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (pNpR) of clinical yfiN/yfiR mutants. ‘pR’ and ‘pN’ contain pMR-yfiR-flag or pGm-yfiprom-N only. E) Attachment of Clin110 ± pME-araC-yfiR and Clin163 relative to PA01.
Mentions: “In vitro” mutagenesis of the yfiBNR operon has revealed several routes to Yfi-mediated SCV formation. Disruption of yfiR induces a strong SCV phenotype [11], and activating mutations can arise in yfiN or yfiB (Figure 2, 4). To determine whether mutations in the yfi genes were responsible for the SCV phenotypes of clinical P. aeruginosa isolates from CF patients, we sequenced the yfiBNR operon of clinically derived SCVs and analyzed the contribution of candidate SNPs. Firstly, 45 clinical SCV strains were pooled and their genomes sequenced (S. Häussler and A. Dötsch, unpublished data). The yfiBNR operon sequence of each pool (representing a consensus sequence drawn from 15 individual SCV genome sequences) was compared with that of PA01, the positions of SNPs were identified (Table S3), and non-synonymous SNPs reproduced and introduced into the SCV screening strains described above (for details see Materials and Methods). From a total of 11 mutations identified, seven showed no noticeable effect. However, several mutations were identified in yfiN (G173D, D223N, L227M) or yfiR (C26R) that induced an SCV morphology and greatly enhanced attachment in the screening strain (Figure 2C, 7A, D). The amino acid residues altered in YfiN either matched (G173) or were located in the immediate vicinity of positions (D223, L227) identified by in vitro mutagenesis (Figure 2). These data demonstrate that SCVs arise in the airways of CF patients through the selection of clones carrying mutations in yfi genes that lead to de-repression the Yfi pathway.

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