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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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Activating mutations in YfiN.A) Locations of activating mutations in YfiN. Arrows indicate the positions of activating mutations throughout the structure of YfiN. TM: transmembrane helix. The cartoon is drawn to scale. B) Co-immunoprecipitation of active YfiN alleles with flag-tagged YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum. N-WT shows the DyfiNR screening strain with both yfiN (WT) and yfiR-flag plasmids present. Ctrl. shows ΔyfiNR with pGm-yfiprom-N only. The point mutation present in YfiN is indicated for the remaining lanes, which show YfiN immunoprecipitated from ΔyfiNR strains containing both yfiR-flag and the mutated yfiN plasmids. C) Attachment of the active YfiN alleles is shown relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (p-yfiN p-yfiR). Controls containing the yfiN or yfiR-flag plasmid only are also shown. The point mutation present in YfiN is indicated for each bar. Light grey bars indicate mutants whose activity was compensated for by mutations at the C-terminus of YfiR. Mutants with mid grey bars were compensated for by mutations in the signal sequence, or by uncharacterized mutations, while those with dark grey bars were not compensated for during YfiR mutagenesis. The domain locations of mutants are indicated with TM, PAS etc. D) Cartoon showing the locations of activating substitutions (blue) on a homology model of the YfiN PAS domain (residues 44–154). The PAS model is based on the CitA periplasmic domain (see Materials and methods). E) Surface representation of the YfiN PAS model. The locations of activating mutants on the proposed homodimer interface are shown in light blue, those at the possible YfiR binding site are shown in dark blue. F) The locations of activating substitutions (blue) on a homology model of the YfiN HAMP domain (residues 183–236). N and C termini are marked in D) and F). The HAMP model is based on the Aer2 HAMP structure (see Materials and methods).
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ppat-1002760-g002: Activating mutations in YfiN.A) Locations of activating mutations in YfiN. Arrows indicate the positions of activating mutations throughout the structure of YfiN. TM: transmembrane helix. The cartoon is drawn to scale. B) Co-immunoprecipitation of active YfiN alleles with flag-tagged YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum. N-WT shows the DyfiNR screening strain with both yfiN (WT) and yfiR-flag plasmids present. Ctrl. shows ΔyfiNR with pGm-yfiprom-N only. The point mutation present in YfiN is indicated for the remaining lanes, which show YfiN immunoprecipitated from ΔyfiNR strains containing both yfiR-flag and the mutated yfiN plasmids. C) Attachment of the active YfiN alleles is shown relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (p-yfiN p-yfiR). Controls containing the yfiN or yfiR-flag plasmid only are also shown. The point mutation present in YfiN is indicated for each bar. Light grey bars indicate mutants whose activity was compensated for by mutations at the C-terminus of YfiR. Mutants with mid grey bars were compensated for by mutations in the signal sequence, or by uncharacterized mutations, while those with dark grey bars were not compensated for during YfiR mutagenesis. The domain locations of mutants are indicated with TM, PAS etc. D) Cartoon showing the locations of activating substitutions (blue) on a homology model of the YfiN PAS domain (residues 44–154). The PAS model is based on the CitA periplasmic domain (see Materials and methods). E) Surface representation of the YfiN PAS model. The locations of activating mutants on the proposed homodimer interface are shown in light blue, those at the possible YfiR binding site are shown in dark blue. F) The locations of activating substitutions (blue) on a homology model of the YfiN HAMP domain (residues 183–236). N and C termini are marked in D) and F). The HAMP model is based on the Aer2 HAMP structure (see Materials and methods).

Mentions: If YfiR represses YfiN activity through direct binding to its periplasmic PAS domain, it should be possible to isolate constitutively active YfiN variants that fail to bind YfiR. The positions of these activating residue substitutions would consequently provide insights into the mechanism of YfiN function and the binding interface of YfiN and YfiR. Previously, similar experiments have been successfully used to probe the structure-function relationship of the P. fluorescens DGC WspR [54], [55]. To identify YfiR-insensitive YfiN alleles, a screening system was designed in which yfiN and yfiR-flag are expressed from two separate plasmids in a ΔyfiNR background (see Materials and Methods). A pool of yfiN variants was produced by XL-1 red mutagenesis of the yfiN plasmid and screened for mutants that induced an SCV phenotype in the ΔyfiNR tester strain containing a plasmid-borne copy of yfiR. Sequencing identified the locations of twenty independent, activating yfiN mutations. Two residues were identified in the first transmembrane helix, ten were located towards the N-terminal end of the periplasmic PAS domain, four were found in the second transmembrane helix, and four towards the C-terminal end of the HAMP domain (Figure 2A). No mutations were found in the GGDEF domain. Since most of these mutations were isolated several times independently, we assume that the screen was approaching saturation (Table 1).


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)

Activating mutations in YfiN.A) Locations of activating mutations in YfiN. Arrows indicate the positions of activating mutations throughout the structure of YfiN. TM: transmembrane helix. The cartoon is drawn to scale. B) Co-immunoprecipitation of active YfiN alleles with flag-tagged YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum. N-WT shows the DyfiNR screening strain with both yfiN (WT) and yfiR-flag plasmids present. Ctrl. shows ΔyfiNR with pGm-yfiprom-N only. The point mutation present in YfiN is indicated for the remaining lanes, which show YfiN immunoprecipitated from ΔyfiNR strains containing both yfiR-flag and the mutated yfiN plasmids. C) Attachment of the active YfiN alleles is shown relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (p-yfiN p-yfiR). Controls containing the yfiN or yfiR-flag plasmid only are also shown. The point mutation present in YfiN is indicated for each bar. Light grey bars indicate mutants whose activity was compensated for by mutations at the C-terminus of YfiR. Mutants with mid grey bars were compensated for by mutations in the signal sequence, or by uncharacterized mutations, while those with dark grey bars were not compensated for during YfiR mutagenesis. The domain locations of mutants are indicated with TM, PAS etc. D) Cartoon showing the locations of activating substitutions (blue) on a homology model of the YfiN PAS domain (residues 44–154). The PAS model is based on the CitA periplasmic domain (see Materials and methods). E) Surface representation of the YfiN PAS model. The locations of activating mutants on the proposed homodimer interface are shown in light blue, those at the possible YfiR binding site are shown in dark blue. F) The locations of activating substitutions (blue) on a homology model of the YfiN HAMP domain (residues 183–236). N and C termini are marked in D) and F). The HAMP model is based on the Aer2 HAMP structure (see Materials and methods).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375315&req=5

ppat-1002760-g002: Activating mutations in YfiN.A) Locations of activating mutations in YfiN. Arrows indicate the positions of activating mutations throughout the structure of YfiN. TM: transmembrane helix. The cartoon is drawn to scale. B) Co-immunoprecipitation of active YfiN alleles with flag-tagged YfiR. Immunoblot of boiled M2 resin samples with anti-YfiN antiserum. N-WT shows the DyfiNR screening strain with both yfiN (WT) and yfiR-flag plasmids present. Ctrl. shows ΔyfiNR with pGm-yfiprom-N only. The point mutation present in YfiN is indicated for the remaining lanes, which show YfiN immunoprecipitated from ΔyfiNR strains containing both yfiR-flag and the mutated yfiN plasmids. C) Attachment of the active YfiN alleles is shown relative to ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag (p-yfiN p-yfiR). Controls containing the yfiN or yfiR-flag plasmid only are also shown. The point mutation present in YfiN is indicated for each bar. Light grey bars indicate mutants whose activity was compensated for by mutations at the C-terminus of YfiR. Mutants with mid grey bars were compensated for by mutations in the signal sequence, or by uncharacterized mutations, while those with dark grey bars were not compensated for during YfiR mutagenesis. The domain locations of mutants are indicated with TM, PAS etc. D) Cartoon showing the locations of activating substitutions (blue) on a homology model of the YfiN PAS domain (residues 44–154). The PAS model is based on the CitA periplasmic domain (see Materials and methods). E) Surface representation of the YfiN PAS model. The locations of activating mutants on the proposed homodimer interface are shown in light blue, those at the possible YfiR binding site are shown in dark blue. F) The locations of activating substitutions (blue) on a homology model of the YfiN HAMP domain (residues 183–236). N and C termini are marked in D) and F). The HAMP model is based on the Aer2 HAMP structure (see Materials and methods).
Mentions: If YfiR represses YfiN activity through direct binding to its periplasmic PAS domain, it should be possible to isolate constitutively active YfiN variants that fail to bind YfiR. The positions of these activating residue substitutions would consequently provide insights into the mechanism of YfiN function and the binding interface of YfiN and YfiR. Previously, similar experiments have been successfully used to probe the structure-function relationship of the P. fluorescens DGC WspR [54], [55]. To identify YfiR-insensitive YfiN alleles, a screening system was designed in which yfiN and yfiR-flag are expressed from two separate plasmids in a ΔyfiNR background (see Materials and Methods). A pool of yfiN variants was produced by XL-1 red mutagenesis of the yfiN plasmid and screened for mutants that induced an SCV phenotype in the ΔyfiNR tester strain containing a plasmid-borne copy of yfiR. Sequencing identified the locations of twenty independent, activating yfiN mutations. Two residues were identified in the first transmembrane helix, ten were located towards the N-terminal end of the periplasmic PAS domain, four were found in the second transmembrane helix, and four towards the C-terminal end of the HAMP domain (Figure 2A). No mutations were found in the GGDEF domain. Since most of these mutations were isolated several times independently, we assume that the screen was approaching saturation (Table 1).

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