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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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Compensatory YfiR alleles.A) Immunoblots with M2 antiserum, showing levels of compensatory YfiR-flag variants in whole cell lysates. pyfiR: ΔyfiNR pMR-yfiR-flag, ΔyfiNR: strain without vector, pyfiN/pyfiR: ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag, lanes 1–13: ΔyfiNR pGm-yfiprom-N with compensatory pMR-yfiR-flag plasmids, proposed activating mutations are highlighted in bold. Mutants in lane 1, 4–8, 10–13 harbor mutations in the signal peptide that enhance expression, see also Table 2. B) Cartoon showing the locations of activating substitutions (green) on a homology model of YfiR (comprising residues 68–190). N and C termini are marked. The YfiR model is based on multiple structures (see Materials and methods). C) Surface representation of the YfiR model. The locations of activating mutants are shown in green, hydrophobic residues forming the possible YfiN binding surface are shown in dark blue.
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ppat-1002760-g003: Compensatory YfiR alleles.A) Immunoblots with M2 antiserum, showing levels of compensatory YfiR-flag variants in whole cell lysates. pyfiR: ΔyfiNR pMR-yfiR-flag, ΔyfiNR: strain without vector, pyfiN/pyfiR: ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag, lanes 1–13: ΔyfiNR pGm-yfiprom-N with compensatory pMR-yfiR-flag plasmids, proposed activating mutations are highlighted in bold. Mutants in lane 1, 4–8, 10–13 harbor mutations in the signal peptide that enhance expression, see also Table 2. B) Cartoon showing the locations of activating substitutions (green) on a homology model of YfiR (comprising residues 68–190). N and C termini are marked. The YfiR model is based on multiple structures (see Materials and methods). C) Surface representation of the YfiR model. The locations of activating mutants are shown in green, hydrophobic residues forming the possible YfiN binding surface are shown in dark blue.

Mentions: To probe the YfiN-YfiR interaction in more detail, we undertook a screen for compensatory yfiR alleles, i.e. alleles that would restore wild-type (WT) colony morphology in the presence of some of the activated YfiN variants introduced above. Following PCR mutagenesis of yfiR, twenty-one alleles were isolated across eight yfiN mutant backgrounds (Table 2). Several yfiR alleles were independently isolated in several constitutive yfiN backgrounds, resulting in a total of fourteen unique, compensatory yfiR alleles, most of which cluster in the secretion signal sequence or in the C-terminal region of the protein (Table 2, Figure 3B). As the signal peptide is cleaved following export of YfiR into the periplasm, mutations in this region are not predicted to affect the final protein structure. Rather, these mutations might boost YfiR levels in the periplasm through increased translation or export of the protein. This was confirmed by immunoblot analysis demonstrating that signal peptide mutants indeed produced higher overall levels of YfiR than wild-type (Figure 3).


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)

Compensatory YfiR alleles.A) Immunoblots with M2 antiserum, showing levels of compensatory YfiR-flag variants in whole cell lysates. pyfiR: ΔyfiNR pMR-yfiR-flag, ΔyfiNR: strain without vector, pyfiN/pyfiR: ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag, lanes 1–13: ΔyfiNR pGm-yfiprom-N with compensatory pMR-yfiR-flag plasmids, proposed activating mutations are highlighted in bold. Mutants in lane 1, 4–8, 10–13 harbor mutations in the signal peptide that enhance expression, see also Table 2. B) Cartoon showing the locations of activating substitutions (green) on a homology model of YfiR (comprising residues 68–190). N and C termini are marked. The YfiR model is based on multiple structures (see Materials and methods). C) Surface representation of the YfiR model. The locations of activating mutants are shown in green, hydrophobic residues forming the possible YfiN binding surface are shown in dark blue.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002760-g003: Compensatory YfiR alleles.A) Immunoblots with M2 antiserum, showing levels of compensatory YfiR-flag variants in whole cell lysates. pyfiR: ΔyfiNR pMR-yfiR-flag, ΔyfiNR: strain without vector, pyfiN/pyfiR: ΔyfiNR pGm-yfiprom-N, pMR-yfiR-flag, lanes 1–13: ΔyfiNR pGm-yfiprom-N with compensatory pMR-yfiR-flag plasmids, proposed activating mutations are highlighted in bold. Mutants in lane 1, 4–8, 10–13 harbor mutations in the signal peptide that enhance expression, see also Table 2. B) Cartoon showing the locations of activating substitutions (green) on a homology model of YfiR (comprising residues 68–190). N and C termini are marked. The YfiR model is based on multiple structures (see Materials and methods). C) Surface representation of the YfiR model. The locations of activating mutants are shown in green, hydrophobic residues forming the possible YfiN binding surface are shown in dark blue.
Mentions: To probe the YfiN-YfiR interaction in more detail, we undertook a screen for compensatory yfiR alleles, i.e. alleles that would restore wild-type (WT) colony morphology in the presence of some of the activated YfiN variants introduced above. Following PCR mutagenesis of yfiR, twenty-one alleles were isolated across eight yfiN mutant backgrounds (Table 2). Several yfiR alleles were independently isolated in several constitutive yfiN backgrounds, resulting in a total of fourteen unique, compensatory yfiR alleles, most of which cluster in the secretion signal sequence or in the C-terminal region of the protein (Table 2, Figure 3B). As the signal peptide is cleaved following export of YfiR into the periplasm, mutations in this region are not predicted to affect the final protein structure. Rather, these mutations might boost YfiR levels in the periplasm through increased translation or export of the protein. This was confirmed by immunoblot analysis demonstrating that signal peptide mutants indeed produced higher overall levels of YfiR than wild-type (Figure 3).

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