Limits...
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.

Show MeSH

Related in: MedlinePlus

Mutational analysis of YfiB.A) The effect of activating yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to PA01 (PA01 ctrl.). ‘YfiB WT’ indicates pME6032-yfiB. The point mutants in YfiB are indicated for the remaining lanes. Those mutations thought to contribute to YfiB activation are marked in bold. The immunoblot shows the levels of YfiB protein present in each strain. B) Left: Cartoon showing the locations of activating substitutions (red) on a homology model of YfiB (comprising residues 27–168). The YfiB model is based on the Omp/Pal structure (see Materials and methods for details). The N terminus and peptidoglycan binding site are marked. Right: Surface representation of the YfiB model. The locations of activating mutants are shown in red, hydrophobic residues forming the possible YfiR binding surface are shown in dark blue. C) Co-localization of YfiB and YfiR at the outer membrane. Immunoblots of fractionated soluble and membrane samples with anti-YfiB and M2 antisera as shown. ‘YfiB WT’ indicates ΔyfiBNR Tn7::yfiR-flag containing pME6032-yfiB. ‘PG-’ indicates the same background strain containing pME-yfiB-PG- (YfiB containing the D102A and G105A substitutions), while ‘F48S’ contains the hyperactive yfiB F48S plasmid. D) The effect of different yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to pME6032 only (ctrl.). ‘PG-’ mutants ± F48S or L43P mutations as indicated. In ‘LA-’ mutants, the lipid anchor is missing, the signal peptide has been replaced with that from YfiR. ‘ΔBNR LA-’ indicates the yfiB LA- mutant in the ΔyfiBNR strain background. The immunoblot shows the levels of YfiB protein present in each strain. E) Colony morphologies on LB Congo-red agar upon over-expression of yfiB mutants.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3375315&req=5

ppat-1002760-g004: Mutational analysis of YfiB.A) The effect of activating yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to PA01 (PA01 ctrl.). ‘YfiB WT’ indicates pME6032-yfiB. The point mutants in YfiB are indicated for the remaining lanes. Those mutations thought to contribute to YfiB activation are marked in bold. The immunoblot shows the levels of YfiB protein present in each strain. B) Left: Cartoon showing the locations of activating substitutions (red) on a homology model of YfiB (comprising residues 27–168). The YfiB model is based on the Omp/Pal structure (see Materials and methods for details). The N terminus and peptidoglycan binding site are marked. Right: Surface representation of the YfiB model. The locations of activating mutants are shown in red, hydrophobic residues forming the possible YfiR binding surface are shown in dark blue. C) Co-localization of YfiB and YfiR at the outer membrane. Immunoblots of fractionated soluble and membrane samples with anti-YfiB and M2 antisera as shown. ‘YfiB WT’ indicates ΔyfiBNR Tn7::yfiR-flag containing pME6032-yfiB. ‘PG-’ indicates the same background strain containing pME-yfiB-PG- (YfiB containing the D102A and G105A substitutions), while ‘F48S’ contains the hyperactive yfiB F48S plasmid. D) The effect of different yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to pME6032 only (ctrl.). ‘PG-’ mutants ± F48S or L43P mutations as indicated. In ‘LA-’ mutants, the lipid anchor is missing, the signal peptide has been replaced with that from YfiR. ‘ΔBNR LA-’ indicates the yfiB LA- mutant in the ΔyfiBNR strain background. The immunoblot shows the levels of YfiB protein present in each strain. E) Colony morphologies on LB Congo-red agar upon over-expression of yfiB mutants.

Mentions: YfiB is predicted to be an outer-membrane lipoprotein with a PAL-like peptidoglycan (PG) binding domain. Overproduction of YfiB leads to YfiN-dependent SCV formation [11]. How this effect is exerted on YfiN is not clear and no detailed model for YfiB function in P. aeruginosa exists. To investigate the function of YfiB, a screen was conducted for activating mutants that induced an SCV phenotype in PA01 without overproduction of the protein. A total of 20 yfiB alleles were isolated, each containing one or more amino acid substitutions. All activating yfiB alleles caused increased surface attachment and biofilm formation (Figure 4A). Strikingly, while mutations were distributed throughout the sequence of yfiB, at least one substitution was found between residues 35 and 55 in all cases. These affected a total of seven positions, five of which were also isolated as single activating substitutions (Figure 4A). Alleles with single mutations in the YfiB N-terminus generally had strong effects that were weakened by the presence of secondary mutations (for example, YfiB L43P produces higher attachment than L43P alleles containing additional mutations). Together this argued that the majority of mutations leading to YfiBNR activation cluster in this region of the YfiB protein. Some of these variants had very strong activating effects despite showing severely reduced stability (Figure 4A). Three further, similar substitutions (I40F, V42M, and E45G) were found in conjunction with other singly isolated activating mutations. While neither the V42M nor the E45G substitutions contributed positively to YfiB activity, the YfiB-I40F-F48L allele produced a far stronger phenotype than F48L alone, suggesting that the I40F mutation also contributes to YfiB activation (Figure 4A). When the locations of the activating mutations were plotted onto a 3-D homology model of YfiB, they clustered around the first helix of the PAL domain (Figure 4B). Interestingly, activating residues in YfiB surround a predicted surface-exposed hydrophobic region, similar to that seen in the model of YfiR (Figure 3C). This hydrophobic patch is highly conserved in YfiB homologs, but absent in the YfiB structural homolog OprL [61], [62]. Most notably, W55, predicted to form the core of the hydrophobic binding site, is very highly conserved, only replaced in a small minority of cases with phenylalanine (note that the YfiB W55L mutation forms a weak SCV in PA01).


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)

Mutational analysis of YfiB.A) The effect of activating yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to PA01 (PA01 ctrl.). ‘YfiB WT’ indicates pME6032-yfiB. The point mutants in YfiB are indicated for the remaining lanes. Those mutations thought to contribute to YfiB activation are marked in bold. The immunoblot shows the levels of YfiB protein present in each strain. B) Left: Cartoon showing the locations of activating substitutions (red) on a homology model of YfiB (comprising residues 27–168). The YfiB model is based on the Omp/Pal structure (see Materials and methods for details). The N terminus and peptidoglycan binding site are marked. Right: Surface representation of the YfiB model. The locations of activating mutants are shown in red, hydrophobic residues forming the possible YfiR binding surface are shown in dark blue. C) Co-localization of YfiB and YfiR at the outer membrane. Immunoblots of fractionated soluble and membrane samples with anti-YfiB and M2 antisera as shown. ‘YfiB WT’ indicates ΔyfiBNR Tn7::yfiR-flag containing pME6032-yfiB. ‘PG-’ indicates the same background strain containing pME-yfiB-PG- (YfiB containing the D102A and G105A substitutions), while ‘F48S’ contains the hyperactive yfiB F48S plasmid. D) The effect of different yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to pME6032 only (ctrl.). ‘PG-’ mutants ± F48S or L43P mutations as indicated. In ‘LA-’ mutants, the lipid anchor is missing, the signal peptide has been replaced with that from YfiR. ‘ΔBNR LA-’ indicates the yfiB LA- mutant in the ΔyfiBNR strain background. The immunoblot shows the levels of YfiB protein present in each strain. E) Colony morphologies on LB Congo-red agar upon over-expression of yfiB mutants.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002760-g004: Mutational analysis of YfiB.A) The effect of activating yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to PA01 (PA01 ctrl.). ‘YfiB WT’ indicates pME6032-yfiB. The point mutants in YfiB are indicated for the remaining lanes. Those mutations thought to contribute to YfiB activation are marked in bold. The immunoblot shows the levels of YfiB protein present in each strain. B) Left: Cartoon showing the locations of activating substitutions (red) on a homology model of YfiB (comprising residues 27–168). The YfiB model is based on the Omp/Pal structure (see Materials and methods for details). The N terminus and peptidoglycan binding site are marked. Right: Surface representation of the YfiB model. The locations of activating mutants are shown in red, hydrophobic residues forming the possible YfiR binding surface are shown in dark blue. C) Co-localization of YfiB and YfiR at the outer membrane. Immunoblots of fractionated soluble and membrane samples with anti-YfiB and M2 antisera as shown. ‘YfiB WT’ indicates ΔyfiBNR Tn7::yfiR-flag containing pME6032-yfiB. ‘PG-’ indicates the same background strain containing pME-yfiB-PG- (YfiB containing the D102A and G105A substitutions), while ‘F48S’ contains the hyperactive yfiB F48S plasmid. D) The effect of different yfiB mutants, expressed from pME6032 in ΔyfiBNR Tn7::yfiNR, on attachment is shown relative to pME6032 only (ctrl.). ‘PG-’ mutants ± F48S or L43P mutations as indicated. In ‘LA-’ mutants, the lipid anchor is missing, the signal peptide has been replaced with that from YfiR. ‘ΔBNR LA-’ indicates the yfiB LA- mutant in the ΔyfiBNR strain background. The immunoblot shows the levels of YfiB protein present in each strain. E) Colony morphologies on LB Congo-red agar upon over-expression of yfiB mutants.
Mentions: YfiB is predicted to be an outer-membrane lipoprotein with a PAL-like peptidoglycan (PG) binding domain. Overproduction of YfiB leads to YfiN-dependent SCV formation [11]. How this effect is exerted on YfiN is not clear and no detailed model for YfiB function in P. aeruginosa exists. To investigate the function of YfiB, a screen was conducted for activating mutants that induced an SCV phenotype in PA01 without overproduction of the protein. A total of 20 yfiB alleles were isolated, each containing one or more amino acid substitutions. All activating yfiB alleles caused increased surface attachment and biofilm formation (Figure 4A). Strikingly, while mutations were distributed throughout the sequence of yfiB, at least one substitution was found between residues 35 and 55 in all cases. These affected a total of seven positions, five of which were also isolated as single activating substitutions (Figure 4A). Alleles with single mutations in the YfiB N-terminus generally had strong effects that were weakened by the presence of secondary mutations (for example, YfiB L43P produces higher attachment than L43P alleles containing additional mutations). Together this argued that the majority of mutations leading to YfiBNR activation cluster in this region of the YfiB protein. Some of these variants had very strong activating effects despite showing severely reduced stability (Figure 4A). Three further, similar substitutions (I40F, V42M, and E45G) were found in conjunction with other singly isolated activating mutations. While neither the V42M nor the E45G substitutions contributed positively to YfiB activity, the YfiB-I40F-F48L allele produced a far stronger phenotype than F48L alone, suggesting that the I40F mutation also contributes to YfiB activation (Figure 4A). When the locations of the activating mutations were plotted onto a 3-D homology model of YfiB, they clustered around the first helix of the PAL domain (Figure 4B). Interestingly, activating residues in YfiB surround a predicted surface-exposed hydrophobic region, similar to that seen in the model of YfiR (Figure 3C). This hydrophobic patch is highly conserved in YfiB homologs, but absent in the YfiB structural homolog OprL [61], [62]. Most notably, W55, predicted to form the core of the hydrophobic binding site, is very highly conserved, only replaced in a small minority of cases with phenylalanine (note that the YfiB W55L mutation forms a weak SCV in PA01).

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