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YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa.

Malone JG, Jaeger T, Spangler C, Ritz D, Spang A, Arrieumerlou C, Kaever V, Landmann R, Jenal U - PLoS Pathog. (2010)

Bottom Line: The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis.Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy.This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland. jacob.malone@unibas.ch

ABSTRACT
During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

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The YfiBNR complex.A) Membrane localization of YfiB and YfiR. Membrane fractionation was carried out with PA01 yfiR-M2. The separate fractions are labeled as follows: C  =  whole cell sample, L  =  cell lysate, S  =  soluble fraction, M  =  membrane fraction. YfiB localizes to the membrane fraction, YfiR-M2 to the soluble fraction. B) Periplasmic localization of YfiR. Periplasmic fractionation was carried out with PA01 yfiR-M2 pAD6Ω, and YfiR-M2 and GFP were detected by immunoblot analysis. GFP localizes to the spheroplast (cytosolic/membrane) fraction, while YfiR-M2 localizes to the periplasmic fraction. C) In-vivo crosslinking of YfiR-M2. Whole cell samples of PA01 yfiR-M2, ΔyfiN yfiR-M2, and ΔyfiBN yfiR-M2 mutant strains were crosslinked by addition of formaldehyde and YfiR-M2 detected by immunoblot analysis. 0  =  sample before crosslinking; 20 =  sample taken 20 min after formaldehyde addition; 20* = 20 min sample, boiled to break crosslinks. Black arrows indicate major bands corresponding in size to an YfiR-M2 monomer (20 kDa) and an YfiR-M2 oligomer (40 kDa), respectively. D) Bacterial two-hybrid analysis of YfiN and YfiR interactions. Positive interactions produce a red color on MacConkey indicator plates. Clockwise from top left, the cartoons denote YfiN-YfiN, YfiR-YfiR and HAMP domain-YfiN interactions. E) A model for YfiBNR function. YfiN is a membrane-localized DGC and is subject to product inhibition and control by YfiR. YfiB activates YfiN, possibly by releasing YfiR-mediated repression. OM and IM refer to the outer and inner membranes, respectively and PG refers to the peptidoglycan layer.
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ppat-1000804-g004: The YfiBNR complex.A) Membrane localization of YfiB and YfiR. Membrane fractionation was carried out with PA01 yfiR-M2. The separate fractions are labeled as follows: C  =  whole cell sample, L  =  cell lysate, S  =  soluble fraction, M  =  membrane fraction. YfiB localizes to the membrane fraction, YfiR-M2 to the soluble fraction. B) Periplasmic localization of YfiR. Periplasmic fractionation was carried out with PA01 yfiR-M2 pAD6Ω, and YfiR-M2 and GFP were detected by immunoblot analysis. GFP localizes to the spheroplast (cytosolic/membrane) fraction, while YfiR-M2 localizes to the periplasmic fraction. C) In-vivo crosslinking of YfiR-M2. Whole cell samples of PA01 yfiR-M2, ΔyfiN yfiR-M2, and ΔyfiBN yfiR-M2 mutant strains were crosslinked by addition of formaldehyde and YfiR-M2 detected by immunoblot analysis. 0  =  sample before crosslinking; 20 =  sample taken 20 min after formaldehyde addition; 20* = 20 min sample, boiled to break crosslinks. Black arrows indicate major bands corresponding in size to an YfiR-M2 monomer (20 kDa) and an YfiR-M2 oligomer (40 kDa), respectively. D) Bacterial two-hybrid analysis of YfiN and YfiR interactions. Positive interactions produce a red color on MacConkey indicator plates. Clockwise from top left, the cartoons denote YfiN-YfiN, YfiR-YfiR and HAMP domain-YfiN interactions. E) A model for YfiBNR function. YfiN is a membrane-localized DGC and is subject to product inhibition and control by YfiR. YfiB activates YfiN, possibly by releasing YfiR-mediated repression. OM and IM refer to the outer and inner membranes, respectively and PG refers to the peptidoglycan layer.

Mentions: Epistasis experiments indicated that YfiR plays an important role in regulating YfiN activity, possibly by relaying information from YfiB in the outer membrane to YfiN in the cytoplasmic membrane. Such a mechanism would predict that YfiR is located in the periplasm. To test this and due to the absence of an effective YfiR antibody, yfiR triple-M2 tagged (yfiR-M2) reporter strains were constructed. Insertion of the yfiR-M2 allele into the att-Tn7 locus of a ΔyfiR mutant restored the wild type phenotype, indicating that YfiR-M2 is fully functional (data not shown). The subcellular location of YfiR-M2 was subsequently investigated upon fractionation of cell extracts by ultracentrifugation followed by immunoblot analysis (Figure 4A). Because YfiB is predicted to localize to the outer membrane [70],[72], it was included as a control. As expected, YfiB almost exclusively localized to the insoluble, membrane-associated fraction. YfiR-M2 was found exclusively in the soluble fraction (Figure 4A). Next, a strain expressing both yfiR-M2 and gfp (PA01 yfiR-M2 pAD6-Ω) was subjected to periplasmic extraction by osmotic shock, and the fractions were analyzed by immunoblotting. As expected for a cytosolic protein, GFP was detected in the whole-cell and spheroplast fractions only, confirming that cell fractionation did not result in spheroplast lysis. In contrast, YfiR-M2 was found exclusively in the periplasmic fraction (Figure 4B).


YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa.

Malone JG, Jaeger T, Spangler C, Ritz D, Spang A, Arrieumerlou C, Kaever V, Landmann R, Jenal U - PLoS Pathog. (2010)

The YfiBNR complex.A) Membrane localization of YfiB and YfiR. Membrane fractionation was carried out with PA01 yfiR-M2. The separate fractions are labeled as follows: C  =  whole cell sample, L  =  cell lysate, S  =  soluble fraction, M  =  membrane fraction. YfiB localizes to the membrane fraction, YfiR-M2 to the soluble fraction. B) Periplasmic localization of YfiR. Periplasmic fractionation was carried out with PA01 yfiR-M2 pAD6Ω, and YfiR-M2 and GFP were detected by immunoblot analysis. GFP localizes to the spheroplast (cytosolic/membrane) fraction, while YfiR-M2 localizes to the periplasmic fraction. C) In-vivo crosslinking of YfiR-M2. Whole cell samples of PA01 yfiR-M2, ΔyfiN yfiR-M2, and ΔyfiBN yfiR-M2 mutant strains were crosslinked by addition of formaldehyde and YfiR-M2 detected by immunoblot analysis. 0  =  sample before crosslinking; 20 =  sample taken 20 min after formaldehyde addition; 20* = 20 min sample, boiled to break crosslinks. Black arrows indicate major bands corresponding in size to an YfiR-M2 monomer (20 kDa) and an YfiR-M2 oligomer (40 kDa), respectively. D) Bacterial two-hybrid analysis of YfiN and YfiR interactions. Positive interactions produce a red color on MacConkey indicator plates. Clockwise from top left, the cartoons denote YfiN-YfiN, YfiR-YfiR and HAMP domain-YfiN interactions. E) A model for YfiBNR function. YfiN is a membrane-localized DGC and is subject to product inhibition and control by YfiR. YfiB activates YfiN, possibly by releasing YfiR-mediated repression. OM and IM refer to the outer and inner membranes, respectively and PG refers to the peptidoglycan layer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837407&req=5

ppat-1000804-g004: The YfiBNR complex.A) Membrane localization of YfiB and YfiR. Membrane fractionation was carried out with PA01 yfiR-M2. The separate fractions are labeled as follows: C  =  whole cell sample, L  =  cell lysate, S  =  soluble fraction, M  =  membrane fraction. YfiB localizes to the membrane fraction, YfiR-M2 to the soluble fraction. B) Periplasmic localization of YfiR. Periplasmic fractionation was carried out with PA01 yfiR-M2 pAD6Ω, and YfiR-M2 and GFP were detected by immunoblot analysis. GFP localizes to the spheroplast (cytosolic/membrane) fraction, while YfiR-M2 localizes to the periplasmic fraction. C) In-vivo crosslinking of YfiR-M2. Whole cell samples of PA01 yfiR-M2, ΔyfiN yfiR-M2, and ΔyfiBN yfiR-M2 mutant strains were crosslinked by addition of formaldehyde and YfiR-M2 detected by immunoblot analysis. 0  =  sample before crosslinking; 20 =  sample taken 20 min after formaldehyde addition; 20* = 20 min sample, boiled to break crosslinks. Black arrows indicate major bands corresponding in size to an YfiR-M2 monomer (20 kDa) and an YfiR-M2 oligomer (40 kDa), respectively. D) Bacterial two-hybrid analysis of YfiN and YfiR interactions. Positive interactions produce a red color on MacConkey indicator plates. Clockwise from top left, the cartoons denote YfiN-YfiN, YfiR-YfiR and HAMP domain-YfiN interactions. E) A model for YfiBNR function. YfiN is a membrane-localized DGC and is subject to product inhibition and control by YfiR. YfiB activates YfiN, possibly by releasing YfiR-mediated repression. OM and IM refer to the outer and inner membranes, respectively and PG refers to the peptidoglycan layer.
Mentions: Epistasis experiments indicated that YfiR plays an important role in regulating YfiN activity, possibly by relaying information from YfiB in the outer membrane to YfiN in the cytoplasmic membrane. Such a mechanism would predict that YfiR is located in the periplasm. To test this and due to the absence of an effective YfiR antibody, yfiR triple-M2 tagged (yfiR-M2) reporter strains were constructed. Insertion of the yfiR-M2 allele into the att-Tn7 locus of a ΔyfiR mutant restored the wild type phenotype, indicating that YfiR-M2 is fully functional (data not shown). The subcellular location of YfiR-M2 was subsequently investigated upon fractionation of cell extracts by ultracentrifugation followed by immunoblot analysis (Figure 4A). Because YfiB is predicted to localize to the outer membrane [70],[72], it was included as a control. As expected, YfiB almost exclusively localized to the insoluble, membrane-associated fraction. YfiR-M2 was found exclusively in the soluble fraction (Figure 4A). Next, a strain expressing both yfiR-M2 and gfp (PA01 yfiR-M2 pAD6-Ω) was subjected to periplasmic extraction by osmotic shock, and the fractions were analyzed by immunoblotting. As expected for a cytosolic protein, GFP was detected in the whole-cell and spheroplast fractions only, confirming that cell fractionation did not result in spheroplast lysis. In contrast, YfiR-M2 was found exclusively in the periplasmic fraction (Figure 4B).

Bottom Line: The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis.Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy.This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland. jacob.malone@unibas.ch

ABSTRACT
During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

Show MeSH
Related in: MedlinePlus