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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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YfiN-mediated SCVs contribute to persistence in vitro and in vivo.A) Tobramycin survival assay with PA01 wild type (open triangles) and ΔyfiR mutant (open circles). ΔyfiR suppressor colonies are shown as red circles. Statistically significant differences between PA01and ΔyfiR are marked with asterisks (** = p<0.01, * = p<0.05). B) Single infections with PA01 wild type (black symbols) and ΔyfiR mutant (open symbols). Catheter samples (circles) are presented as cfu/ml, tissue samples (squares) as cfu/mg tissue. Statistically significant differences are marked with an asterisk (* = p<0.05). C) Competition experiments with PA01 wild type and ΔyfiR mutant. The graph shows the ratio of wild type to ΔyfiR mutant colonies recovered from catheters (C, circles) and tissue (T, squares) after four and eight weeks, respectively. Values above 1 indicate greater numbers of wild type colonies, values below 1 greater numbers of SCVs.
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ppat-1000804-g007: YfiN-mediated SCVs contribute to persistence in vitro and in vivo.A) Tobramycin survival assay with PA01 wild type (open triangles) and ΔyfiR mutant (open circles). ΔyfiR suppressor colonies are shown as red circles. Statistically significant differences between PA01and ΔyfiR are marked with asterisks (** = p<0.01, * = p<0.05). B) Single infections with PA01 wild type (black symbols) and ΔyfiR mutant (open symbols). Catheter samples (circles) are presented as cfu/ml, tissue samples (squares) as cfu/mg tissue. Statistically significant differences are marked with an asterisk (* = p<0.05). C) Competition experiments with PA01 wild type and ΔyfiR mutant. The graph shows the ratio of wild type to ΔyfiR mutant colonies recovered from catheters (C, circles) and tissue (T, squares) after four and eight weeks, respectively. Values above 1 indicate greater numbers of wild type colonies, values below 1 greater numbers of SCVs.

Mentions: Although the auto-aggregative and slow growing SCV morphotypes are at a considerable disadvantage under conditions that permit rapid growth, they are able to persist in vivo [7]. This phenomenon might be explained by conditions in the host environment (e.g. immune system attack or antimicrobial chemotherapy) that put an even higher burden on rapidly growing, non-adherent strains and thus provide selection for SCVs. To test this, we compared the competitive behavior of PA01 wild type and ΔyfiR SCV strains in vitro and in vivo. When grown in LB the ΔyfiR SCV strain was significantly outperformed by the wild type. Also, suppressors with wild type colony morphology quickly arose in every ΔyfiR sample, forming the majority of the cell population after 18 hours incubation (Figure 7A). The addition of increasing concentrations of tobramycin in the sub-inhibitory range led to a reduction of both wild type and ΔyfiR cell numbers. However, wild type growth decreased at a much steeper rate and at 1.5 µg/ml tobramycin, no significant difference in cfu numbers was observed between the two strains (Figure 7A). Likewise, in the presence of tobramycin the number of suppressors arising from ΔyfiR dropped sharply with no suppressors arising at concentrations above 0.5 µg/ml of the inhibitor. Thus, the fitness disadvantage of the ΔyfiR SCV is strongly reduced in the presence of sub-inhibitory concentrations of tobramycin. Two observations indicate that this effect is not linked to some form of antibiotic tolerance of the ΔyfiR SCV, but rather reflects converging fitness during slow growth under stressful conditions. Firstly, a similar relative increase of ΔyfiR fitness compared to wild type was observed when cells were grown at reduced temperatures (data not shown). Secondly, no differences in MIC were seen between PA01 and ΔyfiR for tobramycin or for any of the other antibiotics tested (Figure S8).


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)

YfiN-mediated SCVs contribute to persistence in vitro and in vivo.A) Tobramycin survival assay with PA01 wild type (open triangles) and ΔyfiR mutant (open circles). ΔyfiR suppressor colonies are shown as red circles. Statistically significant differences between PA01and ΔyfiR are marked with asterisks (** = p<0.01, * = p<0.05). B) Single infections with PA01 wild type (black symbols) and ΔyfiR mutant (open symbols). Catheter samples (circles) are presented as cfu/ml, tissue samples (squares) as cfu/mg tissue. Statistically significant differences are marked with an asterisk (* = p<0.05). C) Competition experiments with PA01 wild type and ΔyfiR mutant. The graph shows the ratio of wild type to ΔyfiR mutant colonies recovered from catheters (C, circles) and tissue (T, squares) after four and eight weeks, respectively. Values above 1 indicate greater numbers of wild type colonies, values below 1 greater numbers of SCVs.
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Related In: Results  -  Collection

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ppat-1000804-g007: YfiN-mediated SCVs contribute to persistence in vitro and in vivo.A) Tobramycin survival assay with PA01 wild type (open triangles) and ΔyfiR mutant (open circles). ΔyfiR suppressor colonies are shown as red circles. Statistically significant differences between PA01and ΔyfiR are marked with asterisks (** = p<0.01, * = p<0.05). B) Single infections with PA01 wild type (black symbols) and ΔyfiR mutant (open symbols). Catheter samples (circles) are presented as cfu/ml, tissue samples (squares) as cfu/mg tissue. Statistically significant differences are marked with an asterisk (* = p<0.05). C) Competition experiments with PA01 wild type and ΔyfiR mutant. The graph shows the ratio of wild type to ΔyfiR mutant colonies recovered from catheters (C, circles) and tissue (T, squares) after four and eight weeks, respectively. Values above 1 indicate greater numbers of wild type colonies, values below 1 greater numbers of SCVs.
Mentions: Although the auto-aggregative and slow growing SCV morphotypes are at a considerable disadvantage under conditions that permit rapid growth, they are able to persist in vivo [7]. This phenomenon might be explained by conditions in the host environment (e.g. immune system attack or antimicrobial chemotherapy) that put an even higher burden on rapidly growing, non-adherent strains and thus provide selection for SCVs. To test this, we compared the competitive behavior of PA01 wild type and ΔyfiR SCV strains in vitro and in vivo. When grown in LB the ΔyfiR SCV strain was significantly outperformed by the wild type. Also, suppressors with wild type colony morphology quickly arose in every ΔyfiR sample, forming the majority of the cell population after 18 hours incubation (Figure 7A). The addition of increasing concentrations of tobramycin in the sub-inhibitory range led to a reduction of both wild type and ΔyfiR cell numbers. However, wild type growth decreased at a much steeper rate and at 1.5 µg/ml tobramycin, no significant difference in cfu numbers was observed between the two strains (Figure 7A). Likewise, in the presence of tobramycin the number of suppressors arising from ΔyfiR dropped sharply with no suppressors arising at concentrations above 0.5 µg/ml of the inhibitor. Thus, the fitness disadvantage of the ΔyfiR SCV is strongly reduced in the presence of sub-inhibitory concentrations of tobramycin. Two observations indicate that this effect is not linked to some form of antibiotic tolerance of the ΔyfiR SCV, but rather reflects converging fitness during slow growth under stressful conditions. Firstly, a similar relative increase of ΔyfiR fitness compared to wild type was observed when cells were grown at reduced temperatures (data not shown). Secondly, no differences in MIC were seen between PA01 and ΔyfiR for tobramycin or for any of the other antibiotics tested (Figure S8).

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