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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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In silico analysis of the YfiBNR system.A) Lineage tree illustrating the distribution of the yfiBNR genes along bacterial lineages. Individual branches indicate separate genera. Genera marked in large, bold type contain species with complete yfiBNR operons. Those marked with an asterisk (*) contain species with yfiB and yfiN only, conserved and in synteny. The remaining genera contain species with two yfi genes conserved and in synteny (usually yfiN and yfiR) and a third yfi homolog elsewhere in the genome. Bacterial classes are indicated with coloured shading. Alpha, Beta etc. refer to the respective proteobacterial class. B-D) Weblogo representations of YfiBNR residue conservation. The height of the letter in each case indicates the degree of conservation at that position. Hydrophobic residues are coloured blue, hydrophilic residues red. Asterisks (*) indicate the sites of activating mutations, while blue underlining indicates those residues suggested to contribute to a hydrophobic protein binding site. B) YfiB residues 33–72. C) YfiN PAS-domain residues 48–87, YfiN HAMP-domain residues 198–237. D) YfiR residues 122–191.
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ppat-1002760-g008: In silico analysis of the YfiBNR system.A) Lineage tree illustrating the distribution of the yfiBNR genes along bacterial lineages. Individual branches indicate separate genera. Genera marked in large, bold type contain species with complete yfiBNR operons. Those marked with an asterisk (*) contain species with yfiB and yfiN only, conserved and in synteny. The remaining genera contain species with two yfi genes conserved and in synteny (usually yfiN and yfiR) and a third yfi homolog elsewhere in the genome. Bacterial classes are indicated with coloured shading. Alpha, Beta etc. refer to the respective proteobacterial class. B-D) Weblogo representations of YfiBNR residue conservation. The height of the letter in each case indicates the degree of conservation at that position. Hydrophobic residues are coloured blue, hydrophilic residues red. Asterisks (*) indicate the sites of activating mutations, while blue underlining indicates those residues suggested to contribute to a hydrophobic protein binding site. B) YfiB residues 33–72. C) YfiN PAS-domain residues 48–87, YfiN HAMP-domain residues 198–237. D) YfiR residues 122–191.

Mentions: Homologs of the YfiB, YfiN, and YfiR proteins were determined and plotted on a 16S rRNA-based phylogenetic tree to represent the taxonomic spread of the system (Figure 8A), for more details see Materials and Methods. 144 genomes were found to contain complete or partial yfiBNR operons. Genera containing complete, conserved yfiBNR operons (total 99) were found in the alpha-, beta- and gamma-proteobacteria, with most examples clustering in the gamma and beta classes. Two types of degenerate yfi operons were also identified. Firstly, operons containing yfiN and yfiB homologs in synteny, but no yfiR, were found in ten predominantly gamma-proteobacterial genomes (Table S4). In most cases, the transmembrane helices and GGDEF output domain of YfiN, as well as the PAL domain of YfiB were conserved. However, no homology was observed for the periplasmic domain of YfiN, and none of the proposed YfiR binding residues were conserved (Figure 8C, D), indicating that these systems function in a markedly different manner to P. aeruginosa YfiBNR. Secondly, 35 genomes were identified containing all three yfi homologs, but with only two of them in synteny (Table S4). Closer examination suggested that the genes in synteny were almost always yfiN and yfiR. The γ-proteobacteria Salmonella and Dickeya contain fully conserved yfiR and yfiN genes but lack yfiB. Interestingly, with growing evolutionary distance from Pseudomonas sp., the general divergence of the Yfi systems increased. Almost all of these operons lacked an yfiB homolog, and in many cases the GGDEF output domain of YfiN was replaced with GGDEF-EAL pairs, histidine-phosphotransfer domains (HPT) or sensor histidine kinases (Table S4). Variation was also seen at the level of signal input. In Thauera sp. MZ1T a system was identified with two YfiR homologs and a single YfiN with a hybrid sensor kinase output. The genomic location of the yfiN-yfiR homologs was highly variable, with some systems part of operons and others found alone. Nonetheless, alignment of PA01 YfiR and YfiN with some of the most divergent homologs revealed that the hydrophobic residues identified above as important for function were conserved throughout (Figure S5), suggesting that the basic concept of YfiR regulation of YfiN remains unchanged even in these highly divergent systems.


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)

In silico analysis of the YfiBNR system.A) Lineage tree illustrating the distribution of the yfiBNR genes along bacterial lineages. Individual branches indicate separate genera. Genera marked in large, bold type contain species with complete yfiBNR operons. Those marked with an asterisk (*) contain species with yfiB and yfiN only, conserved and in synteny. The remaining genera contain species with two yfi genes conserved and in synteny (usually yfiN and yfiR) and a third yfi homolog elsewhere in the genome. Bacterial classes are indicated with coloured shading. Alpha, Beta etc. refer to the respective proteobacterial class. B-D) Weblogo representations of YfiBNR residue conservation. The height of the letter in each case indicates the degree of conservation at that position. Hydrophobic residues are coloured blue, hydrophilic residues red. Asterisks (*) indicate the sites of activating mutations, while blue underlining indicates those residues suggested to contribute to a hydrophobic protein binding site. B) YfiB residues 33–72. C) YfiN PAS-domain residues 48–87, YfiN HAMP-domain residues 198–237. D) YfiR residues 122–191.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375315&req=5

ppat-1002760-g008: In silico analysis of the YfiBNR system.A) Lineage tree illustrating the distribution of the yfiBNR genes along bacterial lineages. Individual branches indicate separate genera. Genera marked in large, bold type contain species with complete yfiBNR operons. Those marked with an asterisk (*) contain species with yfiB and yfiN only, conserved and in synteny. The remaining genera contain species with two yfi genes conserved and in synteny (usually yfiN and yfiR) and a third yfi homolog elsewhere in the genome. Bacterial classes are indicated with coloured shading. Alpha, Beta etc. refer to the respective proteobacterial class. B-D) Weblogo representations of YfiBNR residue conservation. The height of the letter in each case indicates the degree of conservation at that position. Hydrophobic residues are coloured blue, hydrophilic residues red. Asterisks (*) indicate the sites of activating mutations, while blue underlining indicates those residues suggested to contribute to a hydrophobic protein binding site. B) YfiB residues 33–72. C) YfiN PAS-domain residues 48–87, YfiN HAMP-domain residues 198–237. D) YfiR residues 122–191.
Mentions: Homologs of the YfiB, YfiN, and YfiR proteins were determined and plotted on a 16S rRNA-based phylogenetic tree to represent the taxonomic spread of the system (Figure 8A), for more details see Materials and Methods. 144 genomes were found to contain complete or partial yfiBNR operons. Genera containing complete, conserved yfiBNR operons (total 99) were found in the alpha-, beta- and gamma-proteobacteria, with most examples clustering in the gamma and beta classes. Two types of degenerate yfi operons were also identified. Firstly, operons containing yfiN and yfiB homologs in synteny, but no yfiR, were found in ten predominantly gamma-proteobacterial genomes (Table S4). In most cases, the transmembrane helices and GGDEF output domain of YfiN, as well as the PAL domain of YfiB were conserved. However, no homology was observed for the periplasmic domain of YfiN, and none of the proposed YfiR binding residues were conserved (Figure 8C, D), indicating that these systems function in a markedly different manner to P. aeruginosa YfiBNR. Secondly, 35 genomes were identified containing all three yfi homologs, but with only two of them in synteny (Table S4). Closer examination suggested that the genes in synteny were almost always yfiN and yfiR. The γ-proteobacteria Salmonella and Dickeya contain fully conserved yfiR and yfiN genes but lack yfiB. Interestingly, with growing evolutionary distance from Pseudomonas sp., the general divergence of the Yfi systems increased. Almost all of these operons lacked an yfiB homolog, and in many cases the GGDEF output domain of YfiN was replaced with GGDEF-EAL pairs, histidine-phosphotransfer domains (HPT) or sensor histidine kinases (Table S4). Variation was also seen at the level of signal input. In Thauera sp. MZ1T a system was identified with two YfiR homologs and a single YfiN with a hybrid sensor kinase output. The genomic location of the yfiN-yfiR homologs was highly variable, with some systems part of operons and others found alone. Nonetheless, alignment of PA01 YfiR and YfiN with some of the most divergent homologs revealed that the hydrophobic residues identified above as important for function were conserved throughout (Figure S5), suggesting that the basic concept of YfiR regulation of YfiN remains unchanged even in these highly divergent systems.

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