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Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of Motility or sessility in Escherichia coli CFT073.

ABSTRACT: Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli regulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n = 11), DGC (n = 13), PilZ (n = 2), and both PDE and DGC (n = 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolate E. coli CFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenic E. coli. IMPORTANCE Uropathogenic Escherichia coli (UPEC) contains several genes annotated as encoding enzymes that increase or decrease the abundance of the second messenger molecule, c-di-GMP. While this class of enzymes has been studied in an E. coli K-12 lab strain, these proteins have not been comprehensively examined in UPEC. UPEC utilizes both swimming motility and adherence to colonize and ascend the urinary tract; both of these processes are hypothesized to be regulated by the concentration of c-di-GMP. Here, for the first time, in a uropathogenic strain, E. coli CFT073, we have characterized mutants lacking each protein and demonstrated that the uropathogen has diverged from E. coli K-12 to utilize these enzymes to regulate adherence and motility by distinct mechanisms.

SUBMITTER: Spurbeck RR

PROVIDER: S-EPMC3484386 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of Motility or sessility in Escherichia coli CFT073.

Spurbeck Rachel R RR Tarrien Rebecca J RJ Mobley Harry L T HL

mBio 20121009 5

Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli r ...[more]

PMID: 23047748

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Project description:Cyclic diguanosine monophosphate (c-di-GMP) is an important second messenger involved in bacterial switching from motile to sessile lifestyles. In the opportunistic pathogen Pseudomonas aeruginosa, at least 40 genes are predicted to encode proteins for the making and breaking of this signal molecule. However, there is still paucity of information concerning the systemic expression pattern of these genes and the functions of uncharacterized genes. In this study, we analyzed the phylogenetic distribution of genes from P. aeruginosa that were predicted to have a GGDEF domain and found five genes (PA5487, PA0285, PA0290, PA4367, and PA5017) with highly conserved distribution across 52 public complete pseudomonad genomes. PA5487 was further characterized as a typical diguanylate cyclase (DGC) and was named dgcH A systemic analysis of the gene expression data revealed that the expression of dgcH is highly invariable and that dgcH probably functions as a conserved gene to maintain the basal level of c-di-GMP, as reinforced by gene expression analyses. The other four conserved genes also had an expression pattern similar to that of dgcH The functional analysis suggested that PA0290 encoded a DGC, while the others functioned as phosphodiesterases (PDEs). Our data revealed that there are five DGC and PDE genes that maintain the basal level of c-di-GMP in P. aeruginosa IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in animals, humans, and plants. The formation of biofilms by P. aeruginosa is the central mode of action to persist in hosts and evade immune and antibiotic attacks. Cyclic-di-GMP (c-di-GMP) is an important second messenger involved in the regulation of biofilm formation. In P. aeruginosa PAO1 strain, there are around 40 genes that encode enzymes for making and breaking this dinucleotide. A major missing piece of information in this field is the phylogeny and expression profile of those genes. Here, we took a systemic approach to investigate this mystery. We found that among 40 c-di-GMP metabolizing genes, 5 have well-conserved phylogenetic distribution and invariable expression profiles, suggesting that there are enzymes required for the basal level of c-di-GMP in P. aeruginosa This study thus provides putative therapeutic targets against P. aeruginosa infections.

Project description:The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC (adrA in Salmonella) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA, affecting at the same time the inversion of the fim promoter switch (fimS). In the pst mutant, inactivation of yaiC restored fim-dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC, which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model.IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.

Dataset Information

Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of Motility or sessility in Escherichia coli CFT073.

Publications

Enzymatically active and inactive phosphodiesterases and diguanylate cyclases are involved in regulation of Motility or sessility in Escherichia coli CFT073.

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