Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence

Abstract

The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action.

Fig. 1.

Phenotypic analysis of P. aeruginosa PA14 transposon mutants in genes encoding the DGC, PDE, and DGC-PDE domain proteins. (A) Cytotoxic effect of P. aeruginosa PA14 transposon mutants on CHO cells after infection, monitored by the release of LDH. (B) Biofilm phenotypes were measured as the attachment of P. aeruginosa to tubes and formation of the biofilm rings and pellicles after 20-h static incubation at 30°C.

Fig. 2.

Phenotypic analysis of P. aeruginosa PA14 overexpressing genes encoding the DGC, PDE, and DGC-PDE domain proteins. (A) Cytotoxic effect of PA14 transposon mutants on CHO cells after infection, monitored by the release of LDH. (B) Biofilm phenotypes were measured as the attachment of P. aeruginosa to glass tubes and the formation of the biofilm rings and pellicles after 12-h static incubation at 30°C.

Fig. 3.

Analysis of enzymatic activities of proteins with DGC and PDE domains. Extracts of P. aeruginosa PA14, overexpressing genes with DGC or DGC-PDE domains, were fractionated by reverse-phase HPLC, and levels of c-di-GMP were quantified. For the assessment of PDE activity, synthetic c-di-GMP was added to lysates of cultures overexpressing proteins with PDE and DGC-PDE domains, and loss of the c-di-GMP peak in the HPLC analysis was quantified. (A) Representative traces from the HPLC analysis of the PA14 extract with the pMMB67EHGent vector or overexpressing PA5487. Trace of synthetic c-di-GMP. (B) Traces of extracts of reaction mixtures containing synthetic c-di-GMP and lysates of PA14 with pMMB67EHGent vector, overexpressing pvrR, or PA2133.

Fig. 4.

Virulence of P. aeruginosa PA14 strains with mutations in genes encoding the DGC, PDE, and DGC-PDE domain proteins in a murine model of acute infection. Survival of mice after thermal injury and infection by 2 × 10⁶ wild-type and various P. aeruginosa mutants was monitored daily.