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

Departments of *Microbiology and Molecular Genetics and
^‡Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115;
^§Department of Molecular Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114;
^¶Shriners Hospital for Children, 3229 Burnet Avenue, Cincinnati, OH 45229; and
^∥Graduate School of Information Science/Human Informatics and Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency, Nagoya University, Chikusa, Nagoya 464-8601, Japan

↵ ^†H.K. and V.L. contributed equally to this work.
Contributed by Frederick M. Ausubel, December 22, 2005

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.

Footnotes

**To whom correspondence may be addressed. E-mail: ausubel{at}molbio.mgh.harvard.edu or stephen_lory{at}hms.harvard.edu
Author contributions: H.K., V.L., A.B., and S.L. designed research; H.K., V.L., A.B., and A.N.N. performed research; N.L., J.U., S.M., D.G.L., M.H., Y.H., and F.M.A. contributed new reagents/analytic tools; H.K., V.L., A.B., and S.L. analyzed data; and H.K., V.L., A.B., F.M.A., and S.L. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

c-di-GMP,

bis-(3′-5′)-cyclic dimeric GMP;

DGC,

diguanyl cyclase;

PDE,

phosphodiesterase;

TTSS,

type III secretion system;

CHO,

Chinese hamster ovary;

LDH,

lactate dehydrogenase;

DGC-PDE,

diguanyl cyclase-phosphoesterase fusion.