| Pseudomonas aeruginosa is a gram-negative opportunistic pathogen ubiquitous in the environment. P. aeruginosa is an important pathogen in cystic fibrosis (CF), where it develops into a biofilm. Biofilms are surface-attached communities encased in a hydrated matrix. Exopolymeric substances (EPS) comprise the matrix and largely consist of polysaccharides. Alginate has been the paradigm polysaccharide produced by P. aeruginosa , and was considered to be a requirement for biofilm formation. We showed that alginate is not essential for biofilm formation, since an algD mutant incapable of producing alginate forms biofilms identical to the wild type strain. Furthermore, alginate is not required for biofilm antibiotic resistance; and glucose is the primary monomer component of EPS carbohydrates isolated from the wild type PAO1 and algD mutant strains.; In an effort to study an alternative EPS, the psl locus was discovered to be important for early stages of biofilm formation in the wild type PAO1 strain. Additionally, psl contributes to the phenotype of sticky (ST) variants by enhancing autoaggregation in liquid cultures. In concert with a second EPS locus, pel, psl genes are required for wrinkled colony appearance, autoaggregation, and biofilm formation. The importance of cyclic-di-(3'-5') guanylic acid (c-di-GMP) levels in ST isolates was demonstrated, as the over-expression of an EAL domain-containing protein responsible for decreasing c-di-GMP, eliminated the wrinkled colony phenotype, autoaggregation, and biofilm production. Clinical variants of P. aeruginosa isolated from a longitudinal CF collection were discovered to be similar to biofilm variants in gene expression profiles. Furthermore, biofilm formation and autoaggregation in the clinical variants required psl and pel genes as well as elevated c-di-GMP levels. Additionally, compared to their wild type counterparts, both biofilm and clinical ST variants elicited a lower inflammatory response from lung epithelial cells, suggesting a role for the wrinkled phenotype in persistence.; Regulation of psl was addressed using various genetic backgrounds and environmental conditions. psl translation was diminished by over-expression of the post-transcriptional regulatory protein, RsmA, which also resulted in decreased biofilm production. psl translation was increased in an rsmA mutant. Reduced c-di-GMP levels were found to decrease psl at both transcriptional and translational levels. c-di-GMP and RsmA could potentially be linked in a system that modulates biofilm formation and virulence in P. aeruginosa . |
