Genomic and bioinformatic analysis of mucoid Pseudomonas aeruginosa in cystic fibrosis.

摘要

Pseudomonas aeruginosa is the most prevalent pathogen causing chronic respiratory infections in cystic fibrosis (CF). After an initial phase characterized by intermittent infections, a chronic colonization is established in CF upon the conversion of P. aeruginosa to the mucoid, exopolysaccharide alginate overproducing phenotype. The emergence of mucoid P. aeruginosa in CF is associated with excessive pulmonary inflammation, respiratory decline, and poor prognosis. A major pathway of conversion to mucoidy in clinical strains of P. aeruginosa is dependent upon activation of the alternative sigma factor AlgU ( P. aeruginosa σE). Here I carried out studies of AlgU-dependent global expression patterns in P. aeruginosa in order to examine whether additional genes, other than those involved in the production of the mucoid exopolysaccharide alginate, are upregulated during conversion to mucoidy. Towards this end, I applied a combined genomics, bioinformatics, and classical molecular biology approach in conjunction with the available P. aeruginosa genome sequence. These and microarray analyses were used to examine the effect of AlgU activation on global gene transcription. In the course of carrying out these studies, the following has been accomplished: (i) The number of identified, mapped AlgU-dependent promoters has been expanded from the previously known 5 to a current total of 16. (ii) It has been demonstrated that additional systems beyond those required for alginate biosynthesis are induced during conversion to mucoidy. Notably, a specific subset of virulence factors, including proteases and genes responsible for production of secreted small molecular mass toxic products, are induced concomitantly with conversion to mucoidy and likely contribute to P. aeruginosa pathogenesis in cystic fibrosis. (iii) A massive, previously unappreciated, induction of lipoproteins was observed upon conversion to mucoidy. (iv) I have also linked products associated with the conversion to mucoidy with the ability to stimulate inflammation in clinically relevant cells. I have shown that lipopeptides, based on P. aeruginosa lipoprotein genes co-activated with the alginate system, stimulate NF-κB activation and inflammatory cytokine production in human macrophage and primary bronchial epithelial cells. These studies provide new leads that should help explain the excessive inflammatory response in CF.