Cystic fibrosis (CF) patients are predisposed to recurrent respiratory tract infections by the bacterium Pseudomonas aeruginosa. Complications arising from these infections are a major source of morbidity and the leading cause of death in those afflicted. P. aeruginosa strains that initially colonize the lungs are nonmucoid, but over time mucoid variants emerge and this is correlated with a worsening clinical condition for the CF patient. The mucoid phenotype is due to high-level synthesis of a capsular polysaccharide called alginate and overproduction of this virulence factor confers a selective advantage for P. aeruginosa in the CF lung. Thus, the long-term objective of this proposal is to understand the molecular mechanisms responsible for the production of alginate by strains of P. aeruginosa which colonize CF patients. ? ? Most of the genes for alginate production are in a large operon that is transcribed by a tightly controlled promoter (palgD). palgD is activated in mucoid P. aeruginosa isolates but no transcription is detectable from this promoter in nonmucoid strains. This proposal will focus on AlgZ and AlgB, two proteins that are essential for algD activation. In addition, experiments aimed at defining the events leading to mucoid conversion and subsequent algD activation are proposed. Biochemical and genetic approaches will be utilized to address three central questions which constitute the basis of this proposal: (1) What is the mechanism of AIgZ-mediated transcriptional activation of the algD promoter? (2) How does the response regulator AlgB controls algD expression and alginate synthesis? (3) What are the molecular events controlling mucoid conversion in P. aeruginosa? Since the overproduction of alginate correlates with a poor clinical outcome for CF patients colonized with mucoid P. aeruginosa, and since algD activation is a prerequisite for alginate synthesis, a basic understanding of algD transcription is essential for understanding the pathogenesis of P. aeruginosa. This will lead to novel therapies and improve the quality of life for CF patients colonized with mucoid P. aeruginosa.
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