(Taken directly from the application) 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 which 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 required for alginate production are contained in a large operon which 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 characterizing a recently identified DNA binding protein, AlgZ, which is required for activation of palgD. Biochemical and genetic approaches will be utilized to address three central questions which constitute the basis of this proposal: (1) What is the nature of the algZ gene product? This will be approached by cloning algZ and determining the DNA sequence. The gene product will be over-expressed to verify it is sufficient for DNA binding activity. (2) What is the overall contribution of AlgZ to algD expression and alginate production? A novel allelic exchange technique will be used to generate defined algZ mutants. The role of AlgZ in alginate production, algD transcription, and in the control of alginate genes apart from algD will be examined in isogenic wild-type and algZ mutants. (3) By what mechanism(s) does AlgZ activate algD transcription? AlgZ likely activates algD via a protein-protein contact with RNA polymerase. This interaction may be mediated by a co-activator such as IHF or AlgR. The AlgZ binding site will be mutated and placed in combination with mutations in the AlgR or IHF binding sites to determine if AlgZ is synergistically linked with these proteins. A genetic analysis of AlgZ interactions at the algD promoter will be undertaken by isolating and characterizing AlgZ positive control mutants and corresponding pseudo-revertants. The biochemical basis for such interactions will be further examined. 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, an understanding of the mechanism by which AlgZ functions in controlling algD is important 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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (SRC (06))
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Wake Forest University Health Sciences
Schools of Medicine
United States
Zip Code
Xu, Binjie; Wozniak, Daniel J (2015) Development of a Novel Method for Analyzing Pseudomonas aeruginosa Twitching Motility and Its Application to Define the AmrZ Regulon. PLoS One 10:e0136426
Jones, Christopher J; Newsom, David; Kelly, Benjamin et al. (2014) ChIP-Seq and RNA-Seq reveal an AmrZ-mediated mechanism for cyclic di-GMP synthesis and biofilm development by Pseudomonas aeruginosa. PLoS Pathog 10:e1003984
Limoli, Dominique H; Rockel, Andrea B; Host, Kurtis M et al. (2014) Cationic antimicrobial peptides promote microbial mutagenesis and pathoadaptation in chronic infections. PLoS Pathog 10:e1004083
Wang, Shiwei; Parsek, Matthew R; Wozniak, Daniel J et al. (2013) A spider web strategy of type IV pili-mediated migration to build a fibre-like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms. Environ Microbiol 15:2238-53
Jones, Christopher J; Ryder, Cynthia R; Mann, Ethan E et al. (2013) AmrZ modulates Pseudomonas aeruginosa biofilm architecture by directly repressing transcription of the psl operon. J Bacteriol 195:1637-44
Ma, Luyan; Wang, Juan; Wang, Shiwei et al. (2012) Synthesis of multiple Pseudomonas aeruginosa biofilm matrix exopolysaccharides is post-transcriptionally regulated. Environ Microbiol 14:1995-2005
Pryor Jr, Edward E; Wozniak, Daniel J; Hollis, Thomas (2012) Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:985-93
Zegans, Michael E; Wozniak, Daniel; Griffin, Edward et al. (2012) Pseudomonas aeruginosa exopolysaccharide Psl promotes resistance to the biofilm inhibitor polysorbate 80. Antimicrob Agents Chemother 56:4112-22
Pryor Jr, Edward E; Waligora, Elizabeth A; Xu, Binjie et al. (2012) The transcription factor AmrZ utilizes multiple DNA binding modes to recognize activator and repressor sequences of Pseudomonas aeruginosa virulence genes. PLoS Pathog 8:e1002648
Irie, Yasuhiko; Borlee, Bradley R; O'Connor, Jennifer R et al. (2012) Self-produced exopolysaccharide is a signal that stimulates biofilm formation in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 109:20632-6

Showing the most recent 10 out of 46 publications