REGULATION OF ALGINATE AND PSL POLYSACCHARIDE EXPRESSOIN IN CLINICAL STRAINS OF PSEUDOMONAS AERUGINOSA

Pseudomonas aeruginosa is an ubiquitous opportunistic pathogen, which is often the terminal pathogen for patients suffering from cystic fibrosis (CF), a genetic disorder affecting the patient's ability to clear respiratory infections. The P. aeruginosa strains most often recovered from patients suffering from long-term chronic infections are mucoid, overexpressing the polysaccharide alginate. Alternatively, most environmental and acute infection strains are non-mucoid and produce an alternative polysaccharide, designated Psl. Both alginate and Psl polysaccharides promote biofilm formation and thus persistence of P. aeruginosa during infections. We hypothesize that mucoid strains express reduced Psl levels, indicating coordinate regulation between different polysaccharide genes. To test this, we performed immunoblotting with a Psl-specific antibody on two sets of mucoid and nonmucoid isogenic isolates. Mucoid P. aeruginosa strains consistently produce less Psl than their isogenic nonmucoid counterparts. We subsequently evaluated whether one or more of the alginate regulatory gene products repress Psl production in mucoid strains of P. aeruginosa. Psl immunoblots and ELISAs showed that the alginate regulator AmrZ represses Psl production. AmrZ is a DNA binding protein and is thus a candidate for transcriptional repression of the psl operon. DNA binding assays show that AmrZ does bind upstream of the first gene in the psl operon, in a region overlapping with the promoter of this operon. Transcriptional fusions confirm that AmrZ functions as a repressor of psl transcription as well as an activator of alginate genes. These findings support the hypothesis that P. aeruginosa infection in the CF lung involves a progression from nonmucoid strains producing Psl to mucoid strains producing alginate. An evaluation of clinical nonmucoid and mucoid strains of P. aeruginosa illustrated that polysaccharide expression in the clinical setting is more complicated than previously understood, with Psl levels varying in nonmucoid and mucoid strains. Therefore, Psl expression in mucoid strains does not always follow the repression described here, but requires further study in cases involving elevated Psl in mucoid strains. Understanding the genetic and biochemical properties of these isolates may allow us to develop practical therapies that prevent P. aeruginosa colonization or the transition to the mucoid phenotype.

Grantor dc:publisher

Wake Forest University

Year dc:date.issued

2010

Author dc:creator

• Ryder, Cynthia

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10339/30423

OAI identifier oai:identifier

oai:wakespace.lib.wfu.edu:10339/30423