Chronic lung infection by P. aeruginosa occurs in patients with cystic fibrosis (CF) and is the leading cause of morbidity and mortality associated with this disease. A critical virulence factor of P. aeruginosa associated with CF is the over-production of a viscous exopolysaccharide called alginate which gives clinical isolates a mucoid phenotype. The PI has recently shown that induction of mucoidy correlates with induction of sodA, which encodes manganese superoxide dismutase (Mn-SOD). Strikingly, mutants defective in sodA (generated by gene replacement) were alginate stable, even though this phenotype is unstable in wild-type bacteria. In addition, sodA is in an operon with fumC encoding a fumarase in the TCA cycle, and thus both genes are highly expressed in mucoid bacteria. Mutants defective in fumC produce only 40% of the alginate seen in the wild-type CF isolate. Interestingly, the fumC-sodA operon normally undergoes transcriptional activation only under iron deprivation. The central hypotheses of this proposal are that expression of the fumC-sodA operon is important for optimal alginate production and that iron deprivation is an important factor in the conversion to mucoidy in vivo. Others have shown that growth of P. aeruginosa in iron-limited continuous culture increases the frequency of conversion to mucoidy. In contrast, supplementation of iron salts to routine culture media causes a reversion from mucoidy to nonmucoidy. Many iron-regulated genes in bacteria are controlled by Fur (ferric uptake regulatory protein), a global regulator that binds to a consensus iron box sequence immediately upstream of these genes to prevent transcription. The PI has established that fur mutants of nonmucoid P. aeruginosa produce elevated Mn-SOD activity, suggesting that Fur regulates the fumC-sodA operon. Although the iron box was absent in the region immediately preceding fumC and sodA, the operon includes at least one more gene upstream (fagA) and the target DNA to which Fur binds is located there. This operon may include more genes downstream as well. Overall, the data suggests that iron plays an important role in alginate conversion and the FumC-SodA are important for alginate instability. Information derived from the studies proposed herein will advance the current knowledge about the role of these factors in the pathogenesis of P. aeruginosa infections in CF.
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