The role of AlgR in Pseudomonas aeruginosa virulence and the identification of AlgR regulated genes
Description
AlgR is a transcriptional regulator of Pseudomonas aeruginosa previously known to be required for the production of the exopolysaccharide alginate and for twitching motility. This work investigated the role of AlgR in P. aeruginosa virulence and identified novel AlgR regulated genes required for virulence. The initial experiments found differences in sensitivity between PAO1 and its algR mutant to in vitro oxidative stress. It was also found that AlgR was required for virulence in both an acute septicemia infection model and in an acute pneumonia model. The overall affect of AlgR regulated genes on host cytokine transcription in an acute pneumonia model was also examined. To determine what genes AlgR regulated for P. aeruginosa virulence, 2D gel electrophoresis and Affymetrix GeneChip transcriptional profiling experiments were conducted. These studies indicated that the AlgR regulon was potentially quite large, and indicated that AlgR can function as a repressor. This is the first report of AlgR repressing transcription in P. aeruginosa. Two genes identified by the microarray as AlgR regulated, hcnA and ORF PA1557, were selected for follow up studies. Primer extension and S1 nuclease protection assays confirmed AlgR regulated PA1557 and hcnA. In agreement with the transcriptional analysis of the hcnA promoter, HCN production was repressed by AlgR in nonmucoid P. aeruginosa. Because the AlgR binding site is at least partially known, a genome search was done to identify potential AlgR binding sites. This search identified a known virulence factor, pyoverdine, and an uncharacterized ORF, PA0267 as potentially AlgR regulated. Pyoverdine production was repressed by AlgR in the nonmucoid P. aeruginosa. PA0267, which showed highest homology to the chemotaxis regulator CheY2 of Sinorhizobium meliloti, was characterized and found to be a negative regulator of swimming motility. Based on its homology to CheY2 and its function as a regulator of swimming motility, ORF PA0267 was named CheY4. These studies were the first to show that AlgR: (i) can repress transcription, (ii) regulates pyoverdine production, (iii) regulates flagellar motility and (iv) regulates hcnA transcription and HCN production. Taken together, this work discovered a broad role for AlgR in the regulation of P. aeruginosa virulence