AlgR and ANR are transcriptional regulators in the opportunistic pathogen, Pseudomonas aeruginosa. Here we demonstrate these transcriptional regulators are involved in biofilm and hydrogen cyanide (HCN) production. Affymetrix GeneChip analysis of P. aeruginosa PAO1 biofilm revealed a number of genes under ANR control. Two such genes, nirS and ccoN2, were required for aerobic biofilm development. Moreover, biofilms were unable to develop without ANR, and its accessory regulator, DNR. The transcriptome of PSL317(DeltaalgR) biofilm, revealed a number of genes associated with quorum-sensing and HCN production. Microscopy and biofilm assays showed that PSL317 was unable to produce a mature biofilm. The transcriptomes of PAO1 and the algR mutant strain, grown as biofilms, showed differential regulation of hcnA indicating that this gene is controlled by AlgR. Here, we used S1 nuclease protection assays to show that AlgR activates hcnA transcription in mucoid P. aeruginosa. Additionally, HCN quantification revealed that mucoid laboratory strains made 7-fold more HCN than nonmucoid parental strains. Cystic fibrosis (CF) mucoid isolates produced 4.7 mumol HCN/mg protein compared to 2.4 mumol HCN/mg protein in nonmucoid isolates. P. aeruginosa HCN production has been shown to contribute to pathogenesis in C. elegans, but not for host tissue. To examine the impact of HCN on host tissue, we developed a 3-D aggregate model using A549 human lung epithelial cells for the study of P. aeruginosa pathogenesis. Immunohistochemistry revealed an increase of epithelial cell-specific markers, a decrease of cancer-specific markers, and the presence of tight junctions and polarity in the 3-D aggregates. Additionally, mucoglycoprotein production was assayed through staining and mucin-specific antibodies, MUC1 and MUC5A. P. aeruginosa attached to and penetrated A549 monolayers more readily than 3-D aggregates. SEM showed monolayers detached from the surface post-infection, while 3-D aggregates remained attached to microcarrier beads. Moreover, the 3-D A549 aggregates had a greater fold-induction of pro-inflammatory cytokines than monolayer controls. Taken together, these findings suggest that: (i) The transcriptional regulators, AlgR and ANR are required for aerobic biofilm production; (ii) AlgR regulates HCN production in P. aeruginosa and; (iii) A549 3-D aggregates may represent a more physiologically relevant model to examine P. aeruginosa pathogenesis
