(474) Inhibition of the envelope stress response in Pseudomonas aeruginosa is required for bacterial viability

Background: The opportunistic pathogen Pseudomonas aeruginosa forms chronic infections in the lungs of people with cystic fibrosis (CF), leading to a slow decline in lung function and ultimately death. During these decades-long infections, P. aeruginosa undergoes selection in the lung environment. One bacterial genetic adaptation often observed in CF isolates is mutation of mucA. MucA inhibits the envelope stress response sigma factor AlgU. Mutations in mucA lead to AlgU misregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. Due to mutations arising frequently in mucA, it is assumed to be dispensable for bacterial viability. Here we show that, paradoxically, MucA depletion leads to cell death and a portion of mucA is essential for P. aeruginosa viability.

Methods: We modified the standard allelic exchange protocol to create a statistically robust assay for determining if a gene is essential.

Results: We demonstrate that mucA is no longer required in a strain lacking algU and that mucA alleles that encode for proteins that do not bind to AlgU are insufficient for bacterial viability. We show that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Furthermore, we found that overexpression of algU prevents cell growth in the absence of MucA, and that this phenotype can be rescued by overproduction of RpoD, the housekeeping sigma factor.

Conclusions: Together, our results suggest that the unregulated activity of AlgU in the absence of MucA leads to bacterial cell death, suggesting that this interaction may serve as a good therapeutic target. We speculate that essentiality of anti-sigma factors that regulate envelope function may be a widespread phenomenon in bacteria. Our work suggests that this interaction may serve as a good therapeutic target, as eliminating the MucA-AlgU interaction results in either cell death or mutations that render AlgU less active. These outcomes, which result in either dead cells or non-mucoid cells, could be beneficial to CF patients.

Acknowledgements: MCS, DR, BT, and PAJ are supported by the Cystic Fibrosis Foundation. MCS and EKC are supported by a University of Nevada Las Vegas doctoral graduate research assistantship. EAC and PAJ are supported by the National Institutes of Health.

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