Phenylacetyl-CoA, not phenylacetic acid, mediates quorum sensing-regulated virulence in Burkholderia cenocepacia

In many bacteria, phenylalanine and other aromatic compounds are catabolized through phenylacetic acid (PAA) degradation. In this pathway, PAA is converted to phenylacetyl-CoA (PAA-CoA) by a ligase, PaaK, and then epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation to the TCA cycle. As Burkholderia cenocepaciapaa ABCDE mutants release PAA and are attenuated for quorum sensing-regulated virulence in rat and nematode models of infection, overproduction of PAA in B. cenocepacia was linked to the regulation of the LuxIR-like quorum sensing system, CepIR. To further investigate this link, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to wild type and PaaABCDE mutant strains using liquid chromatography/mass spectrometry (LC-MS/MS) and phenotypic assays. Both mutants released high levels of PAA. However, only the PaaABCDE mutant accumulated PAA-CoA intracellularly, had decreased virulence in Caenorhabditis elegans, and was defective for exoprotease activity. Conversely, the PaaK mutant did not accumulate PAA-CoA and was virulent, linking PAA-CoA to the attenuation of virulence in B. cenocepacia. While transcriptional analysis showed no evidence of cepIR downregulation by PAA-CoA, the attenuated pathogenicity of a cepR mutant was dependent on the presence of PAA-CoA in PAA pathway mutants, as it drastically changed to a virulent phenotype upon deletion of paaK but not when paaABCDE was deleted. Our results demonstrate the role of PAA-CoA in quorum sensing-regulated virulence and are consistent with a virulence regulatory mechanism that is evident when CepR is not active.