3-Hydroxyphenylacetic Acid Induces the Burkholderia cenocepacia Phenylacetic Acid Degradation Pathway ? Toward Understanding the Contribution of Aromatic Catabolism to Pathogenesis

Abstract

The phenylacetic acid (PA) degradative pathway is the central pathway by which various aromatic compounds (e.g., styrene) are degraded. Upper pathways for different aromatic compounds converge at common intermediate phenylacetyl-CoA (PA-CoA), which is then metabolized to succinyl-CoA and acetyl-CoA. We previously made a link in Burkholderia cenocepacia between PA degradation and virulence by showing that insertional mutagenesis of paaA and paaE genes, that encode part of a multicomponent oxidase of PA-CoA, results in PA-conditional growth and an attenuated killing phenotype in the Caenorhabditis elegans model of infection. However, insertional mutagenesis of paaK1, which encodes a phenylacetate-CoA ligase, did not result in a PA-conditional growth probably due to the presence of a putative paralog gene paaK2. Recently published crystallographic and enzyme kinetics data comparing the two PaaK ligases showed that PaaK1 is more active than PaaK2 and that the larger binding pocket of PaaK1 can accommodate hydroxylated PA derived molecules such as 3-hydroxyphenylacetic (3-OHPA) acid and 4-hydroxyphenylacetic acid (4-OHPA). The higher activity and broader substrate specificity suggested a more active role in pathogenesis. In this work, we aimed to determine the relevance of PaaK1 activity to the killing ability of B. cenocepacia to C. elegans. Using reporter activity assays, we demonstrate that 3-OHPA activated PA degradation gene promoters of Burkholderia cenocepacia K56-2 in a paaK1-dependent manner, while 4-OHPA had no effect. We compared the pathogenicity of a paaK1 deletion mutant with that of the wild type in C. elegans and observed no differences in the killing ability of the strains. Taken together, these studies suggest that 3-OHPA, but not 4-OHPA, can induce the PA pathway and that this induction is dependent on the paaK1 gene. However, the more active PaaK1 does not play a distinct role in pathogenesis of B. cenocepacia as previously suggested.

Phenylacetyl Coenzyme A, Not Phenylacetic Acid, Attenuates CepIR-Regulated Virulence in Burkholderia cenocepacia

“…To determine the role of each of these molecules in the attenuation of virulence, we characterized a paaK mutant that should produce only PAA and compared it to the paaABCDE mutant. Similar to the reference strain B. cenocepacia J2315 ( 22), K56-2 harbors two similar but not identical phenylacetyl-CoA ligases, PaaK1 and PaaK2 (23). We created markerless deletion mutants of paaK1 (BCAL0404) and paaK2 (BCAM1711).…”

Structural Organization of Enzymes of the Phenylacetate Catabolic Hybrid Pathway

“…PaaK1 has lower K m value for phenylacetate and a more relaxed substrate specificity than PaaK2, which likely arises from its extended aryl substrate pocket [ 20 ]. Nevertheless, the knockout of the paaK1 gene alone does not impair growth and pathogenicity of the microbe [ 21 ].…”

Progress in structural and functional study of the bacterial phenylacetic acid catabolic pathway, its role in pathogenicity and antibiotic resistance

“…The quorum sensing (QS) system that works on the production and detection of signaling molecules is vital in bacterial intercellular communication; further, this system can bind the transcriptional regulator to activate the expression of virulence factors in several opportunistic pathogens. The relationship between the PAA pathway and bacterial pathogenicity has been well described in the opportunistic pathogen Burkholderia cenocepacia , which establishes persistent infections in humans the genetic diseases, cystic fibrosis and pulmonary cystic fibrosis ( Law et al, 2008 ; Imolorhe and Cardona, 2011 ; Pribytkova et al, 2014 ; Lightly et al, 2019 ).…”