A Functional Phenylacetic Acid Catabolic Pathway Is Required for Full Pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans Host Model

Abstract

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of metabolically versatile bacteria that have emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Previously a screen of transposon mutants in a rat pulmonary infection model identified an attenuated mutant with an insertion in paaE, a gene related to the phenylacetic acid (PA) catabolic pathway. In this study, we characterized gene clusters involved in the PA degradation pathway of B. cenocepacia K56-2 in relation to its pathogenicity in the Caenorhabditis elegans model of infection. We demonstrated that targeted-insertion mutagenesis of paaA and paaE, which encode part of the putative PA-coenzyme A (CoA) ring hydroxylation system, paaZ, coding for a putative ring opening enzyme, and paaF, encoding part of the putative beta-oxidation system, severely reduces growth on PA as a sole carbon source. paaA and paaE insertional mutants were attenuated for virulence, and expression of paaE in trans restored pathogenicity of the paaE mutant to wild-type levels. Interruption of paaZ and paaF slightly increased virulence. Using gene interference by ingested doublestranded RNA, we showed that the attenuated phenotype of the paaA and paaE mutants is dependent on a functional p38 mitogen-activated protein kinase pathway in C. elegans. Taken together, our results demonstrate that B. cenocepacia possesses a functional PA degradation pathway and that the putative PA-CoA ring hydroxylation system is required for full pathogenicity in C. elegans.

Synthetic cystic fibrosis sputum medium diminishes Burkholderia cenocepacia antifungal activity against Aspergillus fumigatus independently of phenylacetic acid production

“…S1, step 15 and 18), and BCAL0407 as a homolog of PP_3280, the putatively identified paaJ gene of P. putida KT2440 by our bi-directional BLASTP search. This corroborates our previous annotation where BCAM2568 and BCAL0407 were both identified as candidate paaJ genes (Law et al 2008). …”

“…cenocepacia J2315 (Law et al 2008). However, our bidirectional BLASTP search identified BCAS0364 as the best hit to b1387 (paaZ) from E. coli K-12 MG1655 ( Fig.…”

Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing

“…Another pathway highly induced under CF conditions is involved in phenylacetate (Paa, a byproduct of aromatic catabolism) metabolism although Paa was not detectable in our medium before growth so the reason for this induction is not obvious. However, it was recently shown that mutations in this pathway disrupted Burkholderia virulence in a C. elegans model system (9). Members of the general secretion (Sec) pathway were also induced under CF conditions.…”

Defining a Structural and Kinetic Rationale for Paralogous Copies of Phenylacetate-CoA Ligases from the Cystic Fibrosis Pathogen Burkholderia cenocepacia J2315

“…As such, the initial enzyme of the PAA pathway, phenylacetateCoA ligase (PCL), plays a vital role in regulating entry of potential substrates into the pathway. An unusual feature of the PAA pathway in B. cenocepacia is that it encodes two copies of the pcl gene (paaK1 and paaK2), yet harbors only a single copy of each subsequent enzyme in the pathway (2). Although a rationale for the additional PCL in B. cenocepacia has not been established, studies of related bacterial PCLs suggest the potential for altered kinetics (6 -8).…”

“…For example, the degradation of phenylacetic acid (PAA) 3 is proposed to facilitate B. cenocepacia in establishing lifethreatening infections in cystic fibrosis patients (1). Although the precise mechanism that governs this process is not yet understood, it is speculated that early PAA degradation pathway intermediates play a role in host cell damage (2,3).…”