The Phenylacetic Acid Catabolic Pathway Regulates Antibiotic and Oxidative Stress Responses in Acinetobacter.

The opportunistic pathogen Acinetobacter baumannii is responsible for a wide range of infections that are becoming increasingly difficult to treat due to extremely high rates of multidrug resistance. Acinetobacter's pathogenic potential is thought to rely on a "persist and resist" strategy that facilitates its remarkable ability to survive under a variety of harsh conditions. The paa operon is involved in the catabolism of phenylacetic acid (PAA), an intermediate in phenylalanine degradation, and is the most differentially regulated pathway under many environmental conditions. We found that, under subinhibitory concentrations of antibiotics, A. baumannii upregulates expression of the paa operon while simultaneously repressing chaperone-usher Csu pilus expression and biofilm formation. These phenotypes are reverted either by exogenous addition of PAA and its nonmetabolizable derivative 4-fluoro-PAA or by a mutation that blocks PAA degradation. Interference with PAA degradation increases susceptibility to antibiotics and hydrogen peroxide treatment. Transcriptomic and proteomic analyses identified a subset of genes and proteins whose expression is affected by addition of PAA or disruption of the paa pathway. Finally, we demonstrated that blocking PAA catabolism results in attenuated virulence in a murine catheter-associated urinary tract infection (CAUTI) model. We conclude that the paa operon is part of a regulatory network that responds to antibiotic and oxidative stress and is important for virulence. PAA has known regulatory functions in plants, and our experiments suggest that PAA is a cross-kingdom signaling molecule. Interference with this pathway may lead, in the future, to novel therapeutic strategies against A. baumannii infections. IMPORTANCE Acinetobacter baumannii causes a wide range of infections that are difficult to treat due to increasing rates of multidrug resistance; however, the mechanisms that this pathogen uses to respond to stress are poorly understood. Here, we describe a new mechanism of stress signaling in Acinetobacter that is mediated by the metabolite phenylacetic acid (PAA). We found that disrupting PAA catabolism interfered with A. baumannii's ability to adapt to stress, leading to decreased antibiotic tolerance and hydrogen peroxide resistance. We propose that investigating this stress response could lead to the development of novel therapeutics. In fact, PAA derivatives constitute a group of FDA-approved nonsteroidal anti-inflammatory drugs that could potentially be repurposed as antivirulence therapies to target multidrug-resistant Acinetobacter infections.

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

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

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

Phenylacetic acid (PAA), an intermediate of phenylalanine degradation, is emerging as a signal molecule in microbial interactions with the host. In this work, we explore the presence of phenylalanine and PAA catabolism in 3 microbial pathogens of the cystic fibrosis (CF) lung microbiome: Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aspergillus fumigatus. While in silico analysis of B. cenocepacia J2315 and A. fumigatus Af293 genome sequences showed complete pathways from phenylalanine to PAA, the P. aeruginosa PAO1 genome lacked several coding genes for phenylalanine and PAA catabolic enzymes. High-performance liquid chromatography analysis of supernatants from B. cenocepacia K56-2 detected PAA when grown in Luria-Bertani medium but not in synthetic cystic fibrosis sputum medium (SCFM). However, we were unable to identify PAA production by A. fumigatus or P. aeruginosa in any of the conditions tested. The inhibitory effect of B. cenocepacia on A. fumigatus growth was evaluated using agar plate interaction assays. Inhibition of fungal growth by B. cenocepacia was lessened in SCFM but this effect was not dependent on bacterial production of PAA. In summary, while we demonstrated PAA production by B. cenocepacia, we were not able to link this metabolite with the B. cenocepacia - A. fumigatus microbial interaction in CF nutritional conditions.

The attenuated virulence of a Burkholderia cenocepacia paaABCDE mutant is due to inhibition of quorum sensing by release of phenylacetic acid.

The phenylacetic acid degradation pathway of Burkholderia cenocepacia is active during cystic fibrosis-like conditions and is necessary for full pathogenicity of B. cenocepacia in nematode and rat infection models; however, the reasons for such requirements are unknown. Here, we show that the attenuated virulence of a phenylacetic acid catabolism mutant is due to quorum sensing inhibition. Unlike wild-type B. cenocepacia, a deletion mutant of the phenylacetyl-CoA monooxygenase complex (ΔpaaABCDE) released phenylacetic acid in the medium that favours infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the ΔpaaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild-type, due to phenylacetic acid consumption. In line with reduced detection of acyl-homoserine lactones in spent medium, the ΔpaaABCDE exhibited transcriptional inhibition of the quorum sensing system cepIR. Phenotypes repressed in ΔpaaABCDE, protease activity and pathogenicity against C. elegans, increased with exogenous N-octanoyl-L-homoserine lactone. Thus, we demonstrate that the attenuated phenotype of B. cenocepacia ΔpaaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N-octanoyl-L-homoserine lactone signalling. Further, we propose that active degradation of phenylacetic acid by B. cenocepacia during growth in cystic fibrosis-like conditions prevents accumulation of a quorum sensing inhibiting compound.