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

Abstract

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.

The acquisition of additional feedback loops may optimize and speed up the response of quorum sensing

“…This is even more evident for CepI, whose steady-state concentration is particularly higher in the complete model. Notably the steady-state concentration of the AI (C8HSL) falls well in the range of the one experimentally measured in B. cenocepacia [22]. Besides steady-state concentration, regulatory circuits as the ones implemented in this work can be characterized by their response time (RT), i.e.…”

“…More in detail, in the cluster comprising B. mallei, B. pseudomallei and B. thalilandiensis species, the complete set of cci genes is never found. Instead, the distribution of cci genes in the other cluster (hereinafter cci -group, that includes, among the others, the BCC and the members of the B. glumae/gladioli/plantarii group ) is patchy and includes (22) microbes that harbour more than 50% of the reference cci genes and others (93) possessing less than 50% of the reference cci genes (Figure 2). This pattern suggests a complex evolutionary history for this genomic islands, mainly guided by gain/loss events.…”

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

“…Previous studies in the Gram-negative bacteria P. aeruginosa and B. cenocepacia , have shown that PAA might interact with the quorum-sensing machinery of these strains ( 23 , 45 ). However, in B. cenocepacia , it was discovered that PAA is converted to PAA-CoA to interfere with quorum-sensing-mediated virulence of this strain ( 46 ). We cannot rule out that PAA-CoA, rather than PAA, is the signaling molecule that is responsible for the appreciable phenotypes in Acinetobacter .…”

Methodological tools to study species of the genus Burkholderia

“…Biological reporters are reliable tools in QS studies but, for a more precise identification and quantification of autoinducers, the use of more powerful analytical techniques is often necessary. In this case, liquid chromatography with tandem mass spectrometry (LC–MS/MS) is currently the method of choice, allowing the fine separation of the culture supernatant extracts on a HPLC column, followed by the sensitive detection of signal molecules such as AHLs (Lightly et al 2019 ; Chapalain et al 2017 ; Le Guillouzer et al 2017 ; 2018 ; 2020 ) or BDSF (Waldron et al 2019 ; Cui et al 2019 ) by mass spectrometer.…”

“…To test the catalytic activity of B. cenocepacia CepI, a spectrophotometric assay that measures the formation of the holo -acyl carrier protein (ACP) during the biosynthetic reaction was used. Indeed, through a redox reaction between the released free thiol of ACP and the dichlorophenylindophenol (DCPIP) it is possible to determine the catalytic activity by measuring the absorbance at 600 nm (Scoffone et al 2016 ; Buroni et al 2018 ; Sass et al 2019 ; Lightly et al 2019 ). Similarly, the thioesterase activity of the other main B. cenocepacia QS synthase DsfA (RpfF Bc ), responsible for the BDSF biosynthesis, was assessed using the DCPIP reagent (Spadaro et al 2016 ), or alternatively by checking the holo -ACP release with conformation-sensitive nondenaturing gel electrophoresis (Spadaro et al 2016 ; Waldron et al 2019 ).…”