Quorum sensing (QS) is a cell-to-cell communication mechanism mediated by small diffusible signaling molecules. Previous studies showed that RpfR controls Burkholderia cenocepacia virulence as a cis-2-dodecenoic acid (BDSF) QS signal receptor. Here, we report that the fatty acyl-CoA ligase DsfR (BCAM2136), which efficiently catalyzes in vitro synthesis of lauryl-CoA and oleoyl-CoA from lauric acid and oleic acid, respectively, acts as a global transcriptional regulator to control B. cenocepacia virulence by sensing BDSF. We show that BDSF binds to DsfR with high affinity and enhances the binding of DsfR to the promoter DNA regions of target genes. Furthermore, we demonstrate that the homolog of DsfR in B. lata, RS02960, binds to the target gene promoter, and perception of BDSF enhances the binding activity of RS02960. Together, these results provide insights into the evolved unusual functions of DsfR that control bacterial virulence as a response regulator of QS signal.

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski\'s five rules. It also shows high gastrointestinal absorption and the ability to cross the blood-brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

Outer membrane vesicle (OMV)-delivered Pseudomonas quinolone signal (PQS) plays a critical role in cell-cell communication in Pseudomonas aeruginosa. However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from P. aeruginosa and liposome reduces the competitiveness of Burkholderia cenocepacia, which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in B. cenocepacia through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in B. cenocepacia. Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community.IMPORTANCEQuorum sensing (QS) is a ubiquitous cell-to-cell communication mechanism. Previous studies showed that Burkholderia cenocepacia mainly employs cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) QS systems to regulate biological functions and virulence. Here, we demonstrate that Pseudomonas quinolone signal (PQS) delivered by outer membrane vesicles from Pseudomonas aeruginosa or liposome attenuates B. cenocepacia virulence by targeting the LysR-type regulator ShvR, which regulates the production of the QS signals BDSF and AHL in B. cenocepacia. Our results not only suggest the important roles of membrane-enclosed PQS in interspecies and interkingdom communications but also provide a new perspective on the use of functional nanocarriers loaded with QS inhibitors for treating pathogen infections.

Natural environment serves as a reservoir for Burkholderia cepacia complex organisms, including the highly transmissible opportunistic human pathogen B. cenocepacia. Currently, there is a lack of an effective and quantitative method for B. cenocepacia detection in fresh food and other environmental niches. A quantitative real-time PCR (qPCR) detection method for B. cenocepacia bacteria was established in this study and validated using artificially inoculated fresh vegetable samples. Genome-wide comparative methods were applied to identify target regions for the design of species-specific primers. Assay specificity was measured with 12 strains of closely related Burkholderia bacteria and demonstrated the primer pair BCF6/R6 were 100% specific for detection of B. cenocepacia. The described qPCR assay evaluated B. cenocepacia with a 2 pg μl-1 limit of detection and appropriate linearity (R2 = 0.999). In 50 samples of experimentally infected produce (lettuce, onion, and celery), the assay could detect B. cenocepacia as low as 2.6 × 102 cells in each sample equal to 1 g. The established qPCR method quantitatively detects B. cenocepacia with high sensitivity and specificity, making it a promising technique for B. cenocepacia detection and epidemiological research on B. cepacia complex organisms from fresh vegetables.

Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and pathogenicity: the cis-2-dodecenoic acid (BDSF) system and the N-acyl homoserine lactone (AHL) system. In this study, we reported that oridonin, which was screened from a collection of natural products, disrupted important B. cenocepacia phenotypes, including motility, biofilm formation, protease production, and virulence. Genetic and biochemical analyses showed that oridonin inhibited the production of BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Furthermore, we revealed that oridonin directly binds to the regulator RqpR of the two-component system RqpSR that dominates the above-mentioned QS systems to inhibit the expression of the BDSF and AHL signal synthase-encoding genes. Oridonin also binds to the transcriptional regulator CepR of the cep AHL system to inhibit its binding to the promoter of bclACB. These findings suggest that oridonin could potentially be developed as a new QS inhibitor against pathogenic B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important human opportunistic pathogen that can cause life-threatening infections in susceptible individuals. It employs quorum-sensing (QS) systems to regulate biological functions and virulence. In this study, we have identified a lead compound, oridonin, that is capable of interfering with B. cenocepacia QS signaling and physiology. We demonstrate that oridonin suppressed cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) signal production and attenuated virulence in B. cenocepacia. Oridonin also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that oridonin could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.

It has been demonstrated that quorum sensing (QS) is widely employed by bacterial cells to coordinately regulate various group behaviors. Diffusible signal factor (DSF)-type signals have emerged as a growing family of conserved cell-cell communication signals. In addition to the DSF signal initially identified in Xanthomonas campestris pv. campestris, Burkholderia diffusible signal factor (BDSF) (cis-2-dodecenoic acid) has been recognized as a conserved DSF-type signal with specific characteristics in both signal perception and transduction from DSF signals. Here, we review the history and current progress of the research on this type of signal, especially focusing on its biosynthesis, signaling pathways, and biological functions. We also discuss and explore the huge potential of targeting this kind of QS system as a new therapeutic strategy to control bacterial infections and diseases.

Global transcriptional regulator downstream RpfR (GtrR) is a key downstream regulator for quorum-sensing signaling molecule cis-2-dodecenoic acid (BDSF). As a bacterial enhancer-binding protein (bEBP), GtrR is composed of an N-terminal receiver domain, a central ATPases associated with diverse cellular activities (AAA+) ATPase σ54 -interaction domain, and a C-terminal helix-turn-helix DNA-binding domain. In this work, we solved its AAA+ ATPase domain in both apo and GTP-bound forms. The structure revealed how GtrR specifically recognizes GTP. In addition, we also revealed that GtrR has moderate GTPase activity in vitro in the absence of its activation signal. Finally, we found the residues K170, D236, R311, and R357 in GtrR that are crucial to its biological function, any single mutation leading to completely abolishing GtrR activity.

Burkholderia cenocepacia is among the important pathogens isolated from cystic fibrosis (CF) patients. It has attracted considerable attention because of its capacity to evade host immune defenses during chronic infection. Advances in systems biology methodologies have led to the emergence of methods that integrate experimental transcriptomics data and genome-scale metabolic models (GEMs). Here, we integrated transcriptomics data of bacterial cells grown on exponential and biofilm conditions into a manually curated GEM of B. cenocepacia. We observed substantial differences in pathway response to different growth conditions and alternative pathway susceptibility to extracellular nutrient availability. For instance, we found that blockage of the reactions was vital through the lipid biosynthesis pathways in the exponential phase and the absence of microenvironmental lysine and tryptophan are essential for survival. During biofilm development, bacteria mostly had conserved lipid metabolism but altered pathway activities associated with several amino acids and pentose phosphate pathways. Furthermore, conversion of serine to pyruvate and 2,5-dioxopentanoate synthesis are also identified as potential targets for metabolic remodeling during biofilm development. Altogether, our integrative systems biology analysis revealed the interactions between the bacteria and its microenvironment and enabled the discovery of antimicrobial targets for biofilm-related diseases.

Quorum-sensing (QS) signals are widely employed by bacteria to regulate biological functions in response to cell densities. Previous studies showed that Burkholderia cenocepacia mostly utilizes two types of QS systems, including the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) systems, to regulate biological functions. We demonstrated here that a LysR family transcriptional regulator, Bcal3178, controls the QS-regulated phenotypes, including biofilm formation and protease production, in B. cenocepacia H111. Expression of Bcal3178 at the transcriptional level was obviously downregulated in both the AHL-deficient and BDSF-deficient mutant strains compared to the wild-type H111 strain. It was further identified that Bcal3178 regulated target gene expression by directly binding to the promoter DNA regions. We also revealed that Bcal3178 was directly controlled by the AHL system regulator CepR. These results show that Bcal3178 is a new downstream component of the QS signaling network that modulates a subset of genes and functions coregulated by the AHL and BDSF QS systems in B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important opportunistic pathogen in humans that utilizes the BDSF and AHL quorum-sensing (QS) systems to regulate biological functions and virulence. We demonstrated here that a new downstream regulator, Bcal3178 of the QS signaling network, controls biofilm formation and protease production. Bcal3178 is a LysR family transcriptional regulator modulated by both the BDSF and AHL QS systems. Furthermore, Bcal3178 controls many target genes, which are regulated by the QS systems in B. cenocepacia. Collectively, our findings depict a novel molecular mechanism with which QS systems regulate some target gene expression and biological functions by modulating the expression level of a LysR family transcriptional regulator in B. cenocepacia.

Dioxin compounds are persistent carcinogenic byproducts of anthropogenic activities such as waste combustion and other industrial activities. The ubiquitous distribution of dioxins is global concerns these days. Among of recent techniques, bioremediation, an eco-friendly and cost-effective technology, uses bacteria or fungi to detoxify in dioxins; however, not many bacteria can degrade the most toxic dioxin congener 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD). In this study, the endophytic bacterium Burkholderia cenocapacia 869T2 was capable of TCDD degradation by nearly 95 % after one-week of an aerobic incubation. Through transcriptomic analysis of the strain 869T2 at 6 -h and 12 -h TCDD exposure, a number of catabolic genes involved in dioxin metabolism were detected with high gene expressions in the presence of TCDD. The transcriptome data also indicated that B. cenocepacia strain 869T2 metabolized the dioxin compounds from an early phase (at 6 h) of the incubation, and the initial outline for a general dioxin degradation pathway were proposed. One of the catabolic genes, l-2-haloacid dehalogenase (2-HAD) was cloned to investigate its contribution in dioxin dehalogenation. By detecting the increasing concentration of chloride ions released from TCDD, our results indicated that the dehalogenase played a crucial role in dehalogenation of dioxin in the aerobic condition.