Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In , the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal-2-dodecenoic acid, also called diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMPphosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in and had overlapping effects on both the transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.
Quorum sensing (QS) is widely utilized by bacterial pathogens to regulate biological functions and pathogenicity. Recent evidence has shown that QS is subject to regulatory cascades, especially two-component systems that often respond to environmental stimulation. At least two different types of QS systems regulate pathogenesis in Burkholderia cenocepacia. However, it remains unclear how this bacterial pathogen controls these QS systems. Here, we demonstrate a novel two-component system RqpSR (Regulating Quorum sensing and Pathogenicity), which plays an important role in modulating QS and pathogenesis in B. cenocepacia. We demonstrate strong protein-protein binding affinity between RqpS and RqpR. Mutations in rqpS and rqpR exerted overlapping effects on B. cenocepacia transcriptomes and phenotypes, including motility, biofilm formation and virulence. In trans expression of rqpR rescued the defective phenotypes in the rqpS mutant. RqpR controls target gene expression by direct binding to DNA promoters, including the cis-2-dodecenoic acid (BDSF) and N-acylhomoserine lactone (AHL) signal synthase gene promoters. These findings suggest that the RqpSR system strongly modulates physiology by forming a complicated hierarchy with QS systems. This type of two-component system appears to be widely distributed and coexists with the BDSF QS system in various bacterial species.
Quorum sensing (QS) signals are widely used by bacterial pathogens to control biological functions and virulence in response to changes in cell population densities. Burkholderia cenocepacia employs a molecular mechanism in which the cis-2-dodecenoic acid (named Burkholderia diffusible signal factor [BDSF]) QS system regulates N-acyl homoserine lactone (AHL) signal production and virulence by modulating intracellular levels of cyclic diguanosine monophosphate (c-di-GMP). Thus, inhibition of BDSF signaling may offer a non-antibiotic-based therapeutic strategy against BDSF-regulated bacterial infections. In this study, we report the synthesis of small-molecule mimics of the BDSF signal and evaluate their ability to inhibit BDSF QS signaling in B. cenocepacia. A novel structural analogue of BDSF, 14-Me-C 16:Δ2 (cis-14-methylpentadec-2-enoic acid), was observed to inhibit BDSF production and impair BDSF-regulated phenotypes in B. cenocepacia, including motility, biofilm formation, and virulence, while it did not inhibit the growth rate of this pathogen. 14-Me-C 16:Δ2 also reduced AHL signal production. Genetic and biochemical analyses showed that 14-Me-C 16:Δ2 inhibited the production of the BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Notably, 14-Me-C 16:Δ2 attenuated BDSF-regulated phenotypes in various Burkholderia species. These findings suggest that 14-Me-C 16:Δ2 could potentially be developed as a new therapeutic agent against pathogenic Burkholderia species by interfering with their QS signaling. IMPORTANCE Burkholderia cenocepacia is an important opportunistic pathogen which can cause life-threatening infections in susceptible individuals, particularly in cystic fibrosis and immunocompromised patients. It usually employs two types of quorum sensing (QS) systems, including the cis-2-dodecenoic acid (BDSF) system and N-acyl homoserine lactone (AHL) system, to regulate virulence. In this study, we have designed and identified an unsaturated fatty acid compound (cis-14-methylpentadec-2enoic acid [14-Me-C 16:Δ2 ]) that is capable of interfering with B. cenocepacia QS signaling and virulence. We demonstrate that 14-Me-C 16:Δ2 reduced BDSF and AHL signal production in B. cenocepacia. It also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that 14-Me-C 16:Δ2 could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.
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 transcriptional level was obviously down-regulated in both the AHL-deficient and BDSF-deficient mutant strains comparing 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 co-regulated by the AHL and BDSF QS systems in B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important opportunistic pathogen in humans, which 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.
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