The cis-2-Dodecenoic Acid (BDSF) Quorum Sensing System in Burkholderia cenocepacia

ABSTRACT

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.

INTRODUCTION

Quorum sensing (QS) is a cell-cell communication process that occurs widely in both Gram-positive and Gram-negative bacteria. It is used by bacteria to sense and respond to changes in cell density to regulate group behaviors and many biological functions (1, 2). The QS systems in Gram-positive bacteria typically use either an unmodified or a posttranslationally modified small peptide and a two-component system, which includes a membrane-bound sensor kinase receptor and a cytoplasmic transcription factor that directly alters gene expression (3, 4). Many Gram-negative bacterial species utilize the homologs of the LuxIR proteins first found in Vibrio fischeri to produce acyl-homoserine lactones (AHLs) as QS signal molecules (5, 6), which are the most studied self-induced signal molecules and possess a core N-acylated homoserine lactone (HSL) ring and a 4- to 18-carbon acyl chain (1, 7).

In addition to AHL signals, there are many other QS signaling molecules that have been identified in Gram-negative bacteria, including autoinducer-2 (AI-2) (8), Pseudomonas aeruginosa quinolone signal (PQS) (9), autoinducer in Escherichia coli (AI-3) (10), 2-heptyl-4-quinolone (HHQ) (11), diketopiperazines (12), bradyoxetin (13), diffusible signal factor (DSF) family signals that were first identified in the plant pathogen Xanthomonas campestris pv. campestris (14–16), 3-hydroxypalmitic acid methyl ester (3-OH PAME) (17), and a new signal, anthranilic acid, which was recently identified in Ralstonia solanacearum (18).

Studies have demonstrated that RpfF, RpfC, and RpfG are the key elements of the DSF QS system in X. campestris pv. campestris. RpfF is responsible for the synthesis of DSF, which is characterized as cis-11-methyl-2-dodecenoic acid, and RpfC-RpfG constitutes a two-component system for signal perception and transduction (19, 20). In more detail, RpfC senses and binds to DSF by a membrane-spanning sensor domain, leading to its autophosphorylation within the histidine kinase domain and then phosphotransfer to RpfG (19). The activated HD-GYP domain of RpfG has phosphodiesterase (PDE) activity and is able to degrade cyclic dimeric GMP (c-di-GMP), which is an intracellular second messenger (21, 22). Comparative genomic studies found that the rpfFCG gene cluster is highly conserved, which indicates that the DSF-dependent QS system is widely distributed throughout xanthomonads (23, 24).

In addition to the DSF family signals identified in xanthomonads, Burkholderia diffusible signal factor (BDSF) (cis-2-dodecenoic acid) has been recognized as a novel DSF family signal, which was first found in Burkholderia cenocepacia (25) (Fig. 1). Subsequent studies have elucidated the signaling pathway and regulatory mechanism of this signal, which is definitely specific and different from what we have known. Here, we review the research history and current progress on BDSF signals in recent years. In addition to its role as an intracellular signal, the functions of BDSF in interspecies and interkingdom communications are also described here.

FIG 1.

The mechanism of RpfF_Bc to synthesize BDSF. The synthesis of BDSF by RpfF_Bc (BCAM0581) is divided into two steps: RpfF_Bc first dehydrates 3-hydroxydodecanoyl-ACP to form cis-2-dodecenoyl-ACP and then further cleaves cis-2-dodecenoyl-ACP to obtain BDSF and holo-ACP. RpfR (BCAM0580) interacts with RpfF_Bc, blocks the substrate from entering the channel, and finally inhibits the synthesis of BDSF.

BDSF IS A NOVEL DSF FAMILY SIGNAL

The Burkholderia cepacia complex (BCC) is a group of Gram-negative bacteria that comprises more than 20 closely related species (26, 27). B. cenocepacia is an opportunistic pathogen that constitutes the majority of transmissible and epidemic strains and is highly virulent (28, 29); it can cause a decline in lung function and life-threatening infections in cystic fibrosis patients and immunocompromised individuals (30, 31). It has been shown that the formation of biofilms and the expression of virulence factors are modulated by the QS systems of B. cenocepacia (32, 33). Evidence indicates that the CepIR system is an AHL-based LuxIR QS system and is highly conserved in BCC species (34, 35). It controls the production of extracellular proteases, biofilm maturation, and swarming motility in B. cenocepacia. Increasing evidence suggests that once AHL signals accumulate to the threshold in the environment, they can bind to the CepR protein to form a complex, which causes conformational changes in the CepR protein and then activates or inhibits the expression of the target genes by binding to the promoters (34–37). Moreover, one more AHL-based QS system, CciIR, has also been found in B. cenocepacia (37). Intriguingly, there is an orphan LuxR homolog, CepR2, which is not genetically linked to a cognate AHL synthase-encoding gene, that was also found in B. cenocepacia (38–40).

In 2008, Boon et al. first characterized BDSF as a DSF family QS signal (Fig. 1), which was biosynthesized by B. cenocepacia RpfF (RpfF_Bc). The structure of BDSF is similar but not identical to that of the DSF (cis-11-methyl-2-decenoic acid) signal produced by X. campestris pv. campestris, where BDSF lacks a methyl group at the C-11 position (25). It was further identified that BDSF is a conserved signaling molecule produced by the BCC, with at least nine species producing it as the major DSF family signaling molecule (41). Intriguingly, BDSF not only controls biofilm formation and other AHL-regulated phenotypes (42) but also regulates the synthesis of AHL signals in B. cenocepacia (43), suggesting that the BDSF QS system is the central regulatory system in B. cenocepacia.

THE BIOSYNTHESIS OF THE BDSF SIGNAL IN B. CENOCEPACIA

BCAM0581 catalyzes the production of the BDSF signal in B. cenocepacia.

Protein sequence alignment analysis showed that BCAM0581 (also known as RpfF_Bc) shares 37.2% identity with RpfF, and the two proteins are functionally interchangeable (25). It was revealed that the deletion of BCAM0581 abolished BDSF production in B. cenocepacia, and the phenotypic defect of the rpfF mutant of X. campestris pv. campestris could be restored to the wild-type strain level by the in trans expression of BCAM0581 (25, 41). It was demonstrated that BCAM0581 is a bifunctional crotonase with both dehydratase and thioesterase activities, which enables the direct conversion of the acyl carrier protein (ACP) thioester of 3-hydroxydodecanoic acid into cis-2-dodecenoic acid in B. cenocepacia (23, 44) (Fig. 1). BCAM0581 is the first member of the crotonase superfamily that has been identified to exert desaturase and thioesterase activities to cleave the acyl-ACP thioester bond to release free fatty acids (44).

The RqpSR two-component system controls BDSF production in B. cenocepacia.

As RpfF_Bc (BCAM0581) is the enzyme that catalyzes the production of BDSF signals, our group continued to study the regulatory mechanism of rpfF_Bc expression. We found a novel two-component system, RqpSR, in which RqpS is a signal transduction histidine kinase and RqpR is a response regulator with a DNA-binding domain. It not only controls the QS-regulated phenotypes in B. cenocepacia but also positively controls the production of BDSF and AHL signals by modulating the transcriptional expression levels of cepI and rpfF_Bc (45). The response regulator of this system, RqpR, controls the QS system by directly binding to the promoters of BDSF and AHL signal synthase-encoding genes (45). These findings suggest that the RqpSR system modulates B. cenocepacia physiology and pathogenicity by forming a complicated hierarchy with QS systems. Intriguingly, the RqpSR system appears to be widely distributed and coexists with the BDSF QS system in various bacterial species (45) (Table 1).

TABLE 1.

Homologs of RpfF_Bc, RpfR, RqpS, RqpR, and GtrR in various bacterial species^a

Bacterium	Strain	RpfFBc homolog identity (%)	RpfR homolog identity (%)	RqpS homolog identity (%)	RqpR homolog identity (%)	GtrR homolog identity (%)
Burkholderia
B. cenocepacia	H111	100.0	100.0	100.0	100.0	100.0
B. cenocepacia	J2315	100.0	100.0	99.6	100.0	99.8
B. cepacia	JBK9	96.5	99.9	91.1	99.1	100.0
B. contaminans	FFH2055	95.1	96.3	91.1	99.1	97.6
B. anthina	MSMB0848	99.7	97.3	96.7	98.2	99.1
B. lata	383	94.8	96.3	90.8	NA	97.6
B. seminalis	FL-5-5-10-S1-D0	99.0	96.9	91.1	97.7	98.1
B. pyrrocinia	Lyc2	95.8	96.1	89.7	97.2	97.6
B. dolosa	AU0158	96.9	92.4	84.5	96.3	95.3
B. pseudomultivorans	MSMB0607	94.8	93.3	83.4	95.0	96.1
B. vietnamiensis	G4	94.8	91.2	79.7	93.6	96.3
B. multivorans	ATCC 17616	94.4	93.2	75.6	93.6	97.0
B. ubonensis	MSMB0011	90.2	86.5	77.5	93.6	95.7
B. diffusa	MSMB0010	95.5	96.0	81.2	93.1	95.5
B. latens	RF32-BP12	96.2	91.6	83.0	93.1	95.0
B. stagnalis	MSMB1135	87.8	86.7	76.0	93.1	95.0
B. territorii	A63	95.5	92.5	81.2	92.7	93.0
B. ambifaria	AMMD	94.4	92.1	83.4	92.7	95.5
B. pseudomallei	1026b	NA	33.0	65.7	87.6	99.0
B. oklahomensis	C6786	NA	37.5	58.7	87.2	89.2
B. plantarii	ATCC 43733	NA	43.3	53.1	86.2	89.4
B. gladioli	ATCC 10248	NA	42.9	55.4	85.7	89.9
B. kururiensis	M130	71.4	65.8	49.1	83.5	82.4
B. glumae	BGR1	NA	38.4	53.9	82.9	88.4
B. graminis	C4D1M	71.1	65.9	51.3	82.1	82.5
B. bryophila	376MFSha3.1	70.7	64.9	50.2	82.1	82.1
B. fungorum	ATCC BAA-463	70.7	65.5	52.0	82.1	81.9
B. phymatum	STM815	73.9	66.1	50.6	81.7	37.4
B. caribensis	MBA4	72.5	65.2	52.4	81.2	84.3
B. ginsengisoli	NBRC 100965	71.1	65.3	50.2	80.7	81.0
B. xenovorans	LB400	70.7	66.3	55.0	80.3	82.1
B. acidipaludis	NBRC 101816	NA	39.9	49.8	79.8	75.2
B. sprentiae	WSM5005	NA	61.9	45.8	79.8	38.5
B. phytofirmans	PsJN	71.1	41.2	52.8	79.4	49.3
B. ferrariae	NBRC 106233	NA	41.9	48.7	79.4	75.9
Caballeronia
C. choica	NA	NA	40.5	46.5	76.1	39.8
C. telluris	NA	NA	38.9	44.3	76.1	38.0
C. glathei	NA	NA	36.9	42.4	75.7	38.6
C. jiangsuensis	MP-1	NA	37.8	38.0	75.6	74.2
C. grimmiae	R27	NA	38.5	35.1	70.6	38.9
Other
Mumia flava	MUSC 201	95.1	35.1	91.1	99.1	NA

^aNA, not applicable.

BIOLOGICAL FUNCTIONS OF BDSF SIGNALS

The biological functions of BDSF signals in B. cenocepacia.

It was demonstrated that BDSF controls various biological functions of B. cenocepacia (42, 46, 47). The rpfF_Bc mutant of B. cenocepacia showed reduced virulence and defective functions, including motility and biofilm formation, while all of these phenotypes could be restored to wild-type levels by the addition of BDSF or complementation with RpfF_Bc (42, 48). It was revealed that the BDSF-null mutant was compromised in the expression of virulence factors such as the metalloproteases ZmpA and ZmpB (42, 47, 49, 50). The transcription of the zinc metalloprotease zmpB was downregulated in the rpfF_Bc mutant (47), and the downregulated expression of zmpA in the rpfF_Bc mutant of B. cenocepacia J2315 could be restored when the medium was supplemented with BDSF (42). The identification of BDSF as another QS signal in B. cenocepacia provides more evidence that the bacterial pathogens recruit multiple signaling systems to coregulate virulence gene expression. Intriguingly, the inactivation of rpfF_Bc in B. cenocepacia H111 also resulted in decreased cepI expression and diminished AHL production, suggesting that BDSF is involved in the AHL-dependent QS system by reducing the transcription of cepI, while mutation of the cepIR system did not impair the production of BDSF (43, 47). The integrated analysis of transcriptome sequencing (RNA-seq) and phenotypes showed that the two QS systems not only have specific signal pathways but also form a complex intraspecies signaling network in B. cenocepacia.

The role of BDSF in interspecies and interkingdom communication.

In nature, bacteria are more likely to grow in polymicrobial communities than in monocultures. The development and maintenance of such communities depend on interactions among the members, including interspecies signal transmission (22). Many pathogenic bacteria use cell-cell signaling to regulate the expression of factors contributing to virulence. This may be related to the fine control of intraspecies or interspecies signaling by a range of bacteria. Recently, signaling molecules of the DSF family have been found to be involved in the regulation of pathogenesis and biofilm formation in diverse bacteria—not only in their cognate bacteria but also in unrelated bacteria (25). BDSF isolated from B. cenocepacia was found to be involved in the modulation of virulence, antibiotic resistance, and persistence of P. aeruginosa in the cystic fibrosis airway (51, 52). The exogenous addition of BDSF reduced the transcriptional expression of QS system-related genes and the production of QS signals, including 3-oxo-C₁₂-HSL, PQS, and C₄-HSL, consequently resulting in the downregulation of biofilm formation and virulence factor production of P. aeruginosa (53). Furthermore, BDSF and some of its derivatives inhibit the type III secretion system (T3SS) of P. aeruginosa with stronger activity than that for the inhibition of the QS systems, suggesting that BDSF may interfere with the QS systems and T3SS of P. aeruginosa through two independent signaling pathways. However, the specific mechanism by which BDSF interferes with the QS systems and T3SS of P. aeruginosa still needs further investigation.

Moreover, BDSF was reported to inhibit the dimorphic transition of Candida albicans at physiologically relevant concentrations and thereby to block the biofilm formation of fungi (25, 54). Real-time reverse transcription-quantitative PCR (qRT-PCR) analysis showed that BDSF could downregulate the expression of ALS1 and EAP1, which are involved in C. albicans adhesion, and upregulate the expression of YWP1, which exerts an inverse effect on adherence (55). In addition, BDSF exerted a protective effect in an experimental mouse model of Candida vaginitis by inhibiting virulence factors (56). Further analyses showed that either B. cenocepacia cocultured with C. albicans or the exogenous addition of physiologically relevant levels of BDSF strongly inhibited the formation of C. albicans germ tubes, which suggests that the signal might play a role in cross-kingdom microbial competition in ecosystems (25).

THE SIGNALING PATHWAY OF BDSF IN B. CENOCEPACIA

The signal perception mechanism of BDSF.

Previous studies have shown that RpfF and the two-component system RpfCG are responsible for DSF signal molecule synthesis and signal transduction in X. campestris pv. campestris, respectively (57, 58). RpfF_Bc is the key enzyme that produces BDSF; it possesses homology with RpfF and is highly conserved in all BCC family species (41) (Table 1), but the homologous proteins RpfCG and the rpf gene cluster could not be found in B. cenocepacia, suggesting that there is a different mechanism that is in charge of BDSF signal perception and transduction. Deng et al. identified that BCAM0580 (designated rpfR), which is located next to the BDSF biosynthase gene rpfF_Bc, was responsible for encoding a protein containing a PAS domain, a GGDEF domain, and an EAL domain (59). Their study showed that the deletion of rpfR shared similar phenotypic changes with the rpfF_Bc mutant with an increased intracellular c-di-GMP level. Furthermore, the deletion of rpfF_Bc did not affect the transcription level of rpfR, suggesting that BDSF may influence the activity of RpfR through ligand-protein interactions. This hypothesis was confirmed by isothermal titration calorimetry and circular dichroism (CD) analyses, which showed that BDSF binds to RpfR in vitro with strong affinity and causes a conformational change, and the PAS domain is required for BDSF binding (59) (Fig. 2).

FIG 2.

Molecular models of the sensing mechanisms of DSF and BDSF signals. (A) The chemical structures of DSF, BDSF, and C_12:0. (B) Molecular model of RpfC to sense DSF. RpfC possesses five transmembrane domains, and five central residues (Arg15, Asp17, Ser18, Glu19, and Gln22) are essential for the sensing of DSF. (C and D) Cartoon representation of RpfR_Ct (cyan) in complex with BDSF. RpfR-BDSF hydrophobic interactions are depicted as a smooth cyan contour line. (Panels C and D are reproduced from reference 60.)

In 2019, Waldron et al. reported the X-ray crystal structure of the RpfR homolog of Cronobacter turicensis (RpfR_Ct) in complex with BDSF, which determined in detail the molecular basis of the RpfR receptor to sense, perceive, and specifically bind to its ligands (60). It was found that RpfR_Ct shares 56% identity with RpfR of B. cenocepacia. Compared with C_12:0 and trans-2-dodecenoic acid, the cis-2 double bond of BDSF may contribute to the specificity and affinity for binding with RpfR. Analysis of the crystal structure showed that arginine 187 (Arg187) of RpfR_Ct forms two hydrogen bonds with BDSF, while there was no hydrogen bond formed between RpfR_Ct and C_12:0 (Fig. 2). This conformational difference is the result of the additional H bond mediated by Arg187 with a different rotameric configuration that caused much of the Arg187 side chain to be solvent exposed. This conformational change may play a critical role in regulating phosphodiesterase activity (60), which is described in the next section. In addition, the hydroxyl of Tyr183 interacts with the conserved Asn202 side chain amide N-H and strengthens the interaction between BDSF and Asn202 of RpfR_Ct. Furthermore, BDSF showed a rigid architecture that would reduce entropy loss compared with saturated fatty acids (60). These findings suggest that the cis-2 double bond of BDSF plays a key role in signal perception.

The transduction process of BDSF signals.

c-di-GMP is a ubiquitous second-messenger molecule in bacteria that regulates a variety of physiological functions, including biofilm formation, cell differentiation, the production of pathogenic factors, and so on (61–64). The intracellular c-di-GMP concentration is modulated by the opposing activities of diguanylate cyclase (DGC) and cyclic nucleotide phosphodiesterase (PDE), which in turn are regulated by extra- or intracellular factors (61, 64). DGCs produce c-di-GMP from two molecules of GTP, and PDEs hydrolyze c-di-GMP to linear pGpG or two GMP molecules. The GGDEF motif is essential for the enzymatic activity of DGCs, while PDE activity is associated with the EAL and HD-GYP domains (64–68). Many Gram-negative bacteria sense the population density of the community and control multiple phenotypes in response to changes in the environment through QS and intracellular c-di-GMP systems. For example, RpfC activates the phosphodiesterase activity of RpfG to degrade c-di-GMP after sensing DSF signal molecules in X. campestris pv. campestris (57, 58, 65, 69). By comparing the intracellular c-di-GMP levels between the RpfF_Bc mutant and the wild type, the results showed that the level of intracellular c-di-GMP of the rpfF_Bc mutant was significantly higher than that of the wild-type strain and returned to the level of the wild-type strain when exogenous BDSF was added, indicating that BDSF may be involved in the metabolism of c-di-GMP in some way (59).

The GGDEF and EAL domains usually refer to features of diguanylate cyclase and phosphodiesterase, which are involved in c-di-GMP biosynthesis and degradation, respectively (64). In B. cenocepacia, the phenotypes of the rpfR mutant could not be rescued by complementation with the GGDEF (DGC) domain alone, whereas the EAL (PDE) domain in fact complements the impaired phenotypes, comparably to the full-length protein RpfR, suggesting that BDSF activates the phosphodiesterase activity of RpfR, which causes a decrease in the level of intracellular c-di-GMP (59). Similar results were observed in other BCC species, such as the rpfR mutant strain of Burkholderia lata, which was found to show decreases in the growth rate, swimming motility, AHL production, and pathogenicity in Caenorhabditis elegans but with an increase in biofilm formation, suggesting that rpfR plays a vital role in various phenotypes of BCC species by regulating the level of intracellular c-di-GMP (70). Furthermore, the BDSF-induced allosteric conformational change through its interaction with the PAS domain was the factor that stimulated the c-di-GMP phosphodiesterase activity of RpfR (59), which was consistent with the BDSF-RpfR_Ct complex crystal structure in which the binding of BDSF to RpfR_Ct led to RpfR_Ct-R187 having a different rotameric configuration (60). These results suggest that the BDSF-dependent RpfFR QS system and c-di-GMP are intricately intertwined, and BDSF activates RpfR to degrade intracellular c-di-GMP; subsequently, the signal is transmitted to downstream components to change phenotypes.

RpfR not only acts as a QS signal receptor and a c-di-GMP phosphodiesterase but also inhibits BDSF synthesis by interacting directly with RpfF_Bc. At the PAS-like domain of the RpfR N terminus, there is a highly conserved and undescribed region of RpfR, which refers to the RpfF_Ct interaction domain (FI) (60). The RpfR FI deletion mutant strain produced a lower level of c-di-GMP than the wild-type strain, while the RpfR deletion mutant strain promoted its concentration, which could be explained by the regulation of BDSF on RpfR PDE activity or might be the direct effect of the deletion of FI on the PDE activity of RpfR. The X-ray crystal structure of the RpfF_Bc-RpfR_Ct complex showed a heterohexamer consisting of three RpfR_Ct and RpfF_Bc protomers. Two RpfF_Bc protomers interact with one RpfR_Ct protomer near their homodimerization interfaces and lead to the RpfF_Bc acyl-ACP substrate tunnel being blocked by the proximity of RpfR_Ct and the formation of an interaction interface, consistent with the inhibition of RpfF_Bc thioesterase activity in the presence of RpfR_Ct (60). As the receptor of the BDSF QS signal, RpfR, in turn, can inhibit its synthesis, suggesting that it plays a critical role in the feedback regulation loop. However, the mechanism by which the extracellular level of BDSF significantly decreases after reaching the accumulation peak is still unclear (42).

Intriguingly, Yang et al. showed that RpfR not only possesses c-di-GMP phosphodiesterase activity but also acts as a c-di-GMP sensor (71). Their study identified a global regulator (named GtrR) that was a key downstream component that could interact with RpfR and regulate the expression of genes in B. cenocepacia. An electrophoretic mobility shift assay (EMSA) suggested that RpfR enhanced the binding of GtrR to the target gene promoter; however, the ability of the RpfR-GtrR complex to bind to promoter DNA was reduced by the addition of exogenous c-di-GMP. This is caused by the binding of c-di-GMP to RpfR with an estimated dissociation constant (K_d) of 2.92 ± 0.26 μM (Fig. 3). Given that RpfR exhibited low PDE activity in the absence of BDSF, and c-di-GMP binds with GtrR-RpfR to form a ternary complex to inhibit the regulatory activity of GtrR on target gene expression, we concluded that both BDSF and c-di-GMP are the signal ligands of RpfR, which bind with RpfR to exert different functions. Moreover, both RpfR and GtrR homologs are present in diverse Gram-negative bacteria, suggesting that the BDSF-type QS system is widespread (71) (Table 1). In addition, the GtrR-RpfR complex can regulate the transcription of cepI (71), and the cep system regulator, CepR, can bind to the promoter of Bcal3178, which is a LysR family transcriptional regulator, and enhance the expression of Bcal3178. Bcal3178 controls some QS-regulated functions, but one study revealed that there was no binding between GtrR and the Bcal3178 promoter (72) (Fig. 3).

FIG 3.

Schematic representation of the BDSF-dependent QS systems in B. cenocepacia. RqpSR is a two-component regulatory system. RqpS perceives signals and activates RqpR, which positively regulates the expression of the rpfF_Bc and cepI genes, which are required for the synthesis of BDSF and AHL signals, respectively. When the intracellular c-di-GMP level is high, it combines with GtrR-RpfR to form a ternary complex, which inhibits the regulatory activity of GtrR. When the bacterial density reaches a certain threshold, RpfR combines with BDSF to enhance its PDE activity, decreases the level of c-di-GMP, and further closely integrates with GtrR to regulate gene expression. In addition, activated RpfR binds to RpfF_Bc through the FI domain, which in turn inhibits the synthesis of BDSF (the black arrow represents the BDSF synthesis channel). CepR, the signal receptor of AHLs, can bind to the promoter of Bcal3178 and enhance the expression of Bcal3178, which controls the expression of some QS-regulated genes.

CONCLUDING REMARKS

Previous studies have shown that many bacterial pathogens coordinate the expression of virulence-related genes through QS (2). The QS signaling molecule cis-2-dodecenoic acid, which was also named BDSF, was first identified in B. cenocepacia. It was demonstrated that BDSF is a novel DSF family QS signaling molecule that regulates a variety of phenotypes, including biofilm formation, motility, and virulence factor production, in B. cenocepacia and some other bacterial species (25, 59). Studies have shown that BDSF can be detected in all tested BCC species; additionally, homologs of the BDSF synthase RpfF_Bc, the receptor RpfR, and the key downstream component of the BDSF QS system GtrR exist in many species (Table 1), suggesting that BDSF is an important QS signaling molecule with widespread conservation.

In recent years, due to the abuse of antibiotics, the major challenge for us is to develop novel therapeutic strategies to treat antibiotic-resistant pathogens (73). The DSF-type QS system exists widely in bacteria and plays a vital role in the regulation of pathogenicity, showing great potential for the development of antivirulence therapy by interfering with this kind of QS system to attenuate the virulence of pathogens rather than kill them (73). The development of QS inhibitors as new drugs for the treatment of bacterial infections has attracted great attention (74). Cui et al. reported a QS signal inhibitor (cis-14-methylpentadec-2-enoic acid, also known as 14-Me-C_16:Δ2) that is derived from unsaturated fatty acids and showed great activities to interfere with BDSF signaling and the production of virulence factors but did not inhibit the growth rate of the pathogen (75). Their data showed that 14-Me-C_16:Δ2 targeted the QS signal synthase RpfF_Bc to inhibit the production of BDSF and AHL signals, leading to defects in the pathogenic phenotypes. Remarkably, 14-Me-C_16:Δ2 displayed a similar effect on various Burkholderia species. Likewise, some QS inhibitors have also been applied to other pathogens and showed a marked effect on attenuating QS-regulated virulence (73). In addition, it was previously reported that hosts can monitor QS signals to regulate their own immune response (76). As a QS signal widely distributed in various microorganisms, BDSF is likely to interact with the host, which might be used for the development of new vaccines to enhance the host immune response to control infection in the future.