Cis-2-dodecenoic acid quorum sensing system modulates N-acyl homoserine lactone production through RpfR and cyclic di-GMP turnover in Burkholderia cenocepacia
© Deng et al.; licensee BioMed Central Ltd. 2013
Received: 6 March 2013
Accepted: 27 June 2013
Published: 1 July 2013
Burkholderia cenocepacia employs both N-Acyl homoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) quorum sensing (QS) systems in regulation of bacterial virulence. It was shown recently that disruption of BDSF synthase RpfFBc caused a reduction of AHL signal production in B. cenocepacia. However, how BDSF system influences AHL system is still not clear.
We show here that BDSF system controls AHL system through a novel signaling mechanism. Null mutation of either the BDSF synthase, RpfFBc, or the BDSF receptor, RpfR, caused a substantial down-regulation of AHL signal production in B. cenocepacia strain H111. Genetic and biochemical analyses showed that BDSF system controls AHL signal production through the transcriptional regulation of the AHL synthase gene cepI by modulating the intracellular level of second messenger cyclic di-GMP (c-di-GMP). Furthermore, we show that BDSF and AHL systems have a cumulative role in the regulation of various biological functions, including swarming motility, biofilm formation and virulence factor production, and exogenous addition of either BDSF or AHL signal molecules could only partially rescue the changed phenotypes of the double deletion mutant defective in BDSF and AHL signal production.
These results, together with our previous findings, thus depict a molecular mechanism with which BDSF regulates AHL signal production and bacterial virulence through modulating the phosphodiesterase activity of its receptor RpfR to influence the intracellular level of c-di-GMP.
Quorum sensing (QS) is widely employed by bacterial pathogens to coordinate bacterial group behavior and regulate biological functions such as biofilm formation, motility, virulence, plasmid transfer, and antibiotic production [1, 2]. This regulation mechanism depends on the production and perception of diffusible signal molecules in a cell-density dependent manner [2–4]. At low cell density, bacterial cells produce a basal level of QS signals, which are diffused or transported into extracellular environments. When the cell density reaches a critical concentration, the accumulated signals initiate a set of biological activities in a coordinated fashion. Several types of QS systems have been identified including the most-characterized acylhomoserine lactone (AHL) dependent QS system and the relatively newly identified diffusible signal factor (DSF) dependent QS system [3, 5]. The AHL- and DSF-QS systems are mainly conserved in different Gram-negative bacteria pathogens.
While most bacterial pathogens employ either AHL- or DSF-dependent QS systems in regulation of virulence and biofilm formation [3, 6], the members of the Burkholderia cepacia complex were found to produce both AHL- and DSF-type QS signals [7–9]. In B. cenocepacia, which is an opportunistic pathogen in cystic fibrosis or immunocompromised patients, the AHL-type QS system comprises the AHL synthase CepI, which was shown to catalyze the synthesis of N-octanoyl homoserine lactone (C8HSL, also known as OHL) as a major AHL signal [10, 11], and the AHL receptor CepR. The receptor CepR forms a complex with AHL signals to activate or repress a set of target genes, and thus control a range of biological functions, including virulence, swarming motility and biofilm formation [8, 9].
In addition to the AHL-dependent QS system, a DSF-dependent system has recently been identified in B. cenocepacia[12–15]. The QS signal synthase, RpfFBc, catalyzes the production of BDSF signal (cis-2-dodecenoic acid), which is an analogue of the QS signal DSF (cis-11-methyl-2-dodecenoic acid), originally identified in the plant bacterial pathogen Xanthomonas campestris pv. campestris. Our recent study showed that BDSF acts by interacting with its receptor RpfR, which is a modular protein with PAS-GGDEF-EAL domains . Perception of BDSF by RpfR sharply enhances its c-di-GMP phosphodiesterase activity and consequently causes a reduction in the intracellular level of the second messenger cyclic di-GMP (c-di-GMP) in B. cenocepacia, which consequently affects a range of biological activities, including swarming motility, biofilm formation and virulence .
It has become clear that both AHL and BDSF systems control similar biological functions. Recently, it was reported that there is a direct relationship between the two QS systems as inactivation of BDSF synthase reduces the production of AHL signals in B. cenocepacia[17, 18]. However, how BDSF system affects AHL system remains obscure. In this study, by generating and analyzing single- and double-deletion mutants defective in QS signal production, we showed that BDSF signaling system plays a dominant role in the regulation of AHL QS system and various biological activities in B. cenocepacia. In addition, we have investigated the molecular mechanisms with which BDSF signaling system influencing AHL signal production and unveiled the involvement of the second messenger c-di-GMP. Furthermore, we have determined the relationships of these two QS systems in the cell-cell communication signaling cascade and their impacts on bacterial physiology and virulence.
BDSF system positively regulates AHL signal production
BDSF system positively controls cepI expression at transcriptional level
BDSF system controls AHL signal production and biological functions through regulation of intracellular c-di-GMP level
The cumulative effect BDSF and AHL systems on regulation of bacterial motility, biofilm formation and protease activity
The impact of BDSF and AHL signaling systems on B. cenocepacia H111 pathogenicity
Many bacterial pathogens contain either AHL- or DSF-type QS systems in coordination of bacterial physiology. The human opportunistic pathogen B. cenocepacia is one of the exceptions which contain both BDSF and AHL signaling mechanisms [7, 12, 13, 15, 19, 23]. In this study, we have investigated the relationship of the two QS systems in signaling modulation of bacterial physiology and virulence. Although the recently published results believe that the BDSF and AHL systems control overlap set and specific genes [17, 18], we found that the two QS systems exert cumulative effect on bacterial motility, biofilm formation and virulence factor production (Figure 5A-C). In addition, we showed that BDSF regulates AHL signal production by influencing the c-di-GMP phosphodiesterase activity of its receptor RpfR. Given that both QS systems are widely conserved in the members of B. cepacia complex [7, 10], it would be of great interest to investigate whether the similar cross-talking mechanisms of the AHL and BDSF systems are conserved in other members of the Burkholderia species.
The intracellular signal c-di-GMP is a widely conserved second messenger, which is known to be involved in the regulation of a range of biological activities, including bacterial motility, biofilm formation and virulence factor production [10, 24, 25]. The research progress over the last few years shows that c-di-GMP commonly controls various biological functions through interacting with different receptor or effector proteins, such as PilZ, FleQ, VpsT, LapD, FimX, PelD, and Clp [26–32]. Interestingly, different from this paradigm, the findings from this study have unveiled a new mechanism with which c-di-GMP could influence bacterial physiology. We showed that null mutation of RpfR, which is an one-component BDSF sensor/response regulator containing a BDSF-binding domain and the GGDEF-EAL domains associated with c-di-GMP metabolism , resulted in a similar level of reduction in AHL signal production as the BDSF-minus mutant ΔrpfFBc (Figure 3A). Given that binding of BDSF by RpfR could substantially increases its activity in c-di-GMP degradation , it is rational that increasing c-di-GMP level would lead to down-regulation of the AHL signal production and that decreasing c-di-GMP level would promote AHL signal production. Consisting with the above reasoning, our results showed that in trans expression of the c-di-GMP synthases, WspR from P. aeruginosa or the GGDEF domain of RpfR, in wild type H111 led to decreased AHL production (Figure 4), and that reducing c-di-GMP level in the BDSF-minus mutant ΔrpfFBc by overexpressing either RocR from P. aeruginosa or the EAL domain of RpfR resulted in increased AHL signal biosynthesis (Figure 4). These findings have elucidated a signaling pathway with which the BDSF-type QS system regulates the AHL-type QS system in B. cenocepacia and, additionally, have also further expanded our understanding of the c-di-GMP signaling mechanisms in modulation of bacterial physiology. However, how c-di-GMP controls AHL signal production remains to be further investigated.
The QS signal BDSF controls AHL signal production through regulation of the AHL synthase CepI expression at transcriptional level by modulating the intracellular level of the second messenger c-di-GMP through its novel receptor RpfR. The two QS systems have a cumulative role in regulation of various biological functions, including swarming motility, biofilm formation and virulence factor production. Exogenous addition of either BDSF or AHL signal molecules could only partially rescue the changed phenotypes of the double deletion mutant defective in BDSF and AHL signal production.
Bacterial growth conditions and virulence assays
Bacterial strains and plasmids used in this study
Strain or plasmid
Phenotypes and/or characteristics
Reference or source
Wild type strain H111, Genomovars III of the B. cepacia complex
Wild type strain harboring the expression construct pLAFR3-GGDEF
Wild type strain harboring the expression construct pMLS7-wspR
BDSF-minus mutant derived from H111 with rpfF Bc being deleted
Mutant ∆rpfFBc harboring the expression construct pLAFR3-EAL
Mutant ∆rpfFBc harboring the expression construct pMLS7-rocR
Mutant ∆rpfFBc harboring the expression construct pMLS7-wspR
Mutant ∆rpfFBc harboring the expression construct pMLS7-rpfFBc
Mutant ∆rpfFBc harboring the expression construct pMLS7-cepI
Mutant ∆rpfFBc harboring the expression construct PcepI-lacZ
Deletion mutant with rpfR being deleted
Mutant ∆rpfR harboring the expression construct pMLS7-rpfR
Mutant ∆rpfR harboring the expression construct pMLS7-rpfRAAL
Mutant ∆rpfR harboring the expression construct pMLS7-rpfRGGAAF
Deletion mutant with cepI being deleted
Mutant ∆cepI harboring the expression construct pMLS7-rpfFBc
Double deletion mutant with rpfF Bc and cepI being deleted
Mutant ∆rpfR harboring the expression construct PcepI-lacZ
Insertional mutant of BCAM0227 harboring the expression construct PcepI-lacZ
supE44 ∆lacU169(Φ80lacZ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 λpir
Food source of C. elegans
AHL reporter strain
Lab of Stephen K. Farrand
pMLS7 containing rpfF Bc
pMLS7 containing rpfR
pMLS7-rpfR harboring an E443A amino acid substitution
pMLS7-rpfR harboring a D318A and E319A amino acid substitution
pMLS7 containing cepI
pMLS7 containing wspR
pMLS7 containing rocR
pLAFR3 containing the encoding region of the GGDEF domain of RpfR
pLAFR3 containing the encoding region of the EAL domain of RpfR
pME2-lacZ containing promoter of cepI
Construction of in-frame deletion mutants and complementation strains
The cepI deletion mutant of B. cenocepacia strain H111 was used as the parental strain to generate the in-frame double deletion mutant of rpfF Bc and cepI, following the methods described previously . For complementation analysis, the coding region of WspR was amplified by PCR using the primers listed in Additional file 4: Table S1, and cloned under the control of the S7 ribosomal protein promoter in plasmid vector pMSL7. The resultant construct was conjugated into the rpfF Bc deletion mutant B. cenocepacia H111 using tri-parental mating with pRK2013 as the mobilizing plasmid.
Construction of reporter strains and measurement of β-galactosidase activity
The promoter of cepI was amplified using the primer pairs listed in Additional file 4: Table S1 with HindIII and XhoI restriction sites attached. The resulting products were digested with HindIII and XhoI, and ligated at the same enzyme sites in the vector pME2-lacZ . These constructs, verified by DNA sequencing, were introduced into B. cenocepacia H111 using tri-parental mating with pRK2013. Transconjugants were then selected on LB agar plates supplemented with ampicillin and tetracycline. Bacterial cells were grown at 37°C and harvested at different time points as indicated, and measurement of β-galactosidase activities was performed following the methods as described previously .
Biofilm formation, swarming motility and proteolytic activity assays
Biofilm formation in 96-well polypropylene microtiter dishes was assayed essentially as described previously . Swarming motility was determined on semi-solid agar (0.5%). Bacteria were inoculated into the center of plates containing 0.8% tryptone, 0.5% glucose, and 0.5% agar. The plates were incubated at 37°C for 18 h before measurement of the colony diameters. Protease assay was performed following the previously described method . Protease activity was obtained after normalization of absorbance against corresponding cell density.
Analysis of AHL signals
Bacterial cells were grown in NYG medium to a same cell density in the late growth phase. The supernatants were acidified to pH = 4.0 and extracted using ethyl acetate in a 1:1 ratio. Following evaporation of ethyl acetate the residues were dissolved in methanol. Quantification of AHL signals was performed using β-galactosidase assay with the aid of the AHL reporter strain CF11 as described previously . Briefly, the reporter strain was grown in minimal medium at 28°C with shaking at 220 rpm overnight. The cultures were inoculated in the same medium supplemented with extracts containing AHL signals. Bacterial cells were harvested and β-galactosidase activities were assayed as described in previous section. For TLC analysis, 5 μl of the concentrated AHL extracts were spotted onto 10 × 20 cm RP-18254 s plate (MERCK) and separated with methanol–water (60:40, v/v). The plates were subsequently air dried and overlaid with 50 ml minimal medium containing 0.8% agarose, 50 μg ml-1 X-gal, and 1 ml stocked CF11 culture. The plates were then incubated overnight at 28°C, and AHL is indicated by the presence of a blue spot.
Western blotting analysis
Bacterial cultures were grown in NYG medium overnight and inoculated in the same medium. The refreshed cultures were grown at 37°C to an OD600 of 4.5; and 1 ml of each bacterial culture was collected and centrifuged. The cells were lysed by adding 250 μl celLyticTM B cell Lysis Reagent (Sigma). The concentrations of total protein samples were measured and normalized. Then the samples were denatured by boiling for 10 min and separated by 10% SDS-PAGE. Western blot analysis was performed following the standard protocols .
The funding for this work was provided by the Biomedical Research Council, the Agency of Science, Technology and Research (A*Star), Singapore.
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