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Journal of Bacteriology, June 2007, p. 4320-4324, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.00003-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Control of Quorum Sensing by a Burkholderia pseudomallei Multidrug Efflux Pump{triangledown}

Ying Ying Chan,1 Hao Sheng Bian,1 Theresa May Chin Tan,1 Margrith E. Mattmann,2 Grant D. Geske,2 Jun Igarashi,3 Tomomi Hatano,3 Hiroaki Suga,3 Helen E. Blackwell,2 and Kim Lee Chua1*

Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore,1 Department of Chemistry, University of Wisconsin—Madison, 1101 University Ave., Madison, Wisconsin 53706-1322,2 Chemical Biology and Biotechnology Lab, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan3

Received 2 January 2007/ Accepted 13 March 2007


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ABSTRACT
 
The Burkholderia pseudomallei KHW quorum-sensing systems produced N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxy)-octanoyl-homoserine lactone, N-(3-hydroxy)-decanoyl-homoserine lactone, N-(3-oxo)-decanoyl-homoserine lactone, and N-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the B. pseudomallei BpeAB-OprB efflux pump.


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TEXT
 
Burkholderia pseudomallei, a gram-negative soil bacillus, is the causative agent of melioidosis, a severe and potentially fatal emerging tropical infectious disease. Its intrinsic resistance to many antibiotics is attributed mostly to multiple multidrug efflux pumps, such as AmrAB-OprA, BpeAB-OprB, and BpeEF-OprC, which efflux aminoglycosides, macrolides, chloramphenicol, and trimethoprim (4, 12, 14). We hypothesized that these pumps also efflux physiological compounds that could compromise the fitness of the bacterium when accumulated to high intracellular concentrations and propose that this exerts a positive selection pressure for persistence even in the absence of antimicrobials (11).

B. pseudomallei is reported to produce up to six different types of acyl-homoserine lactones (acyl-HSLs), the composition of which may differ slightly from strain to strain. For instance, strain PP844 secreted N-octanoyl-homoserine lactone (C8HSL), N-(3-hydroxy)-octanoyl-homoserine lactone (3-hydroxy-C8HSL), N-(3-oxo)-octanoyl-homoserine lactone (3-oxo-C8HSL), N-decanoyl-homoserine lactone (C10HSL), N-(3-hydroxy)-decanoyl-homoserine lactone (3-hydroxy-C10HSL), and N-(3-hydroxy)-dodecanoyl-homoserine lactone (3-hydroxy-C12HSL), while strain DD503, an amrAB-oprA efflux pump operon deletion mutant derived from 1026b, secreted only C8HSL, 3-hydroxy-C8HSL, and C10HSL (13, 17-19). It is unclear how acyl-HSLs move across the B. pseudomallei cell membranes and into the extracellular compartment, although in Pseudomonas aeruginosa, the shorter-chain acyl-HSLs appear to do so by diffusion while the longer-chain acyl-HSLs are secreted by multidrug efflux pumps (1, 15). In B. pseudomallei KHW, the expression of bpeAB-lacZ was induced by acyl-HSLs, and the bpeAB mutant failed to secrete any extracellular acyl-HSLs when it was cross-streaked against the JB525 reporter strain (3).

Extracellular secretion of acyl-HSLs is dependent on BpeAB-OprB. In order to ascertain whether a blockage in the efflux mechanism, an inhibition of acyl-HSL synthesis, or both had contributed to the absence of extracellular acyl-HSLs in the bpeAB mutant, we compared the acyl-HSLs produced in cell supernatants of B. pseudomallei before and after the bacterial cells were permeabilized by a freeze-thaw procedure. Wild-type B. pseudomallei KHW, KHW carrying a plasmid overexpressing the BpeR repressor, the bpeAB null mutant, and a complemented bpeAB mutant were used in the comparison (Table 1), and a modified cross-streak bioassay was used for detection of acyl-HSLs (2). The culture supernatants of unpermeabilized wild-type KHW and the complemented bpeAB mutant contained acyl-HSLs but not those of the bpeAB mutant and the bpeR-overexpressing strain, both of which had impaired BpeAB-OprB function (Fig. 1, lanes U). Acyl-HSLs were detected in the cell supernatants of the bpeAB mutant and the bpeR-overexpressing strain only after the bacterial cells were permeabilized by five cycles of freeze-thaw (–80°C for 3 min and 37°C for 1 min). This demonstrates that the bpeAB mutant and the bpeR-overexpressing strain were defective in the extracellular secretion, but not intracellular synthesis, of acyl-HSLs (Fig. 1, lanes P). It was not possible to ascertain from the experiment whether the synthesis of a specific acyl-HSL(s) might be affected, and the bioassay was also not informative about the relative abundance of acyl-HSLs in the wild-type and bpeAB mutant cells.


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  		TABLE 1. Bacterial strains used in this study


Figure 1
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  		FIG. 1. Detection of extracellular and intracellular acyl-HSLs produced by B. pseudomallei with the E. coli JB525 bioassay. Overnight (16-h) cultures of B. pseudomallei in 5 ml of AB medium (6) containing 0.2% glucose were centrifuged at 3,000 rpm for 15 min. Secreted acyl-HSLs were detected by adding 50 µl of the cell supernatant to the wells labeled U on LB agar. The cell pellets, each resuspended in 1 ml of fresh AB medium, were freeze-thawed (five cycles at –80°C for 3 min and 37°C for 1 min) to permeabilize the cells, and 50 µl of the cell extract was then added to the adjacent wells, labeled P. A gradient of acyl-HSLs diffusing from the wells after incubation at 30°C for 20 h was detected as green fluorescence by a row of 2-µl spots of E. coli JB525 when illuminated under UV. The direction of diffusion from the well is indicated by the arrow. E. coli DH5{alpha} was included as a negative control.

BpeAB-OprB is required for efflux but not for influx of acyl-HSLs during quorum sensing. The consensus is that longer-chain acyl-HSL autoinducers produced by gram-negative bacteria, such as N-(3-oxo)-dodecanoyl-homoserine lactone (3-oxo-C12HSL) in P. aeruginosa, probably require active export, while short-chain acyl-HSLs, such as N-butanoyl-homoserine lactone (C4HSL) diffuse freely across the cell membrane (15). Early-log-phase cultures of wild-type B. pseudomallei KHW and the bpeAB mutant were exposed to exogenous [14C]acyl-HSLs, and the intracellular levels of [14C]acyl-HSLs were measured at 30, 60, and 90 min thereafter. After 60 min, the amounts of intracellular [14C]acyl-HSLs were ~2.5-fold higher for the bpeAB null mutant and wild-type cells that carried a plasmid overexpressing the BpeR repressor than for the wild type (Fig. 2). This observation was similar to the accumulation [14C]erythromycin in the bpeAB mutant, which we reported previously (4). The bpeR null mutant that overexpressed bpeAB also accumulated less [14C]acyl-HSLs than did the bpeAB mutant but to levels similar to those of the wild-type (Fig. 2). We attribute the similar levels of accumulation of [14C]acyl-HSLs in the wild type and bpeR mutant to the possibility that bpeAB gene expression may not be linearly correlated with BpeAB-OprB pump activity (Fig. 2). Previous data comparing the susceptibilities of wild-type KHW and the bpeR null mutant to antimicrobial agents had shown an increase of 16-fold in the MIC of gentamicin but of only 2-fold in the MIC of streptomycin in the bpeR null mutant compared to the wild type, although both antibiotics were substrates of BpeAB-OprB (3). It is also possible that, after 60 min of incubation, some of the accumulated 14C-labeled product in the bpeAB mutant and the bpeR-overexpressing strain might have degraded, and the radiolabel detected could have included metabolites of acyl-HSLs that were also dependent on BpeAB-OprB efflux. BpeAB-OprB seemed to be required specifically for the efflux of acyl-HSLs but had no effect on the reentry of acyl-HSLs into the bacterial cell during quorum sensing, during which they are most likely to enter the cells by passive diffusion.


Figure 2
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  		FIG. 2. Intracellular accumulation of exogenous [14C]acyl-HSLs. Radiolabeled [14C]acyl-HSLs were prepared by adding 5 µCi of L-[1-14C]methionine (Sigma) to a 16-h culture of B. pseudomallei in AB medium containing 20 mM glycerol for 30 min. [14C]acyl-HSLs were then extracted from the culture supernatants by acidified ethyl acetate, according to a procedure described previously (16). Two hundred microliters of the [14C]acyl-HSLs was added to early-log-phase cells (optical density at 600 nm, ~0.2), and 0.2-ml aliquots were removed after 30, 60, and 90 min for scintillation counting. The cells were washed three times with saline containing 100 nM C8HSL to remove extracellular radiolabeled acyl-HSLs, air dried, and solubilized in 2 ml of scintillation cocktail (Amersham Biosciences) for liquid scintillation counting with an LS6500 multipurpose scintillation counter (Beckman Instruments Inc., Fullerton, CA). White bars, wild-type B. pseudomallei KHW; black bars, KHW {Delta}bpeAB mutant; stippled bars, KHW bpeR::Km null mutant; gray bars, KHW(pUCP28TbpeR).

Involvement of BpeAB-OprB in the efflux of acyl-HSLs. Using reversed-phase high-performance liquid chromatography (HPLC) to profile acyl-HSLs extracted from the culture supernatants of the wild type and the bpeAB null mutant with acidified ethyl acetate, it was possible to ascertain which acyl-HSL(s) was effluxed by BpeAB-OprB. Seven major peaks were detected in both the cell culture supernatants and freeze-thawed extracts of the wild-type cells (Fig. 3A and B, respectively). Peaks 1, 2, 3, 4, 5, 6, and 7 were identified as L-methionine, C8HSL, C10HSL, 3-hydroxy-C8HSL, N-(3-oxo)-decanoyl-homoserine lactone (3-oxo-C10HSL), 3-hydroxy-C10HSL, and N-(3-oxo)-tetradecanoyl-homoserine lactone (3-oxo-C14HSL), respectively, after comparisons of retention times with those of synthetic acyl-HSL standards and methionine. The compounds eluted in peaks 2 to 7 were also confirmed as acyl-HSLs by applying 50 µl of each fraction in the modified cross-streak bioassay described earlier (data not shown). Strain KHW differed from strain DD503 in the secretion of 3-oxo-C10HSL, 3-hydroxy-C10HSL, and 3-oxo-C14HSL, in addition to the three acyl-HSLs also secreted by DD503 (18). Among these, 3-oxo-C10HSL was unique to KHW. As the AmrAB-OprA efflux pump was defective in DD503, the extracellular secretion of 3-oxo-C10HSL by KHW would suggest that it might be effluxed by AmrAB-OprA, which coincidentally shares similar antimicrobial substrates with BpeAB-OprB (4, 14). Another B. pseudomallei strain, PP844, was also reported to secrete six acyl-HSLs when cultured in tryptic soy broth, although it differed from KHW in its production of 3-oxo-C8HSL and 3-hydroxy-C12HSL, both of which were not detected in KHW and DD503 (13). Although this conclusion is speculative, it is possible that the synthesis of acyl-HSLs, which are derived from methionine and acyl-chain donors, may be affected by culture conditions, a possibility that should be investigated.


Figure 3
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  		FIG. 3. Comparison of intracellular and extracellular acyl-HSLs produced by wild-type B. pseudomallei KHW and the bpeAB null mutant by reversed-phase HPLC. Radiolabeled [14C]acyl-HSLs was extracted from 5-ml cultures of KHW, KHW {Delta}bpeAB mutant, and KHW(pUCP28TbpeR), as described in the legend to Fig. 2. The extract was dried and dissolved in 200 µl of 20% (vol/vol) methanol before the application of 100 µl into a C18 reversed-phase HPLC column. Two-milliliter fractions were collected during elution at a flow rate of 1 ml/min, with an isocratic profile of methanol-water (50:50, vol/vol) for 10 min, followed by a linear gradient of 50 to 90% methanol in water for 25 min and an isocratic profile of 90% methanol over 10 min. (A) [14C]acyl-HSLs secreted into the culture supernatants of untreated stationary-phase bacterial cultures. (B) [14C]acyl-HSLs extracted from freeze-thaw-permeabilized bacterial cells. Peaks 1, 2, 3, 4, 5, 6, and 7 were detected with a Radiomatic 500TR Radioisotope HPLC detector (Perkin-Elmer) and correspond to the elution profiles of L-methionine, C8HSL, C10HSL, 3-hydroxy-C8HSL, 3-oxo-C10HSL, 3-hydroxy-C10HSL, and 3-oxo-C14HSL, respectively. The HPLC profiles of acyl-HSLs extracted from culture supernatants of wild-type KHW and its bpeAB deletion mutant are represented by closed and open squares, respectively. C8HSL, C10HSL, and methionine were purchased from Sigma, while 3-oxo-C10HSL and 3-oxo-C14HSL were synthesized by a microwave-assisted solid-phase synthetic route to acyl-homoserine lactones (8) and 3-hydroxy-C8HSL and 3-hydroxy-C10HSL were synthesized by the Reformatsky reaction (9).

In contrast to wild-type B. pseudomallei KHW, none of the acyl-HSL species was detected in the culture supernatant of the bpeAB mutant, which corroborates the data from the cross-streak bioassays (Fig. 3A). Only C8HSL—none of the other five acyl-HSLs—was detected in the cell extracts of the permeabilized bpeAB mutant (Fig. 3B). The intracellular level of C8HSL, which was reduced in the bpeAB mutant compared to the wild type, may be attributed to a negative impact on the synthesis of C8HSL by BpsI as a result of the lack of quorum sensing in the bpeAB mutant. This is supported by our previous data, which also showed a significant reduction in the expression of bpsI-lacZ in the bpeAB mutant compared to the wild type (3). As the bpeAB mutant synthesized only C8HSL, this would suggest that the BpsIR (PmlIR) quorum-sensing system probably controls the expression of the other bpmIR systems, which produce C10HSL, 3-hydroxy-C8HSL, 3-oxo-C10HSL, 3-hydroxy-C10HSL, and 3-oxo-C14HSL. It might also be inferred that inhibition of the BpeAB-OprB efflux pump would be beneficial, because it enhances the antimicrobial properties of aminoglycoside and macrolide antibiotics and attenuates B. pseudomallei virulence by blocking quorum sensing. BpeAB-OprB is expressed in 24 of the 26 B. pseudomallei isolates that we have tested (data not shown).

Effect of BpeR on the efflux of acyl-HSLs. The bpeR null mutant, which overexpressed bpeAB, differed from the wild type in that it secreted only C8HSL, C10HSL, and 3-hydroxy-C8HSL (Fig. 4A). The absence of intracellular 3-hydroxy-C10HSL, 3-oxo-C10HSL, and 3-oxo-C14HSL in the bpeR mutant would suggest that an optimal level of BpeR repressor, probably via its regulation of bpeAB expression, was necessary to control the synthesis of these acyl-HSLs. The exact mechanism by which BpeR regulates expression of bpeAB-oprB, and whether it might also regulate the expression of another resistance-nodulation-division pump, such as AmrAB-OprA, which we had earlier alluded to in the secretion of 3-oxo-C10HSL, is still unclear. Basal expression of bpeAB was increased in the bpeR null mutant but could still increase further in the presence of erythromycin, a BpeAB-OprB substrate. It is not clear how the BpeR-dependent and BpeR-independent mechanisms interact with each other (3).


Figure 4
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  		FIG. 4. Comparison of reversed-phase HPLC profiles of intracellular and extracellular acyl-HSLs produced by the B. pseudomallei bpeR null mutant and wild-type KHW carrying a plasmid overexpressing the BpeR repressor. (A) [14C]acyl-HSLs extracted from supernatants of untreated stationary-phase bacterial cultures. (B) [14C]acyl-HSLs extracted from bacterial cells permeabilized by repeated freeze-thawing in fresh AB medium. Peak positions 1, 2, 3, 4, 5, 6, and 7 correspond to the elution profiles of methionine, C8HSL, C10HSL, 3-hydroxy-C8HSL, 3-oxo-C10HSL, 3-hydroxy-C10HSL, and 3-oxo-C14HSL, respectively. Both the bpeR null mutant and the wild-type KHW carrying a plasmid overexpressing the BpeR repressor failed to synthesize any 3-oxo-C14HSL (peak 7). The HPLC profiles of acyl-HSLs extracted from culture supernatants of KHW bpeR and KHW(pUCP28TbpeR) are represented by black circles and gray circles, respectively.

Wild-type KHW carrying a plasmid that overexpresses the BpeR repressor showed significantly reduced bpeAB transcription and enhanced susceptibility to gentamicin, streptomycin, and erythromycin, thus suggesting a reduced BpeAB-OprB efflux function in these cells (3). Therefore, the detection of 3-oxo-C10HSL and 3-hydroxy-C10HSL in the supernatants of these stationary-phase cultures was intriguing (Fig. 4A). From the observation that B. pseudomallei DD503 secreted 3-hydroxy-C10HSL only when pmlI, bpmI2, or bpmI3 was overexpressed and 3-oxo-C14HSL only when bpmI2 or bpmI3 was overexpressed, it is plausible that the BpeR repressor might have had an indirect influence on the expression of the autoinducer synthases responsible for syntheses of these two acyl-HSLs. As B. pseudomallei KHW containing a plasmid that overexpressed the BpeR repressor was highly attenuated in cytotoxicity and cell invasion, produced less siderophores and phospholipase C, and was impaired in biofilm formation compared to the wild type, it may be inferred that 3-oxo-C10HSL and 3-hydroxy-C10HSL are probably not essential for the expression of B. pseudomallei virulence and virulence-associated factors (3).

As in the bpeAB mutant, KHW carrying a plasmid that overproduces BpeR also synthesized less C8HSL than did the wild type, but it failed to produce any C10HSL, 3-hydroxy-C8HSL, or 3-oxo-C14HSL (Fig. 4B). This again lends support to the suggestion that the BpsIR system, which produces C8HSL, may be a key player in B. pseudomallei quorum sensing. While the roles of efflux pumps in the export of acyl-HSLs in P. aeruginosa have been described previously, the situation in B. pseudomallei appears to be unique because of the absolute requirement of BpeAB-OprB for extracellular secretion of all the different acyl-HSLs. We have thus identified a nonantibiotic physiological substrate of a multidrug efflux pump that is relevant to B. pseudomallei virulence. The consequence of defective BpeAB-OprB function is the complete inhibition of quorum sensing in B. pseudomallei, resulting in dramatic attenuation in virulence (3). Although the focus of this study was the BpeAB-OprB pump, it would also be prudent to study the roles of the other B. pseudomallei resistance-nodulation-division efflux pumps in the efflux of acyl-HSLs. In P. aeruginosa, reduced synthesis of 3-oxo-C12HSL and N-butanoyl-homoserine lactone (C4HSL), respectively, was associated with overexpression of the MexAB-OprM and MexEF-OprN efflux pumps (7, 10), while reduced production of quorum-sensing-regulated products was associated with an impaired MexGHI-OpmD pump (1). In contrast, the mechanism by which acyl-HSLs enter cells remains unknown, although it is widely believed to occur by passive diffusion.

Inhibition of BpeAB-OprB would be therapeutically beneficial, because it enhances the susceptibility of B. pseudomallei to the commonly used aminoglycoside and macrolide antibiotics and attenuates virulence by preventing quorum sensing. We have previously shown that the addition of C8HSL and C10HSL to mammalian cell cultures was able to restore the invasiveness and cytotoxicity of the bpeAB mutant to wild-type levels (3). Future work should include the identification of inhibitors and quenchers of quorum sensing, as well as inhibitors of BpeAB-OprB, and their evaluation as potential therapeutic agents against B. pseudomallei and other clinically relevant gram-negative bacterial pathogens.


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ACKNOWLEDGMENTS
 
We are grateful to Lian-hui Zhang for helpful discussions.

This work was funded by grants from the National University of Singapore Academic Research Fund (R183-000-111-112) and the National Medical Research Council (R183-000-165-213). H.E.B. acknowledges the NIH (AI063326-01), the Shaw Award Program of the Greater Milwaukee Foundation, the Burroughs Wellcome Fund, and Johnson and Johnson for support of acyl-HSL synthesis in her laboratory. H.E.B. is a research fellow of the Alfred P. Sloan Foundation. G.D.G. acknowledges support through an ACS Division of Medicinal Chemistry Graduate Fellowship (2005-2006).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore. Phone: (65) 65163684. Fax: (65) 67791453. E-mail: bchckl{at}nus.edu.sg Back

{triangledown} Published ahead of print on 23 March 2007. Back


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Journal of Bacteriology, June 2007, p. 4320-4324, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.00003-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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