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Antimicrobial Agents and Chemotherapy, February 2009, p. 580-586, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00995-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Ambuic Acid Inhibits the Biosynthesis of Cyclic Peptide Quormones in Gram-Positive Bacteria

Jiro Nakayama,^1* Yumi Uemura,¹ Kenzo Nishiguchi,¹ Norito Yoshimura,¹ Yasuhiro Igarashi,² and Kenji Sonomoto^1,3

Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan,¹ Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan,² Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan³

Received 25 July 2008/ Returned for modification 1 October 2008/ Accepted 12 November 2008

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Quorum sensing is a cell-density-dependent regulatory system in gram-positive bacteria and is often regulated by cyclic peptides called "quormones," which function as extracellular communication signals. With an aim to discover an antipathogenic agent targeting quorum sensing in gram-positive bacteria, we screened 153 samples of fungal butanol extracts with the guidance of the inhibition of quorum-sensing-mediated gelatinase production in Enterococcus faecalis. Following the screenings, we found that ambuic acid, a known secondary fungal metabolite, inhibited the quorum-sensing-mediated gelatinase production without influencing the growth of E. faecalis. We further demonstrated that ambuic acid targeted the biosynthesis of a cyclic peptide quormone called gelatinase biosynthesis-activating pheromone. Furthermore, ambuic acid also inhibited the biosynthesis of the cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. These results suggest the potential use of ambuic acid as a lead compound of antipathogenic drugs that target the quorum-sensing-mediated virulence expression of gram-positive bacteria.

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Quorum sensing is a common system present in unicellular microorganisms that controls the expression of certain target genes in a cell-density-dependent manner (38). In order to orchestrate this cell-to-cell communication, bacteria often use signal molecules, occasionally called "autoinducers," "pheromones," or "quormones" (we have called them "quormones" in this study). Many gram-positive bacteria employ peptides for quormones, while gram-negative bacteria use nonpeptidic compounds such as N-acylhomoserine lactones in most cases (14, 38). Cyclic thiolactone and lactone peptides are commonly found quormones that are involved in quorum sensing in some gram-positive species (10, 33).

The agr system in staphylococci is the best-studied cyclic peptide-mediated quorum-sensing system (5, 20). This system employs 7- to 9-residue cyclic peptide quormones, called autoinducing peptide (AIP), in which the cysteine residue at the fifth position from the C terminus forms a thiolactone along with the C-terminal -carboxyl group (5). A quormone propeptide is translated from agrD and is processed and cyclized with the aid of a biosynthetic enzyme, namely, AgrB (29). The mature quormone accumulated outside the cell triggers a two-component regulatory system through the activation of AgrC, which is a membrane histidine kinase (4). Eventually, AgrA, which is the response regulator, is phosphorylated and promotes the expression of RNA III, which encodes delta-hemolysin and also acts as a regulatory RNA molecule that controls the gene expression of staphylococcal virulence factors, e.g., hemolysins and toxic shock syndrome toxins (20). The four components, namely, AgrA, AgrB, AgrC, and AgrD, which are involved in agr quorum sensing, are encoded in a gene cluster organized as agrBDCA (20).

An ortholog of an agr-like gene cluster was found in the genome sequences of some gram-positive bacteria including those belonging to the genera Listeria, Clostridium, and Bacillus, suggesting the widespread presence of cyclic-peptide-mediated quorum sensing among gram-positive bacteria (17, 29). For example, a "Lactobacillus agr-like gene module" (lam) was found in the genomic sequence of Lactobacillus plantarum WCFS1 (34). The structure of a putative cyclic peptide quormone was elucidated to be a 5-residue cyclic peptide in which the sulfhydryl group of the N-terminal cysteine residue formed a thiolactone bridge with the C-terminal -carboxyl group (34). The putative cyclic peptide quormones LsrD698 and LsrD826 have been identified from Listeria innocua and are also suspected to function as quormones in Listeria monocytogenes because of the DNA sequence identity between these two species (19).

It is also known that Enterococcus faecalis possesses an fsr quorum-sensing system mediated by an 11-residue cyclic peptide carrying a lactone bridge between the hydroxyl group of serine residue at the third position and the C-terminal -carboxyl group (15, 16, 27, 28). This cyclic peptide quormone secreted from each cell triggers the expression of two extracellular pathogenicity-related proteases, namely, gelatinase and serine protease, and was named gelatinase biosynthesis-activating pheromone (GBAP) (15). Although the molecular mechanism of GBAP biosynthesis has been suggested to be similar to that of the agr quormone, the organization of open reading frames involved in GBAP biosynthesis is somewhat different. It has been demonstrated that the GBAP propeptide is translated from fsrD, which is located in frame in the 5'-end part of fsrB, and that FsrB', an FsrD segment-truncated FsrB, is functional as a cysteine protease-like processing enzyme to generate GBAP from FsrD (17).

Quorum sensing has recently been considered to be an emerging target for antimicrobial drug therapy (2, 6, 39). Compounds targeting quorum sensing can attenuate virulence without displaying any bactericidal or bacteriostatic activity and are proposed to be used as "antipathogenic drugs" (7, 22). For example, macrolides such as azithromycin, which inhibit N-acylhomoserine lactone-mediated quorum sensing but not the growth of Pseudomonas aeruginosa, are known to efficiently alleviate the symptoms of cystic fibrosis and diffuse panbronchiolitis (35-37). Furthermore, several other studies have already revealed the efficacy of inhibitors targeting the N-acylhomoserine lactone-mediated quorum sensing of gram-negative bacteria (7, 23, 30-32).

In the case of gram-positive pathogens, inhibitors of quorum sensing have been investigated mainly with regard to the staphylococcal agr system (6, 13, 20). Lyon et al. successfully developed a rational design of a peptide antagonist of the agr quormone (12, 13). A heptapeptide named RNA III-inhibiting peptide, which is found in the culture filtrates of some staphylococcal strains, and its synthetic analogs are also expected to be antistaphylococcal agents (1). Recently, 3-oxo-C₁₂-homoserine lactone was reported to have successfully inhibited the agr quorum-sensing system (26). It has also been reported that the peptide inhibitor P+1, a proline-containing mimic peptide of the AgrD processing region, inhibited the removal of the N-terminal leader of the quormone peptide, which resulted in the blockage of the agr quorum-sensing system (8).

In order to discover compounds that inhibit E. faecalis quorum sensing, we have recently established a screening system for inhibitors targeting the GBAP-mediated quorum-sensing system, called the fsr system, and found that a peptide antibiotic, namely, siamycin, efficiently attenuates the fsr quorum-sensing system in sublethal concentrations (18). In the present study, we screened secondary fungal metabolites using the same screening system and found that ambuic acid, a known antifungal compound, inhibits the fsr quorum-sensing system through the inhibition of GBAP biosynthesis. We also demonstrated that ambuic acid inhibits the biosynthesis of cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. This suggests the potential application of ambuic acid as an antipathogenic compound to target the quorum-sensing system of gram-positive bacteria.

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Bacterial strains, media, and culture conditions. E. faecalis strain OG1RF was used as a standard gelatinase-positive strain (3). E. faecalis OU510 is a GBAP-negative but GBAP-sensitive strain used for the GBAP assay (18). All E. faecalis strains were cultured in Todd-Hewitt broth (THB) medium (Oxoid, Hampshire, United Kingdom) at 37°C with gentle agitation. The OU510 strain (pQU2200 and pQU2301) was cultured in the presence of chloramphenicol (20 µg/ml) and erythromycin (50 µg/ml). Staphylococcus aureus 12600^T and S. aureus 8325-4(pSB2035) (25) were cultured aerobically in TSB-YE medium (30 g of tryptic soy broth and 6 g of yeast extract per liter) at 30°C or Luria-Bertani broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter) containing 7 µg/ml of chloramphenicol at 37°C, respectively. Listeria innocua ATCC 33090^T was cultured in a chemically defined medium (9) at 30°C with shaking.

Culture of fungi. Fifty-one fungal strains were cultured for the screening. These strains were obtained from the Biotechnology Research Center at Toyama Prefectural University. Each strain was incubated by static culture at 25°C for approximately 1 month in 3 different media, namely, A-3 M (21), potato dextrose agar (39 g/liter) (Difco, Detroit, MI), and M-1-D medium (24). The culture broths were mixed with equal volumes of butanol and shaken for 2 h at room temperature. After centrifugation at 6,500 x g for 20 min, 1 ml of butanol was collected and evaporated to dryness in vacuo. The extract was stored at –20°C in a freezer until the screening assay. For the screenings, the butanol extract (1 ml) was dissolved in 25 µl of methanol, and 5 µl of this solution was added to 0.5 ml of fresh THB medium for the assays of gelatinase production or GBAP production.

For the large-scale production of ambuic acid, the fungal strain KAP-21 was incubated by static culture in A-3 M medium for approximately 1 month.

First screening (assay for inhibition of gelatinase production in E. faecalis OG1RF). The assay for the inhibition of gelatinase production in E. faecalis was performed using a method described previously (18). A culture (3 µl) of E. faecalis OG1RF grown overnight was inoculated into 0.5 ml of THB medium containing the tested sample and was cultured for 5 h at 37°C. The culture supernatant was then collected after centrifugation at 9,100 x g for 5 min, and 25 µl of this culture supernatant was subjected to the gelatinase assay using azocoll as a substrate as described previously (15, 18). Briefly, 25 µl of E. faecalis culture supernatant was added to 0.5 ml of azocoll suspension, incubated for 4 h with constant mixing (170 rpm), and centrifuged at 20,000 x g for 5 min, and the optical density at 540 nm (OD₅₄₀) of the supernatant was then measured.

Second screening (assay for the inhibition of GBAP production in E. faecalis OG1RF). The assay for the inhibition of GBAP production in E. faecalis was performed using a method described previously (18). A culture (3 µl) of E. faecalis OG1RF grown overnight was inoculated into 0.5 ml of fresh THB medium containing the tested sample. After 5 h of cultivation, the cells were removed by centrifugation at 9,100 x g for 5 min; this was followed by filtration through a cellulose acetate filter (0.2 µm) (DISMIC-13CP; Advantec Toyo, Tokyo, Japan), and 100 µl of the filtrate was added to 400 µl of fresh THB medium. A culture (3 µl) of E. faecalis OU510 grown overnight was inoculated into the conditioned medium and cultured for 5 h. The culture supernatant was then subjected to the gelatinase assay, and the GBAP activity was represented by the induced gelatinase activity (OD₅₄₀).

Purification of ambuic acid. The butanol extract of the KAP-21 culture (2 liters) was dissolved in 50 ml of 20% (vol/vol) acetonitrile and then applied onto a Sep-Pak Vac C₁₈ cartridge column (35 ml, 10 g; Waters, Milford, MA). After washing with 100 ml of 20% acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid (TFA), the column was eluted with 100 ml of 40%, 60%, and 80% acetonitrile containing 0.1% TFA. The fraction of 60% acetonitrile was evaporated, lyophilized, and redissolved in 3 ml of 20% acetonitrile containing 0.1% TFA. The solution was divided into six aliquots, and each aliquot was subjected to reverse-phase high-performance liquid chromatography (Inertsil ODS-3, 20 by 150 mm; GL Sciences Inc., Tokyo, Japan). The column was washed with 20% acetonitrile in 0.1% TFA for 5 min at 10 ml/min and then developed by a gradient of 20% to 80% acetonitrile in 0.1% TFA for 30 min at the same flow rate. A single peak obtained at 27 min was collected, evaporated, and lyophilized. Finally, 3.2 mg of ambuic acid was obtained in the form of white powder and was confirmed to possess inhibitory activity against gelatinase production discussed in the above-mentioned assay.

Mass spectrometry. The high-performance liquid chromatography-purified fraction of ambuic acid was loaded onto an electrospray ionization-time of flight mass spectrometer (Accutof T100LC; Jeol, Tokyo, Japan), and the following observations were recorded: the spectrum was obtained in the positive-polarity mode, the capillary temperature was 260°C, the needle voltage was 2.0 kV, the orifice voltage was 70 V, and the ring lens voltage was 10 V. A series of molecular ions were observed at m/z 315 (M – 2H₂O + H)⁺, 333 (M – H₂O + H)⁺, 351 (M + H)⁺, 701 (2 M + H)⁺, and 1,051 (3 M + H)⁺.

NMR spectroscopy. Nuclear magnetic resonance (NMR) spectra were measured using a Bruker Avance 500 spectrometer at 25°C. The purified ambuic acid (1.1 mg) was dissolved in 490 µl of deuterated methanol. The chemical shifts were referenced to the residual solvent signals (_H = 3.31; _C = 49.8).

Effect of ambuic acid on GBAP signaling. To examine the effects of ambuic acid on GBAP signaling, E. faecalis OU510 was cultured in the presence of GBAP and ambuic acid. A culture of E. faecalis OU510 (3 µl) grown overnight was inoculated into 0.5 ml THB containing 100 µM ambuic acid, and synthetic GBAP was added at various concentrations; the bacteria were cultured for 5 h. The culture supernatant was then subjected to the gelatinase assay.

Effect of ambuic acid on FsrD processing. Hexahistidine-tagged DNA was translationally fused to the 3' end of fsrD by PCR by using primers 8048-f (5'-CTGCAGGCATGCGGTAC-3'), 8048-rhis6 (5'-CCTAAAAAGAATATTGAAAAACATCATCATCATCATCATTGAGACTAGTC-3'), and pQU2300 (17) as a template. The PCR product was digested by SpeI and was then self-ligated using a DNA ligation kit (Takara, Kyoto, Japan). The resultant plasmid, pQU2301, was introduced into strain OU510, which already contains plasmid pQU2200 (17), by electroporation, and a transformant was selected on THB agar medium containing erythromycin (50 µg/ml) and chloramphenicol (20 µg/ml).

In order to examine the inhibitory effects of ambuic acid on GBAP biosynthesis, a culture of OU510(pQU2200 + pQU2301) (6 µl) grown overnight was inoculated into 1.0 ml THB containing ambuic acid at various concentrations. After 2 h, 1.0 µl of nisin (25 µg/ml; Sigma) was added to the culture, and 3 h later, the cells and culture supernatant were separated after centrifugation at 9,100 x g for 5 min. The GBAP activity in the culture supernatant was measured as described above. The cells were boiled in 25 µl of a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer (0.1 M Tris-HCl [pH 6.8] containing 24% [wt/vol] glycerol, 8% [wt/vol] SDS, 0.2 M dithiothreitol, and 0.02% [wt/vol] bromophenol blue) and were then centrifuged at 20,000 x g for 5 min. The supernatant (20 µl) was subjected to Tris-Tricine SDS-PAGE on a 15% polyacrylamide gel. After running, the gel was subjected to Western blot analysis using an ECL Plus Western blotting detection system (GE Healthcare, Buckinghamshire, United Kingdom) using an anti-histidine tag antibody (penta-His-horseradish peroxidase conjugate; Qiagen, Hilden, Germany). An identical gel was stained with Coomassie blue R250 to ensure that equal amounts of total protein were loaded into each lane (data not shown).

Inhibitory effect of ambuic acid on agr expression in S. aureus. agr expression was quantified by using a dual reporter strain, S. aureus 8325-4(pSB2035), as described previously (25). Cultures of S. aureus 8325-4(pSB2035) grown overnight were diluted 1/20 into fresh medium after cells were washed with equal volumes of fresh medium and grown for 2 h before the culture was again diluted 1/20 into fresh medium and grown for a further 2 h. Finally, these bacterial cultures were diluted 1/50 into 200 µl of fresh medium containing no or various concentrations of ambuic acid and cultured in a 96-well titer plate with shaking. After 7 h, the OD₆₂₀ was measured by use of a microtiter plate reader (Immuno Mini NJ-2300; Nihon InterMed, Tokyo, Japan). The cells were then harvested by centrifugation at 13,000 x g for 2 min, washed twice in an equal volume of phosphate-buffered saline (PBS), and then suspended with 600 µl of PBS. The green fluorescent protein (GFP) fluorescence of each sample was measured by use of a fluorescence spectrophotometer (excitation at 490 nm and emission at 525 nm) (F-7000; Hitachi Hightechnologies). In order to examine direct effect of ambuic acid on GFP fluorescence, after harvesting cells cultured without ambuic acid, 200 µM of ambuic acid was added to the washed cells in PBS, and the fluorescence was monitored for 15 min.

LC/MS. In order to monitor the biosynthesis of cyclic peptide quormones, the culture supernatant of each bacterial strain was analyzed by liquid chromatography/mass spectrometry (LC/MS). A culture of S. aureus 12600^T (20 µl) grown overnight was inoculated into 2 ml of TSB-YE medium and cultured for 15 h. After cells were removed by centrifugation at 13,000 x g for 5 min, the culture supernatant was filtered using a 0.22-µm filter, and 40 µl of the filtrate was applied onto an LC/MS system (Agilent HP1100 liquid chromatograph, Agilent Zorbax Eclipse XDB-C₁₈ 2.1- by 50-mm column, and Jeol Accutof T100LC mass spectrometer). The column was eluted at a flow rate of 0.2 ml/min at 30°C with a linear gradient of acetonitrile (20% to 40% in 20 min after 5 min of 20%) in 0.05% aqueous TFA solution. The eluates were directly loaded into the electrospray ionization-time of flight mass spectrometer. Mass analyses were performed under the following conditions: positive polarity, capillary temperature of 260°C, needle voltage of 2.0 kV, orifice voltage of 70 V, and ring lens voltage of 10 V. After scanning for molecular ions derived from column eluates in the m/z range of 100 to 2,000, an extracted ion chromatogram was plotted with detector counts in the mass range m/z 961 to 962.

A culture of Listeria innocua (25 µl) grown overnight was inoculated into 5 ml of chemically defined medium and cultured for 15 h. After the cells were removed by centrifugation at 12,000 x g for 5 min, the culture supernatant was filtered, and the culture filtrate was loaded onto a Sep-Pak Plus C₁₈ cartridge column (360 mg; Waters), washed with 5 ml of 10% acetonitrile containing 0.1% TFA, and then eluted with 2 ml of 60% acetonitrile containing 0.1% TFA. The eluate was evaporated to dryness and then redissolved in 100 µl of 30% acetonitrile, and 40 µl of this solution was applied for LC/MS as mentioned above, with the exception that the gradient was from 10% to 36% acetonitrile in 0.05% aqueous TFA solution for 40 min. The monitor m/z ranged from 699 to 700 and from 827 to 828.

Inhibitory effect of ambuic acid on hemolysin production in S. aureus. Various concentrations of ambuic acid in 50% acetonitrile (5 µl) were spotted onto a sheep blood agar plate (Kohjinbio, Osaka, Japan), and the solvent was dried under atmospheric pressure. The culture of S. aureus 12600^T grown overnight was then diluted a thousandfold with fresh TSB-YE medium, and 0.2 µl of this diluted solution was spotted onto the agar plate. The plate was incubated at 37°C, and the zone of clearance surrounding the spots was observed and photographed after 16 h, 24 h, and 48 h.

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Screening of inhibitors targeting fsr quorum sensing from fungal culture supernatants. Fifty-one strains from our fungal culture collection were cultured in three different media. The butanol extracts of 153 cultures were subjected to the first and second screenings. In the first screening, the standard gelatinase-producing strain, namely, E. faecalis OG1RF, was cultured in the medium containing the fungal butanol extract, and the gelatinase production of the strain was examined. Fourteen extracts showed significant inhibitory effects on gelatinase production without a strong inhibition of bacterial growth. In the second screening, E. faecalis OG1RF was cultured in a similar manner. GBAP activity in the culture supernatant was examined using E. faecalis OU510 as an indicator strain; this strain lacks GBAP biosynthesis but can produce gelatinase in response to GBAP. All 14 samples showed inhibitory effects on GBAP production. A butanol extract of an unidentified fungus, KAP-21, cultured in A-3 M medium showed clear and stable inhibitory activity and was eventually selected for further studies.

Identification of KAP-21A. The inhibitor produced by KAP-21, temporarily termed KAP-21A, was purified and subjected to structural analysis. Mass spectrometric results indicated that the molecular mass of KAP-21A was 350 kDa. ¹³C NMR spectra with a proton-decoupling pulse sequence or distortionless enhancement by polarization transfer sequence at a pulse angle of 135° indicated two methyl, six methylene, five methine, and six quaternary carbon atoms. According to chemical shifts in these spectra, the presence of four hydroxy methines, two olefinic carbons, one ketone, and one carboxyl group was suggested. Thus, we estimated the molecular formula to be C₁₉H₂₇O₇, which corresponded to a molecular mass of 368 kDa. The difference between the estimated molecular mass and that observed from the mass spectrum suggested one dehydration and the molecular formula to be C₁₉H₂₆O₆. In addition to the one-dimensional NMR spectra, a series of two-dimensional NMR analyses, namely, ¹H-¹H double-quantum-filtered correlation spectroscopy, ¹H-¹H total correlation spectroscopy, ¹³C-¹H heteronuclear single quantum coherence spectroscopy, and ¹³C-¹H heteromolecular multiple bond connectivity (HMBC), were performed. By analysis of these spectra coupled with the database search in SciFinder Scholar (American Chemical Society, Washington, DC) using the molecular formula, KAP-21A was revealed to be ambuic acid, which is a known antifungal compound. The results of proton and ¹³C assignment are shown in Table 1. The chemical shifts of all protons were almost identical to those of ambuic acid (11), and the resonances observed in the series of two-dimensional NMR experiments agreed with the chemical structure of ambuic acid (Fig. 1).

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TABLE 1. Chemical shifts of 13C and 1H and HMBC of KAP-21A (ambuic acid)

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FIG. 1. Structure of KAP-21A (ambuic acid) (11).

Mode of action of ambuic acid. Fig. 2 shows the effect of various concentrations of purified ambuic acid on the growth and gelatinase production of E. faecalis OG1RF. Ambuic acid had a 50% inhibitory concentration of approximately 10 µM and inhibited the production of gelatinase, but it did not show a marked inhibitory effect on the growth of E. faecalis OG1RF in the tested concentration range lower than 1 mM.

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FIG. 2. Effect of various concentrations of ambuic acid on the cell growth and gelatinase production of E. faecalis OG1RF. E. faecalis OG1RF was grown for 5 h in the presence of ambuic acid at the indicated concentrations; the cell density in the culture supernatant was measured at the OD660 (open circles), and the gelatinase activity was measured at the OD540 (closed circles), as described in Materials and Methods. The data are presented as averages ± standard deviations of experiments performed in duplicate.

In order to examine the effect of ambuic acid on GBAP signaling, GBAP-negative strain OU510 was cultured with GBAP at various concentrations and in the presence or absence of ambuic acid, and the induced gelatinase activity was measured (Fig. 3). The gelatinase induction was not found to have been inhibited by ambuic acid. This suggests that ambuic acid does not inhibit GBAP signaling but rather inhibits GBAP biosynthesis.

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FIG. 3. Effect of ambuic acid (100 µM) on gelatinase production in E. faecalis OU510 induced by GBAP at various concentrations. E. faecalis OU510 was grown for 5 h in the presence of the indicated concentrations of synthetic GBAP with (open circles) or without (closed circles) 100 µM ambuic acid. The gelatinase activity (OD540) and growth (OD660) in the culture supernatant were then measured in duplicate experiments. The value of gelatinase activity/growth was calculated for each experiment, and the average values were plotted. Standard deviations in all experiments were less than 0.1.

The effect of ambuic acid on GBAP biosynthesis was further investigated at the peptide level. Hexahistidine-tagged FsrD (His₆-FsrD) was expressed with or without FsrB'. In the absence of FsrB', His₆-FsrD was detected by the Western blotting technique using an anti-histidine tag antibody, while the His₆-FsrD band disappeared upon expression with FsrB' (Fig. 4A). This suggested that the proteolytic processing of His₆-FsrD was performed by FsrB'. However, when cultured with ambuic acid, this proteolytic processing as well as the GBAP activity were clearly inhibited (Fig. 4B).

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FIG. 4. Effect of ambuic acid on processing of FsrD by FsrB' (A) and GBAP production (B). E. faecalis OU510(pQU2200+pQU2301) (indicated as B' + D) or E. faecalis OU510(pQU2301) (indicated as D) was cultured in the absence or presence of ambuic acid (A) After cultivation for 5 h (induction by nisin for 3 h), the cells were collected and boiled in an SDS-PAGE sample buffer, and the cell extracts were analyzed by Western blot analysis using anti-histidine tag antibody. (B) The cell growth (OD660) (white bar) and GBAP activity in the culture supernatant (OD540) (black bar) were also measured.

Inhibitory effect of ambuic acid on agr expression in S. aureus. RNA III is the pivotal effector molecule in the agr regulon acting primarily at the level of gene transcription. pSB2035 is a dual-reporter plasmid carrying the GFP gene (gfp) and the bacterial luciferase gene (lux) under an RNA III promoter (P3), allowing the in vivo measurement of agr expression (25). When pSB2035 was introduced into an agr⁺ strain, bioluminescence and fluorescence were observed in response to endogenous quormone (25). By using the reporter strain S. aureus 8325-4(pSB2035), the effect of ambuic acid on agr quorum sensing was investigated. Ambuic acid did not show a significant direct inhibitory effect on GFP fluorescence (Fig. 5, inset). Ambuic acid inhibited expressions of both GFP (Fig. 5) and luciferase (data not shown) at the same inhibitory concentrations as those observed for E. faecalis quorum sensing. This suggested that ambuic acid was also effective on agr quorum sensing in S. aureus.

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FIG. 5. Effect of ambuic acid on agr expression in S. aureus 8325-4(pSB2035). The strain was grown in the absence or presence of ambuic acid. After 7 h, the OD620 and GFP fluorescence (fluorescence at 490 nm) were measured. The experiment was done in triplicate, and averages were graphed with standard deviations. In order to examine the direct effect of ambuic acid on GFP activity, after harvesting cells that were cultured without ambuic acid, 200 µM ambuic acid was added to the washed cells in PBS, and the fluorescence was then monitored for 15 min (inset).

Inhibitory effect of ambuic acid on biosynthesis of cyclic peptide quormone of other gram-positive bacteria. In order to examine if the agr inhibition of ambuic acid was caused by the inhibition of quormone biosynthesis, quormone production was investigated in S. aureus cells cultured with ambuic acid. The cyclic peptide quormone of S. aureus 12600^T, namely, AIP-I, was clearly detected by LC/MS analysis of the culture supernatant. However, the peak that corresponded to AIP-I disappeared when this strain was cultured with 50 µM ambuic acid (Fig. 6A). This result indicated that ambuic acid inhibited the biosynthesis of AIP-1 in S. aureus.

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FIG. 6. Effect of ambuic acid on production of AIP-I in S. aureus 12600T (A) and LsrD698 and LsrD826 in Listeria innocua ATCC 33090T (B). (A) S. aureus 12600T was grown for 15 h, and the culture filtrate was applied for LC/MS analysis. The liquid chromatography eluate was monitored at m/z 961, which corresponds to the molecular ion of AIP-I (B). Listeria innocua ATCC 33090T was grown for 15 h, and the partially purified culture filtrate was applied for LC/MS analysis. The liquid chromatography eluate was monitored at m/z 699 and m/z 827, which corresponded to the molecular ions of LsrD698 and LsrD826, respectively.

The effect of ambuic acid was also examined with regard to quormone biosynthesis in Listeria. In the culture supernatant of Listeria innocua ATCC 33090^T, two peaks corresponding to putative cyclic peptide quormones, namely, LsrD698 and LsrD826, were detected at m/z 699 and 827, respectively. When cultured in the presence of 100 µM ambuic acid, these two peaks disappeared, suggesting that ambuic acid also inhibits the biosynthesis of cyclic peptide quormones in Listeria innocua (Fig. 6B).

Influence of ambuic acid on hemolysin production in S. aureus. It is well known that hemolysin production is regulated by the agr quorum-sensing system in staphylococci. Figure 7 shows the different concentrations of ambuic acid solution that were spotted onto sheep blood agar, and hemolysin-producing S. aureus 12600^T was then inoculated onto each site. In the control, at 16 h after inoculation, a hemolytic zone (Fig. 7, white ring) around the bacterial colony was observed. The area of the hemolytic zone became smaller with an increase in the concentration of ambuic acid, and almost no hemolytic zone was observed on the site spotted with 5 µl of 20 mM ambuic acid, while the size of the colony did not differ from that of the control. The inhibitory effect of ambuic acid gradually decreased with time until 48 h, as shown in Fig. 7.

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FIG. 7. Effect of ambuic acid on hemolysin production in S. aureus 12600T. Five microliters of ambuic acid at the indicated concentrations was spotted onto a sheep blood agar plate, and after drying, a culture (0.2 µl) of S. aureus 12600T grown overnight and diluted a thousandfold was spotted. The plate was incubated at 37°C for the indicated period.

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In our previous screening study, we had found siamycin, an actinomycete secondary metabolite, to be a potent inhibitor of fsr quorum sensing (18). The fact that siamycin inhibited gelatinase production in E. faecalis even in the presence of high GBAP concentrations suggested that siamycin targets GBAP signaling. In contrast, the inhibitory effect of ambuic acid on gelatinase production was eliminated by the addition of GBAP at a physiological concentration. This suggests that ambuic acid targets GBAP biosynthesis.

Our previous genetic study suggested that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme required for the biosynthesis of GBAP from FsrD (17). Indeed, as shown in Fig. 4A, FsrD processing as well as GBAP activity were observed when fsrB' and fsrD were coexpressed homologously in GBAP-negative strain OU510. However, in the presence of ambuic acid, the production of GBAP as well as FsrD processing were inhibited. This suggests that ambuic acid targets the posttranslational process of GBAP biosynthesis.

FsrB' showed significant amino acid sequence similarity to staphylococcal AgrB (17, 28). Previous papers (8, 29) demonstrated that AgrB is involved in the processing at the C-terminal end of mature AIP in AgrD and that the processing at the N-terminal end is performed by type I signal peptidase. A search-based examination of the amino acid sequence of AgrB revealed a number of molecules similar to AgrB that were encoded in the genomes of gram-positive bacteria (17, 27, 40). This suggests that cyclic peptide quormones are commonly biosynthesized in a variety of gram-positive bacteria to regulate gene expression in a cell-density-dependent fashion. This study demonstrated that ambuic acid inhibited the production of staphylococcal AIP and putative cyclic peptide quormones of Listeria innocua. This suggests that ambuic acid targets a common point in the biosynthesis process of cyclic peptide quormone. With this type of mode of action, ambuic acid could be a lead compound of antipathogenic drugs targeting cyclic peptide quormone-mediated quorum sensing in a wide range of gram-positive bacteria. Indeed, ambuic acid inhibited not only GBAP-mediated gelatinase biosynthesis in E. faecalis but also AIP-mediated hemolysin production in S. aureus. However, even at high concentrations, the inhibitory effect was not substantial or sustainable. It would be necessary and possible to develop more potent inhibitors based on ambuic acid as a lead compound.

 
This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (grant no. 17580068 and 19658034); the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant no. 16087203); the Kato Memorial Bioscience Foundation; and the Waksman Foundation of Japan.

We thank Paul Williams at University of Nottingham for providing S. aureus 8325-4(pSB2035).

 
* Corresponding author. Mailing address: Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Phone: 81-92-642-3020. Fax: 81-92-642-3021. E-mail: nakayama{at}agr.kyushu-u.ac.jp

Published ahead of print on 17 November 2008.

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Antimicrobial Agents and Chemotherapy, February 2009, p. 580-586, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00995-08
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