Anti-quorum Sensing Activities of Selected Coral Symbiotic Bacterial Extracts From the South China Sea

Abstract

The worldwide increase in antibiotic-resistant pathogens means that identification of alternative antibacterial drug targets and the subsequent development of new treatment strategies are urgently required. One such new target is the quorum sensing (QS) system. Coral microbial consortia harbor an enormous diversity of microbes, and are thus rich sources for isolating novel bioactive and pharmacologically valuable natural products. However, to date, the versatility of their bioactive compounds has not been broadly explored. In this study, about two hundred bacterial colonies were isolated from a coral species (Pocillopora damicornis) and screened for their ability to inhibit QS using the bioreporter strain Chromobacterium violaceum ATCC 12472. Approximately 15% (30 isolates) exhibited anti-QS activity, against the indicator strain. Among them, a typical Gram-positive bacterium, D11 (Staphylococcus hominis) was identified and its anti-QS activity was investigated. Confocal microscopy observations showed that the bacterial extract inhibited the biofilm formation of clinical isolates of wild-type P. aeruginosa PAO1 in a dose-dependent pattern. Chromatographic separation led to the isolation of a potent QS inhibitor that was identified by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy as DL-homocysteine thiolactone. Gene expression analyses using RT-PCR showed that strain D11 led to a significant down-regulation of QS regulatory genes (lasI, lasR, rhlI, and rhlR), as well as a virulence-related gene (lasB). From the chemical structure, the target compound (DL-homocysteine thiolactone) is an analog of the acyl-homoserine lactones (AHLs), and we presume that DL-homocysteine thiolactone outcompetes AHL in occupying the receptor and thereby inhibiting QS. Whole-genome sequence analysis of S. hominis D11 revealed the presence of predicted genes involved in the biosynthesis of homocysteine thiolactone. This study indicates that coral microbes are a resource bank for developing QS inhibitors and they will facilitate the discovery of new biotechnologically relevant compounds that could be used instead of traditional antibiotics.

Keywords: HPLC-MS-NMR; S. hominis; anti-quorum sensing; coral microbes; marine drug.

Figure 1

Screening of anti-QS strains on biosensor plates containing reference strain C. violaceum ATCC 12472 and filter paper for sample detection. Water, LB medium and furanone (diluted 10 times with DMSO) were used as blank, negative and positive controls, respectively. The absence of purple or formation of a pigment inhibition was considered to indicate a potential QS inhibitor. The red arrows refer to the positive anti-QS strains and the pigment inhibition can be observed on a clear background on the plate. Notes: the number indicated the test isolate strains. H samples come from Heilong Island, and D samples come from Daming Island.

Figure 2

(A) Bacterial cell count of the flask incubation assay. The five test isolates were D11 (Staphylococcus hominis), D35 (Staphylococcus warneri), H1 (Lysinibacillus fusiform), D12 (Bacillus cereus), and H12 (Vibrio alginolyticus). C. violaceum ATCC 12472 was incubated for 16 h, and 100 μl of the bacteria, adjusted to OD_600nm of 0.1 (approximately 1 × 10⁸ CFU/ml), were spread on LB plates. The growth inhibition were compared with control. Data are presented as the logarithm of mean CFU ± SD. (B) Inhibition of violacein production by test strains. Violacein production was measured spectrophotometrically as described in the Materials and Methods. Data are presented as mean ± SD of absorbance at 585 nm. Asterisks indicate a statistically difference between experimental groups and control groups (^*P < 0.05; ^**P < 0.01).

Figure 3

(A) Biofilm dispersal activity (crystal violet assay) of extract from isolate Staphylococcus hominis D11. Different concentrations of bacterial extract (1–10 μg/ml) were tested against the widely used biofilm-forming reference strain P. aeruginosa PAO1. Experiments with “extract + DMSO,” “lose QSI ability extract + DMSO,” “DMSO only,” and “no extract + no DMSO” were considered as test control, negative control (1), negative control (2), and blank control, respectively. Asterisks indicate a statistically significant difference (^*P < 0.05; ^**P < 0.01) between experimental groups and control groups. Data are presented as mean ± SD (n = 3). (B) Effect of anti-QS compounds on growth of P. aeruginosa PAO1. Bacteria were grown in LB media with (dotted line) and without (solid line) D11 strain extract (10 μg/ml). The extract did not affect specific bacterial growth rate or bacterial abundance. Flow cytometry results (inset picture) show the count of bacterial cells. Data are presented as mean ± SD (n = 3).

Figure 4

Confocal scanning laser microscopy (CLSM) z-stack 3-D images of P. aeruginosa PAO1 biofilm architecture in the presence (10 μg/ml)or absence (0 μg/ml) of D11 extract in media with 2% glucose. Data shown are early stage (12 h) biofilm structure of P. aeruginosa PAO1 in control group (A) and treatment group (B), and the post-stage (36 h) biofilm structure of P. aeruginosa PAO1 in control group (C) and experimental group (D). In these images, live bacterial cells produced green fluorescence, whereas dead cell sproduced red fluorescence.

Figure 5

Quantification of biofilm formation of P. aeruginosa PAO1 (taking 36 h as example) using COMSTAT software, including (A) bio-volume, (B) average thickness, and (C) roughness coefficient. Error bars indicate SD (n = 3). Asterisks indicate a statistically significant difference (^*P < 0.05; ^**P < 0.01) between experimental groups and control groups.

Figure 6

Analysis of active fraction showing anti-QS activity. (A) Pre-HPLC analysis of S. hominis D11 extract. The chromatogram shows the five main active peaks from S. hominis D11. The insert picture in (A) is the re-test of the anti-QS activity of the five peak compounds. (B) GC chromatograms and ESI-MS/MS of the active fraction (peak 2) of S. hominis D11 extract. Peaks are a function of intensity measured in milli-absorption units over time in minutes. (C) ¹H-NMR spectrum of compound in Methanol-d₄ at 400 MHz. (D) ¹³C-NMR spectrum of compound in Methanol-d₄ at 100 MHz. ¹H-NMR (Methanol-d₄, 400 MHz) δ:4.25 (1H, dd, J = 12.9, 7.0 Hz, H-2), 3.53 (1H, td, J = 11.6, 5.2 Hz, H-4), 3.45 (1H, ddd, J = 11.6, 7.2, 1.1 Hz, H-4), 2.80 (1H, dddd, J = 12.2, 6.7, 5.2, 1.4 Hz, H-3), 2.21 (1H, m, H-3). ¹³C-NMR (Methanol-d₄, 100 MHz) δ:204.0 (C-1), 59.3 (C-2), 30.6 (C-3), 28.6 (C-4). The insert picture in (D) is the structure of DL-homocysteine thiolactone (redrawn by ChemBioDraw Ultra 12.0).

Figure 7

Effect of DL-homocysteine thiolactone on violacein production by C. violaceum ATCC 12472. (A) Percentage of inhibition of the violacein pigment after incubation with different concentrations of DL-homocysteine thiolactone. A dose-response effect in the production of violacein was observed in a series of concentrations (0.0625, 0.125, 0.25, 0.5, and 1.0 μg/mL) of DL-homocysteine thiolactone. Data show the mean (±SD) of three independent experiments. (B) Anti-QS activity of pure DL-homocysteine thiolactone at different concentrations. A, 0.0625 μg/ml; B, 0.125 μg/ml; C, 0.25 μg/ml; D, 0.5 μg/ml; and E, 1.0 μg/ml. LB medium, DMSO only and furanone (dissolved in DMSO, 1.0 μg/ml) were used as blank, negative and positive controls, respectively.

Figure 8

Expression profiling of some anti-QS regulatory genes from P. aeruginosa PAO1 with D11 extract measured by real-time PCR. The mRNA expression of these genes in the absence of extract served as a control. Results are based on three independent experiments and error bars represent means ± SD (n = 3). Asterisks indicate a statistically significant difference (^*P < 0.05; ^**P < 0.01) between experimental groups and control groups.