Anti-Quorum Sensing Activity of the Traditional Chinese Herb, Phyllanthus amarus

The discovery of quorum sensing in Proteobacteria and its function in regulating virulence determinants makes it an attractive alternative towards attenuation of bacterial pathogens. In this study, crude extracts of Phyllanthus amarus Schumach. & Thonn, a traditional Chinese herb, were screened for their anti-quorum sensing properties through a series of bioassays. Only the methanolic extract of P. amarus exhibited anti-quorum sensing activity, whereby it interrupted the ability of Chromobacterium violaceum CVO26 to response towards exogenously supplied N-hexanoylhomoserine lactone and the extract reduced bioluminescence in E. coli [pSB401] and E. coli [pSB1075]. In addition to this, methanolic extract of P. amarus significantly inhibited selected quorum sensing-regulated virulence determinants of Pseudomonas aeruginosa PA01. Increasing concentrations of the methanolic extracts of P. amarus reduced swarming motility, pyocyanin production and P. aeruginosa PA01 lecA∷lux expression. Our data suggest that P. amarus could be useful for attenuating pathogens and hence, more local traditional herbs should be screened for its anti-quorum sensing properties as their active compounds may serve as promising anti-pathogenic drugs.


Introduction
The emergence of more multidrug resistant pathogens at an alarming rate is now a major concern to the public health care and policy makers in the medical industry. The continuous misuse of broad-spectrum antibiotics has caused bacteria to develop resistance hence leading towards a continuous decline in the availability of antibiotics that are able to work against these pathogens. Quorum sensing (QS) is a cell-cell signaling system whereby bacteria produce signalling molecules, termed autoinducers, that coordinate the production of cell density-dependent regulatory factors as well as the expression of virulence factors in pathogens [1,2]. Thus, targeting the QS system to attenuate bacterial virulence and pathogenicity is a promising alternative without the use of antibiotic. Proteobacteria primarily utilize the LuxI/LuxR type QS-signalling system where N-acyl-homoserine lactones (AHLs) are the signalling molecules whereas post-translationally modified peptides regulate QS systems in Gram-positive bacteria [3].
Phyllanthus amarus Schumach. & Thonn ( Figure 1) is a small woody shrub most commonly found growing in uncultivated or wastelands in tropical regions. P. amarus has been tested and shown to be anti-diabetic [16], hepatoprotective [17], anti-inflammatory [18] and chemoprotective [19], along with many other medicinal properties, but its anti-QS properties had not been tested. In this study, we report for the first time the anti-QS properties of P. amarus and its methanolic crude extract is shown to inhibit selected virulence factors of P. aeruginosa PAO1.

Identification of Plant Sample, Deposition of Voucher Specimen, and Extract Preparations
P. amarus samples were collected from wastelands around the grounds of University Malaya. Voucher specimens of P. amarus Schumach. & Thonn. (family: Phyllanthaceae) (KLU 47768) were deposited in the Herbarium of University of Malaya. Collected samples were washed with sterile distilled water and rinsed with 70% (v/v) ethanol prior to drying in a 40 °C oven for three days. Dried samples were ground using an industrial grade blender. Powdered plant samples were treated sequentially with organic solvents (hexane, chloroform and methanol) of increasing polarity (in a ratio of 1:10 w/v). Samples were soaked in each solvent for three days with shaking at 220 rpm. The extracts of each solvent were filtered using Whatman No.1 filter paper. This step was repeated twice to ensure all of the fine powder was completely removed before proceeding to the removal of solvents using a rotary evaporator (EYELA, Tokyo, Japan). The crude extracts were then dried in a fume hood and later placed in a desiccator. Stock solutions of 10 mg/mL were made using 100% DMSO (Merck KGaA, Darmstadt, Germany) and later diluted to (in mg/mL) 1, 2, 3, 4 and 5 using ultrapure water (Mili-Q, Merck KGaA, Darmstadt, Germany) prior to use. The remaining crude extracts were stored at −20 °C.

Bacterial Growth
To rule out the possibility of the plant extracts having antibacterial activity against the biosensor cells that might lead to a false anti-QS result, we first determined the antibacterial activity using a previously reported method [22] with some modifications. Briefly, the optical density at 600 nm (OD 600 ) of overnight grown cultures of C. violaceum CV026, P. aeruginosa PAO1, E. coli [pSB401] and E. coli [pSB1075] were adjusted to OD 600 of 0.1. Next, 230 µL of the bacterial culture and 20 µL of the plant extracts were placed in each well of the 96-well microtitre plate. The microtitre plates were incubated at the optimum temperature of the tested bacteria and the OD 600 were measured every 30 min for 24 h by using Tecan luminometer (Infinite M200, Mannerdorf, Switzerland).

QS Inhibition
Bioassay was performed as described by Tan et al. [13] with several modifications. Briefly, overnight CV026 culture (15 mL) was seeded into warm molten LB agar (200 mL) and supplemented with C6-HSL to a final concentration of 0.25 mg/mL. The mixture was swirled gently to ensure complete mixing before being poured into Petri dishes. Wells were made using sterile 1 µL white pipette tips. Next, 20 µL of plant extracts of various concentrations (1-5 mg/mL) were loaded in the wells and DMSO (10%-50% (v/v)) served as negative controls that corresponded to each of the plant extracts concentration used in the bioassay. The plates were then incubated overnight in an upright position at 28 °C. A ring of colourless and turbid (halo zone) growth on a purple background indicated anti-QS properties of the extracts [23].

Quantification of Bioluminescence
Bioluminescence was quantified using biosensor strains E. coli [pSB401] and E. coli [pSB1075], and inhibition of P. aeruginosa PAO1 lecA expression was performed using P. aeruginosa PAO1 lecA::lux. Luminescence from these biosensors were performed as previously described [24].

Pyocyanin Quantification Assay
Pyocyanin from P. aeruginosa PAO1 was quantified using methods as described previously [13,14,25]. Briefly, overnight grown culture of P. aeruginosa PAO1 were adjusted to OD 600 of 0.1. Next, plant extract (250 µL) was mixed well with PAO1 cultures (4.75 mL) and incubated at 37 °C for 18 h. To extract pyocyanin from the cultures (5 mL), the bacterial cultures were extracted with chloroform (3 mL) and vortexed vigorously. The chloroform layer was then transferred to a new, sterile polypropylene tube and re-extracted with 1 mL HCl (0.2 M). After centrifugation, the top layer (HCl) was removed and the absorbance was measured at 520 nm using UV-visible spectrophotometer (Cary 60 UV-Vis Spectrophotometer, Agilent, Santa Clara, CA, USA). DMSO of 10%-30% (v/v) were used as negative controls.

Swarming Assay
P. aeruginosa PAO1 swarming plates were prepared as follows: glucose (1% w/v), Bacto agar (0.5% w/v), Bacto peptone (0.5% w/v), and yeast extract (0.2% w/v) as described previously [14]. An overlay of 10 mL swarming agar was made prior to adding an additional layer of 5 mL swarming agar seeded with 250 µL of plant extract. Once the agar had solidified, 2 µL of overnight P. aeruginosa PAO1 was inoculated in the middle of the agar and incubated at 37 °C for 16 h. Impaired swarming motility of P. aeruginosa PAO1 indicates anti-QS properties of the plant extract.

Statistical Analysis
All the results represented were means ± standard deviations of three independent experiments carried out in triplicates. The significance of all the data was tested using Student's t-test (p < 0.05) using the GraphPad Prism software.

Screening of Plant Extracts Anti-QS Properties Using Chromobacterium Violaceum CV026
C. violaceum CV026 is a double Tn5 mutant that is only able to produce the purple pigment violacein in the presence of an exogenously supplied short chain AHL [20]. In this study, only the methanolic extract of P. amarus exhibited a notable halo zone on the purple CV026 lawn.
Hexane and chloroform extracts of P. amarus did not cause any observable halo zone (data not shown). Figure 2 shows that the formation of halo zone was observable at 1 mg/mL and increased in diameter as the concentration increased to 5 mg/mL. On the other hand, DMSO (negative control) did not show any bactericidal or anti-QS effect. Therefore, formation of halo zone indicated anti-QS activity of the methanolic extract.

Bacterial Growth
A bacterial growth curve (OD 600 ) was determined over a period of 24 h to rule out any antibacterial properties of the methanolic extracts of P. amarus that may inhibit growth of C. violaceum CV026 and P. aeruginosa PAO1. Figure 3 confirms that the methanolic extract did not show inhibition against the growth of both the bacterial strains used in this study although this plant has been shown to exhibit antibacterial activity by previously reported work [26].

Methanolic Extract of P. amarus Inhibited lux-Based Biosensors
Methanolic extract of P. amarus was further tested for its anti-QS properties against both long and short chain AHLs using lux-based biosensors, namely E. coli [pSB401] and E. coli [pSB1075], which respond to short and long chain AHLs, respectively, by producing bioluminescence [21]. The methanolic extract of the plant significantly reduced the luminescence produced by both the biosensor strains with increasing concentration as compared to DMSO (30% v/v, negative control). The methanolic extract of P. amarus was more effective at reducing the luminescence produced by E. coli [pSB401] as the inhibition was observable at the lowest concentration of 1 mg/mL ( Figure 4). As for E. coli [pSB1075], luminescence was greatly reduced starting at 3 mg/mL. 3.3.2. Inhibition of P. aeruginosa PAO1 lecA Expression P. aeruginosa produces a variety of exoproducts to sustain its survivability and maintain its virulence. Among the exoproducts released are two types of soluble carbohydrate-binding cytotoxic protein termed lectins, LecA (PA-IL) and LecB (PA-IIL) [27]. LecA have been studied to have cytotoxic effect towards respiratory cells and playing a role in biofilm formation [28,29]. In order to study the lecA expression of P. aeruginosa, lecA::luxCDABE reporter fusion was constructed by inserting luxCDABE from Photorhabdus luminescens into the lecA gene region of P. aeruginosa [6]. Figure 5 shows that the methanolic extract of P. amarus reduced PAO1 lecA expression with increasing concentration. However, inhibition of lecA expression was significant at 3 mg/mL indicating that higher concentration was required for inhibition.
According to Winzer and co-workers, lecA expression is tightly regulated by the RhlR/C4-HSL QS system. Furthermore, PAO1 mutant rpoS impaired lectin synthesis, adding that both RpoS and Rhl/C4-HSL are needed for LecA synthesis [6]. This indicates that the methanolic extract of P. amarus may have anti-QS effects on the RhlR/C4-HSL QS-system or on the expression of rpoS. However, more work needs to be done to confirm this hypothesis. Figure 5. The inhibition of P. aeruginosa PAO1 lecA expression at concentration of 3 mg/mL. Statistical analysis was done using Student's t-test with p-value < 0.05 being significant (noted with *).

Swarming Assay
Swarming is an uncoordinated surface-associated motility of hyperflagellated and highly motile bacterial cells which functions to colonise a niche [30,31]. In this assay, increasing concentration of methanolic extract of P. amarus inhibited swarming of P. aeruginosa. Swarming inhibition was observable at the lowest concentration of 1 mg/mL (Figure 6c). Swarming of P. aeruginosa was proven to be QS-system mediated whereby lasI/lasR mutants reduced and delayed swarming adding to rhlI/rhlR mutants that completely diminished swarming ability of the bacterium [32]. Rhamnolipid, a lipopeptide biosurfactant produced by P. aeruginosa, plays a crucial role in swarming motility and is QS-regulated [4,33]. Thus, compounds in the plant extract may have acted by interrupting the QS-systems or caused dysregulation during the synthesis of rhamnolipid. Based on earlier studies, swarming characteristics had been identified in other prokaryotes such as Serratia liquefaciens, Escherichia coli, and Salmonella typhimurium [34][35][36]. This suggests that swarming plays a role in bacterial colonisation in an environment or host [33].  (c) (d) (e)

Pocyanin Assay
Pyocyanin is a blue-redox reactive toxic exoproduct produced by P. aeruginosa [7]. Methanolic extract of P. amarus significantly reduced pyocyanin production with increasing concentration as compared to DMSO that served as negative control (Figure 7).
P. aeruginosa was found to be the predominant opportunistic pathogen that colonizes the airways of cystic fibrosis (CF) patients and a primary causative agent in sepsis of burned and immunocompromised patients [7]. Pyocyanin contributes to the persistence of P. aeruginosa infection by causing detrimental effects toward lung epithelial cells and by dysregulating inflammatory response initiated by the host [37,38]. Pyocyanin synthesis is regulated by a complex synchrony of lasR-lasI, rhlR-rhlI, and mvfR-haqQS-system whereby mutations in these systems led to the deficiency of pyocyanin synthesis [4,9,39]. Compounds in the extract may have inhibited the QS-systems at its gene expression level or act as an antagonist against AHL signalling molecules.

Conclusions
Based on the results obtained from this study, it is proven that the methanolic extract of P. amarus possesses anti-QS capabilities as well as being able to attenuate QS-regulated virulence determinants 10% DMSO 1 mg/mL P. amarus *** 20% DMSO 2 mg/mL P.amarus *** 30% DMSO 3 mg/mL P.amarus *** Pyocyanin (OD 520) of P. aeruginosa POA1. Active compounds in the extract may provide a new insight towards discovering potential anti-pathogenic drugs to combat emerging multidrug resistant pathogens. Future work should direct to the isolation and characterisation of active molecule that is responsible for the anti-QS properties of the methanolic extract of P. amarus.