Catechol-derived Inhibitors against Quorum Sensing from Retiboletus kauffmanii

Liman ZHOU, Lin WANG, Fandong KONG, Qingyun MA, Qingyi XIE, Haofu DAI, Youxing ZHAO*

1. Haikou Key Laboratory for Research and Utilization of Tropical Natural Products, Institute of Tropical Bioscience and Biotechnology, CATAS, Haikou 571101, China; 2. Hainan Academy of Tropical Agricultural Resource, CATAS, Haikou 571101, China

Abstract [Objectives] This study aimed to screen anti-quorum sensing (QS) activity from the constituent of Retiboletus kauffmanii fruit body. [Methods] Chromatographic technology was used to isolate compounds, while well diffusion assay was applied to screen anti-quorum sensing activity. [Results] 12 phenolic compounds were purified from extracts, three catechol-derived compounds at sub-inhibitory concentrations were found to inhibit the production of violacein in Chromobacterium violaceum for the first time. [Conclusions] Three catechol analogs isolated from R. kauffmanii fruit body extract were indicated as quorum sensing inhibitors against C. violaceum.

Key words Chromobacterium violaceum, Quorum sensing inhibitor, Retiboletus kauffmanii, Virulence factors

1 Introduction

Quorum sensing (QS) systems take charge of exchanging chemical signals in bacterial populations to adjust the bacterial phenotypes according to the density of bacterial cells[1]. QS signal molecules are vividly termed as "the language of bacteria" and the QS systems widely exist in many kinds of bacteria. They regulate bacterium movement, formation of biofilm, toxicity factors produce and many other pathogenicity correlation biological phenomenons. QS allows bacteria to act as a community, and thus express phenotypes which are beneficial for the group[2]. Quorum sensing inhibitors (QSIs) are supposed to provide an attractive way of combating bacterial infections and antibiotic resistance problem because they are thought to exert a reduced pressure to selective drug-resistant strains[3]. Secondary metabolites of macrofungi fruiting body are important sources of natural products which have new structure and biological activity[4-8]. Therefore, it is necessary to search for new QSIs from macrofungi fruiting body extracts which are rich source of bioactive compounds. Boleaceae is a family of suborders of basidiomycetes, and most of them are edible[9].Retiboletuskauffmaniibelongs to the boletus family and is widely distributed throughout China[10]. There are few studies on the pharmacological activities of the chemical constituents in boletus. So an effort was made to study the biological activities of the chemical components inR.kauffmanii.

Chromobacteriumviolaceumis an opportunistic human pathogen, which can infect humans and cause abscesses and bacteremia[11]. What’s more,C.violaceummay cause relapses and it is multidrug-resistant[12]. Its quorum-sensing system consists of the LuxI/LuxR homologue[13]. In this work, we studied the anti-QS activity againstC.violaceumofR.kauffmaniifruit body extract, obtained using 95% ethanol. As a result, three compounds were identified to have the anti-QS property and these compounds are structurally similar, containing the structure of 1, 2-benzenediol. Hence, catechol-derived natural compounds as novel QSIs are found inC.violaceumfor the first time.

2 Materials and methods

2.1 MaterialsThe strain used in this study isC.violaceumCV026, which is a violacein-negative,cviImini-Tn5 mutant ofC.violaceum. Unless otherwise stated, the strain was grown on LB broth and incubated at 30 ℃.

The body ofR.kauffmaniiwas collected in Nanhua, Yunnan Province, the People’s Republic of China, and authenticed by professor Zeng Niankai, from Hainan Medical College.

2.2 Methods

2.2.1Well-diffusion assays. The well diffusion assay ofC.violaceumCV026 was used according to the previous studies that tested anti-QS activity[14-16]. Inhibition of QS inC.violaceumwill show the reduce of purple pigmentation around the wells which contain the anti-QS compounds.

2.2.2Growth curve analysis based on concentrations. The effect of these compounds on proliferation ofC.violaceumCV026 was evaluated by monitoring the growth curve. The bacteria were cultured overnight and diluted (Optical density,OD600≈0.05), which was then divided into 96-well plates after the addition of compounds at different concentrations. The cultures in 96-well plates were incubated with shaking at 120 rpm. Optical densities of bacterial cultures were measured at 600 nm after 12 h. TheODunits were converted into the corresponding cell dry weight values (DCWs).

2.2.3Quantitative analysis of virulence productivity. Violacein production was induced inC.violaceumCV026 by adding C6-HSL and quantified as previously described[17-19]. Violacein total output was quantified by detecting theOD585value in DMSO. Bacteria cells harvested by centrifugation were resuspended in 1 mL LB broth to monitor cell growth by measuringOD600value. The ratio ofOD585toOD600values was used to evaluate the violacein productivity.

3 Result and analysis

Violacein production inC.violaceumCV026 responding to C6-HSL is under control of QS system, soC.violaceumCV026 is usually used as a direct-viewing QSIs screening strain[14, 20]. In the presence of QSIs, the opaque and colorless zones would be observed around the wells. There were 12 phenolic compounds separated from the crude extracts ofR.kauffmaniifruit body (Fig.1). Of these 12 compounds, three compounds, caffeic acid methyl ester (3), 3, 4-dihydroxy- acetophenone (9) and protocatechuic acid (10), significantly decreased the violacein production inC.violaceumCV026 and showed anti-QS activity (Fig.2).

Note: The compounds with tag (√) showed anti-QS activity in Chromobacterium violaceum.Fig.1 Isolated compounds from Retiboletus kauffmanii fruit body extract

Note: The final concentration of 50 μg kanamycin/mL was added. C30 was used as the positive control at 10 μg (1 mg/mL, 10 μL). To calibrate this screening system, 10 μL of methanol was used as the negative control. Compounds 1-12 were at 50 μg (5 mg/mL, 10 μL ).Fig.2 Effect of compounds isolated from Retiboletus kauffmanii fruit body extract on violacein production in Chromobacterium violaceum CV026

Most of the QSIs could inhibit QS and associated pathogenesis at subinhibitory concentrations[15, 21]. To eliminate false positives from the plate assay, liquid activity detection with the compounds whose concentrations under the MIC should be performed. MIC was defined as the lowest concentration of compounds at which no visible growth of the test strain could be observed[22]. The MIC of these compounds 3, 9, 10 against CV026 were about 6.4 mM, 12.8 mM and more than 25.6 mM, respectively. Growth curve analysis confirmed that CV026 growth kinetics were nearly unaffected by these three compounds at concentrations ranging from 100 to 600 μM, respectively (Fig.3).

Note: The OD units were converted to DCW by an OD-DCW correlation: 1 OD600=0.49 g DCW/l. All of the data are presented as mean±SD (n=3).Fig.3 Effect of the compounds at different concentrations on the growth curve of CV026

Compared with the control cultures, 150 μM and 300 μM of these compounds could reduce violacein in C6-HSL-inducedC.violaceumCV026 cultures (Fig.4). Among them, the inhibitory activity of compound 9 was strongest and it could reduce violacein productivity at 300 μM by 68.89%. Compounds 3 and 10 reduced violacein productivity at 300 μM by up to 47.05 and 11.88%, respectively. So we further confirmed that these compounds did not inhibit growth and only inhibited violacein productivity. In other words, these three compounds could inhibit the quorum sensing system inC.violaceum.

Note: All of the data are presented as mean±SD (n=3). *P<0.01, **P<0.001.Fig.4 Violacein production and growth by Chromobacterium violaceum CV026 with and without C6-HSL at a final concentration of 500 nM with compounds at different concentrations

Overall, three catechol-derived compounds separated from

R.kauffmaniifruit body extract exhibited the anti-QS activity againstC.violaceum. It was interesting to note that they were very similar in structure. All of them contain the structure of 1, 2-benzenediol, namely catechol. Compared to the other nine phenolic compounds, the structure of these three compounds told us that catechol in structure was essential for the anti-QS activity. Interesting, in our experiment catechol didn’t show anti-QS activity againstC.violaceum(data not shown). It might be due to the catechol metabolism in bacteria, for example, the ortho-cleavage pathway mediated by catechol 1, 2-dioxygenase (C12O) and the meta-cleavage pathway mediated by catechol 2, 3-dioxygenase (C23O)[23-24]. The ring cleavage products from the catechol metabolism would be transformed into tricarboxylic acid (TCA) cycle[25]. Catechol might be metabolized but some catechol analogs could not. It had been reported before that pyrogallol and its analogs could antagonize bacterial quorum sensing inVibrioharveyiwhich had an AI-2 quorum sensing system[26]. Pyrogallol and its analogs were structurally analogs of catechol, too. Our data indicated that catechol analogs might serve as basis for developing therapeutic molecules that could effectively inhibit QS, and demonstrated the utility of catechol-derived compounds for mitigating bacterial virulence.

4 Conclusions

Small molecular natural products are the main source of quorum sensing inhibitor discovery. The screening of active components from the fungusR.kauffmaniiled to the isolation of 12 phenolic compounds. Three catechol-derived compounds, caffeic acid methyl ester, 3,4-dihydroxy-acetophenone and protocatechuic acid, were found to inhibit the production of violacein inC.violaceumfor the first time, indicating these catechol analogs maybe considered to develop as quorum sensing inhibitors againstC.violaceum.