Inhibition of Quorum Sensing and Virulence in Serratia marcescens by

Jan 4, 2019 - School of Environmental and Biological Engineering, Nanjing University of Science and Technology , Nanjing , Jiangsu 210094 , People's ...
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Agricultural and Environmental Chemistry

Inhibition of quorum sensing and virulence in Serratia marcescens by hordenine Jin-Wei Zhou, Ling-Yu Ruan, Hong-Juan Chen, Huai-Zhi Luo, Huan Jiang, Junsong Wang, and Ai-Qun Jia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05922 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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Inhibition of Quorum Sensing and Virulence in Serratia

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marcescens by Hordenine

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Jin-Wei Zhou,†,‡,§ Ling-Yu Ruan,‡,§ Hong-Juan Chen,ξ,§ Huai-Zhi Luo,†,‡ Huan

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Jiang,†,‡ Jun-Song Wang,*,‡ Ai-Qun Jia*,†,‡

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7

Laboratory of Tropical Biological Resources of Ministry Education, Hainan

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University, Haikou 570228, China;

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State Key Laboratory of Marine Resource Utilization in South China Sea, Key

School of Environmental and Biological Engineering, Nanjing University of Science

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and Technology, Nanjing 210094, China;

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ξ

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210023, China.

State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing

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ABSTRACT

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Serratia marcescens NJ01 is a pathogenic bacterium isolated from the diseased tomato

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leaves. Here, we report on the development of a tomato-S. marcescens host-pathogen

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system as a model to evaluate the effects of hordenine on quorum sensing (QS)-

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mediated pathogenicity under native conditions. Exposure to hordenine at 25, 50, and

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100 μg/mL significantly inhibited the production of acyl-homoserine lactones and the

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formation of biofilms. Hordenine treatment notably enhanced the susceptibility of the

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preformed biofilms to ciprofloxacin by reducing the production of extracellular

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polysaccharides (EPS), destroying the architecture of biofilms, and changing the

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permeability of membranes, as evidenced by the scattered appearance and dominant

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red fluorescence in the combination-treated biofilms. Furthermore, the addition of

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hordenine affected the production of virulence factors, influenced the intracellular

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metabolites, and down-regulated the expressions of QS- and biofilm-related genes. The

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plant infection model indicated that hordenine could significantly attenuate the

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pathogenicity of S. marcescens NJ01 in tomato plants. Thus, hordenine could act as a

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potential pesticide or pesticide accelerant in treating crop infections.

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KEYWORDS: hordenine, Serratia marcescens, quorum sensing, biofilm, virulence

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Tomato (Lycopersicon esculentum) is one of the most popular vegetables worldwide

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due to its excellent nutrients, outstanding processing quantities, and global distribution

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and consumption.1 In 2011, global tomato production reached ~160 million tons.2

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However, tomato yield can be severely affected by various crop pathogens, with

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pathogenic bacteria posing serious threat.3 The most common bacterial diseases of

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tomato crops are bacterial wilt and bacterial spot, both of which are caused by Gram-

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negative bacteria.3 Serratia marcescens is a pathogenic bacterium that is widespread in

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water, vegetable plants, food products, and medical devices, and thus causes an

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increasing number of crop infections and foodborne illness.4, 5 Studies have indicated

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that S. marcescens is one of the main pathogenic bacteria causing vegetable yellow vine

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disease, which causes inflicts foliar yellowing, wilting, and even vegetable decline.5

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Synthetic pesticides are the most widely used method for defending against bacterial

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crop diseases. However, their continued application has led to adverse impacts on

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human and environmental health, and the development of resistance in pathogenic

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bacteria.6 Therefore, the development of novel control measures for S. marcescens

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diseases without the rampant use of pesticides is an urgent need.

INTRODUCTION

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One possible mechanism of S. marcescens to possess multidrug resistance is

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attributed by biofilm formation.4 Biofilms are microbial communities in which cells are

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embedded in a self-generated matrix consisting of lipids, exopolysaccharides, proteins,

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and nucleic acids that can block the entry of antimicrobial agents into cells.7, 8 Biofilm

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formation by S. marcescens is reported to be closely related to quorum sensing (QS),9 3

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which is a bacterial communication system used to increase cell density and regulate

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gene expression by the binding of receptors and autoinducers.4 N-acyl-L-homoserine

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lactones (AHL) are secreted as major autoinducers in Gram-negative bacteria. Similar

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to other Gram-negative bacteria, S. marcescens produces C4-C8 homoserine lactones

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for mediating biofilm formation, motility, and extracellular product synthesis with

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respect to pathogenicity.10 It has been evidenced that QS-deficient S. marcescens

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showed reduced exoenzyme activity, prodigiosin levels, and biofilm biomass.11,

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Therefore, interfering with the QS system could be a compelling alternative for

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attenuating pathogenicity and protecting the host against infection by pesticide-resistant

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S. marcescens.

12

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In the search for QS inhibitors, dietary phytochemicals have attracted considerable

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interest due to their diverse biological functions and nontoxic nature.13 For example, 3-

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O-methyl ellagic acid (Figure 1) from Anethum graveolens significantly inhibited

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virulence production and biofilm formation in S. marcescens.13 Vanillic acid (Figure 1)

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in kiwifruit markedly diminished the pathogenicity of S. marcescens by regulating

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proteins involved in the synthesis of histidine, S-layers, fatty acid, and flagellin.14 The

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phenolic phytochemical hordenine (Figure 1) is abundant in sprouting barley and is

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known as a vasoconstrictive agent.15 Our previous study showed that hordenine

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possessed potent virulence suppression activity against Pseudomonas aeruginosa by

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downregulating the expressions of QS-related genes.16 However, how about the QS

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inhibitory potential of hordenine on other pathogens, especially those that cause severe

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losses in tomato yield? Interestingly, through extensive screening in the current study, 4

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we found that hordenine exhibited potent QS inhibitory activity against S. marcescens

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NJ01, a pathogenic bacterium isolated from diseased tomato leaves. Herein, for the first

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time, we reported on the development of the tomato-S. marcescens host-pathogen

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system as a model for assessing the anti-virulence potential of hordenine as a pesticide

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or pesticide accelerant under native conditions.

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MATERIALS AND METHODS

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Isolation and Identification of the Spoilage Bacterium. Diseased tomato leaves

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were collected by Prof. Yongyu Li from the Experimental Farm of Fujian Agriculture

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and Forestry University (Fuzhou, China) in July 2018. The bacterial strain was isolated

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using Luria-Bertani (LB, Sangon Biotech, Shanghai, China) agar plates, as described

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in prior research.17 The bacterium was grown at 28 oC for 24 h, after which the colony

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morphology was characterized. Total DNA was extracted using a DNA extraction kit

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(Tiangen Biotech, Beijing, China) and the 16S rRNA sequence was amplified using

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primers

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TACGGCTACCTTGTTACGAC-3′). The 16S rRNA sequence was compared to similar

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sequences in GenBank using BLAST searching.

27F

(5′-GAGAGTTTGATCCTGGCTCAG-3′)

and

1492R

(5′-

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Plant inoculation. A pathogenicity assay was performed according to previous

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research with slight modification.18 Overnight cultures of S. marcescens NJ01 were

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transferred to fresh LB broth (1:1000, v/v) and incubated at 28 °C overnight. The

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cultures were then dropwise added to trays of three-week old tomato (Solanum

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lycopersicum) plants. The control group was treated with phosphate buffer saline (PBS)

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only, without the addition of strain cultures. Pictures were taken after 96 h of infection. 5

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Growth Measurement. The S. marcescens BJ02 strain was purchased from the

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National Center for Medical Culture Collections (CMCC, No. 41002) and the S.

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marcescens FS14 strain (GenBank No. CP005927) was obtained from Prof. Weiwu

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Wang (Nanjing Agricultural University, Nanjing, China). The minimum inhibitory

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concentration (MIC) of hordenine (0.31-10 mg/mL) against S. marcescens was

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determined using the two-fold serially diluted method with an inoculum of 1 × 105

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CFU/mL in Müller-Hinton broth (Sangon Biotech, Shanghai, China).16 For the growth

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curve, overnight cultures of S. marcescens NJ01 were added to 30 mL of LB broth to

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achieve 0.05 at an optical density of 620 (OD620). The cultures were supplemented with

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hordenine at concentrations ranging from 25 to 100 μg/mL and then cultured for a

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further 24 h. Growth was determined at OD620 using a microplate reader (Biotek Elx800,

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USA).

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Analysis of AHL Production. The inhibitory efficiency of hordenine on the

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production of QS signal molecules was quantified by inoculating 0.1% overnight

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cultures of S. marcescens into LB broth.16 After incubation at 28 °C for 24 h, the

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cultures were centrifuged and the supernatant was extracted using the same volume of

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acidified ethyl acetate. The solvent was then eliminated, and residues were dissolved in

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methanol. The species of AHL were determined using liquid chromatography-tandem

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mass spectrometry (LC-MS/MS) according to the retention time of standard chemicals

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and their MS/MS2 spectra.7 AHL levels were normalized to the standard chemicals for

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relative quantification without the need of standard curve.19

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Biofilm Inhibition. Biofilms were cultivated in LB broth supplemented with or 6

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without hordenine in 24-well polystyrene plates (Costar 3524, Corning, USA) using the

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modified method described by Sethupathy et al.14 After 24-h static incubation, cultures

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and planktonic cells were removed and the sessile cells were stained with 0.05% crystal

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violet, the excess of which was then rinsed off using distilled water. After dissolution

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with 95% ethanol, the biofilm biomass was determined by reading OD570.

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To investigate cell viability, biofilms were washed with PBS and digested with

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dextranase (5 U, D8144-Sigma-Aldrich, USA), followed by 30-s sonication as

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described previously.20 The number of viable cells in the treated biofilms were counted

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by plating at 28 °C for 24 h.20

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Biofilm Dispersion. The biofilm dispersion assay was performed according to

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Ramanathan et al.,8 with minor modification. Biofilms were cultivated in LB broth in

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24-well polystyrene plates at 28 °C without shaking. After 24-h cultivation, the cultures

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were removed, and the biofilms were rinsed with PBS and then supplemented with fresh

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LB broth and hordenine, ciprofloxacin (0.3 μg/mL), or their combination. After another

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24-h cultivation, the formed biofilms were washed with PBS and subsequently fixed

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with methanol, stained using crystal violet, solubilized with ethanol, and eventually

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quantified at 570 nm using a microplate reader. For cell viability, sessile cells were

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washed with PBS, digested with dextranase, and then sonicated for 30 s. The number

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of CFU/biofilm was quantified by LB agar plating.

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Microscopy Analysis. Biofilms of S. marcescens NJ01 were cultivated in 24-well

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polystyrene plates with circular glass coverslips, as mentioned above. After cultivation,

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coverslips were washed with PBS, fixed with 2.5% glutaraldehyde, and dehydrated with 7

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ethanol. Samples were then freeze-dried, gold-coated and detected with a scanning

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electron microscope (SEM, JSM6360, JEOL, Tokyo, Japan).

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To determine the biomass of the S. marcescens biofilms, samples were observed

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using confocal laser scanning microscopy (CLSM, Zeiss LSM 700, Carl Zeiss, Jena,

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Germany). Biofilms that formed on the coverslips were washed with PBS and

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subsequently stained with acridine orange (AO) and ethidium bromide (EB) (1:1).

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Excess dye was removed, and the biofilms were washed using PBS. Stained biofilms

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were then visualized by CLSM with a ×63/1.4 numerical aperture oil objective.16 A

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~110 μm (X) ×110 μm (Y) area was screened in 1-μm Z-intervals (Z-stack) via green

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(excitation 488 nm/emission filter 501-545 nm), and red (excitation 488 nm/emission

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filter 570-670 nm) channels, respectively. For each group, at least five random areas in

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three independent cultures were selected for image analysis. Three-dimensional

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reconstructions were obtained with ZEISS confocal software (ZEN 2012). The images

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were analyzed using PHLIP (version 0.7) and Image J (NIH, Bethesda, MD, USA)

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software to calculate quantitative mean thickness.21

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Virulence Factors and Competitive Binding Assay. Overnight S. marcescens

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cultures were added to LB broth (1:100, v/v) supplemented with hordenine at increasing

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concentrations (25-100 μg/mL). DMSO and QS inhibitor vanillic acid (100 μg/mL)

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served as the negative and positive controls, respectively.14 After 24-h cultivation at

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28 °C, 75 μL of the supernatant was mixed with 125 μL of buffered azocasein. The

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mixtures were cultivated at 37 °C for 15 min followed by the addition of 600 μL of 10%

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trichloro acetic acid. Protease activity was determined at 440 nm using a microplate 8

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reader after terminating the reaction with 1 M NaOH.9

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Lipolytic activity was assessed using p-nitrophenyl palmitate (pNPP) as described

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previously.22 Briefly, 100 μL of culture supernatant was added to 900 μL of buffered

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substrate containing 0.3% (w/v) pNPP in isopropanol and 0.2% (w/v) sodium

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deoxycholate and 0.1% (w/v) gummi arabicum in 50 mM Na2PO4 buffer. After 1-h

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incubation, 1 mL of 1 M Na2CO3 was supplemented followed by 5-min centrifugation

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at 12,000 rpm. Lipolytic activity was determined at OD410.

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For prodigiosin, 1 mL of culture was centrifuged for 10 min. Cells were harvested

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and supplemented with 1 mL of acidified ethanol (4%, 1 M HCl). The pigments were

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determined at 534 nm using a microplate reader.13

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For the hemolysin assay, the supernatant was mixed with a sheep’s blood suspension

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(1:9, v/v) followed by 1-h incubation at 37 °C. After 10-min centrifugation at 3,000

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rpm, the supernatant was quantified at OD530.9

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Extracellular polysaccharides (EPS) were quantified using the carbohydrate

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estimation method.23 Biofilms attached to the coverslips were washed with PBS and

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then added to 500 μL of 0.9% NaCl and 5% phenol and 2.5 mL of 0.2% hydrazine

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sulphate. After 1-h incubation in the dark, the EPS were quantified at OD490.

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For swarming motility, 1 μL of bacterial culture was inoculated in the swarming

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medium containing 1% peptone, 0.5% NaCl, 0.5% glucose, and 0.5% agar. The plates

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were cultivated at 28 °C for 24 h and the swarming migration zones were determined.22

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For the competitive binding assay, overnight cultures of S. marcescens were 0.1%

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inoculated into LB broth supplemented with 100 μg/mL of hordenine, 5 µM C4-HSL, 9

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5 µM C6-HSL, 100 μg/mL of hordenine and 5 µM C4-HSL, and 100 μg/mL of

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hordenine and 5 µM C6-HSL, respectively. DMSO served as the negative control. After

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24-h cultivation at 28 °C, the competitive binding effect of hordenine with the receptors

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was evaluated by measuring prodigiosin levels, as described above.

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1H

NMR-Based Analysis of Intracellular Metabolites. Overnight, S. marcescens

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NJ01 cultures were added to LB broth (1:100, v/v) with DMSO or 100 μg/mL of

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hordenine for 24 h. After incubation, cells were harvested after 10-min centrifugation

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at 10,000 rpm. Cells were washed with PBS and then homogenized to extract the

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metabolites, as described previously.24 The dried metabolites were dissolved in D2O

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phosphate buffer and then transferred to NMR tubes for NMR analysis (Bruker AV 500

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MHz).24 Metabolites were assigned by referring to publicly accessible metabolomics

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databases.24

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Reactive oxygen species (ROS) and H2O2 measurement. ROS were determined as

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described previously, with minor modification.25 In brief, bacterial strains were cultured

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with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 1 mM, Nanjing

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Jiancheng Bioengineering Institute, Nanjing, China) at 28 °C for 30 min and then

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washed with PBS. The cells were resuspended in 1 mL of PBS and ROS were detected

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at 485 nm for excitation and 525 nm for emission using a Hitachi 2700 fluorescence

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spectrophotometer (Hitachi, Japan).

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For H2O2 assessment, bacterial cultures were pelleted and resuspended with 1 mL of

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PBS. Intracellular H2O2 passed through the membranes and equilibrated with the buffer.

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Cells were then centrifugated at 6000 g for 1 min. The suspension was used for H2O2 10

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measurement using the horseradish peroxidase-scopoletin (Nanjing Jiancheng

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Bioengineering Institute, Nanjing, China) method.26

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Quantitative Real-Time PCR (qRT-PCR). The extraction of total RNA and

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synthesis of first-strand complementary DNA was performed as described previously.16

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The qRT-PCR assay was carried out using the Applied Biosystems 7300 system to

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assess the expressions of QS- and biofilm-related genes (Table 1). The rplU gene of S.

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marcescens was set as the internal control.9

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Pathogenicity Inhibition Assay in Tomato Plants. The S. marcescens infection

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assay was performed as described above. Briefly, overnight cultures of S. marcescens

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NJ01 were diluted 1:1000 into fresh LB broth with or without hordenine (25, 50, and

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100 μg/mL) and incubated at 28 °C overnight. The same amount of DMSO and vanillic

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acid (100 μg/mL) served as the negative and positive controls, respectively. The treated

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cultures were then dropwise added to trays of three-week old tomato plants. The normal

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control group was treated with PBS only, without the addition of strain cultures.

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Pictures and leaf area measurements were taken after 48-h and 96-h infection. Single

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leaf area was calculated by the multiplication of length and width multiplied by a

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corrected coefficient 0.75.27 Whole leaf area of the treated plants was calculated from

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all single leaf areas. Statistical Analysis. Each assay was performed in triplicate and data were presented

239 240

as means ± SD. Statistical significance was determined using SPSS 18.0.

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RESULTS Identification of Spoilage Phytopathogen. Among several phytopathogenic 11

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bacteria isolated from diseased tomato leaves, isolate NJ01 was particularly aggressive,

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producing striking red guttates and showing fast growth compared with other isolates

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(Figure 2A(a)). Thus, isolate NJ01 was chosen for further study. Based on

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morphological characteristics (Figure 2A(b, c)) and 16S rDNA sequence data, NJ01

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was identified as Serratia marcescens (Figure 2B). The sequence was deposited in

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GenBank under accession No. MK092719. Strain NJ01 was a Gram-negative, motile,

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and short rod-shaped bacterium able to produce red pigments (Figure 2A(b, c)). In vivo

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inoculation assays with tomato leaves showed that S. marcescens NJ01 could colonize

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tomato leaves, causing chlorosis and wilting characteristics of the disease (Figure

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2A(e)).

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The Minimum Inhibitory Concentration (MIC) and Growth Profile. Based on

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the doubling dilution assay, the MIC of hordenine for all S. marcescens strains was

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determined to be 2.5 mg/mL. The growth profile of S. marcescens NJ01 indicated that

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at sub-MIC ranging from 25 to 100 μg/mL, hordenine exhibited no significant

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inhibition on bacterial growth (Figure 2C).

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Evaluation of AHL Production. The putative anti-QS capacity of hordenine against

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S. marcescens NJ01 was investigated by determining the AHL levels produced by this

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organism. The HPLC chromatograms of the AHL standards showed retention times of

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3.19 min and 10.27 min corresponding to C4-HSL and C6-HSL, respectively (Figure

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3A, B). MS study of the AHL standards showed [M+H]+ ions at 172.10 and 200.13

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corresponding to C4-HSL and C6-HSL, respectively (Figure 4A(a, c)). In addition, the

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presence of C4-HSL and C6-HSL was confirmed by their MS2 spectra (Figure 4A(b, 12

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d)).28 After 24-h treatment with hordenine, peaks and areas of C4-HSL and C6-HSL

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were notably decreased (Figure 3D, E, F). The levels of C4-HSL and C6-HSL were

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quantified based on the area calculation relative to their standard chemicals (Figure 4B,

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C). Exposure to hordenine at 25, 50, and 100 μg/mL resulted in more than 40%, 60%,

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and 80% reduction in C4-HSL, respectively (Figure 4B). A similar inhibitory effect

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was also observed in C6-HSL (Figure 4C). Thus, these results indicate that hordenine

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may act as a potent QS inhibitor against S. marcescens NJ01.

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Inhibitory Effect on Biofilm Formation. The biofilm inhibitory impact of

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hordenine was investigated by crystal violet assay. Exposure to hordenine at

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concentrations of 25, 50, and 100 μg/mL markedly reduced biofilms by 45%, 58%, and

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66%, respectively (Figure 5A(a)). We also detected viable cells in the treated biofilms.

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Hordenine treatment led to a notable reduction in viable cells relative to the DMSO

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control (Figure 5A(b)). In addition, hordenine also showed an inhibitory effect on

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biofilm formation and viable cells of S. marcescens BJ02 and FS14 (Figure S1).

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Visual confirmation of the potential of hordenine against biofilms was obtained

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through SEM (Figure 5B). SEM images of the DMSO control depicted a dense and

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net-structured biofilm coated with EPS. After hordenine treatment, biofilms were

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significantly reduced. Exposure to hordenine resulted in a scattered appearance and

283

disrupted integrity of the biofilms. To investigate the efficiency of hordenine on biofilm

284

formation visually, biofilms were observed using CLSM. After 100 μg/mL of hordenine

285

treatment, biofilm thickness notably decreased from 12.13 ± 3.02 μm to 3.53 ± 1.25 μm

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(Figure 5C). Biofilm biomass was also significantly reduced by ~70% and an obvious 13

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scattered appearance was presented.

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Disruption of Preformed Biofilms. When used alone, hordenine and ciprofloxacin

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resulted in minor but non-significant reductions in biofilm biomass and number of

290

sessile S. marcescens cells (Figure 6). However, hordenine treatment resulted in a

291

scattered appearance and disrupted integrity of the preformed biofilms, as observed

292

through SEM (Figure 7A) and CLSM (Figure 7B) analysis. Treatment of the

293

preformed biofilms with hordenine combined with ciprofloxacin resulted in a

294

remarkable reduction in S. marcescens biofilm biomass and higher mortality of

295

bacterial cells in the treated biofilms relative to the corresponding single agent

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treatment (Figure 6). Concentration-dependent analysis showed that more than 50% of

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biofilm biomass and sessile cells were eradicated when exposed to hordenine (25-100

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μg/mL) and 0.3 μg/mL of ciprofloxacin (MIC, 0.5 μg/mL).

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Both SEM and CLSM images (Figure 7A, B) also demonstrated the efficiency of

300

hordenine and ciprofloxacin in disrupting the preformed biofilms. SEM analysis of the

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treated biofilms clearly revealed few and scattered remaining cells, with disintegration

302

of the samples and notable reduction in EPS compared with the untreated control

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(Figure 7A). CLSM analysis also evidenced the reduced thickness and altered

304

architecture in the hordenine and ciprofloxacin-treated biofilms (Figure 7B). After

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exposure to hordenine and ciprofloxacin, the thickness of biofilms declined from 13.40

306

± 3.38 μm to 4.07 ± 1.49 μm. The antibiotic agent ciprofloxacin penetrated the biofilms

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and killed the cells, as evidenced through the dominant red fluorescence (representing

308

dead cells) in the hordenine/ciprofloxacin combination groups (Figure 7B(f, g, h)). 14

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Interference of Virulence Factors. Hordenine was investigated for its QS inhibitory

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potential against S. marcescens virulence factors. Protease, which is a vital virulence

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factor controlled by QS, can affect host immune responses.14 Chemicals suppressing

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the secretion of protease can be employed to potentiate the host’s innate immune

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response. Exposure to hordenine at 100 μg/mL resulted in a 65% inhibition in protease

314

activity compared with the untreated control (Figure 8A). This is more effective than

315

QS inhibitor vanillic acid, whose application resulted in a 33% inhibition in protease

316

activity.14 In addition, hordenine also showed an inhibitory effect on protease activity

317

of S. marcescens BJ02 and FS14 (Figure S2A). Lipolytic enzymes are involved in

318

degrading the phospholipid bilayer and mediating cell signaling pathways of the host.14

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In the current study, levels of lipase were notably decreased after treatment with

320

hordenine. Reductions of 60% and 40% in lipase levels were evidenced under the 100

321

μg/mL hordenine and vanillic acid treatments, respectively (Figure 8B).

322

Prodigiosin is a prominent red pigment produced by S. marcescens and is essential

323

for invasion, survival, and pathogenicity.29 The synthesis of prodigiosin is under the

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control of QS. As presented in Figure 8C, a concentration-dependent reduction in

325

prodigiosin was observed after treatment with hordenine. Exposure to 100 μg/mL of

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hordenine resulted in the inhibition of ~70% of prodigiosin production, which was more

327

potent than that of vanillic acid (50%). Hordenine also showed an inhibitory effect on

328

prodigiosin production of S. marcescens BJ02 and FS14 (Figure S2B). Hemolysin,

329

another well-studied virulence factor secreted by S. marcescens, was also significantly

330

reduced (Figure 8D). At 100 μg/mL, hordenine reduced the production of hemolysin 15

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by more than 70% compared with 40% by vanillic acid. This inhibitory effect was also

332

observed in S. marcescens BJ02 and FS14 (Figure S2C).

333

EPS are a vital ingredient in biofilms and play important roles in maintaining

334

cohesion, obtaining nutrition, and blocking entry of antimicrobial agents into cells.9

335

Our results showed significantly reduced production of EPS with hordenine treatment

336

(Figure 8E). At concentrations of 25, 50, and 100 μg/mL, EPS were reduced by 35%,

337

50%, and 70%, respectively. When treated with 100 μg/mL of vanillic acid, a nearly

338

40% reduction in EPS was observed. In addition, we also assessed the effect of

339

hordenine on swarming motility and obtained a similar inhibitory effect (Figure 8F, G).

340

The competitive binding assay showed that the exogenous addition of C4-HSL or

341

C6-HSL significantly promoted the production of prodigiosin (Figure S3). This result

342

further confirmed the presence of C4-HSL and C6-HSL in the cultures of S. marcescens

343

NJ01. In addition, treatment with hordenine combined with C4-HSL or C6-HSL

344

significantly reduced the inhibitory effect of hordenine on prodigiosin production. This

345

indicated that hordenine could compete with AHL for binding receptor.

346

Intracellular Metabolites.

1

H NMR-based metabolic analysis was used to

347

investigate the metabolites involved in membrane composition, antioxidation, protein

348

synthesis, and energy metabolism (Table 2). A significant decrease in ethanolamine and

349

glutamate, and marked increase in isoleucine, leucine, valine, succinate, and fumarate

350

were detected in the hordenine-treated group. Details on the metabolites, including

351

assignments, chemical shifts, and fold changes, are presented in Table 2.

352

ROS and H2O2 measurement The effects of hordenine on ROS and H2O2 16

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production were shown in Figure 9A. Treatment with hordenine at 100 µg/mL

354

significantly enhanced the levels of ROS and H2O2. This indicated that the S.

355

marcescens NJ01 strain underwent severe oxidative damage after hordenine treatment.

356

QS and Biofilm-Related Gene Expressions. qRT-PCR was performed to assess the

357

effects of hordenine on the transcriptional levels of QS-mediated genes fimA, fimC, flhD,

358

and bmsA, which are responsible for fimbriae production, adherence, motility, and

359

biofilm formation, respectively (Figure 9B). Results indicated that hordenine treatment

360

resulted in a notable down-regulation in the expressions of fimA (~1.3-fold), fimC

361

(~0.7-fold), flhD (~2.3-fold), and bmsA (~1.8-fold). Similarly, the expressions of pigA

362

and pigB, two genes involved in the biosynthesis of prodigiosin, were also clearly

363

reduced after exposure to hordenine. In addition, we investigated the expressions of

364

genes involved in detoxifying enzymes. Results indicated that the expressions of sodB

365

and zwf encoding superoxide dismutases (SOD) and NADPH-generating glucose-6-

366

phosphate dehydrogenase (GPD), respectively were significantly inhibited (Figure 9B).

367

Pathogenicity Inhibition Assays in Tomato. We determined the efficiency of

368

hordenine in vivo and investigated its physiological relevance with respect to bacterial

369

pathogenesis in vegetable plants. QS inhibitors have been shown to markedly attenuate

370

the virulence of Pectobacterium carotovora in bean and potato rot.30 In this study, we

371

focused on assessing the efficiency of hordenine on tomato plant infection. As presented

372

in Figure 10, hordenine treatment at 25, 50, and 100 μg/mL did not show an increase

373

in chlorosis, stunting, or cell death compared with the PBS control, and thus had no

374

inhibitory effect on plant growth. After 48-h inoculation with the untreated S. 17

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marcescens cultures, leaves derived from tomato plants wilted and withered (Figure

376

10Bb). Some leaves were chlorotic, the whole plant ultimately withered, and growth

377

was significantly suppressed at 96-h postinfection (Figure 10Bc). However, leaves

378

inoculated with vanillic acid and hordenine-treated bacteria showed a significant

379

reduction in virulence (Figure 10C, D, E, F). Leaf area analysis indicated that

380

hordenine treatment resulted in a marked reduction in leaf loss in comparison to the

381

DMSO and vanillic acid-treated groups (Figure 10G). Therefore, these results indicate

382

that hordenine can significantly attenuate the pathogenicity of S. marcescens in

383

vegetable plants.

384



385

Many plants and microorganisms produce QS inhibitors for self-protection and

386

competition with invading organisms.14 Recently, greater attention has been paid to QS

387

inhibitors from edible sources due to their non-toxic nature and multiple functions in

388

attenuating pathogenicity.31 In this study, hordenine, a phenolic phytochemical from

389

sprouting barley, was evaluated for its potential to inhibit QS-regulated virulence in the

390

phytopathogen S. marcescens NJ01. Hordenine showed potent QS-inhibitory effects

391

against S. marcescens NJ01, as evidenced through a notable decrease in AHL levels,

392

reduction in virulence factors, inhibition of biofilms, and down-regulation of QS- and

393

biofilm-related gene expressions.

DISCUSSION

394

Previous research has shown that S. marcescens utilizes AHL as QS signals to

395

mediate the expressions of a battery of genes involved in a variety of physiological

396

activities including virulence production and biofilm formation.19 QS mutants of S. 18

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marcescens exhibit deficiencies in biofilm formation as well as prodigiosin and

398

extracellular enzymes production.11, 12, 32 Due to the importance of QS in pathogenicity,

399

we first determined the effects of hordenine on AHL secretion. Our results confirmed

400

the presence of C4-HSL and C6-HSL in S. marcescens NJ01 cultures4 and showed a

401

significant reduction in AHL levels, thus revealing a potent inhibitory potential of

402

hordenine against the QS system of S. marcescens. In addition, we investigated the

403

impact of hordenine on S. marcescens NJ01 biofilm formation. Results showed a

404

significant inhibition in biofilm formation after hordenine treatment. This result is

405

consistent with that of Jayathilake et al.,33 who demonstrated that inhibition of QS can

406

affect bacteria competition and biofilm formation for mixed bacteria strains. We next

407

determined the expressions of biofilm-related genes. The expression of bsmB was

408

prominently suppressed, which was well-correlated with the biofilm formation assay.

409

Hordenine treatment led to a minor but non-significant reduction in the preformed

410

biofilms. This result was similar to our previous study in which hordenine exerted a

411

weak inhibitory effect on the preformed biofilms of Pseudomonas aeruginosa PAO1.16

412

These results thus reveal a sophisticated relationship between QS and biofilms. It is

413

worth noting that, after hordenine treatment, biofilms were scattered and flat and

414

resembled the biofilms formed by QS-deficient mutants.34 We therefore speculated that

415

the architecture of the preformed biofilms was destroyed. As QS-regulated EPS are an

416

important ingredient in biofilms and act as a barrier to the entry of antimicrobial agents

417

into cells,33,

418

findings revealed that a 100 μg/mL dose of hordenine resulted in a nearly 70% reduction

35

we investigated the efficiency of hordenine on EPS synthesis. Our

19

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419

in EPS, which was much more effective than petroselinic acid in reducing EPS of S.

420

marcescens.8 Our results are similar to those of Jayathilake et al.,33 who demonstrated

421

by computational modeling that inhibition of QS can affect EPS production.

422

Ethanolamine is a vital component of cellular membranes and is involved in

423

maintaining membrane permeability.36 The decreased level of ethanolamine in the

424

present study indicated that membrane permeability was significantly affected (Table

425

2). Given the capacity of hordenine to destroy the architecture of biofilms by reducing

426

EPS production, the effectiveness or susceptibility of antibiotics might be enhanced.

427

Therefore, we examined the inhibitory effect of hordenine in combination with

428

ciprofloxacin against the preformed biofilms of S. marcescens was determined. Results

429

indicated that hordenine remarkably increased the susceptibility of ciprofloxacin

430

against S. marcescens biofilms. The changed biofilm architecture and membrane

431

permeability facilitated the penetration of ciprofloxacin into the treated biofilms and

432

thus the killing of cells, as evidenced by the dominant red fluorescence in the

433

combination groups. The enhanced effect of hordenine and ciprofloxacin in

434

combination is similar to that of phenol, 2,4-bis(1,1-dimethylethyl), which shows

435

significantly increased susceptibility of S. marcescens towards gentamicin.9 The

436

combination of QS inhibitor and conventional antibiotic is a promising approach for

437

eradicating preformed biofilms and curbing infection magnitude.37 Pan and coworkers

438

suggested that the QS inhibitor BF8 reverts the antibiotic tolerance of P. aeruginosa

439

persister cells.38 Persisters might be one of the main causes for therapeutic failure and

440

recurrent infections. The capacity of hordenine to sensitize persisters could enhance 20

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antibiotic efficacy, reduce antibiotic dosage, and attenuate the risk of antibiotic

442

resistance.

443

S. marcescens produces a range of QS-mediated virulence factors, including protease,

444

lipase, prodigiosin, hemolysis, and swarming motility for invasion and infection

445

dissemination. Therefore, interfering with the production of virulence factors could be

446

an efficient approach in attenuating the pathogenicity of S. marcescens.39 Protease

447

possesses the capacity to induce inflammatory and immune responses, whereas lipase

448

is involved in cytolytic activity.13, 40 Labbate et al.32 showed that bsmB is responsible

449

for the production of protease and lipase. In this study, the reduced productions of

450

protease and lipase were in accordance with the suppressed expression of bsmB.

451

Prodigiosin and hemolysin are well-depicted virulence factors of S. marcescens and

452

play vital roles in host invasion and pathogenicity.29 As the biosynthesis of prodigiosin

453

is mediated by the pig gene cluster,41 the expressions of prodigiosin-related genes were

454

investigated. The down-regulation of pig genes was well correlated with the notable

455

reduction of prodigiosin. Attachment is the first step of biofilm formation and is closely

456

related to fimbriae production and swarming motility. Fimbriae is mediated by fimA

457

and fimC and plays vital roles in surface attachment and colonization.22 In S.

458

marcescens, the QS system is governed by swrI and swrR.32 The autoinducer C4-HSL

459

is synthesized by SwrI and then binds to SwrR to induce the expression of a range of

460

proteins involved in biofilm maturation and swarming motility.42 In addition, flagella-

461

controlled swarming motility is governed by flhD and contributes to biofilm formation

462

by enhancing cell surface attachment.43 Here, the inhibition of swarming motility and 21

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463

biofilm formation were well correlated with the significant reduction in C4-HSL and

464

QS-related gene expressions, further revealing the reduced pathogenicity of S.

465

marcescens.

466

As a precursor of the major natural antioxidant glutathione, which combats oxidative

467

injury, the marked decrease in glutamate could be attributed to the synthesis of

468

glutathione and counteraction of the deleterious effects of oxidative stress (Table 2).44

469

QS was reported to enhance the expressions of SOD and NADPH-generating glucose-

470

6-phosphate dehydrogenase to counteract ROS.45 The inhibited expressions of sodB

471

and zwf reflected a severely impacted QS of S. marcescens after dosing with hordenine.

472

Branched chain amino acids (BCAAs) isoleucine, leucine, and valine are essential

473

amino acids and substrates and play crucial roles in protein synthesis. The increase in

474

BCAAs indicated a breakdown of normal protein due to dysfunctional QS and

475

enhanced oxidative damage after dosing with hordenine.24 Furthermore, the dramatic

476

increase in succinate and fumarate suggested disturbance of energy metabolism as they

477

are intermediates of the tricarboxylic acid (TCA) cycle. As the most vital metabolic

478

pathway providing energy for organisms, disruption of the TCA cycle can result in

479

energy metabolism disorder, leading to bacterial pathogenicity dysfunction.24

480

We also investigated host-pathogen relationships between vegetable plants and

481

pathogenic bacterium S. marcescens as a pattern for assessing the capacity of hordenine

482

to affect QS-mediated pathogenicity. Results indicated that hordenine was an effective

483

QS inhibitor for attenuating the infection and pathogenicity of S. marcescens on tomato

484

plants. Similar results have been reported by Mandabi et al., who demonstrated that 22

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karrikin treatment significantly reduces leaf loss in Arabidopsis thaliana and attenuates

486

soft rot symptoms in lettuce midriffs upon infection with P. aeruginosa.46 The present

487

study indicated that hordenine could act as a potential anti-virulence agent in crop

488

disease control.

489



490

Supporting Information

491

The Supporting Information is available free of charge on the ACS Publications website

492

at DOI:

493

Inhibitory effects of hordenine on S. marcescens BJ02 and FS14 biofilm formation;

494

inhibitory effects of hordenine on S. marcescens BJ02 and FS14 virulence factors;

495

competitive binding assay of hordenine and AHL on prodigiosin production

496



497

Corresponding Author

498

*A.-Q.J: E-mail: [email protected]. Tel: +86 25 84303216. Fax: +86 25

499

84303216.

500

*J.-S.W: E-mail: [email protected].

501

ORCID

502

Ai-Qun Jia: 0000-0002-8089-6200

503

Author Contributions

504

§

505

Notes

506

The authors declare no competing financial interest.

ASSOCIATED CONTENT

AUTHOR INFORMATION

J.W.Z., L.Y.R, and H.J.C. contributed equally to this work.

23

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508

This work was supported by grants from the National Key Research and Development

509

Program of China (2017YFD0201401), National Natural Science Foundation of China

510

(41766606), Six Talent Peaks Project in Jiangsu Province, and Fundamental Research

511

Funds for the Central Universities (30916011307).

ACKNOWLEDGEMENTS

512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 24

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(1) Prasanna, R.; Chaudhary, V.; Gupta, V.; Babu, S.; Kumar, A.; Singh, R.; Shivay, Y.

531

S.; Nain, L. Cyanobacteria mediated plant growth promotion and bioprotection against

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Fusarium wilt in tomato. Eur. J. Plant Pathol. 2013, 136, 337-353.

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(2) Vos, C. M.; Yang, Y.; De Coninck, B.; Cammue, B. P. A. Fungal (-like) biocontrol

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organisms in tomato disease control. Biol. Control 2014, 74, 65-81.

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(3) Jan, P. S.; Huang, H. Y.; Chen, H. M. Expression of a synthesized gene encoding

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cationic peptide cecropin B in transgenic tomato plants protects against bacterial

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diseases. Appl. Environ. Microbiol. 2010, 76, 769-775.

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(4) Morohoshi, T.; Shiono, T.; Takidouchi, K.; Kato, M.; Kato, N.; Kato, J.; Ikeda, T.

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Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-

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acylhomoserine lactone. Appl. Environ. Microbiol. 2007, 73, 6339-6344.

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(5) Bruton, B. D.; Mitchell, F.; Fletcher, J.; Pair, S. D.; Wayadande, A.; Melcher, U.;

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Brady, J.; Bextine, B.; Popham, T. W. Serratia marcescens, a phloem-colonizing,

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squash bug-transmitted bacterium: causal agent of cucurbit yellow vine disease. Plant

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Dis. 2003, 87, 937-944.

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(6) Soylu, E. M.; Kurt, S.; Soylu, S. In vitro and in vivo antifungal activities of the

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Int. J. Food Microbiol. 2010, 143, 183-189.

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(7) Zhou, J.; Bi, S.; Chen, H.; Chen, T.; Yang, R.; Li, M.; Fu, Y.; Jia, A. Q. Anti-biofilm

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E.; Huang, C. T.; Fredericks, J.; Burnett, S.; Stewart, P. S.; McFeters, G.; Passador, L.;

662

Iglewski, B. H. Quorum sensing in Pseudomonas aeruginosa controls expression of

663

catalase and superoxide dismutase genes and mediates biofilm susceptibility to

664

hydrogen peroxide. Mol. Microbiol. 1999, 34, 1082-93.

665

(46) Mandabi, A.; Ganin, H.; Krief, P.; Rayo, J.; Meijler, M. M. Karrikins from plant

666

smoke modulate bacterial quorum sensing. Chem. Commun. 2014, 50, 5322-5.

667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 31

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Figure captions:

684

Figure 1. Chemical structures of hordenine, C4-HSL, C6-HSL, vanillic acid, and 3-O-

685

methyl ellagic acid.

686 687

Figure 2. Isolation and identification of S. marcescens NJ01 and effects of hordenine

688

on its growth. (A) Isolation and characterization of S. marcescens NJ01 and its in vivo

689

infection assay in tomato leaves. (a) bacteria isolated from diseased tomato leaves; (b)

690

purified strain of S. marcescens NJ01; (c) application of Gram’s stain in identifying S.

691

marcescens NJ01; (d) inoculation of tomato leaves with PBS without strain; (e)

692

inoculation of tomato leaves with S. marcescens NJ01. (B) Evolutionary relationships

693

between S. marcescens NJ01 and other related strains. (C) Growth profile of S.

694

marcescens NJ01 following exposure to 25, 50, and 100 μg/mL of hordenine for 24 h

695

in LB broth. DMSO served as the negative control. Error bars represent standard

696

deviations of three measurements.

697 698

Figure 3. HPLC chromatograms of C4-HSL and C6-HSL secreted by S. marcescens

699

treated with (C) DMSO, (D) 25 μg/mL of hordenine, (E) 50 μg/mL of hordenine, and

700

(F) 100 μg/mL of hordenine, respectively. (A) Standard chemical of C4-HSL (4 µM).

701

(B) Standard chemical of C6-HSL (15 µM). C4-HSL and C6-HSL were identified

702

according to the retention time of standard chemicals.

703 704

Figure 4. Identification and quantification of C4-HSL and C6-HSL by LC-MS/MS 32

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chromatograms. C4-HSL and C6-HSL were identified according to MS/MS2 spectra.

706

(A) MS spectra of C4-HSL and C6-HSL. (a) full MS spectra of C4-HSL; (b) MS2

707

spectra of C4-HSL; (c) full MS spectra of C6-HSL; (d) MS2 spectra of C6-HSL. (B)

708

Quantitative analysis of C4-HSL treated with 25, 50, and 100 μg/mL of hordenine,

709

respectively, based on the area calculation relative to standard chemical of C4-HSL (4

710

µM). (C) Quantitative analysis of C6-HSL treated with 25, 50, and 100 μg/mL of

711

hordenine, respectively, based on the area calculation relative to standard chemical of

712

C6-HSL (15 µM). Statistical differences were determined by ANOVA followed by

713

Tukey-Kramer test. *** p < 0.001 versus DMSO control.

714 715

Figure 5. Inhibitory effect of hordenine on S. marcescens NJ01 biofilm formation. (A)

716

Quantification of biofilms using (a) crystal violet staining and (b) cell viability methods.

717

(B) SEM images of biofilms treated with (a) DMSO, (b) 25 μg/mL of hordenine, (c) 50

718

μg/mL of hordenine, and (d) 100 μg/mL of hordenine. (C) CLSM images of biofilms

719

treated with (a) DMSO, (b) 25 μg/mL of hordenine, (c) 50 μg/mL of hordenine, and (d)

720

100 μg/mL of hordenine. (a′-d′) represent the corresponding three-dimensional biofilm

721

structures. Statistical differences were determined by ANOVA followed by Tukey-

722

Kramer test. *** p < 0.001 versus DMSO control.

723 724

Figure 6. Effects of hordenine and ciprofloxacin against preformed S. marcescens NJ01

725

biofilms using (A) crystal violet staining and (B) cell viability methods. Statistical

726

differences were determined by ANOVA followed by Tukey-Kramer test. *** p < 0.001 33

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versus the corresponding control.

728 729

Figure 7. SEM (A) and CLSM (B) images of preformed biofilms treated with (a)

730

DMSO, (b) 0.3 μg/mL of ciprofloxacin, (c) 25 μg/mL of hordenine, (d) 50 μg/mL of

731

hordenine (e) 100 μg/mL of hordenine, (f) 0.3 μg/mL of ciprofloxacin and 25 μg/mL of

732

hordenine, (g) 0.3 μg/mL of ciprofloxacin and 50 μg/mL of hordenine, and (h) 0.3

733

μg/mL of ciprofloxacin and 100 μg/mL of hordenine, respectively.

734 735

Figure 8. Inhibitory effects of hordenine on virulence factor production. (A) Protease

736

activity. (B) Lipase activity. (C) Prodigiosin levels. (D) Hemolysin levels. (E) EPS

737

levels. (F) and (G) Swarming motility. Vanillic acid (VAN) served as the positive

738

control and DMSO served as the negative control. Statistical differences were

739

determined by ANOVA followed by Tukey-Kramer test. *** p < 0.001 versus DMSO

740

control.

741 742

Figure 9. Effects of hordenine on reactive oxygen species (ROS) production and gene

743

expression. (A) ROS and H2O2 production. (B) Expressions of genes involved in QS,

744

biofilm, and antioxidation. Statistical differences were determined by ANOVA followed

745

by Tukey-Kramer test. * p < 0.05, ** p < 0.01, *** p < 0.001.

746 747

Figure 10. Efficiency of hordenine on tomato plant infections after 48 and 96 h. (A)

748

Normal group treated with PBS only. (B) Inoculation with S. marcescens treated with 34

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DMSO. (C) Inoculation with S. marcescens treated with vanillic acid (VAN). (D), (E),

750

and (F) represent inoculation with S. marcescens treated with 25, 50, and 100 μg/mL of

751

hordenine, respectively. Images of (a), (b), and (c) indicate 0, 48, and 96 h post-

752

inoculation, respectively. (F) Quantification of leaf areas at 0, 48, and 96 h post-

753

inoculation. Statistical differences were determined by ANOVA followed by Tukey-

754

Kramer test. * p < 0.05 versus DMSO control. ** p < 0.01 versus DMSO control. ***

755

p < 0.001 versus DMSO control.

756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 35

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771 772

Table 1. PCR Primers for qRT-PCR. Gene fimA

fimC

flhD

bsmB

pigA

pigC

sodB

zwf

Primer direction

Sequence (5′−3′)

forward

TTAGCCTGGAGAAATGTGAAGC

reverse

GGCAGAGTAGAGCCGTTGTTAT

forward

AGCAGTTCAACACCTCCTTCAT

reverse

CGGATATTTACCCGGCAGA

forward

CCTCCGCGATGTTCCGTCTTG

reverse

GGTCAGGCGTTCGATGGTCTG

forward

CGGAAGTGACGCTGGAACACG

reverse

TGCTGCTGTTGATGGTGTAATCGG

forward

ATGGCTTTATGGGCGTGTC

reverse

TGAAGGTCAGTTCGCTCCAC

forward

TTCGTCACAAACCGCACTATT

reverse

CGTCTTTCACCGCCCATT

forward

CTGCTGACCGTTGACGTGTGG

reverse

CGCTGCGAAGGTCCAGTTGAC

forward

GAGAAGGTGAAGGTACTGCGTTCG

reverse

TTCGGTGCTGCTGCTCTTGTTC

forward

CAACACCGAGTAAGCGAAGG

reverse

ACGAAAGGAACGCCGATT

rpsL

773 774 775 776 777 778 36

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Amplicon size (bp) 145

216

149

150

117

186

108

156

140

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779 780

Table 2. Identified Metabolites Involved in Membrane Composition, Antioxidation,

781

Protein Synthesis, and Energy Metabolism. No.

Metabolites

Assignments

Chemical shifta (ppm)

Fold changeb

P valuec

1

Ethanolamine

N-CH2, CH2

3.15(t), 3.83(t)

-0.33

*

2

Glutamate

β-CH2, α-CH2, N-CH

2.4(m)

-0.61

**

3

Isoleucine

δ-CH3, β-CH3

0.94(t)

0.32

*

4

Leucine

δ-CH3, CH2

0.96(t)

0.33

***

5

Valine

CH3, CH3

1.0(d),1.05(d)

0.23

*

6 7

Succinate Fumarate

CH CH

2.41(s) 6.53(s)

0.48 0.33

* *

782

a

Multiplicity: (s) singlet, (d) doublet, (t) triplet, (q) quartets, (m) multiplets. bColor

783

coded according to the log2(fold): Red and blue represent the increased and decreased

784

metabolites, respectively, in hordenine-treated group. cP values were calculated based

785

on a parametric Student’s t test or a nonparametric Mann−Whitney test and were

786

corrected by BH (Benjamini−Hochberg) methods. Numbers of symbol * denoted extent

787

of significance: * p < 0.05, ** p < 0.01, and *** p < 0.001.

788 789 790 791 792 793 794 795

37

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796 797

TOC graphic

798

799 800 801 802 803 804 805 806

38

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