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iTRAQ-Based Proteomics Revealed the Bactericidal Mechanism of Sodium New Houttuyfonate against Streptococcus pneumoniae Xiao-Yan Yang, Tianyuan Shi, Gaofei Du, Wanting Liu, XingFeng Yin, Xuesong SUN, Yunlong Pan, and Qing-Yu He J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02147 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 30, 2016

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Journal of Agricultural and Food Chemistry

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iTRAQ-Based Proteomics Revealed the Bactericidal Mechanism of

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Sodium New Houttuyfonate against Streptococcus pneumoniae

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Xiao-Yan Yang1, 2, §, Tianyuan Shi2, §, Gaofei Du2, Wanting Liu2, Xing-Feng Yin2,

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Xuesong Sun2, Yunlong Pan1,* and Qing-Yu He2,* 1

5 2

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The First Affiliated Hospital of Jinan University, Guangzhou, 510632, China;

Key Laboratory of Functional Protein Research of Guangdong Higher Education

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Institutes, Institute of Life and Health Engineering, College of Life Science and

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Technology, Jinan University, Guangzhou 510632, China

9 10 11 12 13 14 15 16 17

§

Equal contributors.

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*Correspondence should be addressed to: Prof. Qing-Yu He, Tel & Fax:

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+86-20-85227039,

21

+86-20-38374151, E-mail: [email protected].

E-mail:

[email protected];

Prof.

Yunlong

Pan,

Tel:

22 1

ACS Paragon Plus Environment


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Abstract

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Sodium New Houttuyfonate (SNH), an addition product of active ingredient

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Houttuynin from the plant Houttuynia cordata Thunb, inhibits a variety of bacteria,

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yet the mechanism by which it induces cell death has not been fully understood. In the

27

present study, we utilized iTRAQ-based quantitative proteomics to analyze the protein

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alterations in Streptococcus pneumoniae (S. pneumoniae) in response to SNH

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treatment. Numerous proteins related to the production of reactive oxygen species

30

(ROS) were found to be up-regulated by SNH, suggesting that ROS pathways may be

31

involved as analyzed via bioinformatics. As reported recently, cellular reactions

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stimulated by ROS including superoxide anion (O2▪－), hydrogen peroxide (H2O2) and

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hydroxyl radicals (OH▪) have been implicated as one of the mechanisms whereby

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bactericidal antibiotics kill bacteria. We then validated that SNH killed S. pneumoniae

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in a dose-dependent manner accompanied with the increasing level of H2O2. On the

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other hand, addition of catalase, which can neutralize H2O2 in cells, showed a

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significant recovery in bacterial survival. These results indicate that SNH indeed

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induced H2O2 formation to contribute to the cell lethality, providing new insights into

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the bactericidal mechanism of SNH and expanding our understanding of the common

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mechanism of killing induced by bactericidal agents.

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Keywords: Streptococcus pneumoniae, Sodium new houttuyfonate, iTRAQ, H2O2,

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Bactericidal mechanism

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Introduction

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One of the most common causes of community-acquired pneumoniae is the infection

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of Streptococcus pneumoniae, a Gram-positive and catalase-negative bacterium 1. The

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bacterium also causes many other serious diseases including meningitis, otitis media,

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bacteremia and sepsis, especially among children, elderly people, patients with AIDS

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and other immunocompromised individuals, posing a major threat to human health

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worldwide 2. Due to the increasing incidence of antibiotic resistant clones and the

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limitations of exiting vaccines, it is urgent to screen for novel drugs to combat

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pneumococcal infection.

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Reactive oxygen species (ROS) including superoxide anion (O2▪－), hydrogen

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peroxide (H2O2) and hydroxyl radicals (OH▪), are produced as by-products of the use

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of oxygen in fundamental enzymatic reactions and metabolism pathways 1. S.

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pneumoniae can generate up to 2.0 mM H2O2 through the pyruvate oxidase (SpxB),

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contributing to its virulence and deleterious effects on itself 1. The overproduced ROS

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have numerous adverse effects on bacterial DNA damage, protein degradation and the

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peroxidation of the cell membrane lipids 3. Recently, several studies pointed out that

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ROS contribute to the cell death of bactericidal antibiotics 4-7.

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Plant is the source of great interest for curing bacterial infection, by providing us 8

, andrographolide

9

berberine 10

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with a set of antimicrobial agents, such as artesunate

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and antimicrobial peptides 11. Houttuynin is the primary constituent in the volatile oil

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of Houttuynia cordata Thunb, an important herbal medicine widely used as the

3



12

, antiviral

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and antibacterial

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anti-inflammatory

agent for numerous years in

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China. It was also used to prevent severe acute respiratory syndrome (SARS) in 2003

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15

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houttuyfonate (SH) and sodium new houttuyfonate (SNH), were synthesized and have

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been approved by China Food and Drug Administration to be used in clinic for the

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treatment of purulent skin infections, respiratory tract infections, including pneumonia

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and acute or chronic bronchitis

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effectively inhibit a variety of bacteria, including Staphylococcus aureus, Bacillus

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subtilis and Pseudomonas aeruginosa

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can,

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Staphylococcus aureus (MRSA). However, despite its widespread and effective use,

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the mechanism of SNH against bacteria remains unknown.

. Because houttuynin is chemically unstable, its addition products, sodium

alone

and

synergizing

16, 17

. Previous studies indicated that SH and SNH

with

18, 19

. Recently, Lu et al reported

antibiotics,

combat

15

that SNH

Methicillin-Resistant

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In the present study, we found that SNH killed S. pneumoniae in a

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dose-dependent manner, then utilized iTRAQ-based quantitative proteomics to dissect

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the global protein alteration of S. pneumoniae in response to short-term SNH exposure.

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Bioinformatics analysis of the proteomic data suggested that SNH may induce H2O2

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formation, and we detected that the H2O2 level indeed increased after SNH treatment.

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Adding catalase to neutralize H2O2 greatly attenuated SNH-induced cell death. These

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observations implicate that ROS were induced in S. pneumoniae by the presence of

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SNH, contributing significantly to the cell death.

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Materials and methods

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1. Bacterial strain and sodium new houttuyfonate

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S. pneumoniae D39 strain NCTC 7466 was obtained from National Collection of

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Type Culture (NCTC, London, UK). S. aureus strain ATCC 29213 was purchased

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from American Type Culture Collection (ATCC). Sodium new houttuyfonate (SNH)

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was purchased from Chembest company (Shanghai, China).

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2. Growth conditions, MIC and MBC assays

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For all experiments, S. pneumoniae was cultured in THY medium (Todd-Hewitt broth

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(Oxiod, UK) with 0.5% yeast extract) or grown on Columbia agar (Difco, USA)

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containing 5% sheep blood (Ruite, China) at 37°C in 5% CO2. S. aureus was grown in

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tryptic soy broth at 37°C with shaking at 200 rpm. The minimum inhibitory

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concentration (MIC) and minimum bactericidal concentration (MBC) of SNH were

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measured by the broth micro-dilution method based on guidelines in the literature 20-22.

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S. pneumoniae (5 × 106 CFU/mL) was incubated with SNH at concentrations of

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twofold serial dilutions (range from 1.56 µM to 200 µM) in a 24 well microplate (1

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mL per well) at 37°C in 5% CO2 for 24 h. The bacterial concentration in each well

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was determined at 600 nm using spectrophotometry (Thermo, USA), the

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concentration of SNH with no visible bacteria growth (OD600 < 0.1) was recorded as

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the MIC

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counting and the concentration of SNH corresponding to the well that produced a

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99.99% kill relative to the starting inoculum was taken as the MBC

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(Sigma, USA) was used as a positive control.

22

. Then the wells with no visible bacteria growth were taken into colony

20

. Ampicillin

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3. Time–kill curve studies

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Time-kill assays of S. pneumoniae treated by SNH (corresponding to 0, 1/8 × MIC,

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1/4 × MIC, 1/2 × MIC, 1 × MIC and 2 × MIC) were performed according to the basic

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microbiological techniques protocol

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and viable bacteria were counted after 0, 1, 2, 3, 6, 12 and 24 h of incubation. Colony

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counts were performed by 10-fold serial dilutions in 1×PBS then plating each onto

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Columbia agar containing 5% sheep blood at 37°C in 5% CO2 for 24 h. The viable

114

counts were calculated to give CFU/mL, and time-kill curves were shown by plotting

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log10 CFU/mL against time (h). Ampicillin was used as a positive control. Each

116

time–kill assay was performed in triplicate.

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4. Determination of growth curves of S. pneumoniae

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When bacteria were grown to an optical density (OD600) of approximately 0.3, 1 ×

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MIC SNH was added. For H2O2 quenching, catalase (Beyotime, China) was added at

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a concentration of 2 mg/mL at the same time as the SNH treatment. CFU/mL was

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monitored every hour for 3 h after drug addition. For growth curve assay, S.

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pneumoniae was incubated with SNH (corresponding to 0, 1/16 × MIC, 1/8 × MIC,

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1/4 × MIC, 1/2 × MIC, 1 × MIC and 2 × MIC), OD600 was monitored every hour for

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the first 4 h and every two hours for next 8 h after drug addition.

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5. Cytotoxicity assay

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The human bronchial epithelial cell line HBE was cultivated in DMEM media with

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10% fetal bovine serum (FBS) at 37°C in 5% CO2. The cytotoxicity of SNH in HBE

20

. Bacteria were cultured at 37°C in 5% CO2,

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cells was assessed using LDH Cytotoxicity Assay Kit (Beyotime, China) according to

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the manufacturer’s instructions. Briefly, 5 × 103 HBE cells were seeded into a sterile

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96-well plate in triplicate and incubated at 37 °C with 5% CO2 for 12 h, followed by a

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48 h incubation in dark with different amounts of SNH (0, 25, 50, 100 and 200 µM).

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LDH releasing agent was added at 1 hour before the end of the incubation as the

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positive control. After incubation at 37 °C for 1 h, the supernatant of each well was

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collected and incubated with LDH working buffer, then the concentration of LDH in

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each well was detected in a microplate reader at 490 nm.

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6. Protein sample preparation

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S. pneumoniae was grown to an optical density (OD600) of approximately 0.3, then

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exposed to 1/4 × MIC (6.25 µM) of SNH, and samples were taken at 0, 1 and 2 h after

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addition of the SNH. Bacteria were harvested using centrifugation at 8,000 g for 10

140

min at 4 °C, and washed three times with 1 × PBS, then lysed in SDS lysis buffer by

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intermittent sonication. The concentrations of the cellular proteins were measured by

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BCA Protein Assay Kit (Thermo, USA).

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7. iTRAQ Labeling, SCX fractionation and LC-ESI-MS/MS analysis

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Proteins from each sample (200 µg) were processed and subjected to iTRAQ (isobaric

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tags for relative and absolute quantitation) labeling using the AB SCIEX iTRAQ

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reagents Multiplex kit according to the manufacturers’ instructions. Briefly, proteins

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were first reduced and alkylated, then digested with trypsin using FASP (filter aided

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sample preparation) method and finally labeled with 114 (SNH treated 0 h), 116 (SNH

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treated 1 h) and 117 (SNH treated 2 h) isobaric tags. The labeled peptides were

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incubated at room temperature for 2 h and then pooled and dried using vacuum

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centrifugation. The iTRAQ-labeled peptide mixture was dissolved in 100 µL of high

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pH buffer A (20 mM NH4CHO2 containing 4% (v/v) acetonitrile, pH 10.0) and

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separated using reverse-phase UPLC with an Ultremex SCX column (4.6 mm × 250

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mm, 5 µm, Phenomenex, USA). In brief, iTRAQ-labeled peptide mixture was eluted

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with a gradient of 5-60% acetonitrile in 20 mM ammonium formate (pH 10.0) for 65

156

min at a flow-rate of 0.8 mL/min. The peptide elution was monitored at 214 nm. After

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5 minutes, the eluted peptides were collected every minute, pooled into ten fractions

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and then lyophilized.

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Dried fractions from the high pH reverse-phase separations were resuspended

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with buffer A (5% acetonitrile, 0.1% formic acid) and detected using an AB SCIEX

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Triple-TOF 5600 mass spectrometer (AB SCIEX, USA) coupled with a Nanospray III

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source and a pulled quartz tip. The identification parameters were used according to

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previous report 23.

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8. Downstream processing and analysis of proteomics data

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The acquired raw data files (.wiff) of each fraction were combined to perform

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searching against S. pneumoniae D39 protein database, and the proteins were

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identified and quantified using ProteinPilot™ Software 4.5. The search parameters

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were set as follows: Sample Type, iTRAQ 4 plex (Peptide Labeled); Cys. Alkylation,

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Iodoacetic acid; Digestion, Trypsin; Instrument, Triple-TOF 5600; ID Focus,

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Biological modifications; Database, S. pneumoniae D39_.fasta; Search Effort,

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Thorough; Detected Protein Threshold [Unused ProtScore (Conf)] >1.30 (95.0%).

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The protein-level quantitative dataset of 1087 proteins was produced by removing

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proteins with only one unique peptides and those not quantified in all of the three

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replicate experiments. A protein defined as differentially expressed protein (DEP)

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should match with the criteria: the fold change had to be greater than 1.50 or less than

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0.67 with p < 0.05 in SNH treated 1 h or 2 h from biological triplicate experiments.

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Volcano plot and protein hierarchical analysis were performed using Matlab.

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Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment

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analysis was performed on the lists of DEPs to identify over-represented biological

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pathways using Blast2GO program, and visualized with Cytoscape.

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9. Real-Time quantitative PCR (RT-qPCR)

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RNA was prepared from S. pneumoniae D39 treated with 1/4 × MIC (6.25 µM) of

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SNH for 0, 1 h and 2 h. Total RNA was obtained using TRIZOL method according to

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previous protocol

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value of gyrB amplified from the corresponding sample and calculated using the

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2−∆∆Ct method 25. The primer sequences are listed in Table S1.

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10. Concentration measurement of hydrogen peroxide (H2O2)

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To measure H2O2 levels, bacteria were removed by centrifugation at 8,000 g for 5 min

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at 4 °C, then supernatants were filtered through a 0.22 µm filter and used for analysis

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in accordance with the Amplex Red hydrogen peroxide/peroxidase assay kit

24

. The fold changes of selected genes were normalized to the Ct

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(Invitrogen, USA). The final value was normalized to give µM/106 CFU.

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11. Statistical analysis

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Data were analyzed using two-tailed unpaired Student t-test, and expressed as mean ±

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SEM. Statistical analysis was conducted using GraphPad Prism 5.0. For all

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comparisons, p < 0.05 was considered as significant.

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Results

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1. Bactericidal activity of SNH against S. pneumoniae

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The bacteriostatic and bactericidal activities of SNH against S. pneumoniae were

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investigated using MIC and MBC. We measured that the MIC value of SNH against S.

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pneumoniae is 25 µM, and MBC value also is 25 µM. According to previous reports 26,

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the bactericidal activity was defined as the MBC value ≤ 4 × MIC, the value (both

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MIC and MBC equals 25 µM) indicated that SNH is bactericidal. The time-kill curves

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of SNH and ampicillin for S. pneumoniae are displayed in Figure 1; ampicillin was

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used as control in this study as it is well-known broad-spectrum bactericidal that

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inhibits cell wall synthesis. The result confirmed the bactericidal nature of SNH, as 1

206

× MIC, or mostly 2 × MIC was sufficient to kill 99.99% bacteria with 1 h of

207

incubation. In contrast, the time for 1 × MIC or 2 × MIC of ampicillin to kill 99.99%

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bacteria is 6 h. These results indicated that the bactericidal rate of SNH is faster than

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ampicillin.

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2. No toxicity of SNH to host cells

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While good bactericidal activity is basic for a potential antimicrobics, SNH must also

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display low or no toxicity towards host cells. Hence, the cytotoxicity of SNH against

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human bronchial epithelial cell line HBE was determined by LDH assay. As shown in

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Figure 2B, SNH at 200 µM (corresponding to 8 × MIC of SNH against S. pneumoniae)

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exhibited no toxicity towards HBE cells over a 24 h period, suggesting that SNH is

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selectively toxic against S. pneumoniae.

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3. iTRAQ-based proteomic analysis on S. pneumoniae in response to SNH

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In order to investigate the bactericidal mechanism of SNH against S. pneumoniae,

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proteomic analyses were conducted using iTRAQ-based LC-MS/MS approach to

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obtain large scale data sets in three biological replicates. According to the time-kill

221

curves (Fig. 1A) and growth curves (Fig. 2A), 1/4 × MIC doses of SNH were applied

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to treat S. pneumoniae, with a consideration to minimize bacterial cell death caused by

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SNH and thus to observe real changes in the abundance of proteins. Bacteria were

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harvested at 0, 1 h and 2 h after treatment with SNH (6.25 µM) so that they had

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sufficient time to produce proteins to counteract the inflicted damage while

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minimizing the secondary unspecific effects.

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A total of 1087 proteins (Table S2) had quantitative values available from

228

triplicate biological experiments and were used for downstream analyses. The volcano

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plot for the 1087 proteins after SNH-treated 1 h and 2 h are shown in Figures 3A&B.

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The proteins with the fold changes greater than 1.50 or less than 0.67 with p < 0.05

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were considered as DEPs. As shown in Figure 3C, 135 proteins (100 up-regulations

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and 35 down-regulations) were found significantly changed in the samples with

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SNH-1 h treatment, while more DEPs (159 proteins including 82 up-regulations and

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77 down-regulations) were identified in the samples with SNH-2 h treatment. Only 75

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DEPs were overlapped in the two groups, with 60 and 84 proteins uniquely and

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differentially expressed in SNH-1 h or -2 h treatments, respectively (Fig. 3D). In order

237

to comprehensively analyze the protein changes in response to the SNH incubation,

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all the 219 proteins (Table S3) that belong to the union set of DEPs in both treatment

239

groups were subjected to bioinformatics analysis.

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4. Classification of the identified DEPs

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To gain insights into the biological pathways of the 219 DEPs, hierarchical clustering

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of these proteins was performed using Matlab to group the data into those proteins

243

increasing in abundance following SNH treatment and those decreasing in abundance

244

(Fig. 4A). KEGG pathways enrichment analysis indicated that the proteins generally

245

down-regulated in SNH treatment are enriched in pathways associated with ribosome,

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fatty acid biosynthesis and RNA polymerase, while the up-regulated proteins are

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enriched for propanoate metabolism, oxidative phosphorylation, other glycan

248

degradation, bacterial secretion system, pyruvate metabolism and pyrimidine

249

metabolism.

250

As shown in Figure 4, 27 ribosomal proteins responsible for the protein

251

biosynthesis (Fig. 4B), three RNA polymerases (RpoB, RpoC, RpoZ) (Fig. 4D), and

252

eight proteins (AccB, AccC, AccD, FabD, FabF, FabG, FabH and FabK) involved in

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fatty acid biosynthesis (Fig. 4E) decreased their expression following SNH treatment.

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These data suggest that RNA, protein and fatty acid biosynthesis were impaired in the

255

bacterium after being exposed to SNH. Two possibilities may lead to this result. One

256

is that SNH may directly inhibit the RNA, protein and fatty acid biosynthesis, and the

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other is that SNH may stimulate the production of ROS and indirectly interfere the

258

RNA, protein and fatty acid biosynthesis, then eventually induced cell death.

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Moreover, seven proteins involved in pyruvate metabolism were up-regulated

260

after SNH treatment (Fig. 4E), which is compatible with the second possibility. In

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particular, pyruvate oxidase (SpxB), an enzyme converting pyruvate to hydrogen

262

peroxide (H2O2) 27, was up-regulated by 2.56 folds and 3.52 folds in SNH-1 h and -2 h

263

treatment, respectively. The up-regulation of spxB in mRNA level was further

264

validated by RT-qPCR analysis (Fig. 5A). Also, non-heme iron-containing ferritin

265

(SPD_1402, homologous with Dpr) involved in hydrogen peroxide stress defense

266

(Table S3 & Fig. 5A) and phosphocarrier protein HPr (PstH) that transports phosphate

267

were increased after SNH treatments. In addition, we observed the significant

268

up-regulation of chaperonin proteins involved in the regulation of misfolded proteins

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(DnaJ, GroS, GrpE) (Table S3). All these observations suggest that ROS production

270

was stimulated by SNH to induce cell death.

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5. H2O2 formation induced by SNH contributing to cell death

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To support the hypothesis that SNH may simulate the production of high ROS and

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indirectly interfere the RNA, protein and fatty acid biosynthesis, then eventually lead

28

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to cell death, we measured the levels of H2O2 in the supernatants from various SNH

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treatments (corresponding to 0, 1/8 × MIC, 1/4 × MIC, 1/2 × MIC and 1 × MIC). As

276

expected, we observed cellular death with 1/4 × MIC, 1/2 × MIC and 1 × MIC SNH

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(Fig. 1A), accompanied with the increase of H2O2 production (Fig. 5B). Furthermore,

278

the concentration of H2O2 in supernatant at 2 h incubation with SNH was more than

279

that with 1 h incubation. These results indicate that SNH induced H2O2 formation in

280

time- and concentration-dependent manners, which are in accord with the time-kill

281

curves.

282

To further explore the biological significance of H2O2 contribution to cell death,

283

we exploited catalase that can neutralize H2O2 to the experiment. We found that S.

284

pneumoniae treated simultaneously with SNH and catalase (2 mg/mL) showed a

285

significant increase in cell survival, near to the survival level of no SNH treatments

286

(Fig. 5C). This increase in bacterial survival suggests that catalase almost eliminated

287

the SNH bactericidal effect, indicating that H2O2 was indeed an important mediator in

288

the bactericidal action of SNH. These results provided evidence that H2O2 plays a

289

significant role in SNH-induced cell death.

290

Discussion

291

SNH inhibits a variety of bacteria, yet its anti-bacterial mechanism remains unknown.

292

In this study, we investigated the bactericidal activity and mechanism of SNH using S.

293

pneumoniae as a model. The MIC and MBC assays combined with time-kill curves

294

showed that SNH killed S. pneumoniae in a dose-dependent manner. We then

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295

employed iTRAQ-based quantitative proteomics analysis to demonstrate that SNH

296

stimulated the expression of the proteins involved in H2O2 formation pathway (Fig. 6),

297

including pyruvate metabolism related proteins (Pta, SpxB, Ldh, PflB) and

298

phosphocarrier protein PstH. Meanwhile, proteins involved in protective responses to

299

ROS, such as non-heme iron-containing ferritin (SPD_1402) and chaperone systems

300

proteins DnaJ, GroS and GrpE, were activated by SNH treatment. On the other hand,

301

SNH suppressed 27 ribosomal proteins responsible for the protein biosynthesis, three

302

RNA polymerases (RpoB, RpoC, RpoZ) involved in RNA biosynthesis and eight

303

proteins (AccB, AccC, AccD, FabD, FabF, FabG, FabH and FabK) involved in fatty

304

acid biosynthesis (Fig. 6).

305

Importantly, the content of H2O2 was greatly increased in the culture supernatant

306

of the bacterium exposed to SNH, while the H2O2 neutralizing enzyme catalase can

307

greatly attenuate the killing by SNH (Fig. 5). Based on these observations, we

308

concluded that SNH induced the formation of H2O2 via SpxB participation in pyruvate

309

metabolism, then elevated H2O2 to result in the damage to RNA, protein and cell

310

membrane lipids and ultimately contributed to the cell death of bacteria (Fig. 6).

311

Interestingly, previous in vitro work has shown that SNH demonstrated

312

antibacterial activity against 103 clinical strains of MRSA, functioning in synergetic

313

effects with oxacillin or netilmicin 15. Here, we also detected the antibacterial activity

314

of SNH against S. aureus by measuring the level of H2O2 in the culture supernatant

315

after SNH treatment. As shown in Figure S1, SNH stimulated the H2O2 production

316

and catalase also greatly decreased the inhibition effect of SNH on S. aureus, a result 15



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317

in agreement with the observation in S. pneumoniae. In addition, SNH analogue SH

318

showed the antibacterial activity against biofilm formation and alginate production of

319

P. aeruginosa 29, echoing the report that ROS production contributes to the killing of

320

biofilm subpopulations in P. aeruginosa exposed to ciprofloxacin

321

results, it seems reasonable to conclude that SNH utilizes a common mechanism of

322

bacteria-killing by inducing the production of ROS responsible for cell lethality.

30

. With these

323

It is somewhat surprising that the bactericidal rate of SNH is better than

324

ampicillin. As shown in time-kill curves (Fig. 1), 1-2 × MIC SNH killed almost all the

325

bacteria in 1 hour, while the equivalent concentration of ampicillin needed 6 hours to

326

kill the bacteria. Bacterial reproduction is very rapid, the generation of most bacteria

327

is about 20-30 min; the shorter time of killing means more effective for the drug to

328

control the infection. Interestingly, the bacteria could recover from the sixth hour post

329

the application of 1/2 × MIC SNH; a possible reason is that some bacteria could not

330

be eliminated by the low concentration of SNH and then became resistant to the drug.

331

However, this phenomenon was not observed during ampicillin application,

332

implicating that the antibacterial mechanisms between SNH and ampicillin may be

333

different. These results provide helpful information for guiding the use of an

334

appropriate concentration of SNH in disease treatment, and also suggest that SNH

335

may be combined with existing antibiotics as an effective strategy to combat bacteria.

336

Bactericidal drug design has generally fallen into three groups: inhibition of DNA

337

replication, protein synthesis and cell-wall turnover, which has yielded great progress

338

in antimicrobial therapy 31. However, the rise of antibiotics resistant bacteria has made 16


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339

it critical for us to develop novel and more effective strategies to kill bacteria. In

340

recent years, the formation of ROS represents one of the major mechanisms to

341

generate cell death. Our results indicated that SNH possesses strong bactericidal

342

activity against S. pneumoniae based on the generation of H2O2. Moreover,

343

proteomics coupled with bioinformatics analysis may reveal drug targets for

344

stimulating ROS formation and thus open-up new avenues for developing new classes

345

of bactericidal drugs.

346

347

ASSOCIATED CONTENT

348

Supporting Information

349

Supporting Information Available: Figure S1, Tables S1, S2 and S3. This material is

350

available free of charge via the Internet at http://pubs.acs.org.

351 352

Funding

353

This work was supported by the National Natural Science Foundation of China

354

(21271086, to Q.-Y. H.), China Postdoctoral Science Foundation (153150, to X.-Y. Y.),

355

and the Open Fund of the First Affiliated Hospital, Jinan University.

356

Notes

357

The authors declare no competing financial interest.

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359

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Figure captions

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Figure 1. Time-kill curves of SNH (A) and ampicillin (B) against S. pneumoniae.

455

Figure 2. Effects of SNH on S. pneumoniae and human bronchial epithelial cell line

456

HBE. (A) SNH inhibited the growth of S. pneumoniae in a dose-dependent manner.

457

The growth curve measurements were taken every hour for the first 4 h and every two

458

hours for next 8 h after drug addition at OD600. (B) SNH exhibited low toxicity

459

towards host cells. HBE cells were treated with the various concentrations of SNH,

460

and the cytotoxicity was determined using LDH Cytotoxicity Assay Kit. The data

461

shown represent the mean of three experiments; error bars indicate SEM.

462

Figure 3. Proteomic changes in S. pneumoniae in response to the treatment with SNH.

463

(A) Volcano plots of DEPs in SNH treatment for 1 h. (B) Volcano plots of DEPs in

464

SNH treatment for 2 h. (C) Numbers of DEPs in the SNH-1 h and -2 h treatments. (D)

465

Venn diagram of the number of DEPs in the SNH-1 h and -2 h treatments,

466

respectively.

467

Figure 4. Classification of 219 proteins that belong to the union set of DEPs in both

468

SNH-1 h and -2 h treatments. (A) Hierarchical cluster analysis was conducted using

469

Matlab for the 219 proteins. The color indicates relative fold changes (red =

470

up-regulated, blue = down-regulated). DEPs were then subjected to KEGG pathways

471

analysis using Blast2GO program, significantly enriched pathways (p < 0.05) were

472

summarized in the tables shown. (B-G) The pathways enriched using Blast2GO,

473

visualized with Cytoscape. Red represents the proteins up-regulated, blue represents 24


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the proteins down-regulated after SNH treatments.

475

Figure 5. SNH induced H2O2 formation that contributed to cell death. (A) RT-qPCR

476

analysis of selected genes, spxB, atpA, spd_1402, fabK and fabG in S. pneumoniae

477

with SNH treatments for 1 and 2 h versus 0 h (control). The relative gene expression

478

was calculated with gyrB as the reference gene. All results represent the relative

479

expression level with SNH treatments for 1 and 2 h versus 0 h (control) (**p < 0.01,

480

***p < 0.001), shown as the means value (± SEM) from three independent

481

experiments. (B) H2O2 production quantified in the culture supernatants at 0, 1 and 2

482

h treatments with different concentrations of SNH (0, 1/8 × MIC, 1/4 × MIC, 1/2 ×

483

MIC and 1 × MIC). Results show mean ± SEM for three experiments. **p < 0.01,

484

***p < 0.001, unpaired Student's t-test. (C) Log change in CFU/mL following

485

exposure to no SNH as the control (circle), 1 × MIC SNH (cross) and 1 × MIC SNH

486

with 2 mg/mL catalase (diamond). Results show mean ± SEM for three experiments.

487

Figure 6. Proposed mechanism of SNH-induced cell death in S. pneumoniae. SNH

488

induced the formation of H2O2 via SpxB participation in pyruvate metabolism, then

489

elevated H2O2 to result in the damage to RNA, protein and cell membrane lipids and

490

finally contributed to cell death.

491 492

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Figure 1

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iTRAQ-Based Proteomics Revealed the Bactericidal Mechanism of

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