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Available online at www.sciencedirect.com

ScienceDirect www.elsevier.com/locate/jprot

A comprehensive proteomic analysis of totarol induced alterations in Bacillus subtilis by multipronged quantitative proteomics Panga Jaipal Reddya , Sandipan Ray a , Gajanan J. Satheb,c , Akshada Gajbhiyed , T.S. Keshava Prasadb , Srikanth Rapoled , Dulal Pandaa , Sanjeeva Srivastavaa,⁎ a

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, Maharashtra, India Institute of Bioinformatics, International Tech Park, Whitefield, Bangalore 560066, India c Manipal University, Madhav Nagar, Manipal 576104, India d Proteomics Lab, National Centre for Cell Science, Ganeshkhind, Pune, Maharashtra, India b

AR TIC LE I N FO

ABS TR ACT

Article history:

The rapid emergence of microbial drug resistance indicates the urgent need for

Received 23 May 2014

development of new antimicrobial agents. Bacterial cell division machinery is considered

Accepted 20 October 2014

as a promising antimicrobial target. Totarol is a naturally existing diterpenoid, which has

Available online 20 November 2014

the ability to restrain bacterial growth by perturbing the cell division. The present study was

Keywords:

totarol treatment to decipher its mechanism of action and possible molecular targets.

conducted to investigate the proteomic alterations in Bacillus subtilis as a consequence of Totarol

Cellular proteome of the totarol treated B. subtilis AH75 strain was analyzed by using

Filamentation

multiple complementary proteomic approaches. After the drug treatment, 12, 38 and 139

Dehydrogenases

differentially expressed (1.5 fold change) proteins were identified using 2-DE, DIGE and

iTRAQ

iTRAQ analyses, respectively. In silico functional analysis of the identified differentially

B. subtilis

expressed proteins indicated a possible effect of totarol on the central metabolism for

Proteomics

energy production, heme biosynthesis and chemotaxis. Interestingly, the primary dehydrogenases, which play a vital role in generating the reducing equivalent, were found to be repressed after totarol treatment indicating an apparent metabolic shutdown. Consequently, multiple cellular assays including resazurin assay and FACS analysis of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining confirmed the effect of totarol on respiratory activity and cellular metabolism. Biological significance The exact mechanism of action of totarol is still unclear and further investigations are essential to identify the molecular/cellular targets of this potential antimicrobial agent. The

Abbreviations: iTRAQ, isobaric tag for relative and absolute quantitation; DIGE, difference gel electrophoresis; CTC, 5-cyano-2,3-ditolyl tetrazolium chloride; FDR, false discovery rate; DAVID, Database for Annotation, Visualization and Integrated Discovery; KOBAS, KEGG Orthology Based Annotation System; MIC, minimum inhibitory concentration; DAPI, 4,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting. ⁎ Corresponding author at: Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400076, India. Tel.: + 91 22 2576 7779; fax: + 91 22 2572 3480. E-mail address: [email protected] (S. Srivastava).

http://dx.doi.org/10.1016/j.jprot.2014.10.025 1874-3919/© 2014 Published by Elsevier B.V.

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present study demonstrates the application of differential proteome to decipher the mechanism of action and molecular targets of totarol in B. subtilis. Our quantitative proteome analysis revealed that totarol induced alterations in the expression levels of 139 proteins (1.5 fold change and ≥2 peptides) in B. subtilis. Findings obtained from this study indicate that totarol treatment leads to metabolic shutdown by repressing the major central metabolic dehydrogenases in B. subtilis. In addition, expression levels of universal chaperone proteins, heme biosynthesis, and ribosomal proteins were found to be altered, which caused the filamentation of the bacteria. To the best of our knowledge, this is the foremost inclusive investigation describing totarol induced alterations in B. subtilis proteome and diverse physiological processes. We anticipate that this in depth proteomic study may contribute to a better understanding of the mode of action of totarol and its primary molecular and cellular targets. © 2014 Published by Elsevier B.V.

1. Introduction Infectious diseases are still the leading global health concerns; even though many synthetic as well as semi-synthetic effective drugs are in practice [1]. The journey of drug discovery started from the “natural products (NPs)” long ago during the ancient civilizations of India, China and other East Asian countries. During the last decade, the synthetic candidates have significantly replaced the NPs as antimicrobial drugs; at the same time excessive and indiscriminate usage of antibiotics has led to the unanticipated alterations in the microbial genome, which introduced a rapid spread of antibiotic-resistant strains. Consequently, the emergence of antibiotic-resistance highlighted again the need for comprehensive research on natural products for drug discovery due to their safe and secure applications [2]. Recently, regulators of cell division machinery have been studied extensively for identification of novel antimicrobial agents [3], and many natural compounds have been screened to evaluate their effects on bacterial cell division and growth. Totarol is a natural diterpenoid extracted from the bark of Podocarpus family and possesses anti-microbial [4], anti-oxidant [5], cytotoxic [6], anti-fungal [7], anti-plasmodial [8] and anti-tumor activities [9]. Kubo et al. extracted six different diterpenoid products from the bark of Podocarpus nagi, investigated their effect against various microorganisms, and reported that totarol is the only active product with potential antimicrobial activity [10]. Subsequently, many researchers have actively focused on the elucidation of the mechanism of action of totarol and demonstrated multiple targets for this promising candidate including inhibition of the energy-coupled respiratory transport [11], prevention of peroxidation of unsaturated fatty acids in the lipid bilayer [5], and hampering the oxidative phosphorylation by acting either as an uncoupler or by inhibiting the crucial enzymes [12]. Recent studies indicated that totarol could also restrain the multidrug efflux pumps [13,14], disturb the phospholipid bilayer permeability [15], and inhibit bacterial cell division by perturbing the FtsZ polymerization [16]. However, the exact mechanism of action of totarol at the molecular level is still obscure, and further investigations are required to identify its molecular and cellular targets. Quantitative analysis of bacterial proteome in the presence and absence of specific antimicrobial agents was found to be

informative for identification of their molecular targets and unraveling their mechanism of action. In the present study, in order to evaluate the cellular effects of totarol, we have selected Bacillus subtilis as the model organism, since it is a widely studied non-pathogenic microorganism, whose genome sequence and physiological vegetative proteome in response to various stress conditions are already reported [17–24]. Here we aimed at deciphering the mechanism of action and possible molecular targets of totarol by using two dimensional electrophoresis (2-DE), 2D-differential in gel electrophoresis (2D-DIGE) and isobaric tags for relative and absolute quantitation (iTRAQ) analysis using LTQ-Orbitrap Velos, Q-TOF and MALDI-TOF/TOF mass spectrometers. Employing these complementary proteomic technologies, we were able to identify overall 1194 proteins (1% FDR). A total of 139 proteins were found to be differentially expressed (69 down-regulated and 70 up-regulated with a ≥1.5 fold change) and were considered for further analysis. In silico analysis involving the differentially expressed proteins indicated the modulation of glycolysis, TCA cycle and heme biosynthesis due to totarol treatment. Additionally, multiple cellular assays including resazurin-based metabolic activity assay and fluorescence-activated cell sorting (FACS) analysis of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining for respiratory activity assay corroborated the effect of totarol on respiratory activity and cellular metabolism. To the best of our knowledge, this is the foremost inclusive investigation describing the totarol induced alterations in B. subtilis proteome.

2. Materials and methods 2.1. Growth curve analysis of B. subtilis under totarol treatment The B. subtilis AH75 strain having a spectinomycin antibiotic marker was grown overnight at 37 °C in Luria Broth (LB) containing 100 μg/mL spectinomycin [25]. This culture was diluted with fresh LB media to maintain the OD600 at 0.05 and subsequently incubated again at 37 °C till the OD600 reached 0.1. B. subtilis growth was measured by monitoring the OD at 600 nm for the untreated control and IC50 (1.5 μM) and MIC (2 μM) totarol treated cultures at 37 °C. The growth curve was plotted with the mean values of triplicate experiments by measuring the OD at 600 nm at every 20 min intervals and continued till 300 min (mid-exponential phase).

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2.2. Microscopic analysis of B. subtilis morphology Totarol (Industrial Research Limited, New Zealand) was dissolved in DMSO to prepare a stock concentration of 10 mM. Two different concentrations of the drug; 1.5 and 2.0 μM (IC50 and MIC, respectively) were added to the bacterial cultures (OD600 = 0.2), and the cultures were further allowed to grow for 2 h in the presence of the antimicrobial agent. The IC50 and MIC values of totarol for B. subtilis 168 were specified following the previous report by Jaiswal et al. [16]. Control cultures were grown under identical experiment conditions, except, only the solvent (DMSO) was added instead of the totarol solution. Once the bacterial cultures reached the mid-exponential phase, both control and totarol treated samples (20 mL each) were harvested by centrifugation at 7000 rpm for 10 min at 4 °C. The harvested cultures were fixed in 2.8% formaldehyde and 0.04% glutaraldehyde at 37 °C; washed 3 times with PBS buffer and resuspended in the same buffer. The nucleoids were stained with 1 μg/mL of DAPI for 20 min in the dark and the morphology of the bacterial cells was observed using a differential interference contrast microscope (Eclipse TE-2000 U microscope; Nikon) at 40× magnification.

2.3. Protein extraction, 2-DE and DIGE analyses Protein extraction from B. subtilis AH75 was performed using the TRIzol protocol reported previously by Reddy et al. [26]. 2-DE and data analyses were performed as described previously [27,28]. Passive rehydration was performed using 24 cm IPG strips (linear pH 4–7; GE Healthcare) for 14 h at RT, and 600 μg of protein was loaded on each strip. The second dimension separation was performed on 12.5% SDS polyacrylamide gels using an Ettan DALTsix electrophoresis unit (GE Healthcare). Triplicate images of the control and totarol treated samples were imported into the Image Master 2D Platinum 7.0 software (GE Healthcare) for comparative analysis. Spots present in all the gels were considered for detailed analysis by using the match tables, histograms and 3D views generated by the software. The differentially expressed and statistically significant (p ≤ 0.05) protein spots present in all the gels were excised and stored at 4 °C for subsequent mass spectrometric analysis. Minimal CyDye labeling and 2D-DIGE were performed as described previously [26,28]. Protein samples extracted from totarol treated and control cultures (three biological replicates) were labeled with CyDyes following the manufacturer's protocol (GE Healthcare). In brief, 60 μg of protein taken from the control and totarol treated samples were labeled with 400 pmol of either Cy3 or Cy5, and internal control containing the equal amount of control and totarol treated samples was labeled with Cy2. Dye swapping was performed for labeling the test and control samples to get rid of dye biasness. Rehydration, IEF and SDS-PAGE settings were similar to classical 2-DE. Gels were scanned using a Typhoon FLX9500 scanner at excitation/emission wavelengths for each of the CyDye [Cy3 (523/580 nm), Cy5 (633/670 nm), and Cy2 (488/ 520 nm)] at 100 μm resolution. DIGE images obtained from FLX9500 were imported into DeCyder software version 7.0 (GE Healthcare). The image loader module was used to crop the gels uniformly and create

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projects consisting of control vs. totarol treated samples. Initially, a batch process module was used to assign the spot boundaries to get the normalized spot volume for individual spots on each gel with respect to the internal control. Batch process allows performing differential in gel analysis (DIA) and generating the files for further biological variation (BVA) analysis. In BVA, all the Cy2 gels were matched with respect to the master gel (internal control having the highest number of spots) and the average volume ratios were compared across the gels. Statistical analysis was performed using the paired t-test. Statistically significant (p ≤ 0.05; present in all replicates) differentially expressed spots were excised from preparative gels for in-gel digestion.

2.4. In-gel digestion and MALDI-TOF/TOF analysis In-gel digestion was carried out as reported by Shevchenko et al. and Reddy et al. with some modifications [26,29]. The gel pieces were washed thrice with a 25 mM ammonium bicarbonate (ABC) buffer and solution A [25 mM ammonium bicarbonate/acetonitrile (ACN) (1:2 v/v)] with intermittent vortexing to remove the CBB stain completely. Then the gel pieces were incubated in a reduction solution (10 mM DTT in 100 mM ABC buffer) for 1 h at 56 °C to break the disulfide bonds followed by addition of an alkylation solution (50 mM IAA in 100 mM ABC buffer), and kept in the dark for 20 min. Again, the gel pieces were washed twice with 25 mM ABC buffer and solution A with intermittent vortexing, and dried completely before trypsin digestion. Trypsin solution (Trypsin Gold; Promega, Madison, USA) was added to the dried gel pieces keeping the ratio of trypsin:protein around 1:10 (w/w). After addition of the trypsin solution, the gel pieces were kept at 37 °C overnight in a dry bath. The digested peptide fragments were extracted with an extraction solution containing a gradient of ACN (50–80%) and 0.1% TFA. Finally, trypsin digested samples were further processed using Zip-Tip C18 pipette tips (Millipore, USA) following the manufacturer's protocol and subjected to a 4800 MALDI-TOF/TOF mass spectrometer (ABSCIEX, Framingham, MA) linked to a 4000 series explorer software (v.3.5.3) as described by Reddy et al. [26,27].

2.5. iTRAQ labeling, SCX and OFFGEL fractionation Soluble protein extraction from B. subtilis was performed using the TRIzol protocol reported previously [26]. For iTRAQ analysis three sets of (each set of sample having a pool of biological triplicates of control and totarol treated protein samples) samples dissolved in a rehydration solution were subjected to buffer exchange (TEAB) using an Amicon Ultra 0.5 mL centrifugal filter (Sigma) to remove the urea, CHAPS and other salts. 60 μg of protein was taken from each set (control and totarol treated) after quantifying the protein with the Quick-Start Bradford reagent (BioRad, USA) and was diluted with a dissolution buffer (TEAB), and 1 μL of denaturant (2% SDS) was added. The reduction reaction was carried out with Tris (2-carboxyethyl) phosphine (TCEP) at 60 °C for 1 h followed by alkylation by using 1 μL of cysteine blocking reagent (MMTS), and the reaction mixtures were vortexed and incubated at room temperature for 10 min. Trypsin digestion was carried out by using sequencing grade trypsin (Promega)

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(trypsin: protein ratio at 1:20) for 16 h at 37 °C. Digested peptides were labeled with the iTRAQ reagent kit (Applied Biosystems Inc., CA, USA) following the manufacturer's protocol as described earlier [30]. Briefly, 2-plex iTRAQ reagents were used for labeling of each set; peptides obtained after trypsin digestion of the control and totarol treated (120 min) samples were labeled with iTRAQ reagents 114 and 115 in the case of LTQ-Orbitrap and Q-TOF analyses, whereas for MALDI-TOF/TOF analysis 114 and 117 iTRAQ reagents were used to label the control and treated samples, respectively. Two sets of the labeled peptides from the control and totarol treated samples were reconstituted in 5 mM potassium phosphate buffer in 25% ACN (pH 2.7), pooled and subjected to strong cation exchange (SCX) chromatography following the same protocol as described earlier [31]. A PolySulfethyl A column (PolyLC, Columbia, MD) (100 × 2.1 mm, 5 μm particles with 300 Å pores) was used for SCX fractionation on an Agilent 1100 series LC system. Dried peptides were reconstituted in solution-A (5 mM KH2PO4 pH 2.7, 30% ACN). A gradient of 50 min from 5 to 40% solution B (350 mM KCl in solution A) with flow rate 300 μL/min was used for fractionation, and a total of 96 fractions were collected. The peptides were cleaned up with C18 STAGE tips and reconstituted in 0.1% formic acid. One set of labeled peptides from the control and totarol treated samples were pre-fractioned using a 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA) with a high resolution (pH 3–10, 24 cm) IPG strip according to the manufacturer's protocol given for peptide fractionation. After OFFGEL fractionation each fraction was cleaned up with C18 STAGE tips before injecting into the LC–MS/MS.

2.6. Nano-LC-Orbitrap–MS/MS, Q-TOF and MALDI-TOF/TOF analysis SCX fractionated samples were used for LTQ-Orbitrap and MALDI-TOF/TOF analysis whereas OFFGEL fractionated samples were used for Q-TOF analysis (Table S1). The LTQ-Orbitrap Velos mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) equipped with Proxeon Easy nLC liquid chromatography was used for LC–MS/MS analysis. Magic C18 AQ reversed phase material (Michrom Bioresources, 5 μm,100 Å) was used to make the in-house chromatographic capillary columns in 100% ACN at a pressure of 1000 psi. A trap column (75 μm × 62 cm) was used at a flow rate of 3 μL/min to enrich the peptides followed by an analytical column (75 μm × 10 cm) at a flow rate of 350 nL/min to resolve the peptides. A linear gradient of 7–35% ACN was used for 60 min to elute the peptides from the analytical column. The mass spectrometry parameters followed in this study include: acquisition of the full scan data with a mass resolution of 60,000 at 400 m/z using the Orbitrap mass analyzer, 20 intense peaks from each MS cycle were selected for MS/MS with a mass resolution of 15,000 for fragmentation at 400 m/z, 40% normalized collision energy for fragmentation with a 30 sec exclusion time. Automatic gain control and filling time were kept 5 × 105 ions and 100 ms for MS, and 1 × 105 ions and 500 ms for MS/MS, respectively. Polydimethylcyclosiloxane (m/z, 445.1200025) ion was used for internal calibration. Quantitative proteome analysis was performed with 1260 Infinity HPLC-nano-chip linked to 6550 Q-TOF iFunnel

technology (Agilent Technologies, USA) having polaris C18A chip (150 mm × 0.075 mm) with 160 nL trap column. A linear gradient of 7–35% ACN was used for 60 min to elute the peptides from the analytical column with flow-rates of 2 μL/min for the capillary pump and 0.5 μL/min for the nano pump. Mass spectrometric analysis was performed online with m/z ranging from 100–3200, MS scan rate of 6 spectra/s, MS/MS scan rate of 3 spectra/s, with top 15 peaks for MS/MS analysis. The cleaned-up SCX fractions were injected into Tempo Nano MDLC system (Eksigent) with 0.5 mm × 2 mm CapTrap™ C18 PepMap guard column (Michrom Bioresources, Auburn, CA) and an Agilent 300 SB C18 (0.075 μm × 150 mm) 3.5 μm, 300 Å Nano column (Agilent Technologies). A linear gradient of 5– 30% ACN for 30 min, 30–50% ACN for 20 min, increase to 80% ACN in 25 min, hold on 80% ACN for 10 min with a flow rate of 300 nL/min. The elution was mixed with MALDI matrix at 800 nL/min flow rate solution (2.5 mg/mL α-cyano-4hydroxycinnamic acid [CHCA] in 80% ACN + 0.1% TFA) using an Ekspot Spotter (Eksigent) and spotted onto 28 × 44 spot arrays on 123 mm × 81 mm Opti-TOF LC/MALDI inserts (ABSCIEX). Spotting time was from 15 to 150 min, with 20 s spot intervals. The ABSCIEX MALDI-TOF/TOF 4800 mass spectrometer equipped with a 200 Hz repetition rate Nd:YAG laser was used for data acquisition. The strongest precursor first selection order was performed using the following criteria: minimum S/N ratio, 60; maximum precursors per spot — 18, and all MS/MS spectra were acquired with 2 kV collision energy by accumulation of 2500 laser shots.

2.7. Mass spectrometry data analysis and data availability SEQUEST (SCM build 59) search engine configured with Proteome Discoverer 1.4 workflow (Thermo Fischer Scientific, Bremen, Germany) was used for both LTQ-Orbitrap and Q-TOF mass spectrometer data analyses (Table S1). B. subtilis 168 reference protein database obtained from the UniProtKB database (http://www.uniprot.org/) containing 4227 reviewed FASTA sequences were configured with SEQUEST for searching the datasets. The analysis workflow includes the spectrum file addition, spectrum selector, SEQUEST HT analysis, target decoy and PSM validator followed by a reporter ion quantifier. The search parameters include: 20 ppm and 0.1 Da mass tolerances for MS and MS/MS respectively, trypsin as the proteolytic enzyme with one allowed missed cleavage, iTRAQ modification at N-terminal and lysine and alkylation of cysteine as fixed modifications and oxidation of methionine as variable modification. Further, the peptides were extracted using high peptide confidence and top one peptide ranking. False discovery rate (FDR) was calculated using a decoy database by searching the peptide sequence. Peptides passing 1% FDR threshold were considered for quantitation by measuring the intensity of the reporter ions. The average relative intensity of the two reporter ions can give the relative quantity of the protein and unique peptides were considered for quantitation. Quantitation of the identified peptides was normalized on protein median [32]. MALDI TOF/TOF data was analyzed using ProteinPilot™ 3.0 software (ABSCIEX) with the integrated Paragon™ search algorithm and Pro Group™ algorithm. The data analysis parameters were set as follows: Sample type-iTRAQ (peptide

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labeled), fixed modification — Cys alkylation, iTRAQ label at N-terminal and lysine, digestion — Trypsin, MS and MS/MS tolerance — 75 ppm and 0.4 Da, instrument — MALDI-TOF/ TOF 4800 (AB Sciex), taxonomy — B. subtilis; ID focus — biological modifications; database — SwissProt, search effort — thorough, FDR analysis — yes, bias correction — no. The unique peptides (1% FDR) were considered for relative protein quantification. The LTQ-Orbitrap mass spectrometry proteomics data was deposited at the ProteomeXchange Consortium [33] via the PRIDE partner repository with the dataset identifier PXD001097.

2.8. Functional pathway and network analysis Differentially expressed proteins with statistical significance (p ≤ 0.05) identified in 2-DE, DIGE and iTRAQ (only Orbitrap data) were subjected to DAVID (Database for Annotation, Visualization and Integrated Discovery) database version 6.7 (http://david.abcc.ncifcrf.gov/home.jsp) [34,35]. The list of UniProt accession IDs of the up- and down-regulated proteins were uploaded into the DAVID database and mapped against the B. subtilis dataset for pathway analysis using the default settings. The list of UniProt accession IDs of the up and down regulated proteins were further imported into KOBAS 2.0 (KEGG Orthology Based Annotation System) database and pathways were mapped against the B. subtilis using the default settings [36]. In addition, the protein–protein interaction analysis (both physical and functional interactions) was performed using STRING 9.1 database (http://string-db.org/) with default parameters and B. subtilis as the organism of interest [37].

2.9. Resazurin microtiter assay for metabolic activity Resazurin assay was performed following the same protocol as described by Mariscal et al. to check cell viability and metabolic activity [38]. Three different dilutions of the control and 120 min totarol treated B. subtilis cultures with cell population ranging from 106 to 108 cells/mL were analyzed in triplicates. Cell numbers in the control and drug treated cultures were determined by measuring the optical density (OD600). Prior to the assay, bacterial cultures were washed thrice with chilled PBS to remove media. Resazurin dye was used at a concentration of 10 μg/mL and the fluorescence intensity was measured at 590 nm for 30 min with 15 sec intervals in a RT-PCR machine (MyiQ2 System, BioRad, USA). The mean values of the totarol treated and control groups were compared by using Student's t-test and a p-value < 0.05 was considered as statistically significant.

2.10. CTC staining and flow cytometric analysis for respiratory activity A 50 mM stock solution of 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC) was prepared in ultrapure deionized water. The control and 120 min totarol treated (1.5 μM) B. subtilis cultures were harvested and washed twice with PBS and finally dispersed in 900 μL of the same buffer. 100 μL CTC stock was added to the cells to obtain a final concentration of 5 mM. The mixture was kept at 37 °C for 30 min in the dark followed by a counter staining with DAPI (1 μg/mL). A negative control was

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prepared by fixing the cells with 2.8% formaldehyde and 0.04% glutaraldehyde for 30 min on ice before the treatment with CTC [39]. Fluorescence-activated cell sorting (FACS) analysis of B. subtilis cultures (test and controls) incubated with CTC was performed using a flow cytometer (FACS Aria; Becton Dickinson, San Jose, CA, USA). The instrument was operated following the standard parameters; blue fluorescence (515 nm) for DAPI and red fluorescence (630 nm) for CTC on logarithmic scale. Approximately 20,000 events were measured at a flow rate of 1000 cells/s. Analysis of FACS data was performed using Cyflogic flow cytometry data analysis software, version 1.2.1 (http://www.cyflogic.com/).

3. Results 3.1. Effect of totarol treatment on the B. subtilis growth and cell morphology Growth curve analysis of the B. subtilis AH75 was performed by measuring the OD600 every 20 min till 300 min (mid-exponential phase) of growth for the control, IC50 and MIC totarol treated cultures. The control B. subtilis showed progressive growth with time, whereas the IC50 and MIC treated totarol cultures showed declined growth after the drug treatment. The totarol IC50 treated cultures showed reduced growth rate compared to the untreated control, while the totarol MIC treated cultures exhibited virtually no growth (Fig. 1A). Further, the control and totarol treated (IC50 and MIC) B. subtilis AH75 cultures were investigated by fluorescent microscopy to visualize the morphological changes in the bacterial cells due to the totarol treatment. Microscopic analysis revealed that normal cells in the absence of totarol have a typical isolated rod shape appearance (Fig. 1B). Incubation of the cultures for 120 min with the IC50 of totarol (1.5 μM) promotes cell elongation, without affecting the nucleoid segregation, and exhibited subtle consequence on cell viability. Since cell viability was exceedingly affected at the MIC of the drug, IC50 was specified for analysis of the mechanism of action and molecular targets of totarol at the proteome level.

3.2. Alterations in B. subtilis proteome due to totarol treatment revealed by gel-based proteomic analysis The major intention of this study was to investigate the proteomic alterations of B. subtilis in response to the totarol treatment (Fig. 2). Comparative proteomic analysis between the totarol treated (1.5 μM) and untreated B. subtilis cells using 2-DE indicated that around 1000 protein spots were commonly shared between the control and treatment groups, among which, 15 spots exhibited differential expression with statistical significance (p ≤ 0.05). Among the 15 differentially expressed protein spots, 11 spots were down-regulated, while the remaining 4 spots were up-regulated in the totarol treated samples compared to the controls (Table 1). The representative 2-DE gel images of the control and totarol treated samples, 3D views and histograms of the differentially regulated protein spots are shown in Figs. 3A & S1. The protein identification details obtained from the MALDI-TOF/ TOF analysis are presented in Tables 1 and S2.

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2D-DIGE analysis revealed differential expression of 62 protein spots, which were processed further for in-gel digestion and subjected to MALDI-TOF/TOF analysis. A total of 53 proteins were identified (45 unique proteins), among which 24 proteins

were up-regulated and 29 proteins were down-regulated. Succinyl-CoA ligase (ADP-forming) subunit beta, elongation factor Ts, NADH dehydrogenase, dihydrolipoyl dehydrogenase, 60 kDa chaperonin, 50S ribosomal protein L10 and elongation

Fig. 1 – Effect of totarol treatment on the B. subtilis growth and cell morphology. (A). B. subtilis AH75 cultures were grown till OD600 reached to 0.1, and DMSO was added to the untreated control, while IC50 (1.5 μM) and MIC (2 μM) of totarol was added to the other cultures. The growth curve was performed by taking the OD600 at every 20 min till 300 min (mid exponential phase). The growth curve was plotted by taking the mean values obtained from three independent experiments. (B). B. subtilis AH75 strains were grown in the presence (1.5 and 2.0 μM) and absence (control) of totarol for 2 h and the nuclear materials were stained using 1 μg/μL DAPI for 20 min. The fluorescence microscopic images were captured with both DAPI and DIC filters. The DIC images are shown in brown and DAPI images in blue color. The control B. subtilis cells showed normal cell length with one or two nucleoids per cell (I, II). After 2 h of incubation with 1.5 μM (IC50) totarol, most of the cells turned into filamentous structure with multiple nucleoids (III, IV), while, after totarol treatment at the MIC (2.0 μM) cell population was significantly declined per field and elongated filamentous cells were observed (V, VI).

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factor Tu were detected in multiple spots probably due to the presence of various isoforms (Tables 1, S3 and S4). The representative overlapped DIGE gel image of the control and totarol treated samples, 3D views and graphs of few selected differentially regulated protein spots are shown in Figs. 3B & S2.

3.3. Analysis of totarol induced alterations in the B. subtilis proteome using LTQ-Orbitrap The protein extracts used for the DIGE analysis were further analyzed using iTRAQ-based quantitative proteomics (Fig. 2). Pooled protein samples from the control (n = 3) and 120 min totarol treated (n = 3) cultures were used for comparative quantitative proteomic analysis using iTRAQ. A total of 1096 proteins were identified based on high peptide confidence, top

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rank peptides and 1% FDR. Quality of the data was checked by S-curve analysis and polygon plots, where 25% of the total proteins showed differential expression (fold change — 1.5) (Fig. 4A). Relative quantitation values for the entire protein list identified in this study along with sequence coverage, protein score and unique peptide information are provided in the Supplementary information (Tables 2 & S5).

3.4. Comparison of LTQ-Orbitrap data with Q-TOF and MALDI-TOF/TOF and gel-based proteomic data Comparative quantitative proteome analysis of three sets (each set of sample having a pool of biological triplicates of the control and totarol treated protein samples) of protein sample

Fig. 2 – Schematic representation of experimental work-flow for analysis of the alterations in the B. subtilis proteome due to totarol treatment. The triplicates of the control and totarol treated samples (IC50) were pooled and labeled with 114 and 115 (114 and 117 labels were used for MALDI-TOF/TOF analysis (not shown in figure)), respectively. Pre-fractionation was performed using SCX and OFFGEL fractionation and fractionated samples were subjected to high-resolution MALDI-TOF/TOF, nano-LC–Orbitrap and Q-TOF for the protein identification and quantitation. Additionally, proteome of the control and totarol treated samples was analyzed using 2-DE and DIGE.

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Table 1 – Partial list of the differentially expressed proteins of B. subtilis (after totarol treatment) identified in three complementary proteomic techniques (2DE, DIGE and iTRAQ)#. Trend

Name of the protein

Up-regulated U-3 Transcription elongation factor greA U-4 Oligoendopeptidase F homolog U-7 Protein IolS OS U-8 Thioredoxin U-9 NADH dehydrogenase U-10 NADH dehydrogenase U-11 UPF0477 protein yjcG U-12 Alkyl hydroperoxide reductase subunit U-17 UPF0447 protein ywfI Down-regulated D-10 Glyceraldehyde-3-phosphate dehydrogenase 2 D-15 Uncharacterized protein yjlC D-24 10 kDa chaperonin D-25 Succinate dehydrogenase flavoprotein subunit D-28 Glycerol kinase D-29 Flagellin D-30 2-Oxoglutarate dehydrogenase E1 component D-36 Surfactin synthetase thioesterase subunit

M. wt 2-DE DIGE Orbitrap 17.26 77.13 35.14 11.49 55.12 55.12 19.7 20.78 29.54

NS NS NS NS NS NS NS NS NS

2.33 2.07 1.94 1.79 1.78 1.76 1.72 1.72 1.5

37.47

NS

−1.51

15.57 10.16 65.39

NS NS NS

55.08 NS 32.62 −2.9 105.67 −1.5 27.47

−2.6

2.4 1.7 1.3 2.2 1.8 1.8 1.5 1.7 1.9

Biological function#

Molecular component#

Transcription Proteolysis – Cell redox homeostasis Cell redox homeostasis Cell redox homeostasis RNA metabolic process Stress response –

DNA binding Protease activity Oxidoreductase Electron carrier activity Oxidoreductase Oxidoreductase Catalytic activity Peroxidase activity –

−1.8

Glycolysis

Oxidoreductase

−1.64 −2.22 −2.28

−1.6 −1.62 −1.63

– Stress response TCA

– ATP binding Oxidoreductase

−2.17 −2.17 NS

−2.36 −3.0 −2.11

Glycerol metabolism Motility TCA and glycolysis

Kinase/Transferase Structural molecular activity Oxidoreductase

NS

−2.41

Sporulation, stress response Hydrolase

Bold — identified in 2-DE gel; NS — not significant or fold change is less than 1.2 in both up and down; NI — not identified; # — gene ontology obtained from the UniProt database.

using LTQ-Orbitrap, Nano-LC–QTOF and Nano-LC–MALDI-TOF/ TOF allowed identification of 1096, 679 and 299 proteins with 1% FDR respectively (Fig. 2). The S-curve analysis of Q-TOF data indicated differential expression (fold change — 1.5) of 50% proteins (Fig. 4A). Comparative analysis of the findings obtained from the three different mass spectrometric platforms indicated that nearly 90% of proteins identified in Q-TOF and MALDI-TOF/TOF were also identified in the Orbitrap analysis (Figs. 4A, & S3A & S3B). A total of 139 proteins (70 up-regulated and 69 down-regulated) were found to be differentially expressed (fold-change ≥1.5 and identified with ≥2 peptides) after totarol treatment. Proteins exhibiting similar trends of differential expression in at least two of the mass spectrometric analyses were considered for further investigation. However, differentially expressed proteins identified only in LTQ-Orbitrap/ESI-Q-TOF analysis were also considered if those candidates accomplished the selection criteria. Among the 139 differentially expressed proteins, 30 proteins were common between Q-TOF and Orbitrap, 28 proteins were common between Orbitrap and MALDI-TOF/TOF, 15 proteins were common among the three MS analyses, whereas 13 proteins were unique to Q-TOF and 53 proteins were unique to Orbitrap (Fig. 4B). These 139 proteins were considered for further bioinformatics analysis. Besides, we also compared iTRAQ data with the findings obtained from gel-based proteomics. All the significant differentially expressed proteins identified in gel-based proteomic analyses, i.e. 2-DE and DIGE were also observed in iTRAQ-based quantitative proteomics analysis. Fifteen differentially expressed proteins were identified in 2-DE, among which 6 proteins (2 up and 4 down-regulated) were

common between 2-DE and DIGE. All the differentially expressed proteins identified in DIGE were detected with similar trend of altered expression in at least one iTRAQ experiment (Fig. S3C).

3.5. Modulation of pathways and networks in B. subtilis due to totarol treatment DAVID analysis indicated that the differentially expressed proteins were involved in TCA cycle, glycolysis, ribosome and heme biosynthesis (Table S6). KOBAS 2.0 pathway analysis also demonstrated the association of the altered proteins with TCA cycle, heme biosynthesis, ribosome, glycolysis and biotin synthesis (Table S6). STRING 9.1 analysis enriched the differentially expressed proteins into seven groups. These enriched groups are belonging to ribosomal structural units, central metabolism, heme biosynthesis, chemotaxis, stress response and arginine metabolism (Fig. S4).

3.6. Effect of totarol on metabolic and respiratory activities Resazurin assay was performed using the control and totarol treated samples to assess the effect of the drug on cell viability and metabolic activity. Resazurin is a non-fluorescent blue dye, which is converted into pink resorufin by central metabolism enzymes, including the members of the electron transfer reactions in viable cells, with the help of reducing equivalents. Compared to the control B. subtilis cells, the IC50 and MIC totarol treated samples showed reduced conversion of the dye to pink color indicating that metabolism of B. subtilis was reduced significantly in IC50 and MIC totarol treated samples (Fig. 5A).

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255

Fig. 3 – Differentially expressed B. subtilis proteins in response to the totarol treatment identified using 2-DE and 2D-DIGE. (A) Representative 2-DE gel images of the control and totarol treated (IC50) B. subtilis proteome. 600 μg of total B. subtilis proteins were focused on linear pH 4–7 IPG strips (24 cm) and then separated on 12.5% polyacrylamide gels, which were stained with PhastGel™ Blue R Stain. Statistically significant (p ≤ 0.05) differentially expressed proteins are marked on gels with blue (down-regulated) and red arrows (up-regulated). 3D views and bar diagrammatic representations of the differentially expressed proteins (p ≤ 0.05) identified in 2-DE are displayed. Data is represented as mean ± SE (where n = 3). (B) Representative 2D-DIGE images (Cy3–Cy5 overlapped) of B. subtilis proteome in response to the totarol treatment along with the 3D and BVA graph views of a few selected differentially expressed proteins (p < 0.05). Abbreviations: GroL, 60 kDa chaperonin; DnaK, Chaperone protein dnaK; GlpK, Glycerol kinase; SucC, Succinyl-CoA ligase [ADP-forming] subunit beta; SufD, FeS cluster assembly protein; AhpF, NADH dehydrogenase.

Additionally, respiratory or metabolic activity of the drug treated cells were further analyzed by using CTC (5-cyano2,3-ditolyl tetrazolium chloride) staining and flow cytometry. CTC is a non-fluorescent dye, which is converted into redcolored fluorescent formazan in the respiratory active cells. In FACS analysis, the total cell population was determined by using DAPI (1 μg/mL) staining, which stains the DNA and

provides blue fluorescence. Negative control was prepared by fixing the cells with 2.8% formaldehyde and 0.04% glutaraldehyde before treating with the CTC, which artificially disrupts the cell membrane and subsequently respiratory activities. FACS analysis of totarol (IC50) treated cells showed two different kinds of populations, whereas control cells showed a single population. In totarol treated cells, lesser population showed

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similar CTC intensity like control cells, whereas higher population exhibited less intensity than the control, which is found to be similar to the negative control CTC intensity. Compared to the untreated cells, the reduction in CTC fluorescence was significant in 120 min totarol treated cells and in the negative controls indicating the loss of respiratory activity as a consequence of totarol treatment (Fig. 5B).

4. Discussion Totarol is a natural compound containing anti-tumor, antimalarial, anti-fungal and anti-microbial activities [4,7–10]. Previous reports showed that totarol exhibits anti-microbial activity by targeting either phospholipid permeability, respiratory mechanism for oxidative phosphorylation, efflux pumps and cell division [5,11,12,16]. In the present study, for the first time we investigated the effect of totarol on B. subtilis using multiple proteomic approaches to explore its possible cellular targets and mechanism of action. The global soluble proteomic analysis of B. subtilis using three complementary proteomic technologies; 2-DE, DIGE and iTRAQ followed by the bioinformatic analysis of the differentially expressed proteins revealed the alterations of crucial physiological pathways including central metabolism for energy production, heme biosynthesis, and chemotaxis due to totarol treatment. Application of three complementary proteomic

approaches provided a decent coverage of the bacterial proteome. Central metabolism is essential for energy generation, DNA replication and cell division [40–42]. The major observation from our proteomic study indicates that totarol treatment introduced interferences in the central metabolism of B. subtilis. Interestingly, most of the primary dehydrogenases, which participate in the NADH (redox) catalyzing reactions associated with ATP production by generating proton motive force (PMF) or membrane potential (MP) across the membrane, were found to be significantly repressed (Fig. 6). Further, resazurin and CTC assays also indicated significant reduction of metabolic and respiratory activities due to totarol treatment (Fig. 5A and B). Interestingly, nitrate reductase alpha chain (NarG) and nitrate reductase beta chain (NarH) involved in anaerobic respiration were found to be elevated. Nitrite acts as an alternative electron acceptor during anaerobic respiration, when TCA enzymes get inactivated [43]. In addition, an anaerobic marker protein, YwfI was found to be induced in response to totarol, which strongly supports the initiation of anaerobic respiration as a consequence of the drug treatment [44]. The fermentative enzyme, lactate dehydrogenase (Ldh) involved in re-generation of NAD+ was also found to be elevated (~1.45 fold) to balance the energy currency [45]. Interestingly, the enzymes involved in heme biosynthesis, which is the major co-factor involved in electron transporting enzymes and regulates the cellular energy balance, were found to be elevated. The electron transport dehydrogenases are closely

Fig. 4 – (A) S-curve analysis of Orbitrap and Q-TOF data for quality checking. (B) Venn diagram showing the distribution of the differentially expressed proteins (1.5 fold-change and identified with ≥2 peptides) identified by iTRAQ-based quantitative proteomics analysis using LTQ-Orbitrap, Q-TOF and MALDI-TOF/TOF mass spectrometers.

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Table 2 – Partial list of totarol induced differentially expressed proteins identified in iTRAQ analysis and their associated physiological pathways$. Accession

TCA cycle P23129 P07343 P80865 P80886 O34425 P54533

Name of the protein

Gene name

Coverage

Unique peptides

PSM

Totarol/ control (Orbitrap)

Totarol/ control (Q-TOF)

Totarol/ control MALDI-TOF/TOF

OdhA

31.89

21

68

0.47

NA

0.58

FumC SucD

21.43 29

5 5

16 17

0.56 0.61

NA 0.71

0.57 0.54

SucC

47.53

16

66

0.64

NA

0.59

GapB

36.47

7

28

0.55

NA

0.52

bfmBC

19.20

8

23

0.34

0.40

NA

LiaH PonA MurB

63.56 2.74 23.76

20 2 5

90 2 13

11.84 1.82 2.47

1.81 1.49 1.58

4.14 NA NA

Tig wapA yqgA pbpD yocH ywsB lytC

36.08 2.36 30.99 6.89 12.20 12.36 5.24

13 4 3 3 2 2 2

45 4 5 5 3 2 3

1.71 1.69 1.57 0.66 4.21 0.65 0.33

4.14 NA NA NA NA NA NA

NA NA NA NA NA NA NA

HemB HemH HemL

34.26 22.58 21.8

7 6 6

26 16 17

1.54 1.81 1.50

2.54 1.98 1.36

NA NA NA

HemY ywfI

6.17 26.77

2 6

3 20

1.61 1.94

NA 2.11

NA NA

GroS AhpC

42.55 62.57

4 9

28 166

0.61 1.69

0.94 1.09

0.64 1.22

HtrB YceD YwrO ctc YxiE

7.64 37.31 15.43 12.75 47.30

3 5 2 2 5

3 22 5 3 11

2.37 2.20 1.81 0.62 1.68

2.12 1.2 4.47 0.70 2.94

NA NA NA NA NA

Chemotaxis protein CheV Flagellin Methyl-accepting chemotaxis protein McpB Flagellar basal-body rod protein FlgG OS Putative sensory transducer protein YfmS OS

CheV Hag McpB

17.82 63.82 10.57

3 11 5

4 91 10

0.59 0.33 0.59

NA NA NA

NA 0.29 NA

flgG yfmS

15.53 19.58

4 5

16 11

NA 0.63

3.16 NA

NA 0.54

30S 30S 50S 50S 50S 50S 50S 50S

rpsK rpsS rplP rplB rplW rplC rpmI rplF

41.22 50.00 29.86 55.60 38.95 39.23 45.45 53.63

6 6 3 13 5 6 4 7

27 45 18 46 16 31 25 42

0.56 0.52 0.52 0.57 0.63 0.66 NA 0.62

NA NA 0.72 NA NA NA 1.89 0.79

0.54 0.55 0.62 0.53 0.52 0.63 NA 0.66

2-Oxoglutarate dehydrogenase E1 component Fumarate hydratase class II Succinyl-CoA ligase [ADP-forming] subunit alpha Succinyl-CoA ligase [ADP-forming] subunit beta Glyceraldehyde-3-phosphate dehydrogenase 2 OS Dihydrolipoyl dehydrogenase OS

Cell wall synthesis and cell division O32201 Protein LiaH P39793 Penicillin-binding protein 1A/1B P18579 UDP-N-acetylenolpyruvoylglucosamine reductase P80698 Trigger factor Q07833 Wall-associated protein OS P54484 Cell wall-binding protein YqgA OS P40750 Penicillin-binding protein 4 OS O34669 Cell wall-binding protein YocH OS P96729 Cell wall-binding protein YwsB OS Q02114 N-acetylmuramoyl-L-alanine amidase LytC OS Heme biosynthesis P30950 Delta-aminolevulinic acid dehydratase P32396 Ferrochelatase P30949 Glutamate-1-semialdehyde 2,1-aminomutase P32397 Protoporphyrinogen oxidase P39645 UPF0447 protein YwfI OS Stress response P28599 10 kDa chaperonin P80239 Alkyl hydroperoxide reductase subunit C Q9R9I1 Serine protease Do-like HtrB P80875 General stress protein 16U P80871 General stress protein 14 P14194 General stress protein CTC OS P42297 Universal stress protein YxiE Chemotaxis P37599 P02968 P39215 P23446 O06477

Ribosome P04969 P21476 P14577 P42919 P42924 P42920 P55874 P46898

ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal ribosomal

protein protein protein protein protein protein protein protein

S11 OS S19 OS L16 OS L2 OS L23 OS L3 OS L35 OS L6 OS

NA — not significant; $ — complete list has been provided in Supp. Table S2.

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Fig. 5 – Multiple cellular assays validating the effect of totarol on respiratory activity and cellular metabolism. (A). Viability/ metabolic activity assay using resazurin. Control and totarol treated (1.5 μM and 2 μM) B. subtilis cultures were used for the assay. Cultures were diluted with phosphate buffer saline until the concentration reached to 106–108 cells, and then were incubated with resazurin (10 μg/mL). Formation of resorufin was measured at 590 nm. 1.5 and 2 μM totarol treated samples showed significantly reduced metabolic activity with respect to the control, which was considered as fully active. (B). Graphical representation of cell count vs. CTC mean intensity obtained in the FACS analysis of CTC stained control, totarol treated (1.5 μM) and formaldehyde treated (negative control) samples. Both dot plot and histogram views are displayed. CTC mean intensity in totarol treated and negative controls was found to be significantly reduced in comparison to the untreated cells (n = 3).

linked to the primary dehydrogenases in generating the electrochemical gradient with the help of the heme co-factor across the membrane for ATP synthesis [46]. Increased expression levels of the enzymes involved in heme biosynthesis could assist to

rebuild the electron transport system (Fig. 6). The interesting fact is that totarol can inhibit the oxygen consumption and NADH-cytochrome C reductase in Pseudomonas aeruginosa [11]. On the other hand, earlier reports also showed that totarol targets

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259

Fig. 6 – Modulation of essential physiological pathways in B. subtilis due to totarol treatment. Pathway analysis was performed using DAVID, KEGG and KOBAS tools involving the differentially expressed proteins identified in differential proteomic analysis of the control and totarol treated cells. Glycolysis, TCA, heme biosynthesis, motility and respiration were found to be affected. Red indicates down-regulation and blue indicates up-regulation.

the bacterial electron transport chain by acting as an ionophore [11,12] or disturbing the membrane potential [47]. A recent study by Foss et al. demonstrated that many of the cell division inhibitors perturb cell division by disturbing membrane permeability, and also reported that totarol exhibits similar effects [47]. Based on our findings and previous reports, we anticipate that totarol significantly affects aerobic respiration by reducing the oxygen consumption required to maintain PMF or membrane potential across the membrane for both energy production and protein localization [48]. We therefore speculate that the enhanced anaerobic respiration and fermentative process might aid in reviving normal growth. On the other hand, the enzymes involved in fatty acid synthesis were repressed slightly probably due to the metabolic shutdown or limited availability of precursors. Methyl-accepting chemotaxis proteins (MCPs) are the chemoreceptors on membrane, which are responsible for motility with the help of downstream chemotaxis (Che) proteins and flagellin assembly proteins in response to different chemicals [49]. Our proteomic analysis indicates that totarol treatment significantly repressed the chemoreceptor proteins (McpB, YfmC and YfmR), downstream Che proteins (CheV) and flagellin assembly proteins (Hag and FlgG). A previous study by Maki et al. demonstrated that the elongated cells (filamentous cells)

have the same motility like the normal cells in Escherichia coli [50]. Therefore, we speculate that the reduced motility could be due to the loss of membrane permeability, which provides support to the flagellin, and also due to the metabolic shutdown, which interrupts energy production (Fig. 6). In addition, expression levels of autolysines, which are cell wall enzymes involved in remodeling of the cell wall during cell separation, cell wall metabolism and motility, were also altered [51]. The reduced expression of autolysines could introduce a defect in cell separation during cell division [52]. LiaFSR is a two-component system, where LiaS is a histidine kinase, and LiaR is the response regulator that activates the LiaH, which is involved in protection of cell wall from oxidative stress [53]. Our proteomic analysis revealed induction of LiaH, which is a marker for cell-wall biosynthesis. A similar response of LiaH induction has been reported in lantibiotic treatment, which targets the cell wall [54]. YceC is a cell envelope stress marker protein induced by many cell wall targeting antibiotics. Interestingly, our proteomics data indicated the induction of YceE and YceD proteins from the family of YceC, which points toward the cell wall stress under totarol treatment [54]. Moreover, the expression levels of quite a few proteins involved in protein synthesis (small and large units of the ribosome) were repressed significantly probably due to the

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lower expression of the ribosomal proteins or owing to their degradation to provide nutrients under metabolic shutdown [23]. Our quantitative proteomic analysis indicated differential expression of quite a few proteins involved in proteolysis and protein folding (chaperon system). Universal chaperon systems such as 60 kDa chaperonin (GroEL) and 10 kDa chaperonin (GroES) are highly conserved among the eubacteria and involved in the folding of 85 proteins, including quite a few cell division proteins such as FtsE, FtsA and FtsZ [55]. The reduction in the expression levels of GroEL and GroES due to totarol treatment signifies its effect on protein folding. Moreover, GroEL and GroES localize at the septum position and help in the efficient polymerization and stabilization of the Z-ring, and a defect in the GroEL/ES system leads to cell elongation [56]. Interestingly, Trigger factor was found to be induced under totarol treatment which has a significant role in filamentation [57]. On the other hand, S-adenosyl methionine (SAM) which is a universal methyl transferring co-factor synthesized by S-adenosyl methionine synthase was down-regulated due to the totarol treatment. An earlier study by Newman et al. showed that SAM also plays a central role in cell division machinery by methylating the late cell division proteins such as FtsQ, FtsI, FtsL, and FtsW, which are required for septum formation in E. coli [58]. Moreover, it has been reported earlier that totarol binds to purified FtsZ from Mycobacterium tuberculosis and B. subtilis and perturb the Z-ring assembly dynamics [16]. In summary, this is the foremost study of totarol induced alterations in the B. subtilis proteome. Differential proteomic analysis of the control and totarol treated bacterial cultures revealed the alterations of TCA cycle, heme biosynthesis and ribosomes. Both gel-based and gel-free quantitative proteome analyses indicated alteration of crucial physiological pathways linked to cell division. This drug modulates membrane potential required for localization of the cell division proteins, and consequently causes perturbation of cell division. Findings obtained from our proteomic and cellular analyses corroborated with quite a few known cellular targets reported earlier; while many of the differentially expressed candidates and associated physiological pathways identified in our study were not correlated directly with totarol action previously. This comprehensive proteomic study may contribute to a better understanding of the mode of action of totarol and its primary molecular targets. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2014.10.025.

Conflict of interest The authors have declared that no competing interests exist.

Acknowledgments This research was supported by a start-up grant 09IRCC007 from the IIT Bombay to SS and a CSIR (Council of Scientific and Industrial Research) grant, India to DP. PJR and SR are senior research fellows supported by the IIT Bombay fellowship. GJS is a recipient of the Senior Research Fellowship from CSIR, Government of India. We thank the Center for Research

in Nanotechnology and Science (CRNTS), Indian Institute of Technology Bombay, for providing the fluorescence activated cell sorting (FACS) and LC–ESI-Q-TOF facility. We thank the Department of Biotechnology (DBT) (BT/01/COE/08/05), and the Government of India for research support to the Institute of Bioinformatics (IOB), Bangalore. TSKP is a recipient of the research grant on “Development of Infrastructure and a Computational Framework for Analysis of Proteomic Data” from DBT (BT/01/COE/08/05). We sincerely thank Dr. Richard Losick (Harvard University, Cambridge, MA) for providing us the B. subtilis AH75 strain. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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