Retraction: Pseudopyronine B: A Potent Antimicrobial and Anticancer Molecule Isolated from a Pseudomonas mosselii

[This retracts the article on p. 1307 in vol. 7, PMID: 27617005.].

INTRODUCTION
The discovery of antibiotics has decreased the spread and severity of a broad range of infectious diseases caused by human pathogenic microbes. However, due to the wide spread use of antibiotics, the competence of many introduced antibiotics in the market is being threatened by the rise of microbial pathogens which are resistance to the current clinical chemotherapeutic agents (Cowan, 1999). Human infections owing to multi-drug-resistant microbes is a serious challenge to many patients, especially those, are in hospitals (Arias and Murray, 2009;Fischbach and Walsh, 2009). Infectious diseases account for more than 13 billion human deaths worldwide, which accounts for about 25% of all deaths (Zin et al., 2011). The antibiotic treatments of various infectious diseases are also getting limited due to the reemergence of multi-drug-resistance (MDR) pathogenic microbes, which have been frequently reported from many parts of the world. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important drug resistant pathogen, which is frequently reported by many clinicians worldwide. A part from MRSA and VRSA (vancomycin resistant S. aureus), several other drug resistant and pan-drug-resistant (PDR) microbes especially Gram-negative bacteria, including carbapenem resistant Pseudomonas aeruginosa, Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter sp., and Stenotrophomonas maltophilia, are emerging as an important health problem to human being (Huang et al., 2013). Recently, few novel drugs have been industrialized exactly for treating various Gram-negative MDR/PDR bacteria (Payne et al., 2006;Vaara et al., 2010;Velkov et al., 2010). Many bioactive compounds from natural sources have played an important role in the discovery of many antibiotics which are used clinically. Moreover, 70% of the clinically used antimicrobials are either unchanged natural compounds used directly or derived from natural compounds by synthetic tailoring (Singh et al., 2011). Thus, more investigation should be initiated for discovering and developing novel antimicrobial molecules especially from natural sources is urgently needed.
At present, one in four deaths in the USA is because of various tumors (Fadeyi et al., 2013). When classified within age groups, the tumor is one of the five important reasons of death among humans (males and females) and the single major cause of human death around the world (Fadeyi et al., 2013). By 2016, cancer morbidity may increase more than 10 million worldwide. This growing tendency also indicates a shortage in the various current tumor treatments including surgical operation, chemotherapy, and radiotherapy. Since the normal endurance rates of cancer have remained essentially unaffected in spite of such above mentioned hostile treatments, so there is an urgent requirement for new anticancer drugs with advanced efficiency, and fewer side effects that can be developed at a reasonable price to common people worldwide. Tawiah et al. (2012) reported that "Throughout the various years, natural compounds are the most constantly fruitful basis of diverge bioactive metabolites having several applications in the field of modern human medicine, pharmaceutics and agriculture." Therefore, the search for new antimicrobials from natural sources is an important area of many researchers worldwide. Microorganisms from soils play an important source of many novel antibiotic compounds due to their high abundance and amazing diversity. There are many reports of the production of antimicrobial metabolites by Pseudomonas spp. Some of these antimicrobials have been chemically characterized, and their commercial exploitation is attempted (Zhou et al., 2012).
As mentioned earlier infectious diseases and cancer is the most leading cause of death worldwide. Therefore, novel and potent therapeutic agents are often required to control these diseases. To achieve this goal, several new sources of antimicrobial/anticancer compounds ranging from natural to synthetic are currently being explored worldwide. Therefore in our continuous search for new bioactive secondary metabolites from soil bacteria, Pseudopyronine B was purified from a Pseudomonas mosselii TR strain. The current manuscript also reported the antimicrobial and anticancer activity of Pseudopyronine B.

Chemicals and Media Used in Current Study
All the chemicals and reagents used in the current study were of the finest purity. All the chemicals used for separation and purification of bioactive compounds were from Merck (Mumbai, India). Silica gel (mesh size: 230-400) used for column purification and precoated silica gel 60 plates (GF 254 ) used for thin layer chromatography (TLC) were purchased from Merck (Germany). Various media used for microbiological investigation were purchased from Hi-Media Laboratories Pvt. Ltd, Mumbai, India. The antibiotics ciprofloxacin and amphotericin B, which were used as standard positive control, were procured from Sigma-Aldrich (USA). The Chemsketch Ultra (Toronto, ON, Canada) software was used for drawing the chemical structure.

Bioactive Compound Producing Bacterium
A fluorescent Pseudomonas, named as TR strain was isolated from paddy field of Kuttanad region of Kerala, India was used in the present study. Kuttanad is a unique agroclimatic zone in India where paddy is cultivated below sea level and is blessed with a rich microbial population which is not fully explored for economic cultivation. The strain was identified as a fluorescent Pseudomonas strain through classical identification methods.

Molecular Identification of TR Strain through 16S rDNA Sequencing
Extraction of Bacterial DNA and Sequencing of the 16S Gene A pure culture of bacteria was raised in 10 ml of Luria-Bertani broth (LB) for 18 h to obtain OD value of approximately 0.7 at 600 nm. The bacterial culture broth (1.5 ml) was pelleted in a microfuge tube for 2 min at 12,000 rpm. Total DNA was extracted using standard phenol-chloroform extraction procedures (Sambrook et al., 2001). PCR amplification was done with universal primers (f: 5 -GATTAGATACCCTGGTAGTCCAC-3 and r: 5 -CCCGGGAACGTATTCACCG-3 ) specific for the 16S rDNA gene. The PCR reaction was carried out in a total volume of 50 µl [DNA (100 ng), MgCl 2 (4 Mm), PCR reaction buffer (1X, Genei, Bangalore, India), each deoxyribonucleotide triphosphate (2.5 mM), Taq polymerase (1.5 U, Genei, Bangalore, India) and each primer (40 pmol)]. PCR cycle conditions were: 2 min initial denaturation at 95 • C, followed by 36 cycles of denaturation for 30 s at 95 • C, followed by 1 min primer annealing at 60 • C and 1 min primer extension at 72 • C. Final primer extension step was carried for 2 min at 72 • C. PCR amplification was achieved on a Bio-Rad Thermal Cycler (USA). Amplicons were analyzed in 1% (w/v) agarose gels. The gels were stained with an aqueous solution containing ethidium bromide (0.5 mg/mL) and visualized on a UV transilluminator. Amplicons were purified using the QIA quick purification kit (Qiagen, Valencia, CA, USA) and sequenced. The sequencing was performed on an ABI PRISM 310 Genetic analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) using the universal primers (described for PCR amplification) and the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Applied Biosystems). The 16S rDNA gene sequences (partial) obtained were finally aligned using ABI Prism software (Perkin-Elmer, Applied Biosystems) and compared to sequences retrieved from GenBank Database and BLAST.

Phylogenetic Analysis
The phylogenetic investigation was performed using MEGA 6.0 software program (Molecular Evolutionary Genetics Analysis, Version 6.0; Tamura et al., 2007). The phylogenetic tree topologies were assessed by bootstrap analyses based on 1000 replicates, and final phylogenetic trees were constructed with the neighbor-joining method as reported earlier (Saitou and Nei, 1987). The taxonomic position of the present strain was recognized based on 16S rDNA homology.

Fermentation and Extraction of Crude Bioactive Secondary Metabolites
The bacterial production and solvent extraction of crude bioactive secondary metabolites from TR strain were done according to George et al. (2015) in King's B media (KB). The supernatant was collected according to the method of George et al. (2015) and extracted with ethyl acetate trice (1:1 v/v) in a globe shaped separating funnel (2 L) and the ethyl acetate portion was collected, concentrated and dried in a Buchi rota evaporator (40 • C) for further detailed investigations.
An aliquot of each collected fraction was spotted on the activated TLC plates (silica gel 60; 10 cm × 10 cm). Ethyl acetate and hexane (1:1) was used for developing TLC plates. Spots were positioned by UV and further revealing the TLC plate to iodine vapors. One fraction (tubes 35-37) was obtained as pure. This fraction was evaporated to give a faint white residue (17 mg). Initial bioactivity of this fraction was confirmed by testing against Bacillus subtilis, which was used as indicator test microorganism.

Identification of the Bioactive Compound
The NMR spectra ( 1 H NMR and 13 C) of the pure compound were documented using a Bruker DRX500 NMR spectrometer (Bruker, Rheinstetten, Germany) at room temperature and the chemical shifts are reported relative to the corresponding reference solvent (methanol). HR-ESI-MS of the compound was recorded using a Thermo Scientific Exactive Mass Spectrometer (Thermo Fisher 110 Scientific, USA) with an electrospray ionization mode.

Pathogenic Microbes Used in the Study Bacterial
The following Gram-positive and negative pathogenic bacteria are used for antibacterial study.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
The MIC of the compound was determined according to broth microdilution method as suggested by Clinical and Laboratory Standard Institute, USA (Clinical and Laboratory Standards Institute [CLSI], 2012a). Briefly, the concentration of the fresh overnight culture of test bacteria was adjusted to 1 × 10 5 CFU/ml using a spectrophotometer. Dilutions of inocula were cultured on a Nutrient agar (NA) medium to check the validity of the inoculum and the absence of any contaminations. Different solvent and water dilutions of test compound (0.5-1000 µg/ml) and ciprofloxacin (0.25-250 µg/ml) were placed in the wells containing 150 µl of nutrient broth (NB), followed by the addition of 10 µl of inoculum. After that, the ELISA plates were incubated for 18-24 h at 37 • C. After incubation MIC was determined by measuring the OD at 600 nm. The lowest concentration of test compound that produced a significant inhibition (around 90%) of the growth of the bacteria in comparison with the positive control was identified as the MIC.
About 100 µl from the ELISA wells not displaying any microbial growth in the MIC test were diluted serially using 0.85% saline and plated on NA plates to determine the MBC values. The NA plates were incubated at 37 • C for 24 h. MBC is defined as the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by 99.99%.

Disk Diffusion Experiment of Test Compound
The antimicrobial activity of the pure compound was performed by the disk diffusion assay against the test bacterial pathogens as mentioned by CLSI, USA (Clinical and Laboratory Standards Institute [CLSI], 2012b). The test bacterial strain preserved in NA at 4 • C were sub-cultured in NB to get the working concentration approximately containing 1 × 10 6 CFU/ml. The test compound (MIC concentration) was loaded into a sterile disk (Hi-Media) with 6 mm diameter. Then Mueller-Hinton agar (MHA) plates were swabbed with each test bacterial pathogens and the compound loaded disks were placed along with the control antibiotics disk. Here, ciprofloxacin disks (5 µg/disk) were used as the standard positive control and the plates were incubated for 24 h at 37 • C until bacteria had developed in a confluent film. The antimicrobial property of the test compound and antibiotic was determined by measuring the zone of inhibition (diameter) expressed in mm. The experiment was performed in triplicate sets.

Minimum Inhibitory Concentration
The MIC of test compound against the fungi was done by broth microdilution assay as per the recommendations of Clinical and Laboratory Standard Institute, USA (Clinical and Laboratory Standards Institute [CLSI], 2010, 2012c), with RPMI 1640 growth medium supplemented by L-glutamine, without sodium bicarbonate (all from Sigma-Aldrich) and buffered to pH 7.0 according to the previously reported method (Aravind et al., 2014). The lowest concentration of agents that produced a significant inhibition (90%) of the growth of the test fungi in comparison with the standard positive control was defined as the MIC.

Disk Diffusion Experiment of Test Compound
The compound was screened for their antifungal activity against test fungi by agar disk diffusion experiment as mentioned by CLSI, USA (Clinical and Laboratory Standards Institute [CLSI], 2008[CLSI], , 2009. Briefly, PDA plates were inoculated (0.1 ml) with a spore suspension in 0.85% sterile saline. The concentration of test fungal suspension was adjusted to 1 × 10 5 CFU/ml using sterile saline. The test compound (MIC concentrations) was loaded into a 6-mm diameter sterile filter paper disks (Hi-Media, India), air dried and then placed on the surface of the PDA plates swabbed previously by test fungi. The plates were incubated at 35 • C for 48-72 h. After incubation, the antifungal activity was assessed by measuring the zone of inhibition (diameter) and expressed in millimeter (mm). The assay was performed in triplicate sets.

Anticancer Studies of the Test Compound
Cancer Cell Lines Used in the Study: Its Source and Maintenance and Treatments The following three cancer cell lines were used in the present investigation (1) Non-small cell lung cancer (A549), (2) mouse melanoma cell (B16F10) and (3) liver cancer cell (HepG2) lines and these cell lines were obtained from NCCS (National Centre for Cell Science), Pune, India and maintained throughout the study in Dulbecco's Modified Eagle Medium (DMEM) added with 3 mM of L-glutamine, 100 µg/ml of streptomycin, 100 IU/ml of penicillin, 10% heat inactivated (56 • C) fetal bovine serum (FBS) and 25 mM of HEPES [4-(2-hydroxyethyl)-1piperazineethanesulfonic acid]. The final pH was adjusted to 7.2 by bicarbonate solution at 37 • C in a CO 2 incubator.
Stock solutions (3 mg/500 µl) of the test compound, made in dimethyl sulfoxide (DMSO), were further dissolved in the corresponding medium (DMEM) to the necessary working concentrations. A549, B16F10 and HepG2 cell lines were seeded into ELISA plates and incubated for 24 h at 37 • C in a CO 2 incubator. After the cell adherence, six different concentrations of test compound (0.1, 1, 5, 10, 50, and 100 µg/ml) were added to the wells containing test cancer cell lines, except to the control wells. Control wells contain only DMEM nutrient medium and the test cells. The cultures were incubated for various time periods (24, 48, and 72 h).

Apoptosis Induction Assay Using the Test Compound
Acridine Orange and Ethidium Bromide Staining (AO/EB Staining) Apoptotic morphology of cancer cells after treatment with Pseudopyronine B was investigated by staining the cancer cells with a combination of the fluorescent DNA-binding acridine orange (AO) and ethidium bromide (EB) dyes to decide the viable and non-viable cancer cells in a population. Cells were collected and washed three times with PBS after being incubated with IC 50 concentration of Pseudopyronine B for 24, 48 and 72 h, and were then stained with 100 µg/ml AO/EB stain (Sigma-Aldrich) for 2 min. Then the color and structure of the cancer cells were immediately recorded under an inverted fluorescence microscope (Aravind et al., 2014).

Detection of Morphological Apoptosis with Hoechst 33342 Staining
The morphological apoptosis like chromatin condensation in the test cancer cell lines was observed by nuclear staining dye Hoechst 33342 (Aravind et al., 2014). After treatment with Pseudopyronine B (IC 50 concentration) for 24, 48 and 72 h, the A549 and B16F10 cells were washed with PBS trice and then further fixed with methanol: acetic acid (3:1) for 10 min at room temperature in the dark. Fixed cells were again washed with PBS and stained with 1 µg/ml of Hoechst 33342 stain (10 mg/ml) for 10 min in the dark at room temperature. Changes in the nuclei of cells after staining with Hoechst 33342 were recorded using an inverted fluorescence microscope (stimulation at 350 nm and emission at 460 nm; Leica, Germany).

Annexin V/Propidium Iodide Assay
The annexin V/propidium iodide (PI) method was done according to the recommendation given by manufacturer's (Invitrogen, USA). Briefly, A549 and B16F10 cell lines were seeded into six well plates and incubated for 24, 48, and 72 h with Pseudopyronine B (IC 50 concentration). After incubation, the cells were collected and washed with ice-cold PBS and centrifuged. The pellet was resuspended in 100 µl of binding buffer containing 2.5 µl FITC conjugated Annexin V and 1 µl 100 µl/ml PI and incubated for further 15 min at room temperature in the dark. A total of at least 10000 events were composed and examined by BD flow cytometry (BD Biosciences, San Jose, CA, USA; Liu et al., 2013).

Cell Cycle Analysis of Cancer Cells after Treatment with Test Compound
The cellular DNA content and distribution in the cell cycle were enumerated by BD flow cytometry using PI staining. A549 and B16F10 cells were seeded in the T 25 culture flask and treated with Pseudopyronine B (IC 50 concentration) for the various time periods (24, 48, and 72 h). Cells were collected, washed with PBS twice, and fixed in 70% ethanol at −20 • C. After fixation, the cells were washed in PBS and centrifuged. The pellets thus obtained was treated with RNase (1 mg/ml; Roche, Mannheim, Germany) at 37 • C for 30 min and then incubated with PI for at least 30 min. Cell-cycle analysis was recorded with the FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) and the data were analyzed with Flowjo software.

Determination of Activation of Caspase 3
Caspase-Glo assay kits (Promega) were used to determine the activation of Caspase 3 by following manufacturer's instructions. A549 and B16F10 cells were plated into ELISA plates and incubated for 24 h. After 24 h seeding, cells were treated with test compound (5, 10, and 50 µg/ml) and incubated for 48 h. Subsequently, 100 ml of caspase-3 assay reagent was added to each well. After adding the reagent, the plate was incubated for 1 h in the dark and the luminescence was quantified with the help of a microplate reader (SpectraMax M5, Molecular Devices). The activity of Caspase was expressed as a percentage (%) of the untreated control treatment (DMSO). The experiment was performed in triplicate (Wang et al., 2014).

Proliferation Assay of Normal Lymphocytes
The proliferation of normal lymphocytes when treated with test compound was done as described earlier (Aravind et al., 2014).

Statistical Analysis
Disk diffusion data was presented as mean ± standard deviation. The error bars represent ± SD. taken from three independent experiments. Statistical analysis was performed with SPSS (Version 17.0; SPSS, Inc., Chicago, IL, USA). The significance level was set at P < 0.05.

RESULTS
Based on 16S rDNA Sequencing and Phylogenetic Analysis, Bacteria Was Identified as P. mosselii Molecular characterization of the TR bacterium (P. mosselii) was performed by 16S rDNA gene sequencing. PCR amplification of 16S rDNA gene yielded -1500 bp amplicon. BLAST analysis recorded 99% similarity to P. mosselii 16S rDNA sequence accessible in the NCBI GenBank database and based on this our TR strain was identified as P. mosselii. The partial 16S rDNA gene sequence data have been deposited in the NCBI GenBank nucleotide database under the accession number KF712283. The phylogenetic tree clearly portrayed the relationships our P. mosselii with other Pseudomonas strains used for the present analysis. The present TR bacterial isolate (P. mosselii strain) was very successfully clustered along with other P. mosselii sequences obtained from the NCBI GenBank database further confirming the authenticity of our isolate (Figure 1). The P. mosselii was currently deposited in CSIR-IMTECH (Institute of Microbial Technology, Chandigarh, India).

The Bioactive Compound Was Identified as Pseudopyronine B Based on Detailed Spectral Analyses
Ethyl acetate fraction of P. mosselii yielded one main compound, which having an R f value 0.68. Initial bioactivity of this compound was confirmed by testing the antimicrobial activity against B. subtilis and the compound recorded significant activity (data not shown). The compound was identified based on the spectral data as Pseudopyronine B (Figure 2).

Antibacterial Activity of Pseudopyronine B
The Pseudopyronine B was tested for checking its antibacterial (MIC and MBC) activity against 13 bacterial species using standard CLSI protocol and the values are presented in Table 1. Pseudopyronine B recorded significant antibacterial activity Frontiers in Microbiology | www.frontiersin.org against Gram positive bacteria, while most of the tested Gram negative bacteria recorded no activity. The microorganism that presented highest sensitivity toward Pseudopyronine B was S. epidermis (1 µg/ml), followed by S. aureus (4 µg/ml). The diameter of zone of inhibition obtained from agar disk diffusion experiment was presented in Table 1 and S. epidermis recorded significant diameter of zone of inhibition (33 mm) and is shown in Figure 3. The activity of Pseudopyronine B against S. epidermis was superior to ciprofloxacin, standard antimicrobial agent ( Table 1).

Antifungal Activity of Pseudopyronine B
Antifungal property of Pseudopyronine B against 11 fungi and the MIC values are shown in Table 2. Pseudopyronine B documented good antifungal property especially against plant pathogenic fungi. Pseudopyronine B displayed best MIC value against P. expansum (4 µg/ml), followed by F. oxysporum and R. solani (16 µg/ml). In agar disk diffusion assay P. expansum recorded significant activity (34 mm; Table 2; Figure 3).

Pseudopyronine B Recorded Significant Anti-proliferative Activity as Evidenced by MTT Assay
Anti-proliferative activity of Pseudopyronine B was assessed on non-small cell lung cancer (A549), mouse melanoma cell (B16F10) and liver cancer cell (HepG2) lines. MTT assay was used to evaluate the cell viability after Pseudopyronine B treatment. From the MTT experiment it is clearly evident that the Pseudopyronine B recorded both dose-and timedependent inhibition in the growth of test cancer cell lines, when treated with 100, 50, 10, 5, 1, and 0.5 µg/ml ( Figure 4A).
Out of three cell lines tested, A549 and B16F10 cells recorded best activity. A549 and B16F10 cells turned out to be the most sensitive cell lines and were selected for further apoptotic studies.

Pseudopyronine B Induces Apoptosis in A549 and B16F10 as Evidenced by Acridine Orange-Ethidium Bromide and Hoechst 33342 Staining
The cultured A549 and B16F10 cells were examined for their morphology features after treatment with Pseudopyronine B. It was observed that the Pseudopyronine B is inducing characteristic apoptosis features changes such as nuclear condensation, membrane blebbing and formation of apoptotic bodies, when compared to untreated control as evaluated by phase contrast microscope. Moreover, there was a significant reduction in the number of A549 and B16F10 cells after Pseudopyronine B treatment ( Figure 4B). Acridine orangeethidium bromide staining was done to confirm the nuclear membrane damage, a characteristic feature of apoptosis as observed by yellow/orange coloration in the nuclei of A549 and B16F10 cells treated with Pseudopyronine B (Figure 5). The cells treated with the compound after 24, 48 and 72 h exhibited time depend increase in AO-EB positivity, when compared to untreated control (Figure 5).
The Hoechst 33342 staining of A549 and B16F10 cells recorded a very significant increase in the number of cancer cells showing nuclear condensation and fragmentation after treatment with Pseudopyronine B when examined through phase contrast microscope (Figure 6). Moreover, cancer cells lost its normal structure after treatment with Pseudopyronine B and presented an expansion of the endoplasmic reticulum. Some very specific signs of early stages of apoptosis (vacuolization in the cytoplasm, nucleus shrinkage and fragmentation and chromatin densification) can also be viewed in Pseudopyronine B treated cells through phase contrast microscope (Figure 6).

Effect of Pseudopyronine B on Cell Cycle Analysis
Distribution of cells on the different phases of the cell cycle was investigated by flow cytometry after treatment of A549 and B16F10 cells with several concentrations of Pseudopyronine B for 24, 48, and 72 h. In both A549 and B16F10 Pseudopyronine B treated cells, G1 and G2/M phase were increased were as S phases found to be decreased. In A549 cells at 48 h of incubation control cells possess 68.96 ± 2.94% in G1 phase were as in Pseudopyronine B treated cells have 76.36 ± 2.67%. Control cells in G2/M phase possess 5.88 ± 0.84% were as in Pseudopyronine B treated cells have 11.88 ± 1.04%. In B16F10 cells at 48 h of incubation control cells possess 70.62 ± 3.56% and 72.79 ± 3.21% for Pseudopyronine B treated cells. In G2/M phase control cells possess 17.59 ± 1.59% and 21.12 ± 1.64% for Pseudopyronine B treated cells suggesting that it can induce significant apoptosis in A549 and B16F10 cells (Figure 7).

Pseudopyronine B Induces Apoptotic Events by Altering Membrane Permeability as Evidenced by Flow Cytometry
Annexin V-FITC staining, using flow cytometry was used to detect the morphological changes that occur in the early stages of apoptotic cells (Figure 8). After 48 h of treatment, Frontiers in Microbiology | www.frontiersin.org

Pseudopyronine B Induces Caspase 3 Activation in Cells
Our next attempt was to examine the basic mechanism behind the cytotoxic effect of Pseudopyronine B. For this, we tested the activation of caspase 3 using a colorimetric assay. Pseudopyronine B treated cells and control lysates were prepared and incubated with Ac-DEVD-pNA caspase 3 specific substrate with the reaction buffer and the caspase 3 pNA release was measured using a spectrophotometer at 405 nm. Significant enhancement in the caspase 3 activity was recorded in Pseudopyronine B treated cells when compared with that of control clearly indicating the involvement of caspase 3 in Pseudopyronine B induced apoptotic cell death (Figure 9). The results are given in Figure 9.

Evaluation of Immunomodulatory Properties of Pseudopyronine B
Immunomodulatory activity of Pseudopyronine B was checked by the proliferation of lymphocyte. The proliferation lymphocyte was slightly enhanced in Pseudopyronine B treated cells when compared to that of control cells (untreated). The proliferation index of lymphocyte was detected to be 1.08 for 5 µg/ml of Pseudopyronine B in the presence of PHA (Figure 10).

DISCUSSION
The enormous rise in the value of drug discovery and development is a strong encouragement for many pharmaceutical industries to contempt all but the most economically bioactive  lead molecules (Higginbotham et al., 2013). But, the decreasing quantity of medicines reaching the market is putting strong pressure on the pharmaceutical companies to research and find alternative sources for novel lead molecules (Bennani, 2012). Natural molecules with diverse bioactivity have been used since the beginning of various conventional medicines in early years (Graca et al., 2013). These bioactive molecules are present in all forms of life and are usually produced during the secondary metabolism by almost all organisms. A wide variety of structurally interesting and bioactive secondary metabolites produced by various microorganisms including bacteria, fungi, actinomycetes, etc. have been reported worldwide and many of them are taken by the several pharmaceutical companies as lead drug molecules especially antibiotics (Gao et al., 2012). Several microbes especially from terrestrial soils have an inborn capability of producing many novel natural molecules with various biological properties and this represent rich source of biologically active compounds which may play very important role in drug discovery process for pharma industry (Bode et al., 2002). In the present study, a bacterial strain, named as TR, was isolated from soil during a screening for microbes with potent antimicrobial property. The microbial strain used in the present study was identified as a P. mosselii strain based 16 S rDNA sequencing and BLAST analyses.
There are many literature reports on the production of compounds with antimicrobial properties by Pseudomonas spp. Some of these antimicrobial compounds have been identified Frontiers in Microbiology | www.frontiersin.org FIGURE 5 | Analysis of A549 and B16F10 cell lines to detect apoptosis by Acridine orange/ethidium bromide staining. In this method live cells were detected as green color, whereas the apoptosis cells documented orange-red due to the co-staining of acridine orange with ethidium bromide stain due to the loss of membrane integrity by the action of Pseudopyronine B.
chemically and the structure has been reported (Zhou et al., 2012). Several fluorescent Pseudomonas spp. have currently received world-wide interest due to the production of a board range of bioactive compounds with antibiotics properties such as phenazine-1-carboxylic acid, pyoluteorin, phenazine-1carboxamide, viscosinamide and tesin (Chin-A-Woeng et al., 2003;Hu et al., 2005;Huang et al., 2009) and several bioactive enzymes. In this study, P. mosselii exhibit remarkable antimicrobial property especially against a broad range of plant pathogenic fungi, Gram-positive and negative human pathogenic bacteria. So far, many antibiotics have been isolated and identified from Pseudomonas species, and most of them were from P. aeruginosa, which is the most studied species of Pseudomonas. P. mosselii is one of the Pseudomonas species that has not been studied extensively. In the present study we have isolated Pseudopyronines B from P. mosselii and the isolation of Pseudopyronine B from P. mosselii is reported here for the first time.
The isolation of Pseudopyronines A and B is previously reported from marine sponge associated Pseudomonas species collected from the Fiji islands (Singh et al., 2003(Singh et al., , 2011Kong et al., 2005) and Pseudomonas sp. associated with entomopathogenic nematode (Grundmann et al., 2012). These compounds showed significant MICs against Gram-positive bacteria, including S. aureus, B. subtilis, methicillin-resistant S. aureus and Enterococcus faecium (MIC value: 2-4 µg/ml) and reasonable activity against E. faecalis and S. pneumoniae (MIC value: 16-64 µg/ml) with being the more active of the two compounds (Giddens et al., 2008;Singh et al., 2011). Similar results were observed in our study also (Table 1), where in our study also Staphylococcus spp. recorded best activity. Antimycobacterial activity of pseudopyronines against M. tuberculosis H37Rv is also well-reported in the literature (Giddens et al., 2008). Pseudopyronines are powerful and comparatively selective parasitic protozoa (Leishmania donovani) inhibitors (Blunt et al., 2010). These reports clearly portrait the role of Pseudopyronines in modern drug discovery process. The antifungal activity of Pseudopyronines especially Pseudopyronine B is not reported in literature. In the present manuscript the antifungal activity of Pseudopyronine B especially against plant pathogenic fungi is reported for the first time.
Bioactive compounds especially from natural have played a significant role over several years in the growth of various anticancer drugs. Many natural compounds and their derivatives have been successfully categorized according to the standard collection of several cancer drugs, such as paclitaxel, vinblastine, Frontiers in Microbiology | www.frontiersin.org  and vincristine (Zhong et al., 2012). Newman et al. (2003) reported that more than 50% of the novel compounds approved between 1982 and 2002 were derived directly or indirectly from natural compounds. This clearly indicated that natural compound play a profound role in the development of various anticancer drugs. Even though cancer is the leading cause of mortality worldwide and most of the chemotherapeutic compounds already in clinic have been reported to exhibit severe toxicity to normal cells, accompanied by other unwanted side effects to our body. Moreover, most of these compound are highly expensive, mutagenic, and carcinogenic (Cao et al., 2010). Therefore, it is very important to find out low toxic anti-cancer agents from natural sources. In this study, Pseudopyronine B was isolated from natural source and was tested for its cytotoxicity effect on A549, B16F10, and HepG2 cancer cell lines. We showed that Pseudopyronine B effectively inhibited cell growth of A549 and B16F10. When compared to that of A549 and B16F10 cells, Pseudopyronine B treated HepG2 cells recorded less activity. Alterations in the apoptosis and its related signaling pathways have a vital role in the development of tumor (Cao et al., 2010). Pseudopyronine B treated A549a and B16F10 cells exhibited typical morphological features of apoptosis including membrane flip-flop, cell shrinkage, higher uptake of stain with apoptotic markers and finally end up in severe DNA damage. The faults in apoptosis pathway are supposed to be one of the important causes Frontiers in Microbiology | www.frontiersin.org of human cancer (Thompson, 1995). Thus, a compound which induces apoptosis in cancer cells are one of the most efficient approaches in treating cancer (Lowe and Lin, 2000). The exact mode of action of Pseudopyronine B remains unclear. Using acridine orange-ethidium bromide and Hoechst staining assay, we observed that Pseudopyronine B induced apoptosis and DNA damage in A549 and B16F10 cell lines. Interesting in all most all cases, cellular DNA is the major goal of many chemotherapeutic drug molecules, which directly or indirectly attach to DNA. These drugs also obstruct DNA metabolism and inhibit the action of DNA polymerases and/or topoisomerases, which intern inhibit cell replication. Usually apoptosis occurs, following cellular DNA damage. Cysteinecontaining aspartate-specific proteases (caspases) in the cells play a very important role in the activation of various apoptotic signaling pathways. So far, 10 members have been identified in humans. Caspase-8 and caspase-9 are two initiator caspases which are capable of transducing apoptosis signals by direct activation of downstream executioner caspase-3 (Chen et al., 2003). Caspase-3, a protein on the common path of cell apoptosis, is one of the most important members and the key executor of cell apoptosis. Caspase-3 usually exists in the cytoplasm in the form of an inactive zymogen. When activated by the many external apoptosis signals, caspase-3 can induce the inactivation of many key proteases in the cytoplasm, cell nucleus, and cytoskeleton, and finally cause the apoptosis of cells. In the current study, cleavage of caspase-3 and up regulation of its cleaved form in A549 and B16F10 cells treated with Pseudopyronine B revealed Pseudopyronine B-induced apoptosis occurred through the caspase-3-dependent pathway.
Cellular apoptosis (programmed cell death) is a common form of cell death induced by many anticancer compounds/drugs (Vaux and Korsmeyer, 1999). Apoptosis cells are well-categorized by various morphological defects, such as condensation of chromatin, membrane flip-flop, and the formation of apoptotic body. These changes in cancer cells were finally leads to DNA fragmentation which was observed as a ladder on agar gel electrophoresis (Desagher and Martinou, 2000;Nonpunya et al., 2014). Apoptosis is mainly induced by a caspase cascade or translocation of apoptosis inducing factors (Nonpunya et al., 2014). There are two important pathways in the activation of caspase, which are the cell surface death receptor (extrinsic pathway) and mitochondrial initiator (intrinsic pathway). Caspase-3 and caspase-7 is the "execute" caspase for the apoptotic induction, while caspase-8 and caspase-9 are the critical caspases and signify the activation of the extrinsic and intrinsic pathways, respectively (McConkey, 1998;Law et al., 2014). The activation of caspases plays a significant role during apoptotic cell death; especially, the caspase 3 activation and activation of this caspase is an important step in the apoptosis procedure (Elmore, 2007). Interestingly Pseudopyronine B recorded enhanced activation of caspase 3, which may play an important role in apoptosis in A549 and B16F10 cells.

CONCLUSION
Here, it is highly evident from the present findings that a P. mosselii which produces a powerful bioactive substance (Pseudopyronine B), active against bacteria especially Grampositive. The present study contributes to the quest for novel antimicrobial compounds, a vital strategy in emerging alternative therapies to treat many infections caused by pathogenic microbes. Pseudopyronine B also recorded significant antifungal activity against plant pathogenic fungi. Isolation of Pseudopyronine B from P. mosselii and antifungal activity is reported here for the first time. Although, the present study delivers some valuable basic information about Pseudopyronine B from P. mosselii and further detailed investigation are needed to determine their potential for clinical applications.
In the present study, we confirmed that Pseudopyronine B inhibiting cell growth in a dose-and time-dependent manner. We also demonstrated that the cell death is due to apoptosis. To our best knowledge, this is the first investigation reporting the anticancer activity of Pseudopyronine B, which has the potential to be evaluated as a novel anticancer drug. Further studies are warranted to decipher the molecular mechanisms by which Pseudopyronine B modulates programmed cell death in various cancer cells. More over the results in the present study clearly point out the potential therapeutic property of Pseudopyronine B as an anticancer agent.

AUTHOR CONTRIBUTIONS
SNK performed experiments, analysis, or interpretation of data and manuscript preparation; SA performed anticancer section; JJ performed purification of compound; GG helped in manuscript preparation; TS performed anticancer section; RL performed purification of compound; BDK designed the work and manuscript correction.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Molecular epidemiology of Staphylococcus aureus bacteremia in a single large Minnesota medical center in 2015 as assessed using MLST, core genome MLST and spa typing

Staphylococcus aureus is a leading cause of bacteremia in hospitalized patients. Whether or not S. aureus bacteremia (SAB) is associated with clonality, implicating potential nosocomial transmission, has not, however, been investigated. Herein, we examined the epidemiology of SAB using whole genome sequencing (WGS). 152 SAB isolates collected over the course of 2015 at a single large Minnesota medical center were studied. Staphylococcus protein A (spa) typing was performed by PCR/Sanger sequencing; multilocus sequence typing (MLST) and core genome MLST (cgMLST) were determined by WGS. Forty-eight isolates (32%) were methicillin–resistant S. aureus (MRSA). The isolates encompassed 66 spa types, clustered into 11 spa clonal complexes (CCs) and 10 singleton types. 88% of 48 MRSA isolates belonged to spa CC-002 or -008. Methicillin-susceptible S. aureus (MSSA) isolates were more genotypically diverse, with 61% distributed across four spa CCs (CC-002, CC-012, CC-008 and CC-084). By MLST, there was 31 sequence types (STs), including 18 divided into 6 CCs and 13 singleton STs. Amongst MSSA isolates, the common MLST clones were CC5 (23%), CC30 (19%), CC8 (15%) and CC15 (11%). Common MRSA clones were CC5 (67%) and CC8 (25%); there were no MRSA isolates in CC45 or CC30. By cgMLST analysis, there were 9 allelic differences between two isolates, with the remaining 150 isolates differing from each other by over 40 alleles. The two isolates were retroactively epidemiologically linked by medical record review. Overall, cgMLST analysis resulted in higher resolution epidemiological typing than did multilocus sequence or spa typing.

Introduction
Staphylococcus aureus is responsible for a high percentage of hospital-and communityacquired infections worldwide. It is also a leading cause of bacteremia, often associated with metastatic infections and significant morbidity and mortality. The epidemiology of S. aureus infection has changed over the past decade and a half, with methicillin-resistant S. aureus (MRSA) being increasingly identified in community settings. This has led to interest in attempting to understand the genetic background of the pathogen across different geographic regions and care settings [1][2][3]. Since a retrospective, population-based, cohort study was done to evaluate initial episodes of S. aureus bacteremia (SAB) occurring in adult residents of Olmsted County, Minnesota, from 1998 through 2005 [4], there has been little data about the epidemiology of SAB from this area, and there has been no genetic characterization of involved isolates. In hospital settings, S. aureus may be transmitted from patient to patient via healthcare worker hands, contaminated equipment or through environmental contamination. Focused infection prevention and control (IPAC) measures guided by epidemiological investigations are necessary to prevent nosocomial transmission. To inform IPAC strategies within individual institutions, it is helpful to understand the molecular epidemiology of infection, including whether particular strains are prevalent, and whether or not there is evidence of patient to patient transmission.
In recent years, numerous tools have become available for typing of S. aureus, ranging from fingerprint-based methods such as pulsed-field gel electrophoresis (PFGE), to PCR-based methods such as multilocus variable-number tandem repeat analysis, to sequence-based methods such as multilocus sequencing typing (MLST), and most recently, to whole genome sequencing (WGS) [5]. The most widely used molecular typing method for defining MRSA epidemiology has traditionally been PFGE [6]. However, PFGE results can be challenging to compare between laboratories and to interpret; furthermore, PFGE is low throughput, is not suitable for long-term epidemiological investigations and assesses a limited amount of the microbial genome [7,8]. A low mutation rate of the sequence fragments of seven housekeeping genes makes MLST most suitable for long-term and global epidemiological studies [9]. DNA sequencing of short sequence repeats of the polymorphic X region of the protein A gene, spa, consisting of a variable number of 21-to 27-bp sequences, is an alternative method for typing S. aureus [10,11]. For MRSA, characterization of the resistance-conferring mobile genetic element, SCCmec is another method of typing. Recently, WGS-based typing has been made possible through next-generation DNA sequencing (NGS) technology; this approach allows detection of single nucleotide polymorphisms and core genome MLST (cgMLST), among analytical approaches. Through mapping of genome-wide variations, this approach theoretically provides optimal resolution to infer phylogenetic relatedness. WGS has been used in evolutionary studies, outbreak investigations, and phylogeographic distribution analyses [12][13][14].
The aim of the present study was to use WGS to investigate the molecular epidemiology of S. aureus isolates causing bacteremia in a single large Minnesota medical center. We also compared spa typing using data extracted from WGS to PCR/Sanger sequencing-based spa typing, and WGS-generated MLST-to cgMLST-based typing.

Patient population
All episodes of S. aureus bacteremia in patients !16 years of age at the Mayo Clinic, in Rochester, Minnesota (2,059 hospital beds) from January 2015 through December 2015 were considered for inclusion. Isolates were prospectively collected. Only the first episode was analyzed in cases of recurrent SAB during the study period. Isolates associated with polymicrobial infection or suspected contamination, as well as those with insufficient medical record data were excluded. This non-interventional study was approved by the Mayo Clinic Institutional Review Board with a waiver of informed consent (IRB 15-001990).

Data collection and definitions
Patient demographics and clinical characteristics were recorded. Community-onset infections were defined as those either present or incubating at the time of hospital admission, or for which the first positive culture was obtained within 48 hours of admission. Community-onset infections were further classified as healthcare-associated (HCA) or community-associated (CA). If a patient fulfilled any of the following criteria, SAB was classified as HCA [15,16]: (1) presence of an invasive device at the time of admission or at onset of infection; (2) history of MRSA infection or colonization in the antecedent 6 months; (3) history of surgery, hospitalization, dialysis, or residence in a long-term care facility in the antecedent 12 months; or (4) use of systemic antibiotics within 3 months prior to SAB. A case was classified as hospital onset (HO) if the first S. aureus blood culture was obtained 48 hours or more after hospital admission.

Identification and antimicrobial susceptibility testing
The BD BACTEC™ FX system (Becton, Dickinson and Company, Franklin Lakes, NJ) was used for blood culture. Blood culture bottles showing possible staphylococci were tested with the FilmArray 1 BCID panel (bioMérieux, Marcy l'Étoile, France), which includes rapid detection of mecA and S. aureus. Identification of S. aureus was by standard methods (biochemicals or matrix-assisted laser desorption ionization time-of-flight mass spectrometry); antimicrobial susceptibility testing was performed using agar dilution, with results interpreted using the Clinical and Laboratory Standards Institute guidelines [17]. Antibiotics tested included clindamycin, mupirocin, oxacillin, rifampin, trimethoprim-sulfamethoxazole, vancomycin, and daptomycin, with ceftaroline additionally tested against MRSA.
spa typing based on PCR/Sanger sequencing Staphylococcal DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA). spa typing was performed with PCR followed by Sanger sequencing, as previously described [18]. Assignment of spa type and Based Upon Repeat Pattern (BURP) analysis for determination of spa clonal complex (spa CC) were performed using Ridom StaphType (Ridom GmbH, Germany).

DNA sequencing for WGS and cgMLST
Staphylococcal DNA was prepared for WGS using lysostaphin or achromopeptidase, the Maxwell 1 16 Tissue DNA Purification kit (Promega Corporation, Madison, WI) and the Genomic DNA Clean & Concentrator™ kit (Zymo Research Corporation, Irvine, CA), performed according to the manufacturers' instructions. WGS was performed on a MiSeq (Illumina, Inc., San Diego, CA) using the paired-end mode (2x250) in the Mayo Clinic Medical Genome Facility. Reads were trimmed using Trimmomatic. FASTQ files were imported into SeqSphere+ software version 2.3 (Ridom GmbH) for analysis. de novo assembly and application of a S. aureus cgMLST scheme was done using SeqSphere+ with default parameters and with 1,861 queried target genes, as described previously [19]. If genomes did not contain !95% of the 1,861 cgMLST target genes with a minimum of ten times global local coverage, WGS was repeated.

Determination of spa, MLST and SCCmec types from WGS data
In addition to performing a specific PCR followed by Sanger sequencing, spa type was determined using SeqSphere+ for spa typing with the WGS data. The WGS data was also used for MLST and SCCmec type determination. For MLST, seven target genes (arcC, aroE, glpF, gmk, pta, tpi, yqiL) were analyzed [20], also in SeqSphere+. If there was no matched allele in Seq-Sphere+, the sequence of the particular gene(s) from the S. aureus MLST scheme was manually extracted from the WGS data and compared to references in NCBI and the S. aureus MLST database (http://pubmlst.org/saureus) to assign the classical sequence type (ST). Novel sequences were submitted to the S. aureus MLST database http://pubmlst.org/saureus. The associated clonal complex (CC) was calculated using the eBURST algorithm (http://saureus. mlst.net/eBURST/), with CCs defined using a criterion of six common alleles [21]. SCCmec typing was determined by assessing ccr and mec gene complexes from WGS data. Sequences have been deposited in the NCBI Sequence Read Archive under bioproject number PRJNA384623.

Results
Of a total of 176 non-duplicate SAB isolates during the study period, 152 were included after excluding 13 which were associated with polymicrobial infection, 6 which were considered possible contaminants and 3 with insufficient medical record data. Two isolates failed to provide sufficient WGS data after multiple attempts. 63% were from male patients; the mean patient age was 61±16.8 (± standard deviation) years. Comparison of antimicrobial susceptibility of MSSA and MRSA 54% of MRSA isolates and 14% of MSSA isolates were resistant to clindamycin. Clindamycin resistance was present 6% of CA isolates, 27% of HCA isolates, and 44% of HO isolates. Fewer than 5% of MRSA isolates were resistant to rifampin, mupirocin, or trimethoprim-sulfamethoxazole (Table 1). All MRSA isolates were susceptible to vancomycin, daptomycin, and ceftaroline.
Compared to PCR/Sanger sequencing-based spa typing, WGS-based spa typing revealed 26 isolates with apparently discrepant results. Using SeqSphere+, spa genes were not detected in 2 isolates and 24 isolates showed apparently different results than PCR/Sanger sequencing.
When sequences were compared manually however, sequence differences were not confirmed for 18 isolates whereas they were confirmed in 6. For the 18 isolates for which the differences were not confirmed, WGS sequencing results showed several nucleotide differences in 12 isolates, and possible insertion, deletion or substitution errors in 6 isolates.

MLST from WGS
152 isolates were subjected to MLST analysis; one locus was not found in one isolate, which was not further analyzed. Using the S. aureus MLST database (http://pubmlst.org/saureus), there were 31 sequence types. The most prevalent sequence type was ST5, represented by 36 isolates (24% of all isolates); the next prevalent clone was ST8, represented by 24 isolates (16% of all isolates). Eighteen STs were divided into 6 CCs by eBURST and 13 STs were singletons. The singletons did not belong to any CC, and differed from all STs in the dataset at two or more MLST loci.

SCCmec typing from WGS
Two SCCmec types were found among the 48 MRSA isolates, type II (56%) and type IV (44%). SCCmec type II isolates were all CC5, and SCCmec type IV was the most common type in CC8 (57%).
cgMLST from WGS   Fig 2 shows a minimum spanning tree based on the cgMLST allelic profiles with overlaid MLST clonal complex (CC) groupings. cgMLST enabled higher resolution within CCs than did MLST or spa typing (Fig 2).
By cgMLST analysis, there were 9 allelic differences between 2 isolates (MZ51 and MZ81), with the remaining 150 isolates differing from each other by over 40 allelic differences. MZ51 and MZ81 were both HO-MRSA bacteremia isolates, recovered from patients with peripheral inserted central catheters. The two bacteremias occurred 55 days apart. The patients from whom the isolates derived were both cared for on the same service and both had care provided by the same nurse practitioner. A phylogenetic dendrogram of cgMLST data was generated to visualize the relatedness among isolates compared to MLST and PCR/Sanger sequencing-based spa typing (Fig 3).  MSSA, methicillin-susceptible S. aureus; MRSA, methicillin-resistant S. aureus a spa Types with fewer than five repeats were excluded. spa types were grouped into the same spa CC if cost six [11]. https://doi.org/10.1371/journal.pone.0179003.t002

Discussion
In a single large Minnesota medical center, over 150 cases of SAB occurred in the year 2015, approximately one third of which were MRSA. spa typing of MSSA revealed wide genotypic diversity; the common MRSA clones were spa CC-002 and CC-008. By MLST analysis, CC45 and CC30 contained only MSSA isolates, and common MRSA clones were CC5 and CC8. cgMLST results yielded higher resolution typing results compared to MLST or spa typing, and demonstrated a single episode of possible hospital-based transmission of S. aureus bacteremia.
Although there are no standardized thresholds for interpreting clonality with cgMSLT, we recently proposed that using the scheme applied herein for S. aureus, 8 allelic differences be considered related, 9-29 allelic differences be considered possibly related and 30 or more allelic differences be considered unrelated [22]. The findings presented herein support the use of these thresholds. Outbreaks of CA-MRSA infections have been described worldwide with successful clones often associated with specific geographical locations [23]. Dominant clones of CA-MRSA in the United States are ST1 (PFGE type USA400) and ST8 (PFGE type USA300), and a common HCA-MRSA clone is ST5 (PFGE type USA100). In our study, only 3 cases were CA-MRSA of which 2 were ST8; the dominant clone for HCA-and HO-MRSA was ST5. Although MRSA isolates were more related with healthcare associated infection, HO MSSA bacteremia was surprisingly frequent. There were three ST398 isolates (t034, t571 in cluster 11 in Fig 1), one of which-MRSA isolate MZ105 (t571)-may have been livestock-associated.
Prevention of S. aureus bloodstream infections is compromised by incomplete understanding of transmission patterns of S. aureus, which in turn reflects, in part, suboptimal resolution offered by conventional typing methods. Analysis of every nucleotide in the genome provides a complete inventory of micro-evolutionary changes, but is impractical for large population samples. The use of cgMLST maps single nucleotide polymorphisms and insertion/deletions across a large number of genes to a reference sequence [24,25]. cgMLST provides higher discrimination than ordinary MLST. In our study, only 2 of 152 isolates had a low number of allelic differences by cgMLST suggesting possible relatedness; these two cases were retroactively determined to be epidemiologically connected. cgMLST was used for the first time with next generation sequencing data for phylogenetic analysis of the German 2011 enterohemorrhagic Escherichia coli O104:H4 outbreak [26]. More recently, cgMLST was used to evaluate a meningococcal outbreak [27]. In a neonatal MRSA outbreak [24], a rapid sequencing platform revealed a distinct cluster of outbreak isolates and clear separation between outbreak and nonoutbreak isolates with same sequence type (ST22). As the costs of sequencing have begun to approach those of conventional methods, high-resolution typing with WGS is now the stateof-the-art tool for routine epidemiological investigations.
We found that traditional spa typing using PCR/Sanger sequencing had better performance than WGS-based spa typing. Bletz et al. suggested that extraction of spa types from WGS with an optimized de novo assembly may enable nearly full compatibility with PCR/Sanger sequencing-based spa typing for MRSA [28]. However, assembly of repetitive areas based on short Illumina NGS read data may be prone to misassembling, resulting in failure or errors [29], as illustrated herein. Although it is not possible to compare data directly since different DNA extracts were used for PCR/Sanger sequencing-and WGS-spa typing herein, spa typing discrepancies between WGS data and traditional spa typing caution against WGS usage for spa typing. We note that we used a paired-end 250 base pair read length protocol for sequencing and that longer reads may improve WGS-based spa typing.
There are several limitations of our study. First, there was some missing data in the cgMLST targets. Second, we analyzed only S. aureus bacteremia patients and thus we cannot assess nosocomial transmission of non-bacteremia events or non-bacteremia to bacteremia events. Third, for our spa typing studies, we did not perform PCR/Sanger sequencing from the same DNA that had been used for WGS.

Conclusions
cgMLST analysis resulted in higher resolution epidemiological typing than did MLST or spa typing. MSSA isolates associated with SAB at a single large Minnesota medical center exhibited wider genotypic diversity than did MRSA isolates. By cgMLST analysis, only two of 152 SAB isolates appeared to be possibly related and conceivably associated with intra-hospital transmission.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Zinc oxide nanoparticles harness autophagy to induce cell death in lung epithelial cells

Although zinc oxide nanoparticles (ZnONPs) are widely used, they have raised concerns of toxicity in humans. Previous studies have indicated that reactive oxygen species (ROS) and autophagy are involved in the cytotoxicity of ZnONPs, but the regulatory mechanisms between autophagy and ROS remain to be elucidated. Herein, we comprehensively investigated the regulatory mechanism of autophagy and the link between autophagy and ROS in ZnONPs-treated lung epithelial cells. We demonstrated that ZnONPs could induce autophagy, and this process could enhance the dissolution of ZnONPs in lysosomes to release zinc ions. Sequentially, zinc ions released from ZnONPs were able to damage not only lysosomes, leading to impaired autophagic flux, but also mitochondria. Impaired autophagic flux resulted in the accumulation of damaged mitochondria, which could generate excessive ROS to cause cell death. We further demonstrated that the inhibition of autophagy by either pharmacological inhibitors or small interfering RNA (siRNA)-mediated knockdown of Beclin-1 and AMP-activated protein kinase could ameliorate ZnONPs-induced cell death. Moreover, we found that lysosomal-associated membrane protein 1/2 (LAMP-1/2), which were the most abundant highly glycosylated protein in late endosomes/lysosomes, exhibited aberrant expression pattern upon treatment with ZnONPs. Intriguingly, LAMP-2 knockdown, but not LAMP-1 knockdown, could exacerbate the ROS generation and cell death induced by ZnONPs treatment. Meanwhile, LAMP-2 overexpression alleviated ZnONPs-induced cell death, suggesting that LAMP-2 was linked to this toxic phenotype induced by ZnONPs. Our results indicate that autophagic dysfunction could contribute to excessive ROS generation upon treatment with ZnONPs in lung epithelial cells, suggesting that modulating the autophagy process would minimize ZnONPs-associated toxicity.

Nanotechnology has made remarkable progress in recent years, and the global nanotechnology market is estimated to reach a trillion dollars annually by 2015. 1 Zinc oxide nanoparticles (ZnONPs) are one of the most important metal oxide nanoparticles and its worldwide production is estimated to be up to 1 million tons per year. 2 They are extensively used not only for industrial/household applications (cosmetics, pigments, coatings, electronic devices, catalysts) 3 but also for clinical purposes. 4 However, because of the large-scale production and increasing utilization, concerns of ZnONPs toxicity is raising. [5][6][7] Airway exposure is the general exposure route besides dermal exposure. The respiratory system is vulnerable as it directly and constantly contacts with the environment; 8 it has been reported that ZnONPs inhalation would cause metal fume fever in humans. 9 Previous studies have revealed several possible reasons for ZnONPs-related toxicity. Among these, ROS and inflammation caused by ZnONPs are most commonly accepted. 8,[10][11][12] Another well-known toxicity-related mechanism is the release of zinc ions from ZnONPs, which can induce organelle damage in the biological environment. [13][14][15] However, the opinions about whether apoptotic cell death is induced by ZnONPs treatment are controversial. This disparity might be due to the differences in ZnONPs characteristics, doses or cells. [16][17][18][19][20] In recent times, autophagy has been considered as a possible mechanism underlying nanomaterial-induced toxicity. 21 Autophagy (specifically macroautophagy) is thought to begin as a phagophore, followed by interaction with Atg5-Atg12 conjugation. Then, LC3 inserts into the extending phagophore membrane to facilitate the engulfment of intracellular cargos (including aberrant organelles, proteins, virus and nanomaterials) in the double-membrane structure referred to as autophagosomes. The autophagosomes may further fuse with lysosomes to form autolysosomes, and the intracellular contents will be degraded in the lysosomes and recycled. [22][23][24] The integrity of lysosomes is crucial for autophagy. Lysosomal acid environment and numerous 1 hydrolases inside lysosomes facilitate intracellular content degradation. The dynamic process of autophagosomes formation, fusion of autophagosomes with lysosomes and intracellular content degradation, is referred to as autophagic flux, which reflects the rate of autophagic degradation. Lysosomal dysfunction can lead to incomplete autophagy. 25 It is reported that ZnONPs might induce autophagy, 16,26 or autophagic cell death, 27 but whether ZnONPs indeed induce autophagy is not addressed. On the other hand, Cho et al. 13 have demonstrated that ZnONPs are dissolved more rapidly in an acidic environment, suggesting that ZnONPs may be dissolved in lysosomes and cause lysosomal destabilization in differentiated THP-1 cells. Mihai et al. 14 have demonstrated that zinc ions accumulates specifically in endosomes and lysosomes of alveolar epithelial cells upon ZnONPs treatment. 14 These results show that ZnONPs can be dissolved in lysosomes and cause lysosomal dysfunction. However, the regulatory mechanism and biological effect of lysosomal dysfunction remain poorly characterized.
Components involved in autophagy are sophisticatedly orchestrated at the molecular level; thus, autophagy can be regulated at multiple steps. The LC3II/LC3I ratio increase or autophagosomes accumulation may result from either autophagy induction or impairment of autophagic flux. 28 Hence, integrated assays should be performed to clarify whether autophagy is induced by ZnONPs. Furthermore, the subsequent effects from autophagic flux blockade should be evaluated cautiously. Mitophagy, autophagy-dependent degradation of the mitochondria, is important for maintaining the integrity of mitochondria and the balance of ROS. 29 When autophagic flux is blocked, the elimination of damaged mitochondria will also be impaired, which might be the nexus of autophagy and ROS.
In the present study, we demonstrated that ZnONPs could be delivered into lysosomes via the autophagy pathway and subsequently dissolved in lysosomes to release zinc ions, which was the crucial factor responsible for the ZnONPsinduced cytotoxicity. Moreover, zinc ions could damage mitochondria and lysosomes, further disrupting the negative feedback mechanisms between ROS and mitophagy, leading to damaged mitochondria accumulation, excessive ROS production and cell death.

Results
Release of zinc ion from ZnONPs induces cell death in respiratory epithelial cells. The characteristics of ZnONPs used in this study were examined by transmission electron microscopy (TEM) (Supplementary Figure S1a), and the results showed that the particle size of ZnONPs was~50 nm and the morphology was roughly spherical. These nanoparticles have been well characterized previously. Roy et al. 30 reported that the zeta potential of this ZnONPs was − 11.5 mV, with dynamic light scattering (DLS) size distribution of an average diameter of 278.8 nm, in culture media supplemented with 10% serum. 30 Respiratory epithelial cells are recognized as the first barrier and vulnerable when exposed to high concentrations of nanoparticles. Because damage of alveolar epithelial cells is the hallmark event of acute lung injury, 31 we chose the human lung adenocarcinoma cell line A549, which is the classical and one of the most widely used alveolar epithelial cell models, for our further experiments. To determine whether ZnONPs could induce cell death on respiratory epithelial cells, we treated A549 cells with 10, 30 and 100 μg/ml ZnONPs. MTS results showed that treatment with ZnONPs induced significant cell death at doses of 30 and 100 μg/ml (Figure 1a).
Dissolution of ZnONPs has been demonstrated as contributing to the cytotoxicity of ZnONPs. Hence, we examined the intracellular concentration of zinc ions using FluoZin-3, a specific indicator for zinc ions. Results of fluorescenceactivated cell sorting (FACS) showed that ZnONPs induced marked elevations of zinc ions signal in A549 cells (Figure 1b). Meanwhile, the mRNA expression level of zinc transporter 1 (ZnT1) was also elevated (Supplementary Figure S2a). To determine whether the elevation of zinc ions concentration was correlated with cell death, zinc ions-chelating agent diethylenetriaminepentaacetic acid (DTPA) and N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) were added 1 h before ZnONPs treatment. Results indicated that chelating of intracellular zinc ions alleviated A549 cell death induced by ZnONPs treatment at doses of 30 and 100 μg/ml (Figure 1c). The discrepant rescue capacity between DTPA and TPEN might be due to the maximum-tolerated nontoxic dose (Supplementary Figures S2b and c). Moreover, DTPA could improve cell viability in a dose-dependent manner (Figure 1d), and might not via affecting the entry of ZnONPs (Supplementary Figure S2d). We also observed that DTPA treatment significantly reduced intracellular zinc ions concentration (Figure 1e). At the same time, we ruled out the possibility of apoptotic cell death after ZnONPs treatment; the evidence contributing to this conclusion included the following: (1) Annexin V-FITC/propidium iodide staining showed no significant apoptosis in ZnONPs-treated A549 cells (Supplementary Figure S3a); (2) ZnONPs treatment had minimal influence on the apoptosis marker including cleaved PARP and cleaved caspase-3 (Supplementary Figure S3b); and (3) pancaspase inhibitor Z-VAD-FMK could not rescue ZnONPs-induced cell death even at a high dose (Supplementary Figure S3c). Taken together, these data demonstrated that zinc ion is the key mediator in ZnONPs-induced non-apoptotic cell death.
ZnONPs induce accumulation of autophagosomes and impairment of autophagic flux. Autophagy has been considered as an important event in nanoparticle-induced cytotoxicity. In particular, ZnONPs have been linked to autophagy. In line with pervious observations, our TEM results showed that ZnONPs were wrapped by cell plasma membrane and formed a phagophore-like structure at 6 h after treatment with ZnONPs ( Figure 2a). Autophagosomes and autolysosomes in A549 cells increased markedly at 24 h after treatment with ZnONPs ( Figure 2b). Meanwhile, western blot analysis results showed that the autophagy marker, LC3B-II/LC3B-I ratio, increased at 6 h and kept increasing during the treatment period with ZnONPs ( Figure 2c) in a dose-dependent manner ( Figure 2d). Interestingly, we also noted that p62 (SQSTM1/sequestome1), a specific substrate degraded by autophagy, 32 increased in ZnONPs-treated cells and exhibited the same pattern as LC3B-II/LC3B-I ratio. These results indicated that ZnONPs might cause impairment of autophagic flux in A549 cells.
We confirmed this notion that the impairment of autophagic flux was induced by ZnONPs in transgenic cells stably expressing mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3). Because GFP fluorescence was not stable in the acidic environment of lysosomes, tfLC3 showed a GFP and mRFP signal (merged as yellow puncta) before the fusion with lysosomes, and exhibited only the mRFP signal subsequently in autolysosomes. 33 Therefore, autophagosomes and autolysosomes are labeled with yellow (i.e., mRFP and GFP) and red (i.e., mRFP only) signals, respectively. 34 As shown in Figures 2e and f, autophagy inducer rapamycin (Rapa) induced more red puncta than untreated cells, suggesting an enhancement of autophagic flux. The specific inhibitor of vacuolar-type H + -ATPase bafilomycin A1 (BAFA1) could prevent the fusion of autophagosomes with lysosomes. By cotreating BAFA1 with Rapa, many yellow puncta were observed, suggesting that the autophagic flux was blocked. Consistent with the above results (Figures 2c and d), yellow puncta increased significantly in ZnONPs-treated A549 cells (Figures 2e and f), confirming that ZnONPs treatment indeed blocked autophagic flux. Autophagosome accumulation accompanied with the increased autophagy substrate could result from either autophagy induction or impairment of autophagic flux. 28 To verify whether autophagy was induced upon ZnONPs treatment, A549 cells were pretreated with BAFA1 1 h before ZnONPs treatment. When lysosomal degradation is inhibited, the amount of LC3B-II is strictly dependent on LC3B-II production, and thus could reflect whether autophagy is induced. 35 Western blot results showed that there were increases in LC3B-II and p62 levels in cells cotreated with ZnONPs and BAFA1, indicating that ZnONPs do indeed induce autophagy (Figure 2g). This phenomenon was further confirmed by treating cells with chloroquine diphosphate salt (CQ), which could elevate the lysosomal pH and impair the fusion of autophagosomes with lysosomes ( Figure 2h). Collectively, our data demonstrate that ZnONPs    Figure S4b). Furthermore, siRNA-mediated knockdown of Beclin-1 (Atg6), which had a central role in autophagy, 37 could rescue the cell viability in ZnONPs-treated cells (Figure 3d). In addition, DTPA treatment could significantly decrease the LC3B-II/LC3B-I ratio and p62 expression in ZnONPs-treated cells, which demonstrated that the release of zinc ions is certainly associated with autophagy (Supplementary Figure S4c). Collectively, our data demonstrate that autophagy induction is positively correlated with the elevation of zinc ions concentration and ZnONPs-induced cell death.
AMPK pathway activation is involved in ZnONPs-induced A549 cell death. The inhibition of the target of Rapa (mTOR) and AMP-activated protein kinase (AMPK) activation are recognized as important regulatory mechanisms of autophagy. 38 Intriguingly, we found that the p-mTOR level was not altered significantly upon ZnONPs treatment, whereas p-AMPKα expression level was markedly upregulated ( Figure 4a). Meanwhile, we found that phosphorylation of protein kinase B/Akt, which was the upstream regulator of mTOR, 39 significantly elevated after ZnONPs treatment (Figure 4b). p-AKT has been shown to rapidly phosphorylate glycogen synthase kinase 3 beta (GSK3β) at serine 9. 40 We also detected that the p-GSK3β(Ser9) expression level was upregulated upon ZnONPs treatment (Figure 4b  demonstrating that ZnONPs could induce the elevation of p-AKT. On the other hand, we performed siRNA-mediated knockdown of AMPK to investigate whether the correlation between AMPKα activation and ZnONP-induced cell death was an epiphenomenon, or whether they were causally linked. We observed that AMPKα knockdown (Figure 4c) caused slight elevation of the cell viability compared with control siRNA-treated cells upon ZnONP treatment (Figure 4d), suggesting that AMPKα activation might be involved in ZnONPs-induced cell death. To determine how the blockade of autophagic flux was induced in ZnONPs-treated cells, we labeled the cells with LysoTracker Red DND-99, a probe accumulated within the acidic lysosomes/late endosomes. FACS results show that treatment with ZnONPs increased the fluorescence signal of LysoTracker Red DND-99, suggesting that the amount of acidic lysosomes increased (Figure 5a). Immunofluorescence results showed that the signal intensity of LAMP-1/2 was slightly diffused in ZnONPs-treated cells (Supplementary Figure S5a). Interestingly, western blot assay data showed that cathepsin B/ D had only a slight change, whereas aberrant LAMP-1/2 expression occurred upon ZnONPs treatment (Figure 5b). Furthermore, we observed that DTPA could ameliorate aberrant LAMP-1/2 expression (Figure 5c) and decrease the amount of lysosomes in cells treated with ZnONPs (Figure 5d), suggesting that zinc ions induced the activation of lysosomes and the aberrant LAMP-1/2 expression. To determine whether LAMPs were involved in ZnONPs-induced cell death, knockdown of LAMP-1/2 was performed before treatment with ZnONPs. Western blot analysis results showed a decrease in LAMP-1 and LAMP-2 expression (Figure 5e, top panel). Further, LAMP-1 or LAMP-2 knockdown had no significant influence on naïve autophagy level (Supplementary Figure S5b) and A549 cell viability (Supplementary Figure S5c). Interestingly, LAMP-2 knockdown, but not LAMP-1 knockdown, increased the ZnONPs-induced cell death (Figure 5e, bottom). More importantly, we observed that the A549 cell line stably expressing LAMP-2-RFP (Figure 5f, top panel) was more resistant than the wild-type A549 cell line with ZnONPs treatment (Figure 5f, bottom panel). We also observed that LAMP-2 expression level was elevated in the Rapainduced autophagy model ( Supplementary Figures S5d and e).
We also investigated additional mechanisms that could be responsible for ZnONPs-induced impairment of autophagic flux. Lysosomal membrane permeabilization, a process induced by the damage of lysosomal membrane integrity, could lead to the leakage of hydrolytic enzyme proteases, such as cathepsin B, D and L, from lysosomal lumen to cytosol. 41,42 However, our immunofluorescence data showed that cathepsin D still colocalized with LAMP-1 after ZnONPs treatment (Supplementary Figure S6a) rather than diffused through the entire cell, which was a typical feature during lysosomal membrane permeabilization. 43 siRNA-mediated knockdown of cathepsin B, one of the most important enzymes in lysosomal membrane permeabilization, 44 did not ameliorate ZnONPs-induced cell death (Supplementary Figure S6b). Treatment with Z-VAD-FMK, which was reported to block the activity of lysosomal proteases, did not alleviate ZnONPs-induced cell death (Supplementary Figure S3c). These data indicated that the impairment of autophagic flux might not result from ZnONPs-induced lysosomal membrane permeabilization. In addition, lysosomal alkalinization is regarded as the major reason for the blockade of autophagic flux in AuNP-treated NRK cells. 35 We stained A549 cells with LysoSensor Green DND-189, an acidotropic dye, which could be used to measure the pH of acidic lysosomes. The FACS data showed that ZnONPs treatment could not affect lysosomal acidic environment in A549 cells (Supplementary Figure S6c).
Impaired autophagic flux results in the accumulation of damaged mitochondria and excessive ROS production. ROS, generated mainly as by-products of mitochondrial respiration, 45 are increasingly identified as a major contributor to the cytotoxicity induced by nanoparticles, including ZnONPs. 8 Mitophagy is triggered for effective removal of damaged mitochondria and is implicated in diminishing excessive ROS. 28 On the other hand, previous studies have reported that LAMP-2 is involved in the fusion of phagosomes with lysosomes, 46,47 and thus it could contribute to the mitophagy regulation. 48 On the basis of these studies and our experimental data, we presume that damaged mitochondria fail to be eliminated as ZnONPs treatment causes aberrant LAMP-2 expression and sequential mitophagy dysfunction.
To verify our hypothesis, we labeled ZnONPs-treated cells with 2'7'-dichlorofluorescin diacetate to detect the intracellular ROS level. The FACS data showed that ZnONPs caused significant elevation in the ROS level (Supplementary Figure S7a). Furthermore, pretreatment with N-acetyl-lcysteine (NAC), a well-known antioxidant and free radical scavenger, ameliorated ZnONPs-induced cell death in a dosedependent manner (Figure 6a). The pretreatment with NAC or DTPA cleared out excessive ROS after ZnONPs treatment ZnONPs are delivered into lysosomes via autophagy, and subsequently dissolved in lysosomes to release zinc ions, which is the crucial factor triggering ZnONPs cytotoxicity. Furthermore, zinc ions (intracellular or extracellular) can damage mitochondria and lysosomes; thereafter, they further disrupt the negative feedback mechanisms of ROS and mitophagy, leading to the accumulation of damaged mitochondria, excessive ROS and finally cell death. Aberrant LAMP-2 expression is probably involved in this process. Inhibition of autophagy, chelating of zinc ions or treatment with antioxidant NAC effectively mitigates ZnONPs-induced cytotoxicity ( Figure 6b). Furthermore, NAC could decrease the LC3B-II/I ratio, p62 and aberrant LAMP-1/2 expression with ZnONPs treatment (Supplementary Figure S7b), suggesting the interplay between ROS and autophagy. These data collectively demonstrate that ZnONPs treatment could induce ROS production and trigger cell death in a ROS-dependent manner.
To determine whether ZnONPs treatment induced mitochondrial damage, we stained A549 cells with tetramethylrhodamine ethyl ester perchlorate (TMRE), a potential-sensitive probe for measuring membrane potential changes in the mitochondria. FACS results showed that ZnONPs treatment decreases the fluorescence signal of TMRE (Supplementary  Figure S7c), indicating that the mitochondria were damaged. On the other hand, TEM images showed the accumulation of aberrant mitochondria in ZnONPs-treated cells compared with untreated cells (Figure 6c). Moreover, we labeled cells with MitoTracker-Green FM, which stains mitochondria regardless of the mitochondrial membrane potential and only reflects the number of mitochondria. FACS results showed an increased fluorescence signal in ZnONPs-treated cells, indicating that ZnONPs treatment caused mitochondrial accumulation in A549 cells (Figure 6d). To investigate whether LAMPs were involved in the regulation of mitophagy and ROS in ZnONPstreated cells, we performed siRNA-mediated knockdown of LAMP experiments. FACS data showed that LAMP-2 knockdown significantly increased the ratio of cells with low signal of TMRE ( Figure 6e) and evoked excessive ROS generation (Figure 6f). However, LAMP-1 knockdown seemed not to be responsible for the removal of damaged mitochondrial accumulation and excessive ROS generation.
In conclusion, we demonstrated that ZnONPs-induced cell death resulted from the cascade of autophagic flux impairment following damaged mitochondrial accumulation and excessive ROS production. This process might attribute to aberrant LAMP-2 expression induced by zinc ions released from ZnONPs in lysosomes. AMPKα activation, but not inhibition of mTOR, was involved in ZnONPs-induced cell death. The inhibition of autophagy, chelating of intracellular zinc ions using DTAP/TPEN or antioxidant NAC treatment can effectively mitigate ZnONPs-induced cytotoxicity (Figure 6g).

Discussion
Previous studies have extensively investigated the toxicity effect of ZnONPs and have proposed potential regulatory mechanisms including ROS production and autophagy induction. However, the detailed regulatory mechanism of autophagy and, more importantly, the interplay between autophagy and ROS in ZnONPs-treated cells are still largely obscure. Our results indicated that ZnONPs subtly harness the two important functions of autophagy in lung epithelial cells: foreign material engulfment and damaged organelles elimination.
First, we demonstrated that ZnONPs induced autophagy, which facilitated ZnONPs to be delivered into lysosomes. The acidic environment of lysosomes would enhance ZnONPs dissolution and the sequential release of zinc ions. 13 The high concentration of zinc ions directly induced cell death, which could be ameliorated by zinc-specific chelating agents such as DTPA or TPEN. Although TPEN was toxic at a concentration higher than 1 μM alone (Supplementary Figure S2c), its synergistic treatment with ZnONP might be quenched, and thus TPEN could be applied at a concentration higher than 1 μM. Our results showed that DTPA inhibited ZnONPsmediated cell death and abrogated intracellular zinc ions; however, this did not rule out chelation of free zinc ions from ZnONPs upon addition to the culture media. Furthermore, we demonstrated that inhibition of autophagy could reduce the release of zinc ions and ameliorate ZnONPs-induced cell death. These results manifested that ZnONPs could use autophagy to release zinc ions and consequently induce nonapoptotic cell death.
More interestingly, we demonstrated that ZnONPs treatment could induce impaired autophagic flux. We observed that the p62 expression level was upregulated during the process of ZnONPs treatment, suggesting the impaired autophagic flux caused by ZnONPs treatment. This notion was further confirmed in the mRFP-GFP-LC3 stably expressing cell line. We found that a significant decrease of red-only puncta and a significant increase of yellow puncta occurred upon ZnONPs treatment, which is the sign of impaired lysosome degradation. Lin et al. 49 showed that ZnONPs could upregulate the expression level of both LC3II and p62 in the human embryonic kidney cell line HEK-293, but they did not further describe the consequent biological effect induced by impaired autophagic flux. Several studies have discussed the relationship between ZnONPs and autophagy in T cells (SupT1 and Jurkat cell lines, primary human T cells), 27 mouse macrophages, 16 and JB6 Cl 41-5a mouse skin epidermal normal cells. 26 However, they did not mention the phenomenon that caused impairment of autophagic flux induced by ZnONPs treatment. To our knowledge, the current study is the first to describe the impaired autophagic flux induced by ZnONPs in lung epithelial cells.
We further explored the possible mechanisms behind the impaired autophagic flux caused by ZnONPs. Unexpectedly, we found that the inhibition of mTOR, a negative regulator of autophagy, was probably not involved in ZnONPs-induced lung epithelial cell death. In contrast, Roy et al. 16 reported that ZnONPs could induce apoptosis by enhancement of autophagy via PI3K/AKT/mTOR inhibition in mouse macrophages. 16 Their results showed that ZnONPs reduced the phosphorylation levels of PI3K, AKT and mTOR in a timedependent manner. 16 However, numerous lines of evidence have shown that zinc ions could rapidly increase the phosphorylation levels of AKT. [50][51][52][53] Our results would support the later opinion as we observed that ZnONPs induced rapid increase of p-AKT (Ser473) and its substrate p-GSKβ (Ser9) (Figures 4c and d). We suspect that the rapid and strong increase of p-AKT could preclude the inhibition of mTOR from induction of autophagy in ZnONPs-treated lung epithelial cells.
In addition to the inhibition of mTOR, AMPK activation is considered as another crucial event in autophagy regulation. [54][55][56] We observed a strong time-dependent increase in p-AMPKα with ZnONPs exposure. AMPKα knockdown experiments further demonstrated the association between AMPK activation and cell death upon ZnONP treatment. AMPK activity can be regulated by AMP/ATP ratios, liver kinase B1, Ca 2+ /calmodulin-dependent protein kinase or TGF-β-activated kinase 1. 57,58 More relevantly, Yu et al. 26 showed that ZnONPs induced an increase in total AMPK and a decrease in the relative ATP amount in normal mouse skin epidermal cells. 26 Although evidence of AMPKα activation could not be accounted for, we showed that AMPKα activation partially contributed to ZnONPs-induced cell death.
In addition, we found that ZnONPs caused aberrant LAMP-1/2 expression. LAMP-2 knockdown or overexpression experiments demonstrated that LAMP-2 was linked to ZnONPs-induced ROS generation and cell death. Our previous study demonstrated that extensive LAMP-1/2 deglycosylation could induce lysosomal membrane permeabilization and cell death upon influenza virus infection. 59 Moreover, recent studies suggested that sugar modification could contribute to the regulation of autophagosomes-lysosomes fusion. 60,61 Therefore, the existence of LAMP-2 deglycosylation and its potential roles in ZnONPs-induced impaired autophagic flux need be further explored.
In summary, we demonstrated that ZnONPs could induce autophagy to facilitate their dissolution in lysosomes, while inhibition of autophagy could decrease intracellular zinc ions concentration and ameliorate cell death. AMPKα activation was involved in ZnONPs-induced cell death. We also showed that ZnONPs could cause impaired autophagic flux, which could lead to ROS production. LAMP-2 appeared to contribute to impaired autophagic flux and to accumulation of damaged mitochondria and cell death in ZnONPs-treated lung epithelial cells. Our study provides a novel insight into the regulation mechanisms of autophagy-lysosomes-mitochondria-ROS axis, which would contribute to a better understanding of the toxicity of nanomaterials.

Materials and Methods
Nanoparticles. Zinc oxide nanopowder was obtained from Sigma-Aldrich (St. Louis, MO, USA) (677450). The particle size of ZnONPs was ⩽ 50 nm (BET data provided by Sigma-Aldrich), which was confirmed by the supplier using X-ray diffraction. We also detected these ZnONPs using TEM, and the results are provided in Supplementary Figure S1a. ZnONPs were suspended in culture medium at a concentration of 3 mg/ml and then sonicated in a sonicator bath for 30 min. The solution was then diluted with a medium to a indicated concentration. The dilutions of ZnONPs were vigorously vortexed for 30 s before cell exposure to avoid nanoparticle agglomeration.
Lentiviruses were packaged in HEK293T cells. To generate stably transfected cell lines, A549 cells were infected with virus for 48 h and then selected using puromycin (Solarbio, Beijing, China).
Transmission electron microscopy. The morphology of A549 cells was observed by TEM as described previously. 62 Briefly, cells were centrifuged at 1000 × g for 5 min after trypsinization, fixed with 4% glutaraldehyde in 0.1M PBS (pH 7.4) for 2 h at 4°C, washed three times with PBS (pH 7.4) and post-fixed with 1% osmium tetroxide in 0.1 M PBS for 1 h at 4°C, dehydrated in a graded series of alcohol and acetone, and then embedded in Epon 816. The ultrathin sections were obtained using a Leica ultramicrotome (Leica Microsystems, Buffalo Grove, IL, USA). The ultrathin sections stained with uranyl acetate and lead citrate were examined with a Hitatch-7500 transmission electron microscope (Hitachi, Tokyo, Japan). For ZnONPs TEM detection, ZnONPs were suspended in deionized water at a concentration of 3 mg/ml and then sonicated in a sonicator bath for 60 min. The solution was then diluted with deionized water to a concentration of 30 μg/ml. The dilutions of ZnONPs were vigorously vortexed for 5 min before TEM detection to avoid nanoparticle agglomeration.
Cell viability assays. Cell viability was determined by the MTS assay (Promega, Madison, WI, USA) as described previously. 63 In the rescue experiments, DTPA, TPEN, 3-MA, Rapa, wortmannin and NAC were added 1 h before, and for the duration of, the stimulation, or at the indicated time points shown in the figure legend. In the siRNA knockdown experiment, A549 cells were transfected with siRNA as described previously, and the MTS assay was performed at 24 h after treatment with nanomaterials.
FACS analysis. For zinc concentration detection, A549 cells were seeded into 6-well plates. After 24 h of ZnONPs treatment, zinc-specific indicator FluoZin-3, AM (Molecular Probe, Waltham, MA, USA) was added to the cell at a final concentration of 500 nM, followed by 30 min incubation at room temperature in the dark. Thereafter, cells were washed three times using the complete medium, followed by an additional incubation for 30 min at room temperature in the dark to allow complete de-esterification of intracellular AM esters.
For ROS detection, 2',7'-dichlorofluorescin diacetate (Sigma-Aldrich) was diluted in FBS-free medium to 20 μM and then added to the cells, followed by 30 min incubation at 37°C.
For lysosome detection, LysoTracker DND-99 (Molecular Probe) and LysoSensor Green DND-199 (Molecular Probe) were diluted in prewarmed complete medium to 50 nM and 1 μM separately, and then were added to cells, followed by 30 min incubation at 37°C.
For measuring membrane potential changes in the mitochondria, TMRE (Sigma-Aldrich) was diluted in prewarmed FBS-free medium to a final concentration of 10 nM, and then was added to cells, followed by 20 min incubation at 37°C. For measuring the mitochondrial mass, MitoTracker-Green FM (Molecular Probe) was diluted in prewarmed FBS-free medium to a final concentration of 100 nM, and then was added to cells, followed by 20 min incubation at 37°C.
For apoptosis detection, the Annexin V-FITC/Propidium Iodide Apoptosis Detection Kit (Beyotime Biotechnology, Jiangsu, China) was used according to the manufacturer's instructions.
All FACS experiments were performed on BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) and results were analyzed using the BD FACS Software (San Jose, CA, USA).
Real-time quantitative PCR analysis. Total RNA was extracted from A549 cells using Eastep Super Total RNA Extraction Kit (Promega), cDNA was Autophagic dysfunction induced by ZnONPs J Zhang et al synthesized from 1.5 μg of total RNA with GoScript Reverse Transcription Kit (Promega) according to the standard protocol provided by the manufacturer. PCR amplification assays were performed with GoTaq Real-time Quantitative PCR Master Mix (Promega) on CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).The relative gene expression levels were calculated using the CT value and normalized to the expression of the TATA-binding protein (TBP) reference gene. The specific primers were synthesized by Sangon (Shanghai, China) and the sequences were as follows: ZnT1 forward, 5′-TGTTGAAGGAGTTGAGGAA-3′; ZnT1 reverse, 5′-TGAATGGTAGTAGCGTGAA-3′; TBP forward, 5′-ATCAGTGCCGTGGTTCGT-3′; TBP reverse, 5′-TTCGGAGAGTTCTGGGATTG-3′.
Confocal microscopy. Native A549 cells or A549 cells stably expressing mRFP-GFP tfLC3 were grown on coverslips in 24-well plates. At the indicated time points after NP treatment or stimulation, the coverslips were washed two times with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. After washing two times with PBS, the cells were permeabilized and blocked with 3% BSA and 0.1% Triton X-100 in PBS for 40 min at room temperature. The coverslips were then incubated with the indicated primary antibody at 4°C overnight and then incubated with Alexa Fluor 488-or Alexa Fluor 568-labeled secondary antibody (Molecular Probes) together with DAPI (Molecular Probes) at room temperature for 1 h. The subcellular localization of each target protein was observed using confocal laser scanning microscopy (Nikon, Tokyo, Japan), and the images were analyzed using NIS-Elements Viewer 4.20 (Nikon, Tokyo, Japan). The primary antibodies used were as follows: LAMP-1 (ab24170; Abcam), LAMP-2 (H4B4; Santa Cruz), cathepsin D (E-7; Santa Cruz).
Statistics analysis. All data are shown as means ± S.E.M. Measurements at single time points were analyzed by analysis of variance, and in case of significance they were further analyzed by a two-tailed t-test. All statistical tests were conducted using Prism 5.0 (GraphPad Software, San Diego, CA, USA). *Po0.05 indicates statistical significance, **P o0.01 indicates high significance, and NS indicates no significance.