- Email: [email protected]
In-vitro assessment of antimicrobial properties and lymphocytotoxicity assay of benzoisochromanequinones polyketide from Streptomyces sp JRG-04
Accepted Manuscript In-vitro assessment of antimicrobial properties and lymphocytotoxicity assay of benzoisochromanequinones polyketide from Streptomyces sp JRG-04 Ganesan Govindarajan, Raju Kamaraj, Karuppiah Balakrishnan, Velayudhan Satheeja Santhi, Solomon Robinson David Jebakumar PII:
S0882-4010(17)30111-0
DOI:
10.1016/j.micpath.2017.06.034
Reference:
YMPAT 2326
To appear in:
Microbial Pathogenesis
Received Date: 6 February 2017 Revised Date:
11 May 2017
Accepted Date: 22 June 2017
Please cite this article as: Govindarajan G, Kamaraj R, Balakrishnan K, Santhi VS, Jebakumar SRD, In-vitro assessment of antimicrobial properties and lymphocytotoxicity assay of benzoisochromanequinones polyketide from Streptomyces sp JRG-04, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.06.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In-Vitro Assessment of Antimicrobial Properties and Lymphocytotoxicity assay of
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Benzoisochromanequinones Polyketide from Streptomyces sp JRG-04
3 Ganesan Govindarajan1*, Raju Kamaraj2, Karuppiah Balakrishnan2, Velayudhan
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Satheeja Santhi1 and Solomon Robinson David Jebakumar1
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Kamaraj University, Madurai - 625021, India
2. Department of Immunology, School of Biological Sciences, Madurai Kamaraj
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1. Department of Molecular Microbiology, School of Biotechnology, Madurai
University, Madurai-625021, India *Corresponding Author
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Department of Molecular Microbiology
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School of Biotechnology
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Madurai Kamaraj University
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Madurai - 625 021, Tamil Nadu, India.
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Telephone: 91-452-2459480
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Fax: 91-452-2459105
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E mail: [email protected]
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Abstract The chromanequinone (BIQ) compound produced by the mangrove estuary
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derived strain, Streptomyces sp. JRG-04 was effective even at low MIC level
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concentration against Methicillin resistant S. aureus and other clinical pathogens. In
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this study, we have investigated the antimicrobial potential of chromanequinone
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compound by using various microscopy and imaging techniques. The flow cytometry
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(FACS) analysis suggested the BIQ aromatic polyketide compound produced by the
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Streptomyces sp. JRG-04 has toxic effect on MRSA cell membrane by increased up
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take of propidium iodide dye. The bacterial imaging analysis by high content screening
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experiment (HCS) revealed the increased number of dead MRSA cells than the live
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MRSA populations with chromanequinone treatment. Furthermore, atomic force
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microscopic study proved the MRSA cell surface ultra-structure changes when the cells
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exposed to chromanequinone compound at 3 h and 6 h. Further, in-vitro
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lymphocytotoxicity effect of chromanequinone compound at different concentrations
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with the combination of complement was performed on human lymphocytes by cell
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lysis assay. Interestingly, we have found that the higher concentration of BIQ
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chromanequinone (10 mg/mL) compound without complement induced apoptosis of
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human lymphocytes. The present investigation reveals that the toxic potential of
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chromanequinone on human lymphocytes might be associated with the complement
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dependent. This study strongly suggests that the chromanequinone compound produced
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by the Streptomyces strain with bioactive property can be developed as a therapeutic
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leads for various pharmaceutical applications.
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Key Words:
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Marine Streptomyces, High content screening, flow cytometry; lymphocytes; Anti-
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lymphocyte serum
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Introduction Emerging multidrug resistant pathogenic bacteria are responsible for causing
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harmful infectious diseases to the humans with high morbidity and mortality. The life
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threatening and most prevalent antibiotics resistant bacteria are Methicillin resistance
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Staphylococcus aureus, Vancomycin-resistant Enterococci, multi drug resistant
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Pseudomonas aeruginosa, Klebsiella pneumonia, Burkholderia cepacia and β-
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lactamase producing bacteria (ESBL) [1,2]. On the other hand, the currently used
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antilymphocytic agents such as cyclosporine A, tacrolimus, Sirolimus are most
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effective but they possess some life threatening complications. In order to overcome
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this problem, there is a need to identify the new pharmaceutical active compounds with
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lower toxicities from marine sources. The secondary metabolites from marine bacteria,
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particularly the members those belonging to the phylum Actinobacteria are capable
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with a wide variety of chemical structures possessing strong biological activities [3].
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The polyketides metabolites are one of the most prevalent class of natural compounds
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which include macrolides, polyethers and aromatics. They exhibit various biological
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activities such as anti-bacterial, anti-fungal, anti-parasitic, immunosuppressive, anti-
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tumor agents and other useful pharmacological activities [4]. Rapamycin, a novel 31
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membered polyketide compound produced by Streptomyces hygroscopicus shows
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various biological activities including antifungal, immunosuppressive and antitumor
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effects [5-7] and currently it is used as effective immune suppressant with less toxicity
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when compared to the FK 506 and cyclosporine A [8]. In the course of drug screening
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programme, a number of studies have been focused towards the isolation and
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characterization of new Streptomyces species from unexplored habitats. The
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unexplored marine environments are pursued as a source of Streptomyces with
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chemically unique secondary metabolites to prevent infectious diseases [9, 10].
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Recently the two polyketides; Actinofuranones A and B isolated from the marine
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derived Streptomyces strain CNQ766 exhibited weak cytotoxicity against mouse
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Splenocyte T-cells and macrophages [11]. In addition, Salinomycin polyketide
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produced by Streptomyces albus has recently identified as potential agent to inhibit the
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leukemia stem cell and epithelial cancer stem cells [12, 13]. Oligomycin F an
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antifungal compound from an unidentified species of Streptomyces had a mixed
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lymphocyte reaction in response to the mitogen Concavalin-A [14]. All these recent discoveries depicted the marine microbial metabolites has the
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diverse pharmacological effect and during the wide bioactive compound screening
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studies, we recently reported a new class of aromatic polyketide compound;
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benzoisochromanequinones (BIQ) from marine-derived Streptomyces sp. JRG-04
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against MRSA and other pathogens with broad spectrum biological effect [15]. The aim
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of the present study is: (a) to investigate the antimicrobial effect of BIQ against MRSA
10
cells by using various microscopy and other complementary imaging techniques, (b) to
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study the effect of BIQ compound on human lymphocytes and erythrocytes towards the
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development of new immune suppressants to minimize the graft rejection.
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MATERIALS AND METHODS
Susceptibility Testing of MRSA by Flow Cytometry and High content Screening
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method
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MRSA cell suspension in log phase culture were (OD600=0.5) treated with
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chromanequinone compound at different Minimal inhibitory concentration (1X MIC,
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3X MIC and 5X MIC) and Dimethyl sulfoxide (DMSO) is used as a negative control
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for 3 hours at 37 °C. After that, the untreated and treated cells were harvested by
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centrifugation at 5000 rpm for 10 minutes and washed twice with PBS. After washing,
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cells were stained with 1µL of DAPI (4', 6-diamidino-2-phenylindole; Sigma) from
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(DAPI 1.0 mg mL-1) stock and incubated at dark for 30 min. Subsequently, the cells
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were stained with propidium iodide (PI 1.0 mg mL-1 stock) and kept in dark condition
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for 5 min. 100 µL of stained cell aliquots were transferred to the Corning Costar black
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well cell culture plate (sigma Aldrich), and live and dead cells were observed under
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high content screening system (live cell imaging). In the flow cytometry analysis, the
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above said concentrations of chromanequinone compound were treated in the log phase
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culture of MRSA cells for 3 hours at 37 °C. After washing with PBS buffer, the cells
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were stained with propidium iodide (PI 1.0 mg mL-1 stock), and finally kept in dark
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condition for 5 min. PI stained cell aliquots were subjected for MRSA cell membrane
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damage by using flow cytometry on FACS Calibur (BD Biosciences, Oxford, UK) and
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the results were analysed with cell Quest Pro software (BD Biosciences, Oxford, UK).
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Time dependent study of MRSA cell membrane damage Broth cultures of S. aureus in log phase (OD600=0.5) were treated with
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antimicrobial chromanequinone compound at specific concentration (3X MIC) and the
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cells were incubated at 37 °C for 5 different time intervals. Subsequent, washing and
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staining methods as described previously as followed.
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Preparation of MRSA cells for Atomic force Microscopic analysis
The log phase growth culture of MRSA cells were centrifuged at 5000 rpm for
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10 min and washed twice with 1.0 mL of PBS buffer. The cell pellet was re-suspended
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in same PBS buffer. This bacterial cell suspension was treated with antimicrobial
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chromanequinone compound at 3X MIC level for two different time intervals such as 3
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hours and 6 hours with antimicrobial compound at 3X MIC level and incubated at 37
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°C for 3 hours and 6 hours and DMSO was used as a negative control. For AFM
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analysis, the samples were diluted at suitable concentration and mounted on the cover
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slip as mentioned previously [16]. The slides were air dried and mounted directly on
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the specimen metal disc. In order to locate the bacteria, the sample specimen was
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scanned at different area by using (Model, APE Research: A100-SGS). For better
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resolution, contact mode micro cantilever was used for the analysis. To determine the
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effect of antimicrobial chromanequinone compound on the MRSA cell membrane,
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approximately five individual bacterial cells were studied (In triplicates). Two
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dimensional (2D) and Three dimensional (3D) images were captured for treated and
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untreated bacterial cells.
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Collection of Blood Sample
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Peripheral blood samples (5mL) were collected from healthy volunteers,
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Institutional ethical clearance was obtained from Madurai Kamaraj University Ethical
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and Review Board Committee (ERC). Then, it was transferred to sterile centrifuge
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were seen (defibrinated) mixing should be gentle, to avoid foaming. Histoprep (Density
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gradient solution), Anti lymphocytic serum (ALS) and Rabbit complement chemical
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were purchased from BAG Health Care, Germany.
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Preparation of Lymphocyte and RBC suspension
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Briefly, 5mL of the blood sample was defibrinated using glass beads, 5-10 mL
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of saline was added to the defibrinated blood. And then, 2.0 mL of Histoprep solution
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was relocated to a sterile serological tube. 4mL of diluted blood (diluted with an equal
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volume of saline) was gently loaded using a Pasteur pipette along the sides of the
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serological tubes without mixing the blood and Histoprep solution (Density gradient
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solution). The tube was centrifuged at 1600 RPM for 20 mins. After centrifugation,
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four distinct layers were observed. In between the top plasma layer Histoprep layer, a
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white puff layer and undermost RBC layer was observed. The white puffy layer
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(interphase) consists of lymphocytes and was carefully transferred into another
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serological tube with the help of Pasteur pipette [17]. The inter phase solution was
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washed with saline and centrifuged at 2000 rpm for 10 min twice. The supernatant was
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discarded and the pellet was diluted using required volume of saline. The undermost
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RBC layer was collected in another fresh serological tube and diluted in the ratio of
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1:20 by using saline.
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Human Lymphocyte toxicity assay
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The methodology described by Terasaki and McClelland [18] was adopted for
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present lymphotoxicity assay. Terasaki trays (60/72 wells, Greiner, USA) were used
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throughout the study. In the first set of experiments, 2.0 µL of freshly prepared
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lymphocytes suspension was added to the each well of Terasaki plate. Then, 4.0 µL of
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purified chromanequinone compound was added to the respective wells at different
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concentration such as (10 mg mL-1 (1X), 5 mg mL-1 (1/2X), 2.5 mg mL-1 (1/4X) and
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1.25 mg mL-1 (1/8X). Anti-lymphocytic serum (ALS) and saline solution were used as
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a positive and negative control respectively. Then, the plate was incubated at 37 °C for
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30 mins. About 5.0 µL of rabbit complement (BAG Health Care, Germany) was added
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to each well. In the second set of experiments, the plate containing lymphocytes were
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treated only with specified concentration of purified chromanequinone compound from
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ACCEPTED MANUSCRIPT different stock (10 mg mL-1 (1X), 5 mg mL-1 (1/2X), 2.5 mg mL-1 (1/4X) and 1.25 mg
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mL-1 (1/8X). In each experiment, Anti-lymphocyte serum (ALS, anti-lymphocyte
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serum, Biotest AG, Germany) and saline were used as a positive and negative control
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respectively. After that, the cells were incubated at 37 °C for 3 h. Subsequently, the
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cells were stained with 2.0 µL of nucleic acid staining dye mix from stock (Acridine
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orange 1 mg mL-1 and Propidium iodide 1 mg mL-1) for 5 mins. Then the cells were
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visualized under fluorescence microscope with 480 nm and 567 nm, 20 X
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magnifications. The experiment was performed in triplicates.
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RBC toxicity assay
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In this experiment, 2.0 µL of human RBC in saline (1: 20) was added to each
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wells of the Terasaki plate and cells were treated with 4.0 µL of purified
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chromanequinone compound from different stock (10 mg mL-1 (1X), 5 mg mL-1
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(1/2X), 2.5 mg mL-1 (1/4X) and 1.25 mg mL-1 (1/8X). The sterilized water was used as
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a negative control. Treated and untreated RBCs were incubated at 37°C for 3 h.
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Subsequently, the plates were visualized under phase contrast microscope (NIKON
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2000) with 20X magnification. The experiment was performed in triplicates.
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RESULTS
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Concentration dependent study of chromanequinone compound on MRSA by
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FACS and High content Screening methods
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In-vitro toxic effect of chromanequinone on MRSA cell membranes was studied with
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combined PI/DAPI dyes by using high content screening and FACS imaging
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techniques. Propidium iodide (PI) is a red-fluorescent dye, which stains the nucleus and
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chromosomes of the dead cells by intercalating between the bases of the nucleic acid.
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However, the DAPI (4', 6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain
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that can easily permeable into the live cell membrane than the dead cells. MRSA cells
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were treated with three different concentrations as follows (1X MIC 1.25µg, 3X MIC
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3.75µg and 5X MIC 6.25 µg) and DMSO used as a negative control.
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ACCEPTED MANUSCRIPT The MRSA cells were affected by higher concentration of chromanequinone
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compound exposure and it caused cell membrane damage and subsequent cell death
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when compared to the control (Fig 1a, b) and definite MIC (Fig 2a, b) levels examined
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by HCS (Fig 3b and 4b). The number of PI positive MRSA cells treated with
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chromanequinone metabolite was determined by flow cytometry. chromanequinone
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compound treatment above MIC concentrations (3X MIC and 5X MIC) induced cell
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membrane damage followed by cell death of 14.8% and 19.4% respectively (Fig 3a and
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4a). This result shows that the lethal activity of chromanequinone compound is attained
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at higher concentration of MIC
levels
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when compared to
control. The
chromanequinone compound exposed MRSA cell also causes the growth inhibition and
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cell membrane breakdown as shown by PI positive cells.
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Time dependent study of MRSA by High content screening method
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The effect of chromanequinone compound on MRSA cell membrane have been
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evaluated by time dependent killing assay using HCS with the help of nucleic acid
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staining dyes both DAPI and PI. Chromanequinone compound at the fixed
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concentration (3 X MIC) was tested at 5 different time points (1h, 3h, 6h, 12h and 24 h)
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shown in Fig 5. In the control experiment, there is an increased number of live cells
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which fluoresce blue in colour and after 1h exposure of chromanequinone compound
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against MRSA, it showed less number of dead cells (red/ yellow florescence) and more
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number of live cells (blue florescence) (Fig 5a and 5b). After 3 h and 6 h incubation,
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the cells showed a drastic change in morphology with increased number of dead cells
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(more permeable of propidium iodide) as in (Fig 5c and 5d). The more number of dead
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cells were seen after 12 h exposure of compound against MRSA followed by complete
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cell death after 24 h exposure (Fig 5e and 5f).
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Assessment of bacterial surface cell morphology changes
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chromanequinone at the concentration of 3XMIC in the log phase of growth
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(O.D600=0.6). The AFM images of untreated MRSA cells were showing round with a
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smooth surface morphology and much undamaged cells (Fig 6a and 6b). A drastic cell
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morphology changes were observed in chromanequinone treated MRSA cells for 3
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hours, and they were shown in Fig 7a and 7b. After 3 h treatment, the cells remain
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The AFM images were obtained for untreated and treated MRSA cells with
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bacterial cell surface with increasing roughened texture on the MRSA cell surface than
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untreated cells. After 6 h exposure, the MRSA cells with chromanequinone compound
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showed uneven cell surfaces when compared to the untreated MRSA cells as shown in
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Fig 8a and 8b. Chromanequinone treated MRSA cells over 3h and 6 h can induced
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considerable topographical changes such as cell shrinkage, cell size and reduced cell
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surface. AFM image analysis revealed that the surface ultra structure of MRSA cells
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treated with chromanequinone were entirely different from the untreated MRSA cells.
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In general, antimicrobial compounds can induce cell membrane damage, cell lysis,
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leakage of intracellular fluids and leads to cell death. Similar to the results of BIQ, a
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naphthoquinone pigment, Shikonin also induced MRSA cell membrane disruption, cell
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lysis, leakage of intracellular cytoplasmic contents and subsequent cell death (19).
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Lymphotoxicity Assay in-vitro
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examined by using nucleic acid binding dye (AO/PI) [20, 21]. Morphological changes
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of the apoptosis can be visualized in either fixed tissue or live cells grown in a culture
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by examining nuclear morphology using vital dyes [22]. Fluorescent microscopy was
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mainly used to study the viability of lymphocytes in response to chromanequinone
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compound treatment with different concentration along with complement for 3 h. Anti-
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lymphatic serum was used as a positive control which kills lymphocytes that are
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considered as 100% cell death at specified concentration Fig 9B. Treatment of
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lymphocytes at 1X concentration in addition with complement shows the 100% dead
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cells (PI stained cells - red in colour) Fig 9C when compared to the negative control an
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untreated cells Fig 9A. More than 75% and 50% of the dead lymphocytes cells were
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observed at 1/2X and 1/4X concentration of chromanequinone Fig 9D and 9E. The
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increased number of live cells (AO stained green cells) were observed at very low
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concentrations of chromanequinone compound exposure Fig 9F.
In presence of complement
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In absence of complement
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Toxic effect of the chromanequinone compound on human lymphocytes was
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examined by previously described method. Lymphocytes were treated with different
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concentration of chromanequinone compound (1/8X, 1/4X, 1/2X and 1X) without
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complement for a duration of 3 h. Lymphocytes cells treated with different
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concentrations (1/8X, 1/4X, 1/2X) of chromanequinone compound results revealed that
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there is an increased number of live cells (green colour), similar to that of negative
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control as shown in Fig 10A, 10D, 10E & 10F. In the case of lymphocytes cells
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treatment with higher concentration (1X) of chromanequinone compound showing,
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there is an increased number of dead cells (red-colored nuclei), similar to those
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obtained with ALS positive control (Fig. 10B and 10C).
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Red blood cell toxicity study
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studied. Red blood cells were treated at different concentration of chromanequinone
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such as 1X, 1/2X and 1/4X for 3 hrs and sterile saline was used as a negative control.
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After incubation, no RBC disruption was observed. The results showed that the
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compound does not reveal any toxic effect to the RBC (Fig 11).
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DISCUSSION
Streptomyces are known to have the ability to produce pharmaceutically
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important secondary metabolites especially antibiotics. In recent days, novel
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compounds with unique structure have been discovered from mangrove actinomycetes
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[23]. BIQ antibiotics with broad spectrum antimicrobial activity was purified and
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characterized from Mangrove derived Streptomyces sp JRG-04 [15]. In the present
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study, flow cytometry and high content screening experiments were employed to detect
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the MRSA cell viability in response to chromanequinone compound with the
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technique which has been applied to study the eukarytotic cells viability, metabolic
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state and identification of antigenic marker of bacteria [24-26]. Currently, flow
4
cytometric methods have been also used to detect the antimicrobial susceptibility of the
5
bacteria based on metabolic activity or membrane integrity using vital dyes [27-30]. In
6
this study, we have showed that the effect of different concentration of
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chromanequinone compound on MRSA cell morphological changes based on PI
8
fluorescent intensity. In the untreated control, the live MRSA cells have intact
9
membranes and are impermeable to propidium iodide (PI) which showed low
10
fluorescent intensity when compared to the treatment at 1X MIC, 3X MIC and 5X MIC
11
levels. The high PI fluorescent intensity was observed only in the case of dead cells and
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compromised membrane cells. The results indicated that the increased numbers of dead
13
cells were observed when increasing the concentration of chromanequinone compound
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and corresponding HCS analysis results also discriminate the live and dead population,
15
with great agreement to flow cytometry results. This study suggested that the viability
16
of MRSA cells is mainly to depend on the dose of chromanequinone compound. In
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addition to that, we have shown the time taken for killing of MRSA cells by
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chromanequinone compound in response to cell membrane damages. This study also
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clearly indicates that there is an increased propidium iodide stained cells (increased
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number of dead cells) when there is increase in the time period.
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Currently, AFM technique is used to study the cell morphology and ultra
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structure of bacteria [31, 32]. In the present study, the AFM imaging technique is
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conducted to determine the action of chromanequinone compound on MRSA cells. The
24
results shown that, the treatment at 3X MIC concentration of chromanequinone
25
compound begin to exert a toxic effect after 3 hours, which induced the substantial
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topographical changes characterized by reduction in size and smoothness of the cells.
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MRSA cells incubated with chromanequinone for 6 hours, showing the complete
28
disorganization of the entire cell structure as shown in Fig 8. Similarly, bacterial
29
morphological changes induced by antibacterial agents were reported previously [33].
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Braga and Ricci [34] also reported that rokitamycin, a macrolides antibiotic start to
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affect the Streptococcus pyogenes cell structure by the formation of large abnormal
32
cells with inadequacy of the chains structure. Hence, the MRSA cell structure
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ACCEPTED MANUSCRIPT disorganization results revealed that, the chromanequinone had inventive antimicrobial
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action against methicillin resistant strain of S. aureus. However, the chromanequinone
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mechanism of action is still unclear, although it changes the ultra structure of the
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bacterial cells, which results in loss of intracellular fluid which leads to cell death.
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Currently, human lymphocytes were most widely used for toxicity analysis of new
6
drugs, particularly the efficiency of the method developed by Terasaki and McClelland
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[18]. Martini et al., [35] have reported that the anti-bacterial flavonoids, 5-hydroxy-7,4-
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dimethoxy flavones from Combretum erythrophyllum was found to be a toxic substance
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to human lymphocytes. In addition, a volatile fatty acid, namely butyric acid produced
10
by some pathogenic microorganisms may suppress more than 90% of the lymphocyte
11
when used at concentration 2.5 mM [36]. Similarly, phenazine derivatives such as
12
pyocyanine pigment produced by Pseudomonas aeruginosa also inhibit the lymphocyte
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proliferation [37, 38]. On the other hand, Hung et al reported that the anthraquinone
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emodin act as a new template for development of effective immunosuppressant with
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vasorelaxant property against transplantation rejection and other autoimmune diseases
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(39).
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Recent reports revealed that some pathogenic E. coli strains harbouring PKS
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Island is responsible for production of a polyketide termed as colibactin which induce
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toxic effects to the lymphocytes. The E. coli producing colibactin that breaks double
20
strand DNA and G2 cell cycle arrest in T lymphocytes was examined by cell membrane
21
integrity [40]. In this present study, we have evaluated the toxic effect of the
22
chromanequinone compound produced by the Streptomyces sp. JRG-04 on human
23
lymphocytes. The data presented here indicated that the cell morphology based
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apoptosis analysis by fluorescence (AO/PI) staining method showed visible
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morphological changes in the nucleus, internal organelle and plasma membrane
26
integrity. The maximum death rate of cells was observed at higher concentration of
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chromanequinone compound exposure followed by lower concentration along with the
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complement. The lymphotoxicity of chromanequinone compound was found to be
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concentration and complement dependent, which is also found to be an inducer of cell
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apoptosis.
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ACCEPTED MANUSCRIPT Lymphocytes treated with higher concentration of chromanequinone compound
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without complement experiments state that there is an increased number of dead cells.
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It reveals, some higher specified concentration of chromanequinone can exert partial
4
cytotoxic effects on human blood lymphocytes and the enhancement of dead cells were
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positively correlated with dosage and is complement dependent. In addition, a series of
6
anthraquinones; the mitoxantrone and its derivative 1,4-bis [(2-aminoethyl)amino]-5, 8-
7
dihydroxy-9,10-anthracenedione
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immunosuppressive activity against T-lymphocytes in a mixed lymphocyte culture
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system and they can be used for organ transplantation (41). Apart from this, a Emodin
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anthraquinone derivative (1,3,8-trihydroxy-6-methyl-anthraquinone) also suppresses
11
the peripheral blood mononuclear cell (PBMC) proliferative responses in dose
12
dependent manner and subsequently decline the production of interleukin in the mixed
13
lymphocyte reaction. The possible mechanism of immune suppressive effect of Emodin
14
were investigated based on structure activity relationship (SAR), it was reported that
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the free beta-hydroxyl group of anthraquinone nucleus play a significant role in the
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immunosuppressive activity (42) by suppression of lymphocyte proliferation and
17
cytokines (43). Similar to emodin, the anthraquinone rhein also exhibited the
18
immunosuppressive activity with different biological activity (44). Celik and Eke [45],
19
reported that the tetracycline polyketide antibiotic can induce genetic damage in human
20
peripheral
21
immunosuppressive polyketide compound has been identified from mangrove
22
endophytic fungus Penicillium sp. ZJ-SY2 [46]. Finally, we have investigated the toxic
23
effect of chromanequinone compound on RBC. The toxicity result indicated that, there
24
was no haemolysis at both higher and lower concentrations of chromanequinone.
25
Saurav and Kannabiran [47] have reported that the assessment of membrane stability,
26
with exposure of new drugs is imperative and erythrocytes can be used as a good model
27
for these membrane stability studies.
(AEAD)
were
induced
lymphocyte
cells
under
in
vitro
conditions.
Recently,
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dihydrochloride
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CONCLUSION
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The assessment of flow cytometry analysis depicted the MRSA cell membrane
31
disruption by taking up more of propidium iodide stain at all three different
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2
results also easily discriminate the live/ dead MRSA cells with the help of nucleic acid
3
stating dye after exposure of chromanequinone. Furthermore, the atomic force
4
microscopic study also showed the altered MRSA cell surface ultra-structure changes
5
when the cells were exposed to chromanequinone compound at two different time
6
points. This study also revealed the human lymphocytotoxicity potential of the BIQ
7
chromanequinone with the combination of complement. Interestingly, the higher
8
concentration of BIQ compound (10 mg mL-1) without complement can also induce
9
toxicity to the human lymphocytes. The toxic potential of BIQ compound on human
10
lymphocytes might be associated with the complement dependent. However, the
11
detailed mechanisms of lymphocytotoxicity need to be elucidated. The red blood cell
12
toxicity assay also indicated that the BIQ compound produced by the Streptomyces sp.
13
JRG-04 is non toxic to the human RBC. Further detailed studies on pharmacological
14
mechanisms of BIQ compound will provide the comprehensive understandings of its
15
efficacy as better drug like lead molecule.
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Acknowledgements
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I would like to acknowledge my mentor and advisor late Professor Dr. SRD
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Jebakumar and I have profited much from his guidance and instructions during my
19
doctoral studies at Madurai Kamaraj University. We also acknowledge the UGC,
20
Government of India for the ‘Research Fellowship in Science for Meritorious Students
21
Scheme and MKU for providing the University Stipendiary Research Fellowship
22
(USRF).
23
Conflict of Interest
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There is no conflict of interest to declare
25
REFERENCES
26 27
AC C
EP
17
1. P.M. Hawkey, A.M. Jones, The changing epidemiology of resistance, J Antimicrob. Chemother. 64 (1) (2009) i3-i10.
14
ACCEPTED MANUSCRIPT 1
2. D. Sharma, T. Kaur, B.S. Chadha, R.K. Manhas, Antimicrobial activity of
2
actinomycetes against multidrug resistant Staphylococcus aureus, E. coli and
3
various other pathogens, Trop. J. Pharm. Res. 10 (6) (2011) 801-808. 3. J. Spizek, J. Novotna, T. Rezanka, A.L. Demain, Do we need new antibiotic?
5
The search for new targets and new compounds, J. Ind. Microbiol. Biot. 37 (12)
6
(2010) 1241-1248.
7 8
RI PT
4
4. R.L. Monaghan, J.S. Tkacz, Bioactive microbial products: focus on mechanism of action, Annu. Rev. Microbiol. 44 (1990) 271-301.
5. R. Calne, S. Lim, A. Samaan, D.S.J. Collier, S. Pollard, D. White, S. Thiru,
10
Rapamycin for immunosuppression in organ allografting, Lancet. (1989) 227.
11
6. J. Douros, M. Suffness, New antitumor substances of natural origin, Cancer. Treat. Rev. 8 (1) (1981) 63–87.
M AN U
12
SC
9
13
7. S. Sehgal, H. Baker, C. Vézina, Rapamycin (AY-22,989), a new antifungal
14
antibiotic. II. Fermentation, isolation and characterization, J. Antibiot. 28 (10)
15
(1975) 727–732.
8. E. Patsenker, V. Schneider, M. Ledermann, H.Saegesser, C. Dorn et al., Potent
17
antifibrotic activity of mTOR inhibitors sirolimus and everolimus but not of
18
cyclosporine A and tacrolimus in experimental liver fibrosis, J. Hepatol. 55 (2)
19
(2011) 388–398
21
9. R. Solanki, M. Khanna, R. Lal, Bioactive compounds from marine actinomycetes, Indian J. Microbiol, 48 (4) (2008) 410-431.
EP
20
TE D
16
10. G.R. Silva-Lacerda, R.C. Santana, M.C. Vicalvi-Costa, E.G. Solidonio, K.X.
23
Sena, G.M. Lima, J.M. Araujo, Antimicrobial potential of actinobacteria isolated
24
from the rhizosphere of the Caatinga biome plant Caesalpinia pyramidalis Tul,
25
AC C
22
Genet. Mol. Res. (2006) DOI: 10.4238/gmr.15017488.
26
11. J.Y. Cho, H.C. Kwon, P.G. Williams, C.A. Kauffman, P.R. Jensen, W. Fenical,
27
ActinofuranonesA and B, polyketides from a marine-derived bacterium related
28
to the genus Streptomyces (Actinomycetales), J. Nat. Prod. 69 (3) (2006) 425-
29
428.
30
12. D. Fuchs, V. Daniel, M. Sadeghi, G. Opelz, C. Naujokat, Salinomycin
31
overcomes ABC transporter- mediated multidrug and apoptosis resistance in
15
ACCEPTED MANUSCRIPT 1
human leukemia stem cell-like KG-1a cells, Biochem. Biophys. Res. Commun.
2
394 (4) (2010) 1098-1104. 13. S.Z. Kuo, K.J. Blair, E. Rahimy, A. Kiang, E. Abhold, J.B. Fan, J.
4
WangiRodriguez, X. Altuna, W.M. Ongkeko, Salinomycin induces cell death
5
and differentiation in head and neck squamous cell carcinoma stem cells despite
6
activation of epithelial-mesenchymal transition and Akt, BMC. Cancer.12 (2012)
7
556
RI PT
3
8
14. H. Laatsch, M. Kellner, G. Wolf, Y.S. Lee, F. Hansske, S. Konetschnyrapp, U.
9
Pessara, W. Scheuer, H. Stockinger, Oligomycin F, a new immunosuppressive homologue of oligomycin A, J. Antibiot. 46 (9) (1993) 1334-1341.
SC
10
15. G. Govindarajan, V.S. Santhi, S.R.D. Jebakumar, Antimicrobial potential of
12
phylogenetically unique actinomycete, Streptomyces sp. JRG-04 from marine
13
origin, Biologicals. 42 (6) (2014) 305-311.
M AN U
11
14
16. R.L. Soon, R.L. Nation, P.G. Hartley, I. Larson, J. Li, Atomic force microscopy
15
investigation of the morphology and topography of colistin- heteroresistant
16
Acinetobacter baumannii as a function of growth phase and in response to
17
colistin treatment, Antimicrob. Agents. Chemother. 53 (12) (2009) 4979-4986. 17. A. Boyum, Separation of White Blood cells, Nature. 204 (1964) 793-794.
19
18. P.I. Terasaki, J.D. McClelland, Microdroplet assay of human serum cytotoxins,
20
TE D
18
Nature. 204 (1964) 998-1000.
19. Y.S. Lee, D.Y. Lee, Y.B. Kim, S.W. Lee, S.W. Cha, H.W. Park, G.S. Kim,
22
D.Y. Kwon, M.H.Lee, S.H. Han, The Mechanism Underlying the Antibacterial
23
activity of Shikonin against Methicillin Resistant Staphylococcus aureus, Evid.
24
Based. Complement. Alternat. Med. (2015) DOI 10.1155/2015/520578.
AC C
EP
21
25
20. Z. Darzynkiewicz, G. Juan, X. Li, W. Gorczyca, T. Murakami, F. Traganos,
26
Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death
27 28 29 30 31
(necrosis), Cytometry. 27 (1) (1997) 1-20.
21. M.G. Ormerod, Using flow cytometry to follow the apoptotic cascade, Redox. Report. 6 (5) (2001) 275-287. 22. F.L. Kiechle, X. Zhang, Apoptosis: a biochemical aspects and clinical implications, Clin. Chim. Acta. 326 (2002) 27-45.
16
ACCEPTED MANUSCRIPT 23. M. Ushakiranmayi, M. Vijayalakshmi, P. Sudhakar, N. Krishna, M. Rajesh
2
Kumar, C. Bhujangarao, Y. Venkateswarlu, Bioactive metabolites produced by
3
Streptomyces Cheonanensis VUK-A from Coringa mangrove sediments:
4
isolation, structure elucidation and bioactivity, 3 Biotech. (2016) DOI
5
10.1007/s13205-016-0398-6.
RI PT
1
6
24. H.M. Davey, D.B. Kell, Flow cytometry and cell sorting of heterogeneous
7
microbial populations: the importance of single cell analyses, Microbiol. Rev. 60
8
(4) (1996) 641-696.
10
25. H.N. Shapiro, Microbial analysis at the single-cell level: tasks and techniques, J. Microbiol. Methods. 42 (1) (2000) 3-16.
SC
9
26. G. Nebe-von-Caron, P.J. Stephens, R.A. Badley, Bacterial detection and
12
differentiation by cytometry and fluorescent probes, Proc. R. Microbiol. Soc. 34
13
(1999) 321-327.
M AN U
11
14
27. G. Nebe-von-Caron, P.J. Stephens, C.J. Hewitt, J.R. Powell, R.A. Badley,
15
Analysis of bacterial function by multi-colour fluorescence flow cytometry and
16
single cell sorting, J. Microbiol. Methods. 42 (1) (2000) 97-114. 28. D.J. Novo, N.G. Perlmutter, R.H. Hunt, H.M. Shapiro, Multiparameter flow
18
cytometric analysis of antibiotic effects on membrane potential, membrane
19
permeability, and bacterial counts of Staphylococcus aureus and Micrococcus
20
luteus, Antimicrob. Agents. Chemother. 44 (4) (2000) 827-834.
TE D
17
29. R.I. Jepras, F.E. Paul, S.C. Pearson, M.J. Wilkinson, Rapid assessment of
22
antibiotic effects on Escherichia coli by bis-(1, 3-dibutylbarbituric acid)
23
trimethine oxonol and flow cytometry, Antimicrob. Agents. Chemother. 41 (9)
24
(1997) 2001-2005.
AC C
EP
21
25
30. C. Gauthier, Y. St-Pierre, R. Villemur, Rapid antimicrobial susceptibility testing
26
of urinary tract isolates and samples by flow cytometry, J. Med. Microbiol. 51
27
(3) (2002) 192-200.
28
31. T.A. Camesano, M.J. Natan, B.E. Logan, Observation of changes in bacterial
29
cell morphology using tapping mode atomic force microscopy, Langmuir. 16
30
(10) (2000) 4563-4572.
31
32. N.P. Mortensen, J.D. Fowlkes, C.J. Sullivan, D.P. Allison, N.B. Larsen, S.
32
Molin, M. J. Doktycz, Effects of colistin on surface ultrastructure and
17
ACCEPTED MANUSCRIPT 1
nanomechanics of Pseudomonas aeroginosa cells, Langmuir. 25 (6) (2009)
2
3728-3733. 33. A.K. Suresh, W.W. Pelletier, J. Moon, B. Gu, N.P. Mortensen, D.P. Allison,
4
D.C. Yoy, T.J. Phelps, M.J. Doktycz, Silver nanocrystallites: Biofabrication
5
using Shewanella oneidensis, and an evaluation of their comparative toxicity on
6
Gram negative and Gram-positive bacteria, Environ. Sci. Technol. 44 (13)
7
(2010) 5210-5215.
RI PT
3
34. P.C. Braga, D. Ricci, Differences in the susceptibility of Streptococcus pyogenes
9
to rokitamycin and erythromycin A revealed by morphostructural atomic force
11
microscopy, J. Antimicrob. Chemother. 50 (4) (2002) 457-460.
35. D.N. Martini, P.R.D. Katerere, N.J. Eloff, Biological activity of five antibacterial
M AN U
10
SC
8
12
flavonoids
from
Combretum
erythrophyllum
13
Ethnopharmacol. 93 (2004) 207-212.
(Combretaceae),
J.
14
36. T. Kurita-Ochiai, K. Fukushima, K. Ochiai, Volatile fatty acids, metabolic by-
15
products of periodontopathic bacteria, inhibit lymphocyte proliferation and
16
cytokine production, J. Dent. Res. 74 (7) (1995)1367-1373. 37. R. Wilson, D.A. Sykes, D. Watson, A. Rutman, G.W. Taylor, P.J Cole,
18
Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and
19
assessment of their contribution to sputum sol toxicity for respiratory epithelium,
20
Infect. Immun. 56 (9) (1988) 2515-2517.
TE D
17
38. K. Kanthakumar, G. Taylor, K.W.T. Tsang, D.R. Cundell, A. Rutman, S. Smith,
22
P.K. Jeffery, P.J. Cole, R .Wilson, Mechanisms of action of Pseudomonas
23
aeruginosa pyocyanin on human ciliary beat in vitro, Infect. Imm. 61 (7) (1993)
AC C
24
EP
21
2848-2853.
25
39. H.C. Huang, C.R. Lee, P.D. Chao, C.C.Chen, S.H Chu, Vasorelaxant effect of
26
emodin, an anthraquinone from a Chinese herb, Eur. J. Pharmacol. 205 (1991)
27
289–294.
28
40. I.P Marcq, D. Martin, G. Payros, G. Cuevas-Ramos, M. Boury, C. Watrin, J.P.
29
Nougayrède, M. Olier, E. Oswald, The Genotoxin Colibactin Exacerbates
30
Lymphopenia and Decreases Survival Rate in Mice Infected With Septicemic
31
Escherichia coli, J. Infect. Dis. 210 (2) (2014) 285-294.
18
ACCEPTED MANUSCRIPT 1
41. B.S. Wang, K.C. Murdock, A.L. Lumanglas, M. Damiani, J. Silva, V.M.
2
Ruszala-Mallon,
3
anthraquinones with their effects on the suppression of immune responses, Int. J.
4
Immunopharmacol. 9 (6) (1987) 733-739.
6
Durr,
Relationship
of
chemical
structures
of
42. Y.X. Liu, N.Y. Shen, C. Liu, Y. Lv, immunosuppressive effects of emodin: An
RI PT
5
F.E,
in vivo and in vitro study, Transplant. Proc. 41 (2009) 1837-1839.
43. H.C. Huang, J.H. Chang, S.F. Tung, R.T Wu, M.L. Foegh, S.H. Chu,
8
Immunosuppressive effect of emodin, a free radical generator, Eur. J. Pharmacol.
9
11 (3) (1992) 559-364.
11
44. F. Hu, F. Xing, G. Zhu, G. Xu, C. Li, J. Qu, I. Lee, L. Pan, Rhein antagonizes P2X7 receptor in rat peritoneal macrophages, Sci. Rep. 5 (2015) 14012.
M AN U
10
SC
7
12
45. A. Celik, D. Eke, The Assessment of Cytotoxicity and Genotoxicity of
13
Tetracycline Antibiotic in Human Blood Lymphocytes Using CBMN and SCE
14
Analysis, in Vitro, Int. J. Hum. Genet. 11 (2011) 23-29.
46. H. Liu, S. Chen, W. Liu, Y. Liu, X. Huang, Z. She, Polyketides with
16
Immunosuppressive activities from Mangrove Endophytic Fungus Penicillium
17
sp. ZJ-SY, Mar. Drugs. 14 (12) (2016) 217.
18
TE D
15
47. K. Saurav, K. Kannabiran, Cytotoxicity and antioxidant activity of 5-(2,4dimethylbenzyl)
pyrrolidin-2-one
extracted
from
20
VITSVK5 spp. Saudi, J. Biol. Sci.19 (1) (2012) 81-86.
marine
Streptomyces
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Figure legends
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Figure 1 a) FACS analysis of untreated MRSA stained with PI and b) HCS image of
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untreated MRSA stained with DAPI/PI.
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Figure 2 a) MRSA treated with at MIC concentration stained with PI and b) MRSA
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treated with at MIC stained with DAPI and PI.
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Figure 3 a) MRSA treated with at 3X MIC concentration stained with PI and b) MRSA
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treated with at 3X MIC stained with DAPI and PI.
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ACCEPTED MANUSCRIPT Figure 4 a) MRSA treated with at 5X MIC concentration stained with PI and b) MRSA
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treated with at 5X MIC stained with DAPI and PI.
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Figure 5 Time dependence of chromanequinone compound effects on MRSA imaged
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by HCS. (a) Control, (b) 1h treated with chromanequinone, followed by (c) 3h, (d) 6h,
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(e) 12h and (f) 24h.
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Figure 6 (a) AFM Images of Untreated MRSA (2D View) and (b) untreated MRSA
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(3D view).
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Figure 7 (a) AFM Images of chromanequinone treated MRSA for 3 hours (2D View)
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and (b) MRSA (3D view).
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Figure 8 (a) AFM Images of chromanequinone treated MRSA for 6 hours (2D View)
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and (b) MRSA (3D view).
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Figure 9 Lymphotoxicity assay with complement imaged by fluorescent microscopy.
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(A) Negative Control, (B) positive control (ALS), (C), (D), (E) and (F)
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chromanequinone compound treatment at 1X, 1/2X, 1/4X and 1/8X respectively.
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Figure 10 Lymphotoxicity assay without complement imaged by fluorescent
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microscopy. (A) Negative Control, (B) positive control (ALS), (C), (D), (E) and (F)
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chromanequinone compound treatment at 1X, 1/2X , 1/4X and 1/8X respectively.
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Figure 11 Phase contrast microscopic images showing the effect of chromanequinone
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on RBC. (A) Negative control, (B), (C) and (D) were chromanequinone treated RBC at
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1X, 1/2X and 1/4X concentrations respectively.
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Highlights This research work is highly credited by studying the role of chromanequinone (BIQ) compound on human lymphocytotoxicity with the combination of complement to
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reveal the toxicity analysis for new drugs. FACS and HCS analysis clearly depict the live
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and dead populations of MRSA with chromanequinone treatment
S0882-4010(17)30111-0
DOI:
10.1016/j.micpath.2017.06.034
Reference:
YMPAT 2326
To appear in:
Microbial Pathogenesis
Received Date: 6 February 2017 Revised Date:
11 May 2017
Accepted Date: 22 June 2017
Please cite this article as: Govindarajan G, Kamaraj R, Balakrishnan K, Santhi VS, Jebakumar SRD, In-vitro assessment of antimicrobial properties and lymphocytotoxicity assay of benzoisochromanequinones polyketide from Streptomyces sp JRG-04, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.06.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In-Vitro Assessment of Antimicrobial Properties and Lymphocytotoxicity assay of
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Benzoisochromanequinones Polyketide from Streptomyces sp JRG-04
3 Ganesan Govindarajan1*, Raju Kamaraj2, Karuppiah Balakrishnan2, Velayudhan
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Satheeja Santhi1 and Solomon Robinson David Jebakumar1
8 9 10
Kamaraj University, Madurai - 625021, India
2. Department of Immunology, School of Biological Sciences, Madurai Kamaraj
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1. Department of Molecular Microbiology, School of Biotechnology, Madurai
University, Madurai-625021, India *Corresponding Author
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Department of Molecular Microbiology
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School of Biotechnology
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Madurai Kamaraj University
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Madurai - 625 021, Tamil Nadu, India.
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Telephone: 91-452-2459480
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Fax: 91-452-2459105
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E mail: [email protected]
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Abstract The chromanequinone (BIQ) compound produced by the mangrove estuary
3
derived strain, Streptomyces sp. JRG-04 was effective even at low MIC level
4
concentration against Methicillin resistant S. aureus and other clinical pathogens. In
5
this study, we have investigated the antimicrobial potential of chromanequinone
6
compound by using various microscopy and imaging techniques. The flow cytometry
7
(FACS) analysis suggested the BIQ aromatic polyketide compound produced by the
8
Streptomyces sp. JRG-04 has toxic effect on MRSA cell membrane by increased up
9
take of propidium iodide dye. The bacterial imaging analysis by high content screening
10
experiment (HCS) revealed the increased number of dead MRSA cells than the live
11
MRSA populations with chromanequinone treatment. Furthermore, atomic force
12
microscopic study proved the MRSA cell surface ultra-structure changes when the cells
13
exposed to chromanequinone compound at 3 h and 6 h. Further, in-vitro
14
lymphocytotoxicity effect of chromanequinone compound at different concentrations
15
with the combination of complement was performed on human lymphocytes by cell
16
lysis assay. Interestingly, we have found that the higher concentration of BIQ
17
chromanequinone (10 mg/mL) compound without complement induced apoptosis of
18
human lymphocytes. The present investigation reveals that the toxic potential of
19
chromanequinone on human lymphocytes might be associated with the complement
20
dependent. This study strongly suggests that the chromanequinone compound produced
21
by the Streptomyces strain with bioactive property can be developed as a therapeutic
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leads for various pharmaceutical applications.
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Key Words:
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Marine Streptomyces, High content screening, flow cytometry; lymphocytes; Anti-
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lymphocyte serum
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Introduction Emerging multidrug resistant pathogenic bacteria are responsible for causing
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harmful infectious diseases to the humans with high morbidity and mortality. The life
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threatening and most prevalent antibiotics resistant bacteria are Methicillin resistance
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Staphylococcus aureus, Vancomycin-resistant Enterococci, multi drug resistant
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Pseudomonas aeruginosa, Klebsiella pneumonia, Burkholderia cepacia and β-
12
lactamase producing bacteria (ESBL) [1,2]. On the other hand, the currently used
13
antilymphocytic agents such as cyclosporine A, tacrolimus, Sirolimus are most
14
effective but they possess some life threatening complications. In order to overcome
15
this problem, there is a need to identify the new pharmaceutical active compounds with
16
lower toxicities from marine sources. The secondary metabolites from marine bacteria,
17
particularly the members those belonging to the phylum Actinobacteria are capable
18
with a wide variety of chemical structures possessing strong biological activities [3].
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The polyketides metabolites are one of the most prevalent class of natural compounds
20
which include macrolides, polyethers and aromatics. They exhibit various biological
21
activities such as anti-bacterial, anti-fungal, anti-parasitic, immunosuppressive, anti-
22
tumor agents and other useful pharmacological activities [4]. Rapamycin, a novel 31
23
membered polyketide compound produced by Streptomyces hygroscopicus shows
24
various biological activities including antifungal, immunosuppressive and antitumor
25
effects [5-7] and currently it is used as effective immune suppressant with less toxicity
26
when compared to the FK 506 and cyclosporine A [8]. In the course of drug screening
27
programme, a number of studies have been focused towards the isolation and
28
characterization of new Streptomyces species from unexplored habitats. The
29
unexplored marine environments are pursued as a source of Streptomyces with
30
chemically unique secondary metabolites to prevent infectious diseases [9, 10].
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Recently the two polyketides; Actinofuranones A and B isolated from the marine
32
derived Streptomyces strain CNQ766 exhibited weak cytotoxicity against mouse
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Splenocyte T-cells and macrophages [11]. In addition, Salinomycin polyketide
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produced by Streptomyces albus has recently identified as potential agent to inhibit the
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leukemia stem cell and epithelial cancer stem cells [12, 13]. Oligomycin F an
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antifungal compound from an unidentified species of Streptomyces had a mixed
3
lymphocyte reaction in response to the mitogen Concavalin-A [14]. All these recent discoveries depicted the marine microbial metabolites has the
5
diverse pharmacological effect and during the wide bioactive compound screening
6
studies, we recently reported a new class of aromatic polyketide compound;
7
benzoisochromanequinones (BIQ) from marine-derived Streptomyces sp. JRG-04
8
against MRSA and other pathogens with broad spectrum biological effect [15]. The aim
9
of the present study is: (a) to investigate the antimicrobial effect of BIQ against MRSA
10
cells by using various microscopy and other complementary imaging techniques, (b) to
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study the effect of BIQ compound on human lymphocytes and erythrocytes towards the
12
development of new immune suppressants to minimize the graft rejection.
13 14 15 16
MATERIALS AND METHODS
Susceptibility Testing of MRSA by Flow Cytometry and High content Screening
17
method
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MRSA cell suspension in log phase culture were (OD600=0.5) treated with
19
chromanequinone compound at different Minimal inhibitory concentration (1X MIC,
20
3X MIC and 5X MIC) and Dimethyl sulfoxide (DMSO) is used as a negative control
21
for 3 hours at 37 °C. After that, the untreated and treated cells were harvested by
22
centrifugation at 5000 rpm for 10 minutes and washed twice with PBS. After washing,
23
cells were stained with 1µL of DAPI (4', 6-diamidino-2-phenylindole; Sigma) from
24
(DAPI 1.0 mg mL-1) stock and incubated at dark for 30 min. Subsequently, the cells
25
were stained with propidium iodide (PI 1.0 mg mL-1 stock) and kept in dark condition
26
for 5 min. 100 µL of stained cell aliquots were transferred to the Corning Costar black
27
well cell culture plate (sigma Aldrich), and live and dead cells were observed under
28
high content screening system (live cell imaging). In the flow cytometry analysis, the
29
above said concentrations of chromanequinone compound were treated in the log phase
30
culture of MRSA cells for 3 hours at 37 °C. After washing with PBS buffer, the cells
31
were stained with propidium iodide (PI 1.0 mg mL-1 stock), and finally kept in dark
32
condition for 5 min. PI stained cell aliquots were subjected for MRSA cell membrane
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damage by using flow cytometry on FACS Calibur (BD Biosciences, Oxford, UK) and
2
the results were analysed with cell Quest Pro software (BD Biosciences, Oxford, UK).
3 4
Time dependent study of MRSA cell membrane damage Broth cultures of S. aureus in log phase (OD600=0.5) were treated with
6
antimicrobial chromanequinone compound at specific concentration (3X MIC) and the
7
cells were incubated at 37 °C for 5 different time intervals. Subsequent, washing and
8
staining methods as described previously as followed.
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Preparation of MRSA cells for Atomic force Microscopic analysis
The log phase growth culture of MRSA cells were centrifuged at 5000 rpm for
12
10 min and washed twice with 1.0 mL of PBS buffer. The cell pellet was re-suspended
13
in same PBS buffer. This bacterial cell suspension was treated with antimicrobial
14
chromanequinone compound at 3X MIC level for two different time intervals such as 3
15
hours and 6 hours with antimicrobial compound at 3X MIC level and incubated at 37
16
°C for 3 hours and 6 hours and DMSO was used as a negative control. For AFM
17
analysis, the samples were diluted at suitable concentration and mounted on the cover
18
slip as mentioned previously [16]. The slides were air dried and mounted directly on
19
the specimen metal disc. In order to locate the bacteria, the sample specimen was
20
scanned at different area by using (Model, APE Research: A100-SGS). For better
21
resolution, contact mode micro cantilever was used for the analysis. To determine the
22
effect of antimicrobial chromanequinone compound on the MRSA cell membrane,
23
approximately five individual bacterial cells were studied (In triplicates). Two
24
dimensional (2D) and Three dimensional (3D) images were captured for treated and
25
untreated bacterial cells.
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Collection of Blood Sample
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Peripheral blood samples (5mL) were collected from healthy volunteers,
29
Institutional ethical clearance was obtained from Madurai Kamaraj University Ethical
30
and Review Board Committee (ERC). Then, it was transferred to sterile centrifuge
5
ACCEPTED MANUSCRIPT tubes containing 8-10 glass beads of 2 mm diameter is carried out until clumps of fibrin
2
were seen (defibrinated) mixing should be gentle, to avoid foaming. Histoprep (Density
3
gradient solution), Anti lymphocytic serum (ALS) and Rabbit complement chemical
4
were purchased from BAG Health Care, Germany.
5
Preparation of Lymphocyte and RBC suspension
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Briefly, 5mL of the blood sample was defibrinated using glass beads, 5-10 mL
7
of saline was added to the defibrinated blood. And then, 2.0 mL of Histoprep solution
8
was relocated to a sterile serological tube. 4mL of diluted blood (diluted with an equal
9
volume of saline) was gently loaded using a Pasteur pipette along the sides of the
10
serological tubes without mixing the blood and Histoprep solution (Density gradient
11
solution). The tube was centrifuged at 1600 RPM for 20 mins. After centrifugation,
12
four distinct layers were observed. In between the top plasma layer Histoprep layer, a
13
white puff layer and undermost RBC layer was observed. The white puffy layer
14
(interphase) consists of lymphocytes and was carefully transferred into another
15
serological tube with the help of Pasteur pipette [17]. The inter phase solution was
16
washed with saline and centrifuged at 2000 rpm for 10 min twice. The supernatant was
17
discarded and the pellet was diluted using required volume of saline. The undermost
18
RBC layer was collected in another fresh serological tube and diluted in the ratio of
19
1:20 by using saline.
20
Human Lymphocyte toxicity assay
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The methodology described by Terasaki and McClelland [18] was adopted for
22
present lymphotoxicity assay. Terasaki trays (60/72 wells, Greiner, USA) were used
23
throughout the study. In the first set of experiments, 2.0 µL of freshly prepared
24
lymphocytes suspension was added to the each well of Terasaki plate. Then, 4.0 µL of
25
purified chromanequinone compound was added to the respective wells at different
26
concentration such as (10 mg mL-1 (1X), 5 mg mL-1 (1/2X), 2.5 mg mL-1 (1/4X) and
27
1.25 mg mL-1 (1/8X). Anti-lymphocytic serum (ALS) and saline solution were used as
28
a positive and negative control respectively. Then, the plate was incubated at 37 °C for
29
30 mins. About 5.0 µL of rabbit complement (BAG Health Care, Germany) was added
30
to each well. In the second set of experiments, the plate containing lymphocytes were
31
treated only with specified concentration of purified chromanequinone compound from
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ACCEPTED MANUSCRIPT different stock (10 mg mL-1 (1X), 5 mg mL-1 (1/2X), 2.5 mg mL-1 (1/4X) and 1.25 mg
2
mL-1 (1/8X). In each experiment, Anti-lymphocyte serum (ALS, anti-lymphocyte
3
serum, Biotest AG, Germany) and saline were used as a positive and negative control
4
respectively. After that, the cells were incubated at 37 °C for 3 h. Subsequently, the
5
cells were stained with 2.0 µL of nucleic acid staining dye mix from stock (Acridine
6
orange 1 mg mL-1 and Propidium iodide 1 mg mL-1) for 5 mins. Then the cells were
7
visualized under fluorescence microscope with 480 nm and 567 nm, 20 X
8
magnifications. The experiment was performed in triplicates.
9
RBC toxicity assay
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In this experiment, 2.0 µL of human RBC in saline (1: 20) was added to each
11
wells of the Terasaki plate and cells were treated with 4.0 µL of purified
12
chromanequinone compound from different stock (10 mg mL-1 (1X), 5 mg mL-1
13
(1/2X), 2.5 mg mL-1 (1/4X) and 1.25 mg mL-1 (1/8X). The sterilized water was used as
14
a negative control. Treated and untreated RBCs were incubated at 37°C for 3 h.
15
Subsequently, the plates were visualized under phase contrast microscope (NIKON
16
2000) with 20X magnification. The experiment was performed in triplicates.
17 18 19
RESULTS
20
Concentration dependent study of chromanequinone compound on MRSA by
21
FACS and High content Screening methods
22
In-vitro toxic effect of chromanequinone on MRSA cell membranes was studied with
23
combined PI/DAPI dyes by using high content screening and FACS imaging
24
techniques. Propidium iodide (PI) is a red-fluorescent dye, which stains the nucleus and
25
chromosomes of the dead cells by intercalating between the bases of the nucleic acid.
26
However, the DAPI (4', 6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain
27
that can easily permeable into the live cell membrane than the dead cells. MRSA cells
28
were treated with three different concentrations as follows (1X MIC 1.25µg, 3X MIC
29
3.75µg and 5X MIC 6.25 µg) and DMSO used as a negative control.
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ACCEPTED MANUSCRIPT The MRSA cells were affected by higher concentration of chromanequinone
2
compound exposure and it caused cell membrane damage and subsequent cell death
3
when compared to the control (Fig 1a, b) and definite MIC (Fig 2a, b) levels examined
4
by HCS (Fig 3b and 4b). The number of PI positive MRSA cells treated with
5
chromanequinone metabolite was determined by flow cytometry. chromanequinone
6
compound treatment above MIC concentrations (3X MIC and 5X MIC) induced cell
7
membrane damage followed by cell death of 14.8% and 19.4% respectively (Fig 3a and
8
4a). This result shows that the lethal activity of chromanequinone compound is attained
9
at higher concentration of MIC
levels
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when compared to
control. The
chromanequinone compound exposed MRSA cell also causes the growth inhibition and
11
cell membrane breakdown as shown by PI positive cells.
12
Time dependent study of MRSA by High content screening method
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The effect of chromanequinone compound on MRSA cell membrane have been
14
evaluated by time dependent killing assay using HCS with the help of nucleic acid
15
staining dyes both DAPI and PI. Chromanequinone compound at the fixed
16
concentration (3 X MIC) was tested at 5 different time points (1h, 3h, 6h, 12h and 24 h)
17
shown in Fig 5. In the control experiment, there is an increased number of live cells
18
which fluoresce blue in colour and after 1h exposure of chromanequinone compound
19
against MRSA, it showed less number of dead cells (red/ yellow florescence) and more
20
number of live cells (blue florescence) (Fig 5a and 5b). After 3 h and 6 h incubation,
21
the cells showed a drastic change in morphology with increased number of dead cells
22
(more permeable of propidium iodide) as in (Fig 5c and 5d). The more number of dead
23
cells were seen after 12 h exposure of compound against MRSA followed by complete
24
cell death after 24 h exposure (Fig 5e and 5f).
25 26 27 28
Assessment of bacterial surface cell morphology changes
29
chromanequinone at the concentration of 3XMIC in the log phase of growth
30
(O.D600=0.6). The AFM images of untreated MRSA cells were showing round with a
31
smooth surface morphology and much undamaged cells (Fig 6a and 6b). A drastic cell
32
morphology changes were observed in chromanequinone treated MRSA cells for 3
33
hours, and they were shown in Fig 7a and 7b. After 3 h treatment, the cells remain
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The AFM images were obtained for untreated and treated MRSA cells with
8
ACCEPTED MANUSCRIPT existed as individual cells. At this time point, slight changes were noticed on the
2
bacterial cell surface with increasing roughened texture on the MRSA cell surface than
3
untreated cells. After 6 h exposure, the MRSA cells with chromanequinone compound
4
showed uneven cell surfaces when compared to the untreated MRSA cells as shown in
5
Fig 8a and 8b. Chromanequinone treated MRSA cells over 3h and 6 h can induced
6
considerable topographical changes such as cell shrinkage, cell size and reduced cell
7
surface. AFM image analysis revealed that the surface ultra structure of MRSA cells
8
treated with chromanequinone were entirely different from the untreated MRSA cells.
9
In general, antimicrobial compounds can induce cell membrane damage, cell lysis,
10
leakage of intracellular fluids and leads to cell death. Similar to the results of BIQ, a
11
naphthoquinone pigment, Shikonin also induced MRSA cell membrane disruption, cell
12
lysis, leakage of intracellular cytoplasmic contents and subsequent cell death (19).
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Lymphotoxicity Assay in-vitro
19
examined by using nucleic acid binding dye (AO/PI) [20, 21]. Morphological changes
20
of the apoptosis can be visualized in either fixed tissue or live cells grown in a culture
21
by examining nuclear morphology using vital dyes [22]. Fluorescent microscopy was
22
mainly used to study the viability of lymphocytes in response to chromanequinone
23
compound treatment with different concentration along with complement for 3 h. Anti-
24
lymphatic serum was used as a positive control which kills lymphocytes that are
25
considered as 100% cell death at specified concentration Fig 9B. Treatment of
26
lymphocytes at 1X concentration in addition with complement shows the 100% dead
27
cells (PI stained cells - red in colour) Fig 9C when compared to the negative control an
28
untreated cells Fig 9A. More than 75% and 50% of the dead lymphocytes cells were
29
observed at 1/2X and 1/4X concentration of chromanequinone Fig 9D and 9E. The
30
increased number of live cells (AO stained green cells) were observed at very low
31
concentrations of chromanequinone compound exposure Fig 9F.
In presence of complement
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In absence of complement
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Toxic effect of the chromanequinone compound on human lymphocytes was
9
examined by previously described method. Lymphocytes were treated with different
10
concentration of chromanequinone compound (1/8X, 1/4X, 1/2X and 1X) without
11
complement for a duration of 3 h. Lymphocytes cells treated with different
12
concentrations (1/8X, 1/4X, 1/2X) of chromanequinone compound results revealed that
13
there is an increased number of live cells (green colour), similar to that of negative
14
control as shown in Fig 10A, 10D, 10E & 10F. In the case of lymphocytes cells
15
treatment with higher concentration (1X) of chromanequinone compound showing,
16
there is an increased number of dead cells (red-colored nuclei), similar to those
17
obtained with ALS positive control (Fig. 10B and 10C).
18 19 20 21
Red blood cell toxicity study
22
studied. Red blood cells were treated at different concentration of chromanequinone
23
such as 1X, 1/2X and 1/4X for 3 hrs and sterile saline was used as a negative control.
24
After incubation, no RBC disruption was observed. The results showed that the
25
compound does not reveal any toxic effect to the RBC (Fig 11).
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DISCUSSION
Streptomyces are known to have the ability to produce pharmaceutically
29
important secondary metabolites especially antibiotics. In recent days, novel
30
compounds with unique structure have been discovered from mangrove actinomycetes
31
[23]. BIQ antibiotics with broad spectrum antimicrobial activity was purified and
32
characterized from Mangrove derived Streptomyces sp JRG-04 [15]. In the present
33
study, flow cytometry and high content screening experiments were employed to detect
34
the MRSA cell viability in response to chromanequinone compound with the
10
ACCEPTED MANUSCRIPT combination of fluorescent staining methods (DAPI/ PI). Flow cytometry, is a
2
technique which has been applied to study the eukarytotic cells viability, metabolic
3
state and identification of antigenic marker of bacteria [24-26]. Currently, flow
4
cytometric methods have been also used to detect the antimicrobial susceptibility of the
5
bacteria based on metabolic activity or membrane integrity using vital dyes [27-30]. In
6
this study, we have showed that the effect of different concentration of
7
chromanequinone compound on MRSA cell morphological changes based on PI
8
fluorescent intensity. In the untreated control, the live MRSA cells have intact
9
membranes and are impermeable to propidium iodide (PI) which showed low
10
fluorescent intensity when compared to the treatment at 1X MIC, 3X MIC and 5X MIC
11
levels. The high PI fluorescent intensity was observed only in the case of dead cells and
12
compromised membrane cells. The results indicated that the increased numbers of dead
13
cells were observed when increasing the concentration of chromanequinone compound
14
and corresponding HCS analysis results also discriminate the live and dead population,
15
with great agreement to flow cytometry results. This study suggested that the viability
16
of MRSA cells is mainly to depend on the dose of chromanequinone compound. In
17
addition to that, we have shown the time taken for killing of MRSA cells by
18
chromanequinone compound in response to cell membrane damages. This study also
19
clearly indicates that there is an increased propidium iodide stained cells (increased
20
number of dead cells) when there is increase in the time period.
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Currently, AFM technique is used to study the cell morphology and ultra
22
structure of bacteria [31, 32]. In the present study, the AFM imaging technique is
23
conducted to determine the action of chromanequinone compound on MRSA cells. The
24
results shown that, the treatment at 3X MIC concentration of chromanequinone
25
compound begin to exert a toxic effect after 3 hours, which induced the substantial
26
topographical changes characterized by reduction in size and smoothness of the cells.
27
MRSA cells incubated with chromanequinone for 6 hours, showing the complete
28
disorganization of the entire cell structure as shown in Fig 8. Similarly, bacterial
29
morphological changes induced by antibacterial agents were reported previously [33].
30
Braga and Ricci [34] also reported that rokitamycin, a macrolides antibiotic start to
31
affect the Streptococcus pyogenes cell structure by the formation of large abnormal
32
cells with inadequacy of the chains structure. Hence, the MRSA cell structure
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ACCEPTED MANUSCRIPT disorganization results revealed that, the chromanequinone had inventive antimicrobial
2
action against methicillin resistant strain of S. aureus. However, the chromanequinone
3
mechanism of action is still unclear, although it changes the ultra structure of the
4
bacterial cells, which results in loss of intracellular fluid which leads to cell death.
5
Currently, human lymphocytes were most widely used for toxicity analysis of new
6
drugs, particularly the efficiency of the method developed by Terasaki and McClelland
7
[18]. Martini et al., [35] have reported that the anti-bacterial flavonoids, 5-hydroxy-7,4-
8
dimethoxy flavones from Combretum erythrophyllum was found to be a toxic substance
9
to human lymphocytes. In addition, a volatile fatty acid, namely butyric acid produced
10
by some pathogenic microorganisms may suppress more than 90% of the lymphocyte
11
when used at concentration 2.5 mM [36]. Similarly, phenazine derivatives such as
12
pyocyanine pigment produced by Pseudomonas aeruginosa also inhibit the lymphocyte
13
proliferation [37, 38]. On the other hand, Hung et al reported that the anthraquinone
14
emodin act as a new template for development of effective immunosuppressant with
15
vasorelaxant property against transplantation rejection and other autoimmune diseases
16
(39).
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Recent reports revealed that some pathogenic E. coli strains harbouring PKS
18
Island is responsible for production of a polyketide termed as colibactin which induce
19
toxic effects to the lymphocytes. The E. coli producing colibactin that breaks double
20
strand DNA and G2 cell cycle arrest in T lymphocytes was examined by cell membrane
21
integrity [40]. In this present study, we have evaluated the toxic effect of the
22
chromanequinone compound produced by the Streptomyces sp. JRG-04 on human
23
lymphocytes. The data presented here indicated that the cell morphology based
24
apoptosis analysis by fluorescence (AO/PI) staining method showed visible
25
morphological changes in the nucleus, internal organelle and plasma membrane
26
integrity. The maximum death rate of cells was observed at higher concentration of
27
chromanequinone compound exposure followed by lower concentration along with the
28
complement. The lymphotoxicity of chromanequinone compound was found to be
29
concentration and complement dependent, which is also found to be an inducer of cell
30
apoptosis.
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ACCEPTED MANUSCRIPT Lymphocytes treated with higher concentration of chromanequinone compound
2
without complement experiments state that there is an increased number of dead cells.
3
It reveals, some higher specified concentration of chromanequinone can exert partial
4
cytotoxic effects on human blood lymphocytes and the enhancement of dead cells were
5
positively correlated with dosage and is complement dependent. In addition, a series of
6
anthraquinones; the mitoxantrone and its derivative 1,4-bis [(2-aminoethyl)amino]-5, 8-
7
dihydroxy-9,10-anthracenedione
8
immunosuppressive activity against T-lymphocytes in a mixed lymphocyte culture
9
system and they can be used for organ transplantation (41). Apart from this, a Emodin
10
anthraquinone derivative (1,3,8-trihydroxy-6-methyl-anthraquinone) also suppresses
11
the peripheral blood mononuclear cell (PBMC) proliferative responses in dose
12
dependent manner and subsequently decline the production of interleukin in the mixed
13
lymphocyte reaction. The possible mechanism of immune suppressive effect of Emodin
14
were investigated based on structure activity relationship (SAR), it was reported that
15
the free beta-hydroxyl group of anthraquinone nucleus play a significant role in the
16
immunosuppressive activity (42) by suppression of lymphocyte proliferation and
17
cytokines (43). Similar to emodin, the anthraquinone rhein also exhibited the
18
immunosuppressive activity with different biological activity (44). Celik and Eke [45],
19
reported that the tetracycline polyketide antibiotic can induce genetic damage in human
20
peripheral
21
immunosuppressive polyketide compound has been identified from mangrove
22
endophytic fungus Penicillium sp. ZJ-SY2 [46]. Finally, we have investigated the toxic
23
effect of chromanequinone compound on RBC. The toxicity result indicated that, there
24
was no haemolysis at both higher and lower concentrations of chromanequinone.
25
Saurav and Kannabiran [47] have reported that the assessment of membrane stability,
26
with exposure of new drugs is imperative and erythrocytes can be used as a good model
27
for these membrane stability studies.
(AEAD)
were
induced
lymphocyte
cells
under
in
vitro
conditions.
Recently,
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CONCLUSION
30
The assessment of flow cytometry analysis depicted the MRSA cell membrane
31
disruption by taking up more of propidium iodide stain at all three different
13
ACCEPTED MANUSCRIPT concentrations of chromanequinone exposure. The high content screening experiment
2
results also easily discriminate the live/ dead MRSA cells with the help of nucleic acid
3
stating dye after exposure of chromanequinone. Furthermore, the atomic force
4
microscopic study also showed the altered MRSA cell surface ultra-structure changes
5
when the cells were exposed to chromanequinone compound at two different time
6
points. This study also revealed the human lymphocytotoxicity potential of the BIQ
7
chromanequinone with the combination of complement. Interestingly, the higher
8
concentration of BIQ compound (10 mg mL-1) without complement can also induce
9
toxicity to the human lymphocytes. The toxic potential of BIQ compound on human
10
lymphocytes might be associated with the complement dependent. However, the
11
detailed mechanisms of lymphocytotoxicity need to be elucidated. The red blood cell
12
toxicity assay also indicated that the BIQ compound produced by the Streptomyces sp.
13
JRG-04 is non toxic to the human RBC. Further detailed studies on pharmacological
14
mechanisms of BIQ compound will provide the comprehensive understandings of its
15
efficacy as better drug like lead molecule.
16
Acknowledgements
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I would like to acknowledge my mentor and advisor late Professor Dr. SRD
18
Jebakumar and I have profited much from his guidance and instructions during my
19
doctoral studies at Madurai Kamaraj University. We also acknowledge the UGC,
20
Government of India for the ‘Research Fellowship in Science for Meritorious Students
21
Scheme and MKU for providing the University Stipendiary Research Fellowship
22
(USRF).
23
Conflict of Interest
24
There is no conflict of interest to declare
25
REFERENCES
26 27
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1. P.M. Hawkey, A.M. Jones, The changing epidemiology of resistance, J Antimicrob. Chemother. 64 (1) (2009) i3-i10.
14
ACCEPTED MANUSCRIPT 1
2. D. Sharma, T. Kaur, B.S. Chadha, R.K. Manhas, Antimicrobial activity of
2
actinomycetes against multidrug resistant Staphylococcus aureus, E. coli and
3
various other pathogens, Trop. J. Pharm. Res. 10 (6) (2011) 801-808. 3. J. Spizek, J. Novotna, T. Rezanka, A.L. Demain, Do we need new antibiotic?
5
The search for new targets and new compounds, J. Ind. Microbiol. Biot. 37 (12)
6
(2010) 1241-1248.
7 8
RI PT
4
4. R.L. Monaghan, J.S. Tkacz, Bioactive microbial products: focus on mechanism of action, Annu. Rev. Microbiol. 44 (1990) 271-301.
5. R. Calne, S. Lim, A. Samaan, D.S.J. Collier, S. Pollard, D. White, S. Thiru,
10
Rapamycin for immunosuppression in organ allografting, Lancet. (1989) 227.
11
6. J. Douros, M. Suffness, New antitumor substances of natural origin, Cancer. Treat. Rev. 8 (1) (1981) 63–87.
M AN U
12
SC
9
13
7. S. Sehgal, H. Baker, C. Vézina, Rapamycin (AY-22,989), a new antifungal
14
antibiotic. II. Fermentation, isolation and characterization, J. Antibiot. 28 (10)
15
(1975) 727–732.
8. E. Patsenker, V. Schneider, M. Ledermann, H.Saegesser, C. Dorn et al., Potent
17
antifibrotic activity of mTOR inhibitors sirolimus and everolimus but not of
18
cyclosporine A and tacrolimus in experimental liver fibrosis, J. Hepatol. 55 (2)
19
(2011) 388–398
21
9. R. Solanki, M. Khanna, R. Lal, Bioactive compounds from marine actinomycetes, Indian J. Microbiol, 48 (4) (2008) 410-431.
EP
20
TE D
16
10. G.R. Silva-Lacerda, R.C. Santana, M.C. Vicalvi-Costa, E.G. Solidonio, K.X.
23
Sena, G.M. Lima, J.M. Araujo, Antimicrobial potential of actinobacteria isolated
24
from the rhizosphere of the Caatinga biome plant Caesalpinia pyramidalis Tul,
25
AC C
22
Genet. Mol. Res. (2006) DOI: 10.4238/gmr.15017488.
26
11. J.Y. Cho, H.C. Kwon, P.G. Williams, C.A. Kauffman, P.R. Jensen, W. Fenical,
27
ActinofuranonesA and B, polyketides from a marine-derived bacterium related
28
to the genus Streptomyces (Actinomycetales), J. Nat. Prod. 69 (3) (2006) 425-
29
428.
30
12. D. Fuchs, V. Daniel, M. Sadeghi, G. Opelz, C. Naujokat, Salinomycin
31
overcomes ABC transporter- mediated multidrug and apoptosis resistance in
15
ACCEPTED MANUSCRIPT 1
human leukemia stem cell-like KG-1a cells, Biochem. Biophys. Res. Commun.
2
394 (4) (2010) 1098-1104. 13. S.Z. Kuo, K.J. Blair, E. Rahimy, A. Kiang, E. Abhold, J.B. Fan, J.
4
WangiRodriguez, X. Altuna, W.M. Ongkeko, Salinomycin induces cell death
5
and differentiation in head and neck squamous cell carcinoma stem cells despite
6
activation of epithelial-mesenchymal transition and Akt, BMC. Cancer.12 (2012)
7
556
RI PT
3
8
14. H. Laatsch, M. Kellner, G. Wolf, Y.S. Lee, F. Hansske, S. Konetschnyrapp, U.
9
Pessara, W. Scheuer, H. Stockinger, Oligomycin F, a new immunosuppressive homologue of oligomycin A, J. Antibiot. 46 (9) (1993) 1334-1341.
SC
10
15. G. Govindarajan, V.S. Santhi, S.R.D. Jebakumar, Antimicrobial potential of
12
phylogenetically unique actinomycete, Streptomyces sp. JRG-04 from marine
13
origin, Biologicals. 42 (6) (2014) 305-311.
M AN U
11
14
16. R.L. Soon, R.L. Nation, P.G. Hartley, I. Larson, J. Li, Atomic force microscopy
15
investigation of the morphology and topography of colistin- heteroresistant
16
Acinetobacter baumannii as a function of growth phase and in response to
17
colistin treatment, Antimicrob. Agents. Chemother. 53 (12) (2009) 4979-4986. 17. A. Boyum, Separation of White Blood cells, Nature. 204 (1964) 793-794.
19
18. P.I. Terasaki, J.D. McClelland, Microdroplet assay of human serum cytotoxins,
20
TE D
18
Nature. 204 (1964) 998-1000.
19. Y.S. Lee, D.Y. Lee, Y.B. Kim, S.W. Lee, S.W. Cha, H.W. Park, G.S. Kim,
22
D.Y. Kwon, M.H.Lee, S.H. Han, The Mechanism Underlying the Antibacterial
23
activity of Shikonin against Methicillin Resistant Staphylococcus aureus, Evid.
24
Based. Complement. Alternat. Med. (2015) DOI 10.1155/2015/520578.
AC C
EP
21
25
20. Z. Darzynkiewicz, G. Juan, X. Li, W. Gorczyca, T. Murakami, F. Traganos,
26
Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death
27 28 29 30 31
(necrosis), Cytometry. 27 (1) (1997) 1-20.
21. M.G. Ormerod, Using flow cytometry to follow the apoptotic cascade, Redox. Report. 6 (5) (2001) 275-287. 22. F.L. Kiechle, X. Zhang, Apoptosis: a biochemical aspects and clinical implications, Clin. Chim. Acta. 326 (2002) 27-45.
16
ACCEPTED MANUSCRIPT 23. M. Ushakiranmayi, M. Vijayalakshmi, P. Sudhakar, N. Krishna, M. Rajesh
2
Kumar, C. Bhujangarao, Y. Venkateswarlu, Bioactive metabolites produced by
3
Streptomyces Cheonanensis VUK-A from Coringa mangrove sediments:
4
isolation, structure elucidation and bioactivity, 3 Biotech. (2016) DOI
5
10.1007/s13205-016-0398-6.
RI PT
1
6
24. H.M. Davey, D.B. Kell, Flow cytometry and cell sorting of heterogeneous
7
microbial populations: the importance of single cell analyses, Microbiol. Rev. 60
8
(4) (1996) 641-696.
10
25. H.N. Shapiro, Microbial analysis at the single-cell level: tasks and techniques, J. Microbiol. Methods. 42 (1) (2000) 3-16.
SC
9
26. G. Nebe-von-Caron, P.J. Stephens, R.A. Badley, Bacterial detection and
12
differentiation by cytometry and fluorescent probes, Proc. R. Microbiol. Soc. 34
13
(1999) 321-327.
M AN U
11
14
27. G. Nebe-von-Caron, P.J. Stephens, C.J. Hewitt, J.R. Powell, R.A. Badley,
15
Analysis of bacterial function by multi-colour fluorescence flow cytometry and
16
single cell sorting, J. Microbiol. Methods. 42 (1) (2000) 97-114. 28. D.J. Novo, N.G. Perlmutter, R.H. Hunt, H.M. Shapiro, Multiparameter flow
18
cytometric analysis of antibiotic effects on membrane potential, membrane
19
permeability, and bacterial counts of Staphylococcus aureus and Micrococcus
20
luteus, Antimicrob. Agents. Chemother. 44 (4) (2000) 827-834.
TE D
17
29. R.I. Jepras, F.E. Paul, S.C. Pearson, M.J. Wilkinson, Rapid assessment of
22
antibiotic effects on Escherichia coli by bis-(1, 3-dibutylbarbituric acid)
23
trimethine oxonol and flow cytometry, Antimicrob. Agents. Chemother. 41 (9)
24
(1997) 2001-2005.
AC C
EP
21
25
30. C. Gauthier, Y. St-Pierre, R. Villemur, Rapid antimicrobial susceptibility testing
26
of urinary tract isolates and samples by flow cytometry, J. Med. Microbiol. 51
27
(3) (2002) 192-200.
28
31. T.A. Camesano, M.J. Natan, B.E. Logan, Observation of changes in bacterial
29
cell morphology using tapping mode atomic force microscopy, Langmuir. 16
30
(10) (2000) 4563-4572.
31
32. N.P. Mortensen, J.D. Fowlkes, C.J. Sullivan, D.P. Allison, N.B. Larsen, S.
32
Molin, M. J. Doktycz, Effects of colistin on surface ultrastructure and
17
ACCEPTED MANUSCRIPT 1
nanomechanics of Pseudomonas aeroginosa cells, Langmuir. 25 (6) (2009)
2
3728-3733. 33. A.K. Suresh, W.W. Pelletier, J. Moon, B. Gu, N.P. Mortensen, D.P. Allison,
4
D.C. Yoy, T.J. Phelps, M.J. Doktycz, Silver nanocrystallites: Biofabrication
5
using Shewanella oneidensis, and an evaluation of their comparative toxicity on
6
Gram negative and Gram-positive bacteria, Environ. Sci. Technol. 44 (13)
7
(2010) 5210-5215.
RI PT
3
34. P.C. Braga, D. Ricci, Differences in the susceptibility of Streptococcus pyogenes
9
to rokitamycin and erythromycin A revealed by morphostructural atomic force
11
microscopy, J. Antimicrob. Chemother. 50 (4) (2002) 457-460.
35. D.N. Martini, P.R.D. Katerere, N.J. Eloff, Biological activity of five antibacterial
M AN U
10
SC
8
12
flavonoids
from
Combretum
erythrophyllum
13
Ethnopharmacol. 93 (2004) 207-212.
(Combretaceae),
J.
14
36. T. Kurita-Ochiai, K. Fukushima, K. Ochiai, Volatile fatty acids, metabolic by-
15
products of periodontopathic bacteria, inhibit lymphocyte proliferation and
16
cytokine production, J. Dent. Res. 74 (7) (1995)1367-1373. 37. R. Wilson, D.A. Sykes, D. Watson, A. Rutman, G.W. Taylor, P.J Cole,
18
Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and
19
assessment of their contribution to sputum sol toxicity for respiratory epithelium,
20
Infect. Immun. 56 (9) (1988) 2515-2517.
TE D
17
38. K. Kanthakumar, G. Taylor, K.W.T. Tsang, D.R. Cundell, A. Rutman, S. Smith,
22
P.K. Jeffery, P.J. Cole, R .Wilson, Mechanisms of action of Pseudomonas
23
aeruginosa pyocyanin on human ciliary beat in vitro, Infect. Imm. 61 (7) (1993)
AC C
24
EP
21
2848-2853.
25
39. H.C. Huang, C.R. Lee, P.D. Chao, C.C.Chen, S.H Chu, Vasorelaxant effect of
26
emodin, an anthraquinone from a Chinese herb, Eur. J. Pharmacol. 205 (1991)
27
289–294.
28
40. I.P Marcq, D. Martin, G. Payros, G. Cuevas-Ramos, M. Boury, C. Watrin, J.P.
29
Nougayrède, M. Olier, E. Oswald, The Genotoxin Colibactin Exacerbates
30
Lymphopenia and Decreases Survival Rate in Mice Infected With Septicemic
31
Escherichia coli, J. Infect. Dis. 210 (2) (2014) 285-294.
18
ACCEPTED MANUSCRIPT 1
41. B.S. Wang, K.C. Murdock, A.L. Lumanglas, M. Damiani, J. Silva, V.M.
2
Ruszala-Mallon,
3
anthraquinones with their effects on the suppression of immune responses, Int. J.
4
Immunopharmacol. 9 (6) (1987) 733-739.
6
Durr,
Relationship
of
chemical
structures
of
42. Y.X. Liu, N.Y. Shen, C. Liu, Y. Lv, immunosuppressive effects of emodin: An
RI PT
5
F.E,
in vivo and in vitro study, Transplant. Proc. 41 (2009) 1837-1839.
43. H.C. Huang, J.H. Chang, S.F. Tung, R.T Wu, M.L. Foegh, S.H. Chu,
8
Immunosuppressive effect of emodin, a free radical generator, Eur. J. Pharmacol.
9
11 (3) (1992) 559-364.
11
44. F. Hu, F. Xing, G. Zhu, G. Xu, C. Li, J. Qu, I. Lee, L. Pan, Rhein antagonizes P2X7 receptor in rat peritoneal macrophages, Sci. Rep. 5 (2015) 14012.
M AN U
10
SC
7
12
45. A. Celik, D. Eke, The Assessment of Cytotoxicity and Genotoxicity of
13
Tetracycline Antibiotic in Human Blood Lymphocytes Using CBMN and SCE
14
Analysis, in Vitro, Int. J. Hum. Genet. 11 (2011) 23-29.
46. H. Liu, S. Chen, W. Liu, Y. Liu, X. Huang, Z. She, Polyketides with
16
Immunosuppressive activities from Mangrove Endophytic Fungus Penicillium
17
sp. ZJ-SY, Mar. Drugs. 14 (12) (2016) 217.
18
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15
47. K. Saurav, K. Kannabiran, Cytotoxicity and antioxidant activity of 5-(2,4dimethylbenzyl)
pyrrolidin-2-one
extracted
from
20
VITSVK5 spp. Saudi, J. Biol. Sci.19 (1) (2012) 81-86.
marine
Streptomyces
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Figure 1 a) FACS analysis of untreated MRSA stained with PI and b) HCS image of
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Figure 2 a) MRSA treated with at MIC concentration stained with PI and b) MRSA
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Figure 3 a) MRSA treated with at 3X MIC concentration stained with PI and b) MRSA
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treated with at 3X MIC stained with DAPI and PI.
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Figure 5 Time dependence of chromanequinone compound effects on MRSA imaged
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by HCS. (a) Control, (b) 1h treated with chromanequinone, followed by (c) 3h, (d) 6h,
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(e) 12h and (f) 24h.
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Figure 6 (a) AFM Images of Untreated MRSA (2D View) and (b) untreated MRSA
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Figure 7 (a) AFM Images of chromanequinone treated MRSA for 3 hours (2D View)
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and (b) MRSA (3D view).
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Figure 8 (a) AFM Images of chromanequinone treated MRSA for 6 hours (2D View)
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and (b) MRSA (3D view).
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Figure 9 Lymphotoxicity assay with complement imaged by fluorescent microscopy.
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(A) Negative Control, (B) positive control (ALS), (C), (D), (E) and (F)
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chromanequinone compound treatment at 1X, 1/2X, 1/4X and 1/8X respectively.
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Figure 10 Lymphotoxicity assay without complement imaged by fluorescent
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microscopy. (A) Negative Control, (B) positive control (ALS), (C), (D), (E) and (F)
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chromanequinone compound treatment at 1X, 1/2X , 1/4X and 1/8X respectively.
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Figure 11 Phase contrast microscopic images showing the effect of chromanequinone
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on RBC. (A) Negative control, (B), (C) and (D) were chromanequinone treated RBC at
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1X, 1/2X and 1/4X concentrations respectively.
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Highlights This research work is highly credited by studying the role of chromanequinone (BIQ) compound on human lymphocytotoxicity with the combination of complement to
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reveal the toxicity analysis for new drugs. FACS and HCS analysis clearly depict the live
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