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SUPPLEMENTARY MATERIAL
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Effects of epigallocatechin gallate on the cell wall structure of
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Mycobacterial smegmatis mc2155
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Tieying Suna#, Biaojie Qinb#, Mingchuan Gaoa, Yuling Yina, Changyuan Wangc, Shizhu Zanga, Xinli Lia ,
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Cuili Zhanga, Yi Xina and Tao Jianga*
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a
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Molecular biology Department, Dalian Medical University, Dalian 116044, China;c Clinical
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Pharmacology Department, College of Pharmacy in Dalian Medical University, Dalian 116044, China.
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Department of Biotechnology, Dalian Medical University, Dalian 116044, China;b Biochemistry and
*Corresponding Author. E-mail:jiangtaodl@163.com.
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Abstract
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Epigallocatechin gallate (EGCG) is the main component of green tea extracts, that
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inhibits the growth of M.smegmatis mc2155, and the mechanism is not clear. This study
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showed the effects of varying concentrations of EGCG on the growth of M. smegmatis
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using ELISA analysis. The changes of EGCG content and EGCG stucture in LB
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medium with mc2155 were identified by HPLC and LC/MS. Transmission electron
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microscopy (TEM) provided a detail characterization of the cell envelope structure. As
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a result, the most optional inhibition concentration was determined to be 20μg.ml-1.
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Mass analysis found that most of EGCG was transferred into its isomeride in LB
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medium while EGCG treatment for 18h, but inhibition effects for mc2155 had yet been
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maintained. There were striking changes in the cell wall envelope profiles after EGCG
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treatment for 18h. The cell wall appeared to have a less electron translucent zone, turn
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rougher and thicker. The results show that EGCG impacts the integrity of
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mycobacterial cell wall, and is likely be a better prophylactic agent against TB.
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Key words: epigallocatechin gallate; M. smegmatis mc2155; antimicrobial effect; the
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cell wall structure
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Experimental
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Antimicrobial circle experiment
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This Drug sensitivity test adopted paper disc agar diffusion method (K-B method) to
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observe antimicrobial functions. Ethambutol (EMB) with final concentration of 48 μg.ml-1
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was regarded as positive control, phosphate salt buffer and ampicillin with concentration of
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100 μg.ml-1 as negative control of antimicrobial effects. EGCG treatments were initiated by
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adding filtered stock solution of EGCG (10 mg ml-1) to achieve the final concentration of
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10 μg ml-1 and 20 μg ml-1 respectively. After using 0.22 μm filter, and then put paper disc
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into every tube with different treated solution, keeping 24 h at 4℃. The plates above
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inverted at 37℃ for 48h after placing at room temperature for 15 min, observing results.
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Repeated the above-mentioned experiment three times, measured the antimicrobial circle
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diameter.
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The effect of EGCG on the growth of M.smegmatis mc2155/ pSMT1
M.smegmatis mc2155 strains electronfected pSMT1 was grown in LB broth containing
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0.05% Tween 80 and 100 μg ml–1 hygromycin at 37 °C. EGCG treatments were initiated by
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adding filtered stock solution of EGCG (10 mg ml–1) to achieve the different final
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concentration. The plasmid DNA called pSMT1 was constructed by Prof..Douglas Young’s
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lab, and it may produce bioluminescence. Bioluminescence has been early employed as a
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reporter to detect the presence of low levels bacterial contamination or infection. Emission
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of light is dependent on the presence of a cofactor, ATP or reduced flavin mononucleotide
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(FMNH2) which is found only in living cells. Dead cells are no longer able to produce the
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cofactor and a corresponding decline in luminescence. In this study, the growth curve of
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M.smegmatis mc2155/pSMT1 was made according to the power of luminescence. A
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calibration curve with M.smegmatis/ pSMT1 confirmed linearity, and 0.1 ml of 1% n-decyl
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aldehyde in DMSO as substrates(Eklund et al. 2010). M.smegmatis mc2155/ pSMT1 was
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seeded in per well of white opaque 96 well plates. Luminescence was measured once every
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four hours until EGCG treatment for 24 h using SpectraMax L chemiluminescence ELISA
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reader.
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Detecting behaviors of EGCG in LB medium through HPLC and LC/MS
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EGCG treatment were initiated to achieve final concentration of 20 μg ml-1, every
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other 6 h to collecte supernatant through centrifuging at 5000 rpm for 10 min, and then
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filtered with 0.22 μm filter. EGCG content in LB medium was analyzed through Binary
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HPLC Pump (JEOL) with Hypersill BDS C18 (250 mm×4.6 mm.5μm) packed
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chromatographic column. With 2% acetic acid as mobile phase A and with
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chromatographic pure acetonitrile as mobile phase B, start at 5% acetonitrile and progress
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30 %,70 % to 100 % acetonitrile over 5-20 min for gradient elution program, set detection
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wavelength as 280 nm ,flow velocity as 0.8 ml/min and column temperature as 35℃.
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LC/MS were acquired to identify behaviors of EGCG in LB medium.
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Transmission electron microscopy (TEM) analysis
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In the early exponential phase of growth (A600≈0.3), EGCG treatments were initiated to
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achieve respectively the final concentration of 10 μg ml-1, 20 μg ml-1 and 50 μg ml-1 in LB
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medium for 18 h. And then the cells centrifuged for 10 min and collected cells for fixing.
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The fixed cells in 2.5% glutaraldehyde were rinsed with 0.2 M phosphate salt buffer
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(pH=7.4), followed by dehydration with gradient alcohol. Cells were embedded with epon
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embedding kit and cut into ultrathin sections. The sections were stained with 2% uranyl
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acetate for 30 min and lead citrate for 20 min and finally examined with a JEM-2000EX
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transmission electron microscope (JEOL).
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Figure S1..Antibacterial circle experiment of green tea extracts and EGCG against
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M.smegmatis mc2155. Effect of different material applied to a blank filter disk on an
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agar plate inoculated with M.smegmatis mc2155; a-c represented respectively
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phosphate salt buffer, 48 μg ml-1 EMB; 100 μg ml-1 Ampicillin; Panel A, 1 and 2
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represented 50μg.ml-1 and 100μg.ml-1 green tea extraction; Panel B, 3 and 4
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represented 10 μg ml-1 and 20 μg ml-1 EGCG.
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Figure S2 The growth curve of M.smegmatis mc2155/pSMT treated by EGCG
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Figure S3-A
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Figure S3-B
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Figure S3-C
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Figure S3 HPLC and LC/MS analysis of EGCG behaviors in LB broth.
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Figure S3-A Detecting the change of EGCG content through HPLC analysis. EGCG
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content in LB broth was gradually decreased over time periods, and it cannot nearly
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be detected while EGCG treatment for 30h.
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Figure S3-B Analyzing the proportion of EGCG isomerides by HPLC. B-1, HPLC analysis
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of EGCG standard; B-2, HPLC analysis of EGCG treated for 18h in LB broth with
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mc2155.
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Figure S3-C The identification of chemical structure of EGCG through LC/MS analysis.
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C-1, mass spectrum of EGCG standard, and the peak was at m/z 457 [M-H]-
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corresponding to EGCG with the relative molecular mass of 458.38. C-2, LC/MS
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analysis of the peak at 3.35 min for EGCG treatment group. One peak was at m/z 453
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[M-4H]- corresponding to EGCG with the relative molecular mass of 458.38, another
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was m/z 382.9 which is not identified. C-3, LC/MS analysis of the peak at 4.45 min
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for EGCG treatment group. A series of peaks at m/z 473.2, 489.4, and 498.9 might
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correspond to ion adducts of EGCG.
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Figure S4. Morphological changes of M.smegmatis mc2155 by EGCG treatment through
TEM. EGCG treatments were initiated to achieve respectively the final concentration
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of 10 μg ml-1(B), 20 μg ml-1 (C) and 50 μg ml-1 (D) in LB medium for 18 h. A, the
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cell envelope structure of M.smegmatis mc2155; B, the cell envelope structure under
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the treatment of 10 μg ml-1 EGCG, and some materials were dropped from the cell
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wall; C, the cell envelope structure under the treatment of 20 μg ml-1 EGCG, and the
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separation of the cytoplasm and cell membrane was appeared; D, the cell envelope
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structure under the treatment of 50 μg ml-1 EGCG, and the cell wall was likely to
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result from structural damage and turn thicker.
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Reference
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Eklund DA, Welin T, Schon O, Stendahl K, Huygen, M. Lerm. 2010. Validation of a
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medium-throughput method for evaluation of intracellular growth of Mycobacterium
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tuberculosis. Clin. Vaccine Immunol. 17:513-517.
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