PMC:6599329 / 29075-38569 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6599329","sourcedb":"PMC","sourceid":"6599329","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6599329","text":"Methods\n\nBacterial strain and growth condition\nE. faecalis SK460, isolated from chronic diabetic ulcer patient was used for quantitative proteomics approach to identify biofilm-associated proteins. The study was approved by the Institutional Human Ethics Committee (Reference number RGCB-IEC No. IHEC/01/2013/11) of Rajiv Gandhi Centre for Biotechnology, Kerala, India. The draft genome sequence of this strain was deposited at NCBI Genbank with accession no. NIXL00000000 [41]. The strain was cultured in Luria Bertani broth (Becton Dickinson, New Jersey, United States) at 37 °C with 150 rpm. The media used for culturing biofilm was Tryptic soy broth (Himedia, Mumbai, India) with 0.75% Glucose.\n\nBiofilm quantification and imaging\nFor confocal imaging, an overnight culture of E. faecalis SK460 was diluted to OD600 = 0.1 and 4 ml of diluted broth was distributed to 6 well microtitre plate (Nunc, Roskilde, Denmark). A sterile square glass coverslip was inserted horizontally in each well and incubated at 37 °C in a static condition for two-time intervals (12 h, 24 h). Biofilm staining was carried out with Syto9 (Invitrogen, California, United States) as described previously [42]. The slides were then observed using a Nikon Eclipse Ti Confocal Laser scanning inverted microscope (Nikon Instruments Corporation, New York, United States). The measurement of biofilm thickness was performed using NIS-Element AR software, version 4.00.04.\n\nCell extract preparation\nThe strain was cultured in different planktonic and biofilm stages to carry out quantitative proteome analysis. The test strain was inoculated in Tryptic soy broth (with 0.75% glucose) and incubated at 37 °C with shaking at 180 rpm. After overnight incubation, the culture was diluted to adjust the optical density to 0.1 at 600 nm.\n\nHarvesting planktonic stage cells\nOne hundred milliliter of diluted culture was dispensed each in 250 ml conical flasks for two different time points (12 h, 24 h) in triplicates and incubated at 37 °C with shaking at 150 rpm. After proper incubation, the cells were pelleted and washed twice with sterile ice-cold phosphate buffered saline (PBS, pH 7.4) and stored at -80 °C till protein isolation.\n\nHarvesting biofilm stage cells\nSimilarly, the diluted culture was dispensed to Cell Culture Treated EasYFlasks, 175 cm2 (Nunc, Roskilde, Denmark) at two-time points (12 h, 24 h) in triplicates and incubated at 37 °C in a static condition. After incubation, the used media along with the planktonic cells were washed off and the flask was rinsed twice with sterile PBS to remove the settled planktonic cells. The biofilm cells were then scraped off, pooled in sterile ice-cold PBS, pelleted and stored at -80 °C till protein isolation.\n\nProtein sample preparation\nProtein isolation from cell pellets of different planktonic and biofilm stages was carried out by bead beating as described previously [43]. Briefly, cell pellets were resuspended in PBS (1 ml PBS per gram of cells) and transferred to 2 ml microcentrifuge tubes containing glass beads (0.5 mm) and protease inhibitor (1 mM Phenylmethyl Sulfonyl Fluoride, PMSF) and incubated on ice for 5 min. The tubes were then placed in a Mini Bead beater (BioSpec Products, USA) and subjected to one-minute pulses at 4200 rpm for three times with 1 min interval. The suspensions were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatant was collected. Protein concentration was estimated by bicinchoninic acid protein assay kit as followed by the manufacturer’s instructions (Thermo Scientific, Massachusetts, United States). Three biological replicates and three technical replicates were used in this experiment. 100 μg of protein (1 μg/μl) from each sample was made up to 100 μl with 50 mM Ammonium bicarbonate and subjected to disulfide reduction, alkylation and in-solution tryptic digestion as followed by previous publication [44]. The digested peptide solution was centrifuged at maximum rpm at 4 °C for 12 min and the supernatant was stored at −20°C until the analysis using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).\n\nLC-MS/MS analysis\nPeptide samples were analyzed by nano-LC/MSE (at elevated energy) using a nanoACQUITY UPLC® system (Waters, Altrincham, UK) coupled to a Quadrupole-Time of Flight (Q/TOF) mass spectrometer (SYNAPT-G2, Waters, Altrincham, UK). Instrument operation and control for both the system was done employing MassLynx4.1 SCN781 software. The peptides were separated by reverse-phase column chromatography in the nanoACQUITY UPLC®. Peptides eluted from the nano-LC were subjected to mass spectrometric analysis on a SYNAPT® G2 High Definition MS™ System (Waters, Altrincham, UK). All analyses were performed using positive mode ESI using a NanoLockSpray™source. The detailed operational set up was followed as in previous publications [44].\n\nData analysis\nProteinLynx Global SERVER™ (PLGS) v2.5.3 (Waters, Altrincham, UK) was used to analyze the acquired ion mobility enhanced MSE spectra for protein identification as well as for the label-free relative protein quantification. Data processing and parameter setups were carried out as followed by [44]. The sequence database of the reference strain E. faecalis V583 (NCBI Reference Sequence: NC_004668.1) was used for database search. The false positive rate (FPR) was set to 4% with a randomized database, appended to the original one. The parameters for protein identification were made in such a way that a peptide was required to have at least one fragment ion match, a protein was required to have at least three fragment ion matches, and two peptide matches for identification. The peptides with 50% or more probability to be present in the mixture and detected with a score above 20, as calculated by the software were selected for proteomic analysis [45]. Data sets were normalized using the ‘internal standard-normalization’ function of PLGS and label-free quantitative analysis was performed by comparing the normalized peak area/intensity of identified peptides between the samples.\n\nAnalysis of physicochemical properties of identified proteome\nThe hydrophobicity of the identified proteins was calculated based on grand average hydropathy (GRAVY) value using the online GRAVY calculator (http://www.gravy-calculator.de/). The GRAVY score was calculated by dividing the sum of hydropathy values of all the amino acids by the protein length [46]. Isoelectric point (pI) and Molecular weight (MW) of the proteins was calculated using compute pI/MW tool of ExPASy Bioinformatics Resource Portal (https://web.expasy.org/compute_pi/).\n\nGene ontology analysis\nThe total proteins from all the stages were analyzed using the Venny tool 2.1.0 to identify proteins specific to the planktonic and biofilm stages. The total proteome of the planktonic stage was obtained by compiling the total proteins identified in the triplicates of two different time points of planktonic stage viz., 12 h and 24 h. Similarly, the total proteome of biofilm stages was compiled. The identified proteins were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID) functional Annotation Bioinformatic analyzer [47, 48]. DAVID generates Expression Analysis Systematic Explorer (EASE) score, a modified Fisher’s Exact P-value for each term and the GO and KEGG pathway terms with a P value ≤0.1 was considered as enriched. Average relative expression values of at least two biological replicates were used and a fold change of more than 30% (ratio of either \u003c 0.70 for downregulation and \u003e 1.3 for upregulation) was considered to be significantly altered levels of expression. Protein-protein interaction networks of differentially overexpressed proteins in biofilm stages were generated using STRING 10.5 (https://string-db.org/) [49] and a confidence view was generated by setting the filter to medium confidence (0.400).\n\nReal-time PCR analysis\nRNA was isolated from planktonic and biofilm stages of E. faecalis SK460 using RNeasy mini kit (Qiagen, Hilden, Germany) as per manufacturer’s protocol involving lysis with lysozyme and finally treated with DNase-1 (Sigma-Aldrich, Missouri, United States). Conventional PCR using 16SrRNA gene was performed to check the DNA contamination. cDNA was synthesized using Prime Script™ 1st strand cDNA synthesis kit according to the manufacturer’s protocol (Applied Biosystems, California, United States) and stored at − 20 °C.\nPrimers of selected genes were designed using PrimerQuest tool of Integrated DNA Technologies (Integrated DNA Technologies, Inc., California, US) and are shown in Additional file 1: Table S1. Power SYBR Green PCR Master Mix (Takara, Kusatsu, Japan) was used for real-time PCR in Applied Biosystems 7900HT Fast Real-Time PCR System as per the manufacturer’s suggestion. The reaction procedure was as follows: incubation at 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s, 49 °C for 15 s and 60 °C for 45 s. Three independent reactions were conducted with triplicates for each gene and reaction mixture without RNA template was used as negative control for each set of primers. Expression analysis of each gene was calculated based on at least two independent experiments. Melting curve analysis was done to verify primer specificity and relative expression values were calculated as 2-∆(CT Target - CT reference) with 23S rRNA gene as housekeeping gene.","divisions":[{"label":"title","span":{"begin":0,"end":7}},{"label":"sec","span":{"begin":9,"end":698}},{"label":"title","span":{"begin":9,"end":46}},{"label":"p","span":{"begin":47,"end":698}},{"label":"sec","span":{"begin":700,"end":1445}},{"label":"title","span":{"begin":700,"end":734}},{"label":"p","span":{"begin":735,"end":1445}},{"label":"sec","span":{"begin":1447,"end":1804}},{"label":"title","span":{"begin":1447,"end":1471}},{"label":"p","span":{"begin":1472,"end":1804}},{"label":"sec","span":{"begin":1806,"end":2204}},{"label":"title","span":{"begin":1806,"end":1839}},{"label":"p","span":{"begin":1840,"end":2204}},{"label":"sec","span":{"begin":2206,"end":2740}},{"label":"title","span":{"begin":2206,"end":2236}},{"label":"p","span":{"begin":2237,"end":2740}},{"label":"sec","span":{"begin":2742,"end":4110}},{"label":"title","span":{"begin":2742,"end":2768}},{"label":"p","span":{"begin":2769,"end":4110}},{"label":"sec","span":{"begin":4112,"end":4858}},{"label":"title","span":{"begin":4112,"end":4129}},{"label":"p","span":{"begin":4130,"end":4858}},{"label":"sec","span":{"begin":4860,"end":6062}},{"label":"title","span":{"begin":4860,"end":4873}},{"label":"p","span":{"begin":4874,"end":6062}},{"label":"sec","span":{"begin":6064,"end":6610}},{"label":"title","span":{"begin":6064,"end":6125}},{"label":"p","span":{"begin":6126,"end":6610}},{"label":"sec","span":{"begin":6612,"end":7986}},{"label":"title","span":{"begin":6612,"end":6634}},{"label":"p","span":{"begin":6635,"end":7986}},{"label":"title","span":{"begin":7988,"end":8010}},{"label":"p","span":{"begin":8011,"end":8532}}],"tracks":[{"project":"2_test","denotations":[{"id":"31253082-29356343-12221388","span":{"begin":474,"end":476},"obj":"29356343"},{"id":"31253082-16332862-12221389","span":{"begin":1185,"end":1187},"obj":"16332862"},{"id":"31253082-26025969-12221390","span":{"begin":2905,"end":2907},"obj":"26025969"},{"id":"31253082-24856245-12221391","span":{"begin":3902,"end":3904},"obj":"24856245"},{"id":"31253082-24856245-12221392","span":{"begin":4854,"end":4856},"obj":"24856245"},{"id":"31253082-24856245-12221393","span":{"begin":5167,"end":5169},"obj":"24856245"},{"id":"31253082-18936059-12221394","span":{"begin":5828,"end":5830},"obj":"18936059"},{"id":"31253082-10806384-12221395","span":{"begin":6422,"end":6424},"obj":"10806384"},{"id":"31253082-19131956-12221396","span":{"begin":7277,"end":7279},"obj":"19131956"},{"id":"31253082-19033363-12221397","span":{"begin":7281,"end":7283},"obj":"19033363"},{"id":"31253082-25352553-12221398","span":{"begin":7895,"end":7897},"obj":"25352553"}],"attributes":[{"subj":"31253082-29356343-12221388","pred":"source","obj":"2_test"},{"subj":"31253082-16332862-12221389","pred":"source","obj":"2_test"},{"subj":"31253082-26025969-12221390","pred":"source","obj":"2_test"},{"subj":"31253082-24856245-12221391","pred":"source","obj":"2_test"},{"subj":"31253082-24856245-12221392","pred":"source","obj":"2_test"},{"subj":"31253082-24856245-12221393","pred":"source","obj":"2_test"},{"subj":"31253082-18936059-12221394","pred":"source","obj":"2_test"},{"subj":"31253082-10806384-12221395","pred":"source","obj":"2_test"},{"subj":"31253082-19131956-12221396","pred":"source","obj":"2_test"},{"subj":"31253082-19033363-12221397","pred":"source","obj":"2_test"},{"subj":"31253082-25352553-12221398","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecbd93","default":true}]}]}}