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Incheon, South Korea

Jung P.E.,Seoul National University | Fong J.J.,Seoul National University | Park M.S.,Seoul National University | Oh S.-Y.,Seoul National University | And 2 more authors.
Journal of Microbiology and Biotechnology | Year: 2014

White-rot fungi of the genus Bjerkandera are cosmopolitan and have shown potential for industrial application and bioremediation. When distinguishing morphological characters are no longer present (e.g., cultures or dried specimen fragments), characterizing true sequences of Bjerkandera is crucial for accurate identification and application of the species. To build a framework for molecular identification of Bjerkandera, we carefully identified specimens of B. adusta and B. fumosa from Korea based on morphological characters, followed by sequencing the internal transcribed spacer region and 28S nuclear ribosomal large subunit. The phylogenetic analysis of Korean Bjerkandera specimens showed clear genetic differentiation between the two species. Using this phylogeny as a framework, we examined the identification accuracy of sequences available in GenBank. Analyses revealed that many Bjerkandera sequences in the database are either misidentified or unidentified. This study provides robust reference sequences for sequence-based identification of Bjerkandera, and further demonstrates the presence and dangers of incorrect sequences in GenBank. © 2014 by The Korean Society for Microbiology and Biotechnology.

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Complementary DNA (cDNA) for human GSDMD was amplified from reverse-transcribed cDNA of HT-29 cells; cDNAs for human GSDMB, human GSDMC and mouse Gsdma3 were synthesized by our in-house gene synthesis facility; cDNAs for human GSDMA and mouse Gsdmd were obtained from Vigene Biosciences (CH892815) and OriGene (MC202215), respectively. The gasdermin cDNAs were inserted into a modified pCS2-3×Flag vector for transient expression in 293T cells and the pWPI lentiviral vector with an N-terminal 2×Flag–HA tag or the FUIGW vector with an N-terminal Flag tag for stable expression in HeLa and iBMDM cells. For recombinant expression in E. coli, the cDNAs were cloned into a modified pET vector with an N-terminal SUMO tag. Truncation mutants of the gasdermins were constructed by the standard PCR cloning strategy and inserted into the pCS2 vector with indicated tags. Expression plasmids for caspase-1, 4, 5 and 11 were previously described4, 9, the caspase-9 plasmid was a gift from X. Wang (National Institute of Biological Sciences, Beijing). cDNAs for human CASP2 and mouse Casp8 are from the Life Technologies Ultimate ORF collection and OriGene (MC200404), respectively. Point mutations were generated by the QuickChange Site-Directed Mutagenesis Kit (Stratagene). All plasmids were verified by DNA sequencing. Antibodies for caspase-1 p10 (sc-515), Myc epitope (sc-789) and GSDMD (sc-81868) were obtained from Santa Cruz Biotechnology. Other antibodies used in this study include anti-HA (MMS-101P, Covance), anti-Flag M2 (F4049), anti-actin (A2066) and anti-tubulin (T5168) (Sigma-Aldrich), rat monoclonal caspase-11 17D9 (NB120-10454, Novus Biologicals), anti-caspase-3 (#9662) and caspase-7 (#12827) (Cell Signaling Technology), IL-1β (3ZD; Biological Resources Branch, National Cancer Institute) and the antibody for detecting endogenous GSDMD (NBP2-33422, Novus Biologicals). Ultrapure LPS from E. coli O111:B4 and poly(dA:dT) were purchased from InvivoGen. LPS (L4524, for priming), TNFα and cycloheximide were purchased from Sigma-Aldrich. SMAC mimetic and the pan-caspase inhibitor zVAD are gifts from the laboratory of X. Wang (National Institute of Biological Sciences, Beijing). Nigericin was purchased from Calbiochem. Recombinant p20/p10 active caspase proteins (caspase-1/2/4/8/9) and lipid A (ALX-581-200-L001) were obtained from Enzo Life Sciences. Cell culture products are from Life technologies and all other chemicals used are Sigma-Aldrich products unless noted. HeLa, HT-29 and 293T cells were obtained from ATCC. C57BL/6 mice-derived wild-type and Tlr4−/−iBMDM cells were kindly provided by K. A. Fitzgerald (University of Massachusetts Medical School, United States) and A. Ding (Weill Cornell Medical College, United States), respectively, and used in our previous studies4, 6, 30. All the cell lines are well-established, commonly used and frequently checked by virtue of their morphological features and functionalities, but have not been subjected to authentication by short tandem repeat (STR) profiling. All the cell lines have been tested to be mycoplasma-negative by the commonly used PCR method. iBMDM, HeLa and 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM); HT-29 cells were grown in McCoy’s 5a modified medium. All media were supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 2 mM l-glutamine. All cells were grown at 37 °C in a 5% CO incubator. Transient transfection of HeLa and 293T cells was performed using the JetPRIME (Polyplus Transfection) or Vigofect (Vigorous) reagents by following the manufacturers’ instructions. For stable expression, lentiviral plasmids harbouring the desired gene were first transfected into 293T cells together with the packing plasmids pSPAX2 and pMD2G with a ratio of 5:3:2. The supernatants were collected 48 h after transfection and used to infect HeLa or iBMDM cells for another 48 h. GFP-positive infected cells were sorted by flow cytometry (BD Biosciences FACSAria II). For siRNA knockdown, 0.5 μl of 20 μM siRNA together with 0.8 μl of INTERFERin reagents (Polyplus Transfection) were used for reverse transfection of iBMDM cells in the 96-well plate format; 5 μl of 20 μM siRNA and 10 μl of INTERFERin reagents were used to transfect HeLa cells in the 6-well plate format. The knockdown was performed for 60 h before subsequent analyses. The knockdown efficiency was assessed by quantitative real-time PCR (qRT–PCR) analyses as previously described4. All siRNA oligonucleotides were synthesized by our in-house facility using the sequences from the MISSION shRNA library (Broad Institute, United States) and their sequences are listed in Supplementary Table 1. Activation of the canonical caspase-1 inflammasomes (the NLRP3, NAIP–NLRC4 and AIM2 inflammasomes) and the non-canonical caspase-11 inflammasome by LPS was performed using the protocols that have been detailed in our previous publications4, 6, 9, 20. For bacteria-induced inflammasome activation, S. typhimurium (wild-type and ΔsipD), B. thailandensis (wild-type and ΔbipB), EPEC (wild-type and ΔescN) were used to infect iBMDM cells and S. typhimurium (wild-type and ΔsifA) was used to infect HeLa cells, as described previously4, 9. To examine cell death morphology, cells were treated as indicated in the 6-well plates (Nunc products, Thermo Fisher Scientific Inc.) for static image capture or in glass-bottom culture dishes (MatTek Corporation) for live imaging. Static bright field images of pyroptotic cells were captured using an Olympus IX71 or a Zeiss Pascal Confocal microscope. The image pictures were processed using ImageJ or the LSM Image Examiner program. Live images of cell death were recorded with the PerkinElmer UltraVIEW spinning disk confocal microscopy and processed in the software Volocity. All image data shown were representative of at least three randomly selected fields. The lentiviral gRNA plasmid library for genome-wide CRISPR-Cas9 screen was obtained from Addgene (#50947)33; amplification of the library and preparation of the lentivirus were performed following the protocol provided by Addgene. In brief, 1 μl of library DNA (10 ng μl−1) was used to transform 25 μl of electrocompetent E. coli (TaKaRa). Transformed colonies (>6 × 107) were scraped off the Luria-Bertani (LB) plates into the media, and plasmids were exacted by using the GoldHi EndoFree Plasmid Maxi Kit (CWBIO). To prepare the virus library, 293T cells in the 15-cm dish were transfected with 25 μg of library DNA together with 15 μg of psPAX2 and 10 μg of pMD2.G. Eight hours after transfection, the media were changed to high-serum DMEM (20% FBS with 25 mM HEPES). Another 40 h later, the media (from twenty 15-cm dishes of transfected cells) were collected and centrifuged at 3,000 r.p.m. for 10 min. The supernatant was filtered through a 0.22-μm membrane and aliquots of 30 ml were stored at −80 °C. In the pilot experiment, the volume of the lentivirus library required for achieving an MOI of 0.3 for infecting the target cell line was determined in the 12-well plate format. For the large scale screen, Tlr4−/− iBMDM cells stably expressing the Cas9 protein were seeded in the 15-cm dish (2 × 106 cells in 20 ml media per dish) and a total of 2 × 107 cells were infected with the gRNA lentivirus library. Sixty hours after infection, cells were re-seeded at a density of 1 × 105 ml−1 in fresh media supplemented with 5 μg ml−1puromycin (to eliminate non-infected cells). After 6 to 8 days, ~3 × 108 cells from five culture dishes were electroporated with LPS to trigger caspase-11-mediated pyroptosis9, or stimulated with LFn–BsaK/protective antigen (PA) to induce caspase-1-mediated pyroptosis4; another 3 × 108 cells were left untreated as the control sample. Each screen was repeated another time. Surviving cells were collected after growing to near 90% confluence and lysed in the SNET buffer (20 mM Tris-HCL(pH 8.0), 5 mM EDTA, 400 mM NaCl, 400 µg ml−1 Proteinase K and 1% SDS). Genomic DNAs of each group of cells were prepared by using the phenol-chloroform extraction and isopropanol precipitation method. The DNA was dissolved in H O (4–5 μg μl−1) and used as the templates for amplification of the gRNA. The gRNAs were amplified by a two-step PCR method using the Titanium Taq DNA polymerase (Clontech Laboratories). In the first step, six 50-μl PCR reactions (each containing 50 μg of genomic DNA template) were performed with the forward primer 50bp-F and the reverse primer 50bp-R; the PCR program used is 94 ° C for 180 s, 16 cycles of 94 ° C for 30 s, 60 ° C for 10 s and 72 ° C for 25 s, and a final 2-min extension at 68 ° C. Products of the first-step PCR were pooled together and used as the template for the second-step PCR. Also six 50-μl PCR reactions (each containing 1 μl of the first-step PCR product) were performed with the forward primer Index-F and one of the reverse primers (Index-R1 to R6): Index-R1 for the control sample, Index-R2 for the replicate control sample, Index-R3 for the caspase-11 screen, Index-R4 for the replicate caspase-11 screen, Index-R5 for the caspase-1 screen and Index-R6 for the replicate caspase-1 screen. The PCR program used is 94 ° C for 180 s, 18 cycles of 94 ° C for 30 s, 54 ° C for 10 s and 72 ° C for 18 s, and a final 2-min extension at 68 ° C. Products of the second-step PCR reactions were subjected to electrophoresis on the 1.5% agarose gel; the DNAs (the 310-bp band) were extracted and sequenced at the HiSeq2500 instrument (Illumina) by using the 50-bp single-end sequencing protocol. The first 19 nucleotides from each sequencing read are the gRNA sequence recovered from the library. The frequency of each gRNA was obtained by dividing the gRNA read number by the total sample read number; the fold of enrichment was calculated by comparing the frequency of each gRNA in the experiment sample with that in the control sample. Sequences for all the primers are listed in Supplementary Table 1. The top 50 gRNA hits from the caspase-11 screen were examined and 18 genes that are conserved in human and mouse were identified for siRNA knockdown validation in HeLa cells. HeLa cells expressed caspase-4 but not caspase-5 (Extended Data Fig. 1b) and respond robustly to cytosolic LPS9, 10. For each gene, a mixture of two independent siRNAs was used and the knockdown efficiency of 12 of those having mRNA expression in HeLa cells was confirmed. Importantly, only siRNAs targeting human GSDMD, besides the control CASP4-targeting siRNA, could efficiently block cytosolic LPS-induced pyroptosis (Extended Data Fig. 1c). When assayed individually, the two GSDMD-targeting siRNAs both showed potent inhibition of HeLa cell pyroptosis (Extended Data Fig. 1d). Human codon-optimized Cas9 (hCas9) and GFP-targeting gRNA-expressing plasmids (gRNA_GFP-T1) were purchased from Addgene. The 19-bp GFP-targeting sequence in the gRNA vector was replaced with the sequence targeting the desired gene by QuickChange site-directed mutagenesis. The target sequences used are AGCATCCTGGCATTCCGAG for mouse Gsdmd and TTCCACTTCTACGATGCCA for human GSDMD. To construct the knockout cell lines, 1 μg of gRNA-expressing plasmid, 3 μg of hCas9 plasmid and 1 μg of pEGFP-C1 vector were co-transfected into 6 × 106 iBMDM or HeLa cells. Three days later, GFP-positive cells were sorted into single clones into the 96-well plate by flow cytometry using the BD Biosciences FACSAria II or the Beckman Coulter MoFlo XDP cell sorter. Single clones were screened by the T7 endonuclease I-cutting assay and the candidate knockout clones were verified by sequencing of the PCR fragments as described previously9. The PCR primers used are listed in Supplementary Table 1. All animal experiments were conducted following the Ministry of Health national guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee at National Institute of Biological Sciences. The Gsdmd knockout mice were generated by co-microinjection of in vitro-translated Cas9 mRNA and gRNA into the C57BL/6 zygotes. Founders with frameshift mutations were screened with T7E1 assay and validated by DNA sequencing. Founders were intercrossed to generate biallelic Gsdmd−/− mice. The gRNA sequence used to generate the knockout mice is AGCATCCTGGCATTCCGAG. C57BL/6 wild-type mice were from Vital River Laboratory Animal Technology Co. and Casp1/11−/− mice were obtained from the Jackson Laboratory. Ripk3−/− mice were a gift from X. Wang (National Institute of Biological Sciences, Beijing). Primary BMDM cells were prepared from 6-week-old male mice (C57BL/6 background) by following a standard procedure as previously described6. For each experimental design, at least two mice were chosen to prepare the BMDM cells for assaying the inflammasome responses; the mice were not randomized and the investigators were not blinded. Relevant cells were treated as indicated. Cell death was measured by the LDH assay using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Cell viability was determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega). To measure IL-1β release, primary BMDM cells were primed with LPS (1 μg ml−1) for 2 h and released mature IL-1β was determined by using the IL-1β ELISA kit (Neobioscience Technology Company). To obtain recombinant human GSDMD, E. coli BL21 (DE3) cells harbouring pET28a-His -SUMO-GSDMD were grown in LB medium supplemented with 30 μg ml−1 kanamycin. Protein expression was induced overnight at 18 ° C with 0.4 mM isopropyl-B-d-thiogalactopyranoside (IPTG) after OD reached 0.8. Cells were harvested and resuspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 20 mM imidazole and 10 mM 2-mercaptoethanol. The His -SUMO-tagged protein was first purified by affinity chromatography using Ni-NTA beads (Qiagen) and the SUMO tag was removed by overnight ULP1 protease digestion at 4 °C. The cleaved GSDMD was further purified by HiTrap Q ion-exchange and Superdex G200 gel-filtration chromatography (GE Healthcare Life Sciences). To obtain the constitutive-active caspase-11 p20/p10 tetramer, cDNAs encoding the p20 large and p10 small subunit were cloned into pET21a with a 6×His tag fused to the C terminus of the p10 subunit. The two subunits were separately expressed in E. coli with 1 mM IPTG induction for 4 h at 30 ° C. Bacteria collected from 1-l culture were resuspended and lysed in 100 ml of lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol) by sonication. Inclusion bodies, obtained by centrifugation of the lysates at 18,000 r.p.m. for 1 h, was washed with 50 ml of Buffer 1 (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 M guanidinium hydrochloride (GdnCl) and 0.1% Triton X-100) and 50 ml of Buffer 2 (50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 1 M GdnCl) twice for each buffer. The washed inclusion bodies were solubilized by stirring in 6 ml of the solubilization buffer containing 6.5 M GdnCl, 25 mM Tris-HCl (pH 7.5), 5 mM EDTA and 100 mM DTT overnight at room temperature. To obtain active p20/p10 tetramers by refolding, 12 ml of above solubilized inclusion body solution containing denatured large and small subunits (molecular ratio, 1:2) were drop-by-drop diluted in 500 ml of refolding buffer (100 mM HEPES, 100 mM NaCl, 100 mM sodium malonate, 20% sucrose, 0.1 M NDSB-201 and 10 mM DTT) and then gently stirred in a nitrogen atmosphere at 16 °C overnight. Protein aggregates were removed by centrifugation at 4,000 r.p.m. for 20 min and the refolded protein supernatants were concentrated and dialysed against a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10 mM 2-mercaptoethanol. The protein was affinity-purified by the Ni-NTA beads and further purified by the Superdex G200 gel-filtration chromatography. Expression and purification of recombinant LFn–BsaK and LFn–FliC proteins were described previously4. Recombinant full-length caspase-4 and caspase-11 were expressed and purified from insect cells also as previously described9. Recombinant PreScission protease (PPase) proteins are routine lab stocks. For cleavage by the p20/p10 tetramers of active caspase, 5 μg of purified recombinant GSDMD was incubated with 1 unit of caspase-1, 2, 4, 8 and 9 or 0.1 μg of caspase-11 in a 25-μl reaction containing 50 mM HEPES (pH 7.5), 3 mM EDTA, 150 mM NaCl, 0.005% (vol/vol) Tween-20 and 10 mM DTT. The reaction was incubated for 60 min at 37 ° C. For cleavage by LPS-activated caspase-4/11, the full-length caspase proteins purified from insect cells were first incubated with LPS, lipid A or MDP for 30 min at 30 ° C; 5 μg of purified recombinant GSDMD was then reacted with the ligand-incubated caspases at 37 ° C for 9 min. Cleavage of GSDMD was examined by Coomassie blue staining of the reaction samples separated on the SDS–PAGE gel.

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