Supporting Information
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Supporting Information Xiang et al. 10.1073/pnas.1321317111 SI Materials and Methods To infect mice, bacterial strains were obtained from the First Antibodies, ELISA Kits, PCR primers, Inhibitors, and siRNAs. Rabbit Affiliated Hospital of Kunming Medical University (China). Two polyclonal antibody against βγ-crystallin fused aerolysin-like different species were used: (i) Escherichia coli ATCC 25922 and protein and trefoil factor complex (βγ-CAT) was produced as (ii) Staphylococcus aureus ATCC 25923. previously described (1). Rabbit polyclonal antibody against Bombina maxima IL-1β and Caspase-1 p20 subunit were produced Bioinformatic Analysis. Protein domain searches were performed by as previously described (2). Rabbit polyclonal antibody against using Motif Scan (http://hits.isb-sib.ch/cgi-bin/PFSCAN) software. caspase-1 p10 (sc-515), IL-1β (sc-7884), NLRP3 (sc-134306), The schematic structure of βγ-CAT was drawn with the DOG 2.0 NLRC4 (sc-99055) ASC (sc-172054), and β-actin (sc-1615) were program (5). Multiple alignments of the amino acid sequences of purchased from Santa Cruz Biotechnology. Rabbit polyclonal an- aerolysin domains were done with Vector NTI 11.5. The aligned tibody against lamp-1 (ab24170) was purchased from Abcam. sequences are vertebrate-derived aerolysin domain-containing Mouse monoclonal antibody against lamp-1 (ab25630) was pur- proteins (Fig. S1A). Prediction of βγ-CAT aerolysin domain 3D chased from Abcam. Rabbit polyclonal antibody against Cathepsin structure was performed by using the I-Tasser server (6, 7). The B (12216-1-AP) was purchased from Proteintech Group. FITC- 3D structure was obtained by multiple threading using the conjugated anti-mouse Gr-1 antibody (108405) and PE-conjugated I-Tasser server (8). A confidence score (C-score) was used to anti-mouseF4/80 antibody (123109) were purchased from Biol- estimate the quality of the computed model. A high C-score egend. means a model with high confidence, which is often in the range of ELISA kits for detecting mouse IL-1β,CXCL1,andCXCL2were −5 to 2. And a C-score greater than −1.5 means a prediction of purchased from R&D Systems. A CytoTox 96 Non-Radioactive Cy- correct folding. The 3D structure of βγ-CAT aerolysin domain was totoxicity Assay kit for LDH assaying was purchased from Promega. compared and presented with aerolysin-like proteins with the Caspase-1 inhibitor Ac-YVAD-cmk (sc-300323) was purchased software PyMOL. from Santa Cruz Biotechnology. Nocodazole, bafilomycin A1, cy- tochalasin D, and CA074-me were purchased from Sigma-Aldrich. Tissue Samples, RNA Extraction, and RT-PCR. Frog tissues were siRNA targeting NLRP3 (sc-45470), NLRC4 (sc-60329), ASC sampled from intact adult frogs. Total RNAs of these tissues were (sc-37282), and Caspase-1 (sc-29922) were purchased from Santa purified by using an RNA Easy kit (QIAGEN) according to the Cruz biotechnology. manufacturer’s instructions. RNA yields and quality were checked by using agarose gel electrophoresis and a spectrophotometer Purification and Characterization of βγ-CAT. βγ-CAT was purified (Thermo). Equal amounts of total RNA were used to synthesize from the skin secretions of B. maxima as previously described the cDNA (TaKaRa) according to the protocol. The synthesized (1). Briefly, lyophilized B. maxima skin secretions were separated first cDNA strand was used as the template for semiquantitative with a DEAE Sephadex A-50 column at pH 7.3. The peak III of or quantitative PCR. The primers used are listed in Table S4. DEAE-Sephadex A-50 column was further separated with a Sephadex G-100 column. The peak II of Sephadex G-100 col- In Vivo Frog Experiments. Bacteria or βγ-CAT in 100 μL of 0.9% umn was applied on an AKTA Mono-Q HR5/5 anion ion ex- NaCl was injected into the peritoneum of the frog. At the in- changecolumnandresultedinseparation of several protein dicated time, the frog was anesthetized with diethyl ether and peaks, and peak I was purified βγ-CAT. The purity of βγ-CAT then intraperitoneally injected (sterile syringe with a 20-G needle) was analyzed with native polyacrylamide gel electrophoresis with 2 mL of 0.9% NaCl prewarmed to 22 °C. The frog was then (PAGE) or SDS/PAGE with silver staining, as shown in Fig. S5. gently massaged, and the injected solution was collected into a sterile tube. The harvested peritoneal lavage fluids were used for Cell Lines. Frog (B. maxima) peritoneal cells were used for in vitro bacterial counting. Cells were separated by centrifugation at 600 × g experiments. The procedure for isolating frog peritoneal cells for 5 min for flow-cytometry analysis. Supernatants were obtained was as previously described (3). Mouse bone marrow-derived by centrifugation at 10,000 × g for 10 min and then dried by ly- macrophages (BMDMs) were isolated from the femurs of mice ophilization and reconstituted to 100 μL. The suspension was used and cultured as described by Riteau, et al. (4). for Western blotting analysis. Bacterial Strains for Infection. To infect frogs, bacteria were iso- For bacterial counting, the peritoneal lavage fluids were serially lated from the skin of live wild frogs. We collected B. maxima diluted by using the 10-fold dilution technique. Using the spread- μ postmetamorphic individuals during the rainy seasons of 2011 plate method, 100 L of each dilution was plated out in duplicate and 2012 in Chuxiong, Yunnan Province, China. Before sam- sets of standard I nutrient medium. The plates were then in- pling, each individual was rinsed three times with 100 mL of cubated at 30 °C for 18 h, and the distinct colonies that de- sterile water to remove transient bacteria. Bacteria from frog veloped were counted. The bacterial numbers were expressed as skin were sampled using sterile cotton-tipped swabs and cultured log colony-forming units (cfu/mL). The same procedure was onto a Standard I nutrient medium (Merck) at 30 °C for up to 72 h. performed for the control. DNA from these bacteria was then extracted from pure cultures To identify frog peritoneal exudate cells (PECs) by flow with a TIANamp Bacteria DNA Kit and then amplified by PCR cytometry, PECs were collected by peritoneal lavage after 4 h, as using universal bacterial 8F and 1492R 16SrRNA primers (8F, described above in the first paragraph. Propidium iodide (Sigma) AGAGTTTGATCMTGGCTCAG; 1492R, GGYTACCTTGTT- was added at a concentration of 1 μg/mL to exclude dead cells ACGACTT). The products were sent to BGI Shenzhen for sequencing and debris. Flow-cytometry analysis was based on forward and and were identified according to similarity with GenBank (www.ncbi. side scatter on a FACSCalibur flow cytometer (Becton Dick- nlm.nih.gov/) and the Ribosomal Database Project (RDP-II) (http:// inson) as previously reported for zebrafish (9). Wright’s stain was rdp.cme.msu.edu/taxomatic/main.spr). The results are shown in Table also used to identify the PEC type that exhibited the greatest S2. A Comamonas sp. strain was used for all frog infections. change. Xiang et al. www.pnas.org/cgi/content/short/1321317111 1of7 Stimulation of Frog B. maxima Peritoneal Cells in Vitro. Frog The peritoneal lavage was harvested and collected in sterile (B. maxima) peritoneal cells were isolated as described above in tubes, and eight serial 10-fold dilutions were made of each sample the section In Vivo Frog Experiments. For short-term culture, the of the homogenates. Then, 100 μL of each dilution was plated cells were cultured in DMEM/F12 (HyClone) supplemented onto blood agar plates. The plates were incubated at 37 °C for with 10% FBS (HyClone). Peritoneal cells acquired from more 20 h; then, the cfus were counted and corrected for the dilution than 30 frogs were mixed to perform all of the in vitro experi- factor. The threshold value of the method was 100 cfus. ments. The cells (4 × 105 cells per mL) were primed with LPS (100 ng/mL) for 2 h, washed with PBS twice, and then cultured in Flow-Cytometry Analysis of Mice Peritoneal Exudate Cells. The PECs the presence or absence of various agents assayed (βγ-CAT or (peritoneal exudate cells) were washed with 1640 medium and pharmacological inhibitors) for the indicated time period. Finally, blocked with 5% normal mouse serum for 10 min at room tem- the cells were lysed for Western blotting, as previously described perature. After the cells were washed with 1640 medium, FITC- (10), and the supernatants were concentrated to 1/20th of the conjugated anti–Gr-1 (Biolegend) and PE-conjugated anti-F4/80 original volume for Western blotting analysis. (Biolegend) were added, and the cells were incubated at room temperature for 15 min. The cells were then analyzed on a FACS- Western Blotting. Prepared protein samples were electrophoresed Calibur flow cytometer (BD), and the Flowjo software was used to in an SDS-polyacrylamide gel and transferred onto a PVDF analyze the data. membrane. The membrane was subsequently blocked with 3% BSA and incubated with the appropriate primary and secondary Antimicrobial Assays. The direct amtimicrobial activity was assayed antibodies. Protein bands were visualized with SuperSignal chem- as previously described (14). Standard bacterial and fungal strains iluminescence reagents (Pierce), as previously described (11). Bands used in antimicrobial assays, Gram-positive bacterial strains were semiquantified with ImageJ software. For data presented as S. aureus (ATCC2592) and Bacillus megatherium, Gram-negative bar graphs, control samples are set as 100%, and percent of change bacterial strains E. coli (ATCC25922), Bacillus pyocyaneus in intensity is reported. Data are representative of at least two to (CMCCB10104), Bacillus dysenteriae, and Klebsiella pneumoniae, three independent experiments. and fungal strains Candida albicans (ATCC2002), Aspergillus flavus (IFFI4015), and Penicillium uticale (IFFI2001) were ob- Native-PAGE and Western Blotting Detection of βγ-CAT.