Materials

Ac-DEVD-CHO was purchased from Selleck Chemicals. ATP2B1 antibody (#ab3528) was purchased from Abcam. Necrosulfonamide (NSA) and SGMS1 antibodies (#ABC732) were purchased from Merck Millipore. β-actin antibody (#4970) was purchased from Cell Signaling Technology, Inc. α-hemolysin, BAPTA-AM, Chloroquine, 2-hydroxypropyl-β-cyclodextrin (HPβCD), EDTA, methyl-β-cyclodextrin (MβCD), sea nettle (Chrysaora quinquecirrha) venom (SN), sphingomyelinase and streptolysin O (SLO) were purchased from Sigma-Aldrich. Caloxin 1B3 (TIPKWISIIQALRGGGSK-amide) and 2A1 peptide (VSNSNWPSFPSSGGG-amide) was prepared by custom synthesis from Mimotopes.

C. fleckeri venom preparation

C. fleckeri were collected from coastal waters near Darwin Harbour (Northern Territory, Australia). Nematocysts were extracted from excised jellyfish tentacles52. The box jellyfish venom was extracted from purified nematocysts, through a process of repeatedly exposing the nematocysts to 0.5 mm glass beads in a bead beater, followed by centrifugation at 3000 rpm for 1 min53. The suspended venom was aspirated, then lyophilised and stored at −80 °C.

Cell culture

Human HAP1 cells were generously provided by Dr. Thijn R. Brummelkamp54. HeLa cells were gift from Dr. Adam R. Cole, Garvan Institute. HAP1 and HeLa cells were cultured in Medium IMDM (Sigma-Aldrich) and DMEM (Sigma-Aldrich), respectively, containing 10% bovine calf serum (BCS; Hyclone Laboratories), 1x GlutaMAX, 100U/ml penicillin G and 100 g/ml streptomycin (Thermo Fisher Scientific) in a humidified atmosphere of 5% CO 2 –95% air at 37 °C and were tested for the absence of mycoplasma contamination.

Mice

All mice in this study were male FVB/NJ mice aged 10–15 weeks obtained from the Animal Resource Centre, WA, Australia. All experiments were approved by the Animal Ethics Committee at the University of Sydney under protocol 1196. Mice were housed in a specific pathogen-free facility on a 12-hour light–dark cycle and standard chow, water and enrichment were provided ad libitum. All in vivo behavioural assays were performed blind to treatment by a single male investigator. Assignment to treatment groups was performed randomly by an investigator blind to the behaviour and health status of the animals.

Cell viability assay

Trypsinised cells (3 × 104) were seeded in each well of a 96-well plate. After 24 h, various concentrations of jellyfish venom were added, and the cells were incubated for an additional 24 h. After incubation, the medium was aspirated from each well and add 150 μl of fresh medium containing a 0.002% solution of resazurin (Sigma-Aldrich) was added to the wells and incubated for 4 h at 37 °C. The absorbance was measured at 570 nm using a microplate spectrophotometer (FLUOstar Omega, BMG Labtech).

Lactate dehydrogenase (LDH) activity in supernatants of cells was assessed according to the protocol of the manufacturer (Thermo Fisher Scientific). Cell death was also determined by intracellular ATP levels using CellTiter-Glo Luminescent Cell Viability Assay (Promega) following the manufacturer’s protocols.

Haemolysis

Mouse red blood cell (RBC) lysis was measured as described47 with modification. Blood was collected by terminal cardiac puncture under isoflurane anaesthesia (2–3%) in MiniCollect EDTA coated tubes (Greiner-Bio-One). Collected blood was washed four times in sterile PBS and recovered by centrifugation at 500 × g for 5 min at 4 °C. Diluted RBC (0.5% in PBS) were treated in triplicate in a 96-well plate and incubate for 1 h at 37 °C. Triplicates of 1% Triton X-100 and PBS alone were used as positive and negative control, respectively. Samples were centrifuged for 5 min at 500 × g to pellet intact RBC, and supernatants were transferred to another 96-well plate. The absorbance of released haemoglobin was measured at 540 nm using a microplate spectrophotometer.

Intracellular calcium measurement

A Fluo-8 Calcium Flux Assay kit (Abcam) was used to measure intracellular calcium influx on a FLUOstar Omega microplate reader following the manufacturer’s protocols. Briefly, trypsinised HAP1 cells (3 × 104) were seeded in each well of a 96-well plate. After 24 h, plates were washed twice in Ca2+-free HBSS supplemented with HEPES buffer (pH 7.2), and then the growth medium was replaced with 100 μl/well of the Fluo-8 dye solution. The plate was incubated at 37 °C for 30 min and then for another 30 min in the dark at room temperature. The loaded cells were then placed in the measurement position in the microplate reader. Changes in fluorescence from the Fluo-8 dye quantify changes in intracellular Ca2+ concentrations (excitation/emission 490/525 nm) after treatment with box jellyfish venom. Ca2+ influx was measured up to 45 min.

Lentivirus production

To generate lentivirus, the human lentiCRISPRv2 plasmid library (Addgene 1000000048) was co-transfected with packaging plasmids pCAG-VSVG and psPAX2 (Addgene plasmids 35616 and 12260, respectively). Briefly, a T-75 flask of 80% confluent 293LTV cells (Cell Biolabs) was transfected in OptiMEM (Thermo Fisher Scientific) using 8 μg of the lentiCRISPRv2 plasmid library, 4 μg pCAG-VSVG, 8 μg psPAX2, 2.5 μg pAdVantage (Promega), 30 μl of P3000 Reagent (Thermo Fisher Scientific), and 30 μl of Lipofectamine 3000 (Thermo Fisher Scientific). Cells were incubated overnight and then media was changed to DMEM (Sigma-Aldrich) with 10% BCS and 1x GlutaMAX (Thermo Fisher Scientific). After 48 h, viral supernatants were collected and centrifuged at 2000 rpm for 10 min to get rid of cell debris. The supernatant was filtered through a 0.45μm ultra-low protein binding filter (Merck Millipore). Aliquots were stored at −80 °C.

Cell transduction using the GeCKO v2 library

HAP1 cells were transduced with the GeCKO v2 library by spinfection. Briefly, 2 × 106 cells per well were plated into a 12-well plate in IMDM media supplemented with 10% BCS and 8 μg/ml polybrene (Sigma-Aldrich). A titrated virus was added in each well along with a no-transduction control. The plate was centrifuged at 2000 rpm for 1 h at 37 °C. After the spin, cells were incubated overnight and then enzymatically detached using TrypLETM Express (Thermo Fisher Scientific). Cells were counted and each well was split into duplicate wells. One replicate treated with 1 μg/ml puromycin (Thermo Fisher Scientific) for 3 days. Percent transduction was determined as cell count from the replicate with puromycin divided by cell count from the replicate without puromycin multiplied by 100. The virus volume yielding a MOI (multiplicity of infection) approximately to 0.4 was used for large-scale screening.

HAP1 jellyfish venom resistance screen

1 × 108 HAP1 cells were transduced as described above using 12-well plates with 2 × 106 cells per well. Puromycin was added to the cells 24 h post transduction and maintained for 7 days. Cells were pooled together into larger flasks after 3 days incubation of puromycin. On day 7, cells were split into treatment conditions in duplicate with a minimum of 2.5 × 107 cells per replicate. Two replicates were cultured in IMDM supplemented with 1 μg/ml jellyfish venom, and one replicate was culture in regular IMDM media. Replicates were either passaged or fresh media was added every 2–3 days. In parallel, untransduced HAP1 cells were treated with 1 μg/ml jellyfish venom to ensure the venom was cytotoxic in each case. Cells were harvested after 14 days of the treatment for genomic DNA analysis.

Genomic DNA sequencing

Genomic DNA (gDNA) extracted from harvested cells with a Blood & Cell Culture Midi kit (Qiagen) was used for PCR reactions as described previously20. Primers used to amplify lentiCRISPR v2 sgRNAs for the first PCR are: sense, 5′-AAT GGA CTA TCA TAT GCT TAC CGT AAC TTG AAA GTA TTT CG-3′ and antisense, 5′-TCT ACT ATT CTT TCC CCT GCA CTG TTG TGG GCG ATG TGC GCT CTG-3′.

A second PCR was performed to attach Illumina adaptors and to barcode samples. The second PCR was done in a 100 μl reaction volume using 5 μl of the first PCR product. Primers for the second PCR include both a variable length sequence to increase library complexity and an 6 bp barcode for multiplexing of different biological samples: sense, 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T (1–9 bp variable length sequence) (6 bp barcode) tct tgt gga aag gac gaa aca ccg-3′ and antisense, 5′-CAA GCA GAA GAC GGC ATA CGA GAT AAG TAG AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT tct act att ctt tcc cct gca ctg t-3′.

Amplification was carried out with 18 cycles for the first PCR and 24 cycles for the second PCR. PCR products from the second PCR were gel extracted, quantified, mixed and sequenced using a HiSeq 2500 (Illumina). The sgRNA sequences against specific genes were recovered after removal of the tag sequences using the Checkout [http://100bp.wordpress.com] and cutadapt (ver. 1.12).

Enrichment of sgRNAs and genes were analysed using MAGeCK55 (Ver.0.5.6) by comparing read counts from cells after jellyfish venom selection with counts from matching unselected cell population to obtain a p-value. P < 0.01 was considered statistically significant.

Gene validation

To validate the candidate genes from screening, sgRNAs from the parent library were cloned into pLentiCRISPRv2 (Addgene plasmid 52961). The control sgRNA was used from the parent library. Lentiviruses were produced as described above and transduced HAP1 or HeLa cells were selected with 1 μg/ml puromycin 24 h post-infection. Two weeks later, puromycin was removed, and cells were allowed to recover for three additional days before analysis. Gene disruption efficiency was verified by western blot. The sequences of the sgRNAs used are in Table S4.

Gene ontology (GO) and pathway enrichment analysis

GO terms and REACTOME pathways enriched in the screen were analysed using the ConsensusPathDB56. The GO level 3–5 categories and a p-value cut-off of 0.05 were selected. The minimum overlap with the input list for pathway analysis was set at two proteins, with p < 0.05.

Western blot analysis

Cells were harvested in lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EDTA, and 0.1% SDS] containing protease inhibitor cocktail (Sigma-Aldrich), and the protein concentrations were determined using the BCA Protein Assay (Thermo Fisher Scientific). The proteins (20 µg) were electrophoresed on 10% SDS-polyacrylamide gels, transferred to PVDF membranes (Amersham Bioscience), and incubated with specific primary antibodies (1:1000 for all primary antibodies) at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; #31460 from Thermo Fisher Scientific) for 1 h and were then visualised with enhanced chemiluminescent substrate (Thermo Fisher Scientific). Uncropped images are shown in Supplementary Fig 5.

Injections

250 ug of Jellyfish venom in 30 μl normal saline (0.9%, Pfizer) alone or in the presence of 20 ul HPβCD (50% W/V) was injected under isoflurane anaesthesia (2–3% mixed with 0.8 to 1 L O 2 , IsoFlo Zoetis) into the mouse footpad. Rapid recovery from anaesthesia was achieved with high flow O 2 .

Spontaneous pain behaviour

Following recovery, mice were placed into plexiglass boxes on a raised clear plexiglass table. Mouse behaviour was recorded from below using a video camera (Panasonic). The first five minutes of video were analysed for flinching behaviour (manually with the assistance of custom behaviour enumeration software) which was the dominant immediate pain behaviour.

Von Frey mechanical allodynia assay

Mice were habituated and tested with 10 probes of ascending filaments (Semmes Weinstein Filaments, North Coast Medical 0.04–2.0 g) on each hind paw in the sural territory. Mouse von Frey thresholds were established on 2 separate days for baseline readings and is defined as the stimulus eliciting reactions in 50% of probes. Mice were tested 1 day following injection of Jellyfish venom into the foot pad.

Necrosis

Necrosis was assessed 3 days following the injection of the venom. Photographs were taken of the mice hind paw and all photographs were scored blind. Necrosis was scored by 3 independent blinded investigators semi-quantitatively from pictures taken at euthanasia (either 3 days following injection or if an ethical endpoint was met). The score for each mouse is an average of the scores of each investigator. The scoring system used was:

0) No evidence of necrosis

1) Mild limited but limited to part of one digit or small portion of paw

2) Mild affecting Multiple digits or affecting paw and digit or entire digit

3) Moderate to severe affecting Multiple digits and paw and/or Autotomy (any loss of a digit)

Swelling

Calipers were used twice daily to assess swelling, swelling scores are from just prior to euthanasia. The measurement was of the largest dorsoventral width.

Histology

Foot pads were dissected and fixed in paraformaldehyde 4% (Sigma) for 24 h then cryoprotected in 30% sucrose and flash frozen embedded in OCT (VWR) in Liquid nitrogen. The resultant tissue was then sectioned on a cryostat at 8–12 µm (Thermo Fisher). For Hoecst staining, tissue was washed in PBS, blocked in 2% BSA, 0.1% Triton X-100, washed and stained using Hoecst (Thermo Fisher 1:2000). Following this, tissue section was imaged at 40X using a Leica DM6000 upright microscope. For Haematoxylin and Eosin staining, PFA fixed frozen Sections were washed in water thoroughly to remove all OCT. The sections were immersed into Harris Haematoxylin for 30–40 s and washed in tap water until clear. The Haematoxylin was blued using Scott’s Bluing solution and washed in tap water. The slides were immersed into ethanol then eosin. Finally, slides were dehydrated into 95% ethanol and 100% ethanol. The slides were dried then cleared into citrasol and cover-slipped using Xylene/citrasol and dried overnight and imaged using a DM6000 upright Leica microscope.

Data analysis

Statistical analysis performed was specified in figure legends. p < 0.05 was considered statistically significant.