293T were obtained from the Cell Bank of the Chinese Academy of Sciences, while Epithelioma papulosum cyprini (EPC) cells were provided by the Freshwater Fisheries Research Institute of Shandong Province. 293T cells were cultured in DMEM medium, which was added with 10% fetal bovine serum (FBS), 10 units/ml penicillin and 10 mg/mL streptomycin at 37 °C in a 5% CO₂ humidified atmosphere. EPC cells were cultured in M199 medium containing 10% FBS, 1% streptomycin and penicillin at 25 °C. Transient transfections in 293T and EPC cells were carried using Lipofectamine 2000 (Invitrogen, USA) and jetPRIME reagent (Polyplus, France) following the manufacturers’ protocols, respectively.

The common carp (about 200 g per fish) were purchased from commercial pond farms in Jinan, China. The fish were cultured in recirculating water (25 ± 2°C) for a minimum of seven days before the experiment and fed twice a day. For tissue expression analysis, three healthy carp with similar size and age were selected and dissected. Ten different tissues, including the liver, foregut, hindgut, brain, skin, head kidney, gills, muscle, heart, and spleen were sequentially acquired. Additionally, in pathogen stimulation group, 500 μL of SVCV (10^4.8 TCID50/100 μL) or A. hydrophila (2 × 10⁸ CFU/ml) were intraperitoneally injected into carp, and the control group was injected with the same volume of M199 medium or PBS. After infection indicated times, the immune-related tissues (liver, spleen, foregut, and hindgut) were collected and immediately storaged at -80°C for subsequent analysis.

The MINK1 gene sequence from common carp was retrieved from the Ensembl genomes database and subsequently cloned using standard molecular biology. The nucleic acid sequence of CcMINK1 were translated into amino acid sequence using BioEdit software and then protein structure of CcMINK1 was predicted using SMART analysis (http://smart.embl-heidelberg.de/). The three-dimensional (3D) structure of MINK1 was further predicted via SWISS-MODEL analysis (https://swissmodel.expasy.org/). For comparative analysis, the MINK1 protein sequence across multiple species was obtained from the NCBI database. The phylogenetic tree was constructed using MEGA7.0 software with the neighbor-joining method. The GenBank accession numbers of all analyzed sequences are provided in Table 1 .

The eukaryotic expression vector encoding CcMINK1-GFP, CcMINK1-Myc, CcMINK1-HA and CcMINK1-Flag were generated using standard molecular biology as described previously (31). In brief, the ORF of CcMINK1 was amplificated and directionally cloned into indicated vector using T4 DNA ligase. Then, the recombinant plasmid was confirmed by TSINGKE Biological Technology and the correct expression vectors were extracted through the Endo-Free Plasmid Mini Kit II (Omega Bio-Tek). The primers used in this study are provided in Table 2 .

Total RNA from tissues samples and cells were isolated via using either RNA simple Total RNA kit (Nobelab) or Trizol reagent following the manufacturers’ protocols. Then, the a Fast Quant Kit (with gDNase) (Nobelab) was used for the reverse transcription of RNA, in line with the manufacturer’s protocol. 2 × SYBR Premix UrTaq II (Nobelab) were applied for the qPCR assay using the LightCycler 96 instrument (Roche). The thermal cycling protocol commenced with an initial step at 95 °C for 120 s, succeeded by 40 cycles of 95 °C for 10 s and annealing/extension at 60 °C for a duration of 30 s. The gene expression levels were normalized to S11 and EF-1α expression, respectively. The details of primers used in the present study are described in Table 2 .

293T cells were transfected with indicated expressing vectors on the figure. After 48 h, the cells lysates were generated on ice using 1%NP-40 lysis buffer supplemented with proteinase inhibitors. For immunoprecipitation assays, cells lysates were incubated with the appropriate affinity gel overnight at 4 °C with gentle rotation. Then, beads were subsequently washed four times with ice-cold 1%NP-40 lysis buffer, the bound proteins were eluted in 2×SDS loading buffer. The whole-cell extracts and ip samples were used for immunoblotting assays.

For immunoblotting analysis, protein was resolved by indicated SDS-PAGE gels and transferred onto PVDF membranes through standard protocols. Membranes were blocked with 5% nonfat milk prior to incubation with primary antibodies: GFP (HUABIO, ET1607-31, 1:20000), HA (Abcam, ab9110, 1:1000), Myc (CST, 2278T, 1:1000) and Flag (Solarbio, K200001M, 1:10000) overnight under 4 °C, respectively. Subsequently, the membrane was washed four using 1×TBST (TBS encompassing 0.1% Tween-20) and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson immunoresearch, USA). Protein band was visualized using Enhanced Chemiluminescence Detection Reagents (Meilunbio, China).

293T or EPC cells were cultured in a 24-well plate with glass slides and transfected with the indicated expression plasmid for 24 h. The cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 30 min. Then, the cells were washed with PBS and DAPI (Beyotime, C1002, 1:1000) was used to stain the nuclear. The stained cells were viewed using a Leica SP8 confocal laser scanning microscope.

Several previous studies have shown that there are two homologs of proinflammatory Caspase, namely Caspase-A and Caspase-B in zebrafish (32–34) and common carp (35). In particular, two transcripts of Caspase-A genes were cloned from common carp, which were named CcCaspase-A1 and CcCaspase-A2 (35). 293T cells were cultured in a 24-well plate and transfected with the indicated expression plasmid for 24 h. The CcCaspase-A1/A2/B enzyme activity was detected using the enzyme activity detection kit (Enzo Life Sciences, USA) and the fluorescence value was detected by multifunctional enzyme-linked immunosorbent assay instrument (FilterMax F3, Austria). Caspase-A1/A2/B enzyme activity = (fluorescence value of experimental group - fluorescence value of control group)/fluorescence value of control group×100%.

LDH cytotoxicity was quantified using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay Kit (Promega). Briefly, 293T cells were seeded in 48-well plates and transfected with corresponding plasmids for 48 h. Following transfection, cells were treated with 10×Lysis Solution for 45 min at 37 °C as positive control. For assay measurements, 33 μL of cell supernatant was transferred to 96-well plate and mixed with an equal volume of reconstituted CytoTox 96^® Reagent. After 30 minutes of protected incubation at room temperature, the reaction was terminated by adding 33 μL of Stop Solution. Absorbance at 492 nm was measured through a FilterMax F3 Multi-Mode Microplate Reader. Cytotoxicity (%) was calculated as experimental LDH release/maximum LDH release × 100%, with background subtraction using untreated cells.

293T cells were transfected with indicated expression plasmids. After 48 h, the cells was lysed and centrifuged at 4 °C, 6000 × g for 5 min. The supernatant was kept for immunoblotting as loading control and the pellet was washed with PBS twice and cross-linked with 2 mM DSS for 30 min at room temperature. After centrifuging at 4 °C, 5000 × g for 10 min, the pellet was resuspended with 50 μL SDS-PAGE loading buffer and determined by immunoblotting analysis with the indicated antibodies.

Phos-tag gels were purchased from FUJIFILM Wako Pure Chemical Corporation and carried out by the same way as immunoblotting assays, except that (1): The Phos-tag gels were soaked in EDTA for 10 min before transfer onto a PVDF membrane to remove Zn²⁺ (2): The Phos-tag gels were soaked transmembrane buffer solution containing 10 mmol/L EDTA and washed twice.

All data are presented as the mean ± standard deviation (SD) from three independent experiments. Data were analyzed using GraphPad Prism 8. Comparisons between two groups were analyzed using the Student’s t test. P-value less than 0.05 was considered statistically significant.

The ORF of MINK1 were cloned and identified from the spleen of common carp, which consisted of 3687 bp and encoded 1229 amino acids. Domain architecture prediction using the SMART online tool demonstrated that CcMINK1 contained a N-terminal S_TKc domain and a C-terrmina CNH domain ( Supplementary Figure 1A ). Subsequently, three-dimensional (3D) architectures modeling of MINK1 proteins across vertebrate species was contrasted using SWISS-MODEL, which showed that the 3D structure of CcMINK1 was similar to that of other species ( Supplementary Figure 1B ). Additionally, the phylogenetic tree was built to investigate the evolutionary relationship of MINK1, which suggested that CcMINK1 was closely related to that of Carassius auratus ( Supplementary Figure 1C ).

To investigate the biological properties of CcMINK1, the subcellular localization of CcMINK1 was evaluated. The recombinant plasmid CcMINK1-mCherry was transfected into 293T and EPC cells. Confocal microscopy analysis revealed that CcMINK1 was exclusively expressed in the cytoplasm of both cell lines ( Figure 1A ). Subsequently, we further delineate the mRNA expression profiles of CcMINK1 in the brain, hindgut, foregut, spleen, heart, muscle, gills, head kidney, liver and skin. qPCR results showed that CcMINK1 was universally expressed across all investigated tissues, and the expression was substantially higher in the brain and hindgut, followed by the foregut and spleen, while skin showed the lowest expression among all tested tissues ( Figure 1B ).

To examine the CcMINK1 expression profiles following pathogen infection, several immune-related tissues were extracted from the common carp at specified time points after SVCV or A. hydrophila infection. Under the stimulation of SVCV, the expression of CcMINK1 in various tissues of common carp significantly increased ( Figure 2 ). In the liver, the CcMINK1 mRNA levels were up-regulated and reached a peak value at 6 h (about 2.0-fold) ( Figure 2A ). In the spleen, hindgut and foregut, CcMINK1 transcript levels initially displayed a decrease at 6 h, then start to increase at 24 h and 48 h, and achieved the highest at 72 h, respectively ( Figures 2B–D ). Similarly, under the infection of A. hydrophila, the CcMINK1 expression level was observed to be increased at indicated points in all examined tissues. In the liver, foregut and hindgut, the CcMINK1 mRNA was induced at 6 h, and arrived the highest level at 72 h ( Figures 2E, G, H ). In the spleen, CcMINK1 expression levels initially displayed a decrease, and achieved the highest at 72 h ( Figure 2F ). In summary, these above results illustrate that CcMINK1 might response to both antiviral and antibacterial immune infection of common carp.

Previous studies have shown that mammals MINK1 interacts with NLRP3 to regulate the assembly of NLRP3 inflammasome (29), while its role in teleost is unclear. To explain the role of CcMINK1 in the regulation of CcNLRP3, we firstly examined the interaction of CcMINK1 and CcNLRP3. Co-ip experiments revealed that CcMINK1 could interact with CcNLRP3 in 293T cells ( Figure 3A ). Similarly, we found that CcMINK1 colocalized with CcNLRP3 in the cytoplasm by confocal microscopy ( Figure 3B ). To further identify which domain of CcMINK1 is responsible for its interaction with CcNLRP3, the recombinant plasmids expressing different domains of CcNLRP3 and CcMINK1 were generated separately ( Figure 3C ). The results of Co-ip revealed that the FIIND, NACHT and PRY-SPRY domain of CcNLRP3 interacted with CcMINK1, but its PYD and LRR domain showed no interaction with CcMINK1 ( Figure 3D ). Additionally, the S_TKC domain of CcMINK1 interacted with CcNLRP3, whereas its CNH domain showed no interaction with CcNLRP3 ( Figure 3E ). Collectively, these results suggested that the S_TKC domain of CcMINK1 directly targeted with the FIIND, NACHT and PRY-SPRY domain of CcNLRP3.

Activated NLRP3 recruits downstream adaptor ASC through the CARD domain, and then ASC assembles into a large protein complex, or “speck”, that is considered to be an upstream readout for inflammasome activation (9). Thus, to determine the functions of CcMINK1 in activation of CcNLRP3 inflammasome, CcMINK1 was co-transfected with CcNLRP3 and CcASC into 293T cells, and DSS crosslink analysis was used to explore the effect of CcMINK1 on CcASC oligomerization. As shown in Figure 4A , CcNLRP3 overexpression markedly boosted the oligomerization of CcASC, and more oligomerization was observed in CcMINK1 overexpressed 293T cells. In addition, immunofluorescence staining showed that CcMINK1 significantly enhanced the speck formation of CcASC mediated by CcNLRP3 both in 293T and EPC cells ( Figures 4B, C , Supplementary Figures 2A, B ). Taken together, these results indicate that CcMINK1 promotes CcNLRP3-mediated the CcASC oligomerization and specks formation.

After being activated, the NLRP3 recruit pro-Caspase-1 via ASC and cleave it to active Caspase-1, which further cleaves pro-IL-1β and GSDMD to mature forms (36). Thus, we investigated whether CcMINK1 modulates the activity of CcCaspases. As shown in Figures 5A–C , CcNLRP3 significantly boosted the CcCaspase-A2 and CcCaspase-B activity, and CcMINK1 overexpressed further enhanced CcNLRP3-induced CcCaspase-A2 and CcCaspase-B activation. However, CcMINK1 did not affect CcNLRP3-mediated CcCaspase-A1 activity. Furthermore, we further examined the cleavage of CcIL-1β. We found that the expression of mature CcIL-1β mediated by CcCaspase-A2 and CcCaspase-B was induced in CcMINK1 overexpressed group. However, CcMINK1 had no effect on CcCaspase-A1-mediated cleavage of CcIL-1β ( Figures 5D–F ). These findings collectively indicate that CcMINK1 plays a regulatory role in CcNLRP3 inflammasome activation, specifically enhancing CcCaspase-A2/B-dependent signaling pathways.

To investigate the role of CcMINK1 in CcGSDME-mediated pyroptosis, the recombinant plasmids of CcMINK1, CcGSDME and CcNLRP3 inflammasome complex were co-transfected into 293T cells, which showed membrane pores and cell membrane rupture phenomenon on the cell membrane, and this phenomenon was more pronounced after overexpression of CcMINK1 ( Figure 6A ). Additionally, the PI staining was applied to assess the level of pyroptosis. The results showed that the number of positively stained cells significantly increased in CcMINK1 overexpressed cells compared to the control group ( Supplementary Figures 3A–C ). At the same time, the LDH release was increased significantly upon overexpression of CcMINK1 compared with only inflammasome proteins ( Figures 6B–D ). Taken together, these results demonstrate that CcMINK1 play a critical role in the CcNLRP3 inflammasome-induced pyroptosis.

NLRP3 oligomerization and phosphorylation is critical steps in inflammasome assembly and activation (37). Hence, to determine the molecular mechanism by which CcMINK1 promotes CcNLRP3 activation, we examined whether CcMINK1 affected CcNLRP3 oligomerization and phosphorylation. CcMINK1-Myc, CcNLRP3-GFP and CcNLRP3-HA was cotransfected into 293T cells, and Co-ip results showed that the CcMINK1 promoted CcNLRP3 self-interaction ( Figure 7A ). Additionally, phos-tag SDS-PAGE was used to determine whether CcMINK1 phosphorylated CcNLRP3. The result showed that the overexpression of CcMINK1 significantly enhanced CcNLRP3 phosphorylation compared to transfection of CcNLRP3 alone ( Figure 7B ). Collectively, these results suggest that CcMINK1 could induce the oligomerization and phosphorylation of CcNLRP3.

Previous studies have shown that MINK1 exhibit several functions that are highly dependent on the kinase activity (38). Thus, we investigated whether CcMINK1 regulates CcNLRP3-mediated inflammasome activation via its kinase activity. We transfected full-length and domain mutants of CcMINK1 into 293T cells. As shown in Figures 8A, B , the full length and S_TKC domain of CcMINK1 significantly enhanced the speck formation of CcASC. However, the CNH domain of CcMINK1 showed no such effect. Additionally, overexpression of full length and S_TKC domain of CcMINK1, but not CNH domain, significantly enhanced CcNLRP3-mediated CcCaspase-A1, CcCaspase-A2 and CcCaspase-B activity ( Figures 8C-E ) and LDH release ( Figured 8F-H ). Meanwhile, phos-tag SDS-PAGE analysis revealed that the full-length CcMINK1 and S_TKC domain mutants both increased CcNLRP3 phosphorylation ( Figure 8I ). Taken together, these results suggest that S_TKC domain of CcMINK1 is necessary and sufficient for promoting CcNLRP3 inflammasome activation.