Zebrafish (Danio rerio H.) were obtained from the Zebrafish International Resource Center and were mated, staged, raised, and processed as described (Westerfield, 2000 #23). The Tg(NFkB-RE:eGFP)^sh235 line, referred to as nfkb:eGFP (19) and Tg(UAS:dn-nfkbiaa)^sd35 (20), has been previously described. The experiments performed complied with the Guidelines of the European Union Council (Directive 2010/63/EU) and the Spanish RD 53/2013. The experiments and procedures were performed as approved by the Bioethical Committee of the University of Murcia (approval no. 669/2020).

STM 12023 (wild type, WT) was used. Overnight cultures in Luria–Bertani (LB) broth were diluted 1:5 in LB with 0.3 M NaCl, incubated at 37°C until 1.5 optical density at 600 nm was reached, and finally diluted in sterile phosphate-buffered saline (PBS). Larvae at 2 days post-fertilization (dpf) were anesthetized in embryo medium with 0.16 mg/ml tricaine, and 10 bacteria per larvae were microinjected in the yolk sac. The larvae were allowed to recover in egg water at 28–29°C and were monitored for clinical signs of disease or mortality over 5 days.

Total RNA was extracted from a pool of 25 zebrafish larvae (3 dpf) with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions and treated with DNase I, amplification grade (1 U/μg RNA; Invitrogen). The SuperScript VILO cDNA Synthesis Kit (Invitrogen) was used to synthesize first-strand cDNA with a random primer from 1 μg of the total RNA at 50°C for 50 min. Real-time PCR was performed with an ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Core Reagents (Applied Biosystems). The reaction mixtures were incubated for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 1 min at 60°C, and finally 15 s at 95°C, 1 min at 60°C, and 15 s at 95°C. For each mRNA, the gene expression was normalized to the ribosomal protein S11 gene (rps11) content in each sample using the Pfaffl method (27). The sequences of the used primers are listed in Supplementary Table S1 . In all cases, each PCR was performed with triplicate samples and repeated at least twice with independent samples.

To study the reporter activity of NF-κB, 2-dpf nfkb:egfp larvae were anesthetized in embryo medium with 0.16 mg/ml buffered tricaine. Images of complete larvae were taken using a Leica MZ16FA fluorescence stereomicroscope. The fluorescence intensity from three biological replicates, each containing several larvae, was obtained and analyzed with ImageJ (FIJI) software.

The CRISPR RNA (crRNA) for zebrafish samhd1 ( Supplementary Table S2 ) and the negative control crRNA (catalog no. 1072544, crSTD), as well as the tracrRNA (trans-activating tracrRNA; catalog no. 1072533), purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA), were resuspended in nuclease-free duplex buffer to 100 µM. Of each, 1 μl was mixed and incubated for 5 min at 95°C for duplexing. After removing from the heat and cooling to room temperature, 1.43 µl of the nuclease-free duplex buffer was added to the duplex [guide RNA (gRNA) and crRNA + tracrRNA], giving a final concentration of 1,000 ng/µl. The injection mix was then prepared by mixing 1 µl of the duplex, 2.55 µl of the nuclease-free duplex buffer, 0.25 µl of Cas9 nuclease V3 (IDT, catalog no. 1081058), and 0.25 µl of phenol red, giving final concentrations of the gRNA duplex (250 ng/µl) and of Cas9 (500 ng/µl). The prepared mix was microinjected into the yolk of one-cell-stage embryos (0.5–1 nl per embryo) using a microinjector (Narishige, Amityville, NY, USA). The same amounts of gRNA were used in all the experimental groups. The efficiency of the gRNA was checked by amplifying the target sequence with a specific pair of primers ( Supplementary Table S1 ) and the TIDE webtool (https://tide.nki.nl/) ( Supplementary Figure S1 ).

pcDNA-IFNphi3 (GenBank accession no. NM_001111083) (21) was microinjected into the yolk of one-cell-stage embryos (1 pg/embryo). The 1-dpf embryos were manually dechorionated and 24 h post-fertilization treated by chemical bath immersion at 28°C. Baricitinib (Bar; MedChemExpress, Monmouth Junction, NJ, USA) was added to the water at 10 µM and BAY11-7082 (MedChemExpress) at 10 nM and renewed every day for the duration of the experiment (6 days). Incubation was carried out in 10-ml plates containing ≈100 larvae/well in egg water (including 60 μg/ml sea salts in distilled water) supplemented with 0.1% dimethyl sulfoxide (DMSO).

All statistical analyses were performed in GraphPad Prism 8. Data are shown as the mean ± SEM and were analyzed using one-way analysis of variance (ANOVA) and Tukey’s multiple range test to determine differences between groups. The differences between two samples were analyzed using Student’s t-test. A log-rank test was used to calculate the statistical differences in the survival of the different experimental groups.

An annotated samhd1 ortholog gene (Gene ID: 553453) to the human gene SAMHD1 was found in chromosome 23 of the zebrafish genome (ENSDARG00000071288.4) ( Figure 1A ). In addition, two zebrafish samhd1 paralogs were also annotated as LOC793232 (Gene ID: 793232) and si:ch211-233g6.2 (Gene ID: 100333959), both located in chromosome 1 (ENSDARG00000099421.2 and ENSDARG00000104626.2), which shared between them 94% of identity, while 58% and 57%, respectively, with the original samhd1 gene. Importantly, the two samhd1-like sequences lacked the SAM domain ( Figure 1A ), which is critical for the regulation and activation of mammalian SAMHD1 (22), suggesting their specialization in other functions. Consequently, in this work, we only focused on the samhd1 gene.

Similarly to Whiters et al. (23), we decided to measure the ISG expression in larval heads, given that the expression of samhd1 was higher in heads than in tails ( Figure 1B ). We next inactivated samhd1 using the CRISPR/Cas9 technology in one-cell-stage embryos (with a knockdown efficiency of 83.1%) ( Supplementary Figure S1 ). We were not able to detect any significant difference in the expression levels of isg15 between the Samhd1-deficient and the control zebrafish larvae ( Figure 1C ). However, the addition of a suboptimal dose of the zebrafish IFN-I ifnphi3 was able to robustly increase the isg15 transcript levels in Samhd1-deficient larvae, but not in their sibling controls ( Figure 1C ). Of note is that a suboptimal dose of 1 pg/egg of IFN-I ifnphi3 was used in the experiment ( Supplementary Figure S2 ).

To study the inflammatory process in Samhd1-deficient zebrafish larvae, the transcriptional activity of NF-κB was analyzed using the reporter line nfkb:eGFP. As seen in Figures 2A, B , Samhd1-deficient larvae showed stronger NF-κB reporter activity than control larvae. As NFKB protects murine models from STM infection and this pathogen is able to hijack NF-κB with its effector protein SpvB (18), we next studied whether Samhd1-deficient zebrafish larvae were more resistant to STM infection than their WT counterparts. The results confirmed this hypothesis, showing that Samhd1-deficient zebrafish larvae were more resistant to STM infection, with almost a 30% higher survival than WT larvae ( Figure 2C ). Moreover, the gene expression analysis after 24 h of infection showed that the transcript levels of nfkb1 significantly increased in Samhd1-deficient infected larvae ( Figure 3A ) compared with control larvae. In addition, the expression of the il1b and isg15 transcripts also increased in Samhd1-deficient larvae following infection ( Figures 3B, C ). Interestingly, the mRNA levels of tnfα and ifng1r from the Samhd1-deficient infected larvae did not overcome those of the control post-infection ( Figures 3D, E ). Finally, the transcript levels of cxcl8a were unaffected by Samhd1 deficiency independently of infection ( Figure 3F ). Collectively, these findings indicate that Samhd1-deficient larvae display a heightened inflammatory state, facilitating a more robust and accelerated response to pathogen infection compared with control larvae.

To further validate the role of NF-κB in the resistance of Samhd1-deficient larvae to infection, we treated the larvae with BAY11-7082, an NF-κB inhibitor, and conducted a challenge with STM. As shown in Figure 4A , although the Samhd1-deficient larvae treated with the NF-κB inhibitor exhibited mortality rates comparable to those of WT larvae, the treatment fully abolished the increased bacterial resistance of Samdh1-deficient larvae ( Figure 4A ). Moreover, we used baricitinib, a JAK1/JAK2 inhibitor, which is able to suppress the IFN-I response in zebrafish larvae (24). Consistent with our expectations, treatment with baricitinib did not alter the susceptibility of Samhd1-deficient larvae to STM infection, which showed a survival rate approximately 30% higher than that of WT larvae and similar to that of untreated Samhd1-deficient larvae ( Figure 4B ). These findings support the hypothesis that the activation of NF-κB is crucial for the enhanced survival observed in Samhd1-deficient larvae.

The above results, together with the critical dual role of macrophages in STM dissemination and clearance (12–16), prompted us to examine whether the activation of NF-κB in the macrophages of Samhd1-deficient larvae mediated their hyperresistance to STM infection. To achieve this, we used the transgenic line Tg(UAS:dn-nfkbiaa), which expresses a dominant negative (DN) form of the nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha a (nfkbiaa) and functions as an inhibitor of NF-κB (20). This line was outcrossed with the Tg(mpeg1:gal4) line, which allows exclusively expressing the DN-Nfkbiaa in macrophages. One-cell-stage embryos were injected with samhd1 crRNA/tracrRNA/Cas9 complexes and subsequently infected with STM, as previously described. Larvae with macrophages expressing DN-Nfkbiaa lost their hyperresistance to infection ( Figure 5A ), behaving similarly to control larvae, indicating that the activation of NF-κB in Samhd1-deficient macrophages is essential for their enhanced resistance to STM infection. Figure 5B shows a schematic model of the restriction of NF-κB activation by Samhd1 in WT macrophages and the hyperresistance of Samhd-1-deficient macrophages to STM infection through the induction of NF-κB activation.