Transferrin knockout reveals a tolerance phenotype against Piscirickettsia salmonis in Atlantic salmon phagocytes.

Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis, is a major challenge in Chilean aquaculture. We evaluated the impact of the vaccine on fish survival, bacterial load, and iron metabolism-related gene expression under field conditions. Atlantic salmon received either a pentavalent inactivated vaccine plus a live attenuated P. salmonis vaccine (SIA) or a tetravalent control vaccine (SS). While survival was similar early (≤ 28 weeks post-seawater transfer), SIA-vaccinated fish showed greater survival by week 41 (85% vs. 52%). Quantitative PCR confirmed a reduced bacterial load in the SIA group during active infection. Expression analysis revealed distinct temporal regulation of iron metabolism genes: ferritin and hepcidin were upregulated in freshwater, whereas transferrin and its receptor were elevated in seawater in the SIA group, suggesting a link between iron homeostasis and vaccine-induced protection. To further investigate the role of transferrin, we generated transferrin-knockout (TF-KO) phagocytes using CRISPR/Cas9. In vitro infection assays revealed that, compared with wild-type (TF-WT) cells, TF-KO cells presented reduced cytopathic effects, decreased formation of P. salmonis-containing vacuoles (PCVs), and improved viability. Surprisingly, no differences in bacterial load or iron-related gene expression were detected between TF-KO and TF-WT cells, indicating that transferrin disruption did not directly alter iron homeostasis. Global transcriptomic analysis revealed 311 differentially expressed genes in TF-KO cells, with functional enrichment in metal-binding and zinc-dependent processes but no direct association with iron metabolism. These findings suggest that transferrin deficiency confers an infection-tolerant phenotype through transcriptional reprogramming unrelated to iron regulation, highlighting novel mechanisms of host‒pathogen interactions and potential avenues for SRS control in aquaculture.