The infection of Aeromonas hydrophila activated Multiple programmed cell death pathways in red blood cells of Clarias fuscus

In contrast to mammalian red blood cells (RBCs), Osteichthyes RBCs contain a nucleus and organelles, suggesting the involvement of more intricate mechanisms, particularly in the context of ferroptosis. In this study, we utilized RBCs from Clarias fuscus (referred to as Cf-RBCs) as a model system. We conducted RNA-seq analysis to quantify gene expression levels in Cf-RBCs after exposure to both Aeromonas hydrophila and lipopolysaccharides. Our analysis unveiled 1326 differentially expressed genes (DEGs) in Cf-RBCs following 4 h of incubation with A. hydrophila, comprising 715 and 611 genes with upregulated and downregulated expression, respectively. These DEGs were further categorized into functional clusters: 292 related to cellular processes, 241 involved in environmental information processing, 272 associated with genetic information processing, and 399 linked to organismal systems. Additionally, notable changes were observed in genes associated with the autophagy pathway at 4 h, and alterations in the ferroptosis pathway were observed at 8 h following A. hydrophila incubation. To validate these findings, we assessed the expression of cytokines (DMT1, TFR1, LC3, and GSS). All selected genes were significantly upregulated after exposure to A. hydrophila. Using flow cytometry, we evaluated the extent of ferroptosis, and the group incubated with A. hydrophila for 8 h exhibited higher levels of lipid peroxidation compared with the 4-h incubation group, even under baseline conditions. An evaluation of the glutathione redox system through GSSG/GSH ratios indicated an increased ratio in Cf-RBCs after exposure to A. hydrophila. In summary, our data suggest that A. hydrophila may induce ferroptosis in Cf-RBCs, potentially by triggering the cystine/glutamate antiporter system (system XC-), while Cf-RBCs counteract ferroptosis through the regulation of the glutathione redox system. These findings contribute to our understanding of the iron overload mechanism in Osteichthyes RBCs, provide insights into the management of bacterial diseases in Clarias fuscus, and offer potential strategies to mitigate economic losses in aquaculture.