The fourth components of the cytoskeleton, septins, are evolutionary conserved GTP – binding proteins distinguished from microtubules and actin filaments by their non-polar filament structure. They play crucial roles in diverse cellular processes, including cytokinesis, vesicle trafficking, membrane remodelling, and host–pathogen interactions. Functionally, septins function as diffusion barriers to create subcellular compartmentalisation and as molecular scaffolds for protein recruitment. Despite their significance, the functional characterization of septins, particularly septin 6 in Indian major carps, is still unknown. In the present study, we cloned the full-length septin 6 cDNA from rohu (Labeo rohita), resulting in a 1203 bp open reading frame encoding 400 amino acids and the phylogenetic analysis revealed it’s high sequence similarity to Anabarilius grahami (99%) and Danio rerio (98.75%). Tissue-specific expression profiling revealed elevated septin 6 expression in the brain, spleen, and gills, while ontogeny analysis showed its upregulation during early developmental stages. Additionally, septin 6 transcript levels were significantly increased in response to three different infection models: Aeromonas hydrophila, poly I:C, and Argulus siamensis, suggesting its crucial role in immune responses. The production and intraperitoneal administration of a recombinant septin 6 protein (~46 kDa) modulated the expression of several immune-related genes, such as pro-inflammatory cytokines, antiviral proteins, antioxidant enzymes, apoptosis regulators, antimicrobial peptides, and toll-like receptors. In vitro studies revealed enhanced Lyso G expression in peritoneal macrophages co-incubated with bacteria and recombinant septin 6. In vivo experiment further conferred significant protection against A. hydrophila (91% relative percent survival) and delayed mortality in A. siamensis infections. Additionally, an indirect ELISA was developed to quantify septin 6 level following A. hydrophila post-infection, and it suggests a significantly higher level of septin 6 in liver, spleen, and brain tissues. Collectively, this study provides the first functional insights into septin 6 in rohu, highlighting their involvement in immune responses and offering potential application in fish health management.
Keywords: Functional analysis, Peritoneal macrophage, Immune response, Septin 6