Molecular identification of NF-κB subunit RelA in Manila clam (Ruditapes philippinarum) and its regulatory role in inflammatory response during Vibrio parahaemolyticus infection.

Abstract

The RelA subunit is a core component of the nuclear factor-kappa B (NF-κB) family and plays a pivotal role in regulating immune and inflammatory responses across vertebrates and invertebrates. However, its functional role remains poorly defined in marine bivalves. In this study, a RelA homolog (RpRelA) was identified and functionally characterized in the Manila clam (Ruditapes philippinarum). The full-length cDNA of RpRelA is 2055 bp, encoding a 684-amino acid protein containing a conserved Rel homology domain (RHD) and an IPT domains. Both its tertiary structure and amino acid sequence are highly conserved across species. Phylogenetic analysis indicated that RpRelA clusters with invertebrate Rel family members. Quantitative real-time PCR (qRT-PCR) revealed that RpRelA is ubiquitously expressed in all examined tissues, with the highest expression levels detected in haemocytes and gills. Following V. parahaemolyticus challenge, both the mRNA and protein levels of RpRelA were significantly upregulated. Consistently, nuclear-cytoplasmic fractionation Western blot and immunofluorescence analyses confirmed its robust nuclear translocation upon bacterial infection. Furthermore, overexpression of RpRelA significantly upregulated pro-inflammatory genes (RpIL-17 and RpTNF) and caused severe gill inflammation, whereas its silencing suppressed their expression and alleviated the inflammatory response. Collectively, these results demonstrate that RpRelA, as a core NF-κB subunit, positively regulates the inflammatory response during V. parahaemolyticus infection in Manila clam, and provide new insights into the immune regulatory mechanism of bivalves against bacterial infection.