Molecular Cloning and Functional Analysis of Lumbricin in Urechis unicinctus.

Lumbricin, a classic proline-rich antimicrobial peptide (PrAMP), has been primarily characterized in terrestrial clitellates, with limited reports in other annelid lineages, leaving a critical knowledge gap regarding its presence and immune roles in marine invertebrates. The echiuran worm Urechis unicinctus lives in microbe-laden intertidal mudflats and depends on innate immunity against pathogens, yet its lumbricin homologs have remained unreported. In this study, we aimed to clone full-length lumbricin cDNA and systematically characterize its molecular traits, tissue distribution, LPS (lipopolysaccharide)-triggered transcription and in vitro antibacterial function. Sequence analysis showed the deduced 67-amino-acid U. unicinctus lumbricin contained 10.4% proline and lacked a signal peptide, retaining the conserved N-terminal cationic patch and C-terminal motifs of earthworm lumbricins. Phylogenetic analysis confirmed its close evolutionary linkage to Eisenia andrei lumbricin-related peptide. Quantitative PCR (qPCR) detected ubiquitous constitutive expression, peaking in the body wall, followed by the hindgut and midgut. LPS induced rapid 3 h upregulation in the body wall for acute local defense, with massive late-stage elevation at 48 h in the hindgut, anal sacs and coelomic fluid. Recombinant lumbricin exerted broad-spectrum growth-inhibitory effects against Gram-positive and Gram-negative bacteria. This study is the first to identify a functional lumbricin homolog in marine non-clitellate annelids (echiurans), which expands the taxonomic range of the lumbricin family and provides new insights into innate immune defense of marine benthic organisms.