Hepcidin from the Chinese Spiny Frog (Quasipaa spinosa) Integrates Membrane-Disruptive Antibacterial Activity with Macrophage-Mediated Protection Against Elizabethkingia miricola

<b>Background/Objectives</b>: Hepcidin is a cysteine-rich antimicrobial peptide that links iron homeostasis and innate immunity in vertebrates, but its functions in amphibians remain poorly understood. The Chinese spiny frog (<i>Quasipaa spinosa</i>) is an economically important species that suffers serious losses from bacterial diseases. This study aimed to identify and functionally characterize a hepcidin homolog (QsHep) from <i>Q. spinosa</i>, focusing on its antibacterial activity, immunomodulatory effects on primary macrophages, and protective efficacy against <i>Elizabethkingia miricola</i> infection. <b>Methods</b>: The <i>QsHep</i> gene was cloned and analyzed, its tissue distribution and inducible expression were examined by qRT-PCR, and the synthetic peptide was tested for antimicrobial, membrane-disruptive, and immunomodulatory activities in vitro, as well as for in vivo protection in an <i>E. miricola</i> infection model. <b>Results</b>: <i>QsHep</i> encodes a typical preprohepcidin with a signal peptide, prodomain, and a conserved mature peptide containing eight cysteine residues. <i>QsHep</i> was widely expressed, with the highest levels in liver, and was significantly upregulated in liver and spleen following bacterial challenge. Synthetic QsHep displayed broad-spectrum antibacterial activity, including strong inhibition of <i>E. miricola</i>, and induced dose-dependent membrane damage in <i>E. miricola</i>. QsHep showed no obvious cytotoxicity but significantly enhanced chemotaxis, phagocytic activity, and respiratory burst in primary macrophages. In vivo, QsHep treatment markedly improved the survival of <i>E. miricola</i>-infected frogs in a dose-dependent manner. <b>Conclusions</b>: QsHep is an amphibian hepcidin that combines membrane-disruptive antibacterial activity with the activation of macrophage effector functions and confers significant protection against bacterial infection in vivo. These findings expand our understanding of hepcidin-mediated innate immunity in amphibians and highlight QsHep as a promising peptide candidate for controlling bacterial diseases in frog aquaculture.