Identification and functional characterization of the astacidin family of proline-rich host defence peptides (PcAst) from the red swamp crayfish (Procambarus clarkii, Girard 1852)

Abstract

This study reports the identification of four novel proline-rich antimicrobial peptides (PR-AMP) from the transcriptome of the red swamp crayfish Procambarus clarkii. The newly identified putative peptides (PcAst-1b, -1c, −2 and −3), which are related with the previously identified hemocyte-specific PR-AMP astacidin-1, are encoded by the multi-genic astacidin gene family. The screening of available and proprietary transcriptomes allowed to define the taxonomical range of distribution of this gene family to Astacoidea and Parastacoidea. The antimicrobial properties of three synthetic PcAst peptides (PcAst-1a, -1b/c and −2), were characterized against reference bacteria or multidrug resistant clinical isolates, and their cytotoxicity was evaluated towards human transformed cell lines. The antimicrobial activity ranged from potent and broad-spectrum, in low-salt medium, to poor, whereas it was generally low in full nutrient broth. No significant toxic effects were observed on cultured human cells. RNA-seq data from 12 different tissues indicated a strong specificity for haemocytes under naïve physiological condition, with moderate expression (5-fold lower) in gills. Quantitative real time PCR revealed a rapid (within 2 h) and significant up-regulation of PcAst-1a (Astacidin 1) and PcAst-2 expression in response to LPS injection. Due to the variation in antimicrobial potency and inducibility, the roles of the other astacidins (PcAst-1b, -1c and −3) need to be further investigated to determine their significance to the immune responses of the red swamp crayfish.