Identification of a Double-ß-Defensin with Multiple Antimicrobial Activities in a Marine Invertebrate.

ß-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian ß-defensin 11 (AvBD11) is unique, with two ß-defensin motifs with a broad range of antimicrobial activities. However, a double-sized ß-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-ß-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to ß-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD in vivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-ß-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.

A Dicer2 from Scylla paramamosain activates JAK/STAT signaling pathway to restrain mud crab reovirus.

A Dicer2 gene from Scylla paramamosain, named SpDicer2, was cloned and characterized. The full length of SpDicer2 mRNA contains a 121 bp 5'untranslated region (UTR), an open reading frame (ORF) of 4518 bp and a 3' UTR of 850 bp. The SpDicer2 protein contains seven characteristic Dicer domains and showed 34%-65% identity and 54%-79% similarity to other Dicer protein domains, respectively. The mRNA of SpDicer2 was high expressed in hemocytes, intestine and gill and low expressed in the eyestalk and muscle. Moreover, expression of SpDicer2 was significantly responsive to challenges by mud crab reovirus (MCRV), Poly(I:C), LPS, Staphylococcus aureus and Vibrio parahaemolyticus. SpDicer2 was dispersedly presented in the cytoplasm except for a small amount in the nucleus. SpDicer2 could activate SpSTAT to translocate from the cytoplasm to the nucleus, and significantly increase the transcription activity of the wsv069 promoter, suggesting that SpDicer2 activated the JAK/STAT pathway. Furthermore, silencing of SpDicer2 in vivo increased the mortality of MCRV infected mud crab and the viral load in tissues and down-regulated the expression of multiple components of Toll, IMD and JAK-STAT pathways and almost all the examined immune effector genes. These results suggested that SpDicer2 could play an important role in defense against MCRV via activating the JAK/STAT signaling pathways in mud crab.