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A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of

The Ras GTPase superfamily, including more than 100 members, plays a vital role in a number of cellular processes, such as cytoskeleton recombination, gene expression, and signaling pathway regulation. Some members of the superfamily participate in innate immunity in animals. However, there have been few studies of RhoA on this aspect. In the present study, we identified a RhoA GTPase in the shrimp Marsupenaeus japonicus and named it MjRhoA. Expression of MjRhoA was significantly upregulated in hemocytes and heart of shrimp challenged with Vibrio anguillarum. Overexpression of MjRhoA in shrimp caused the total bacterial number to decrease significantly and knockdown of MjRhoA increased the bacterial number obviously, with a consequent decline in shrimp survival. These results confirmed the antibacterial function of MjRhoA in shrimp. Further study showed that rate of phagocytosis of hemocytes was decreased in MjRhoA-knockdown shrimp. Interestingly, we observed that MjRhoA was translocated onto the hemocyte membrane at 1 h post V. anguillarum challenge. The expression levels of the β-integrin-mediated phagocytosis markers ROCK2 and Arp2/3 declined significantly after knockdown of MjRhoA. These results suggested that the antibacterial function of MjRhoA was related to β-integrin-mediated phagocytosis in shrimp. Our previous study identified that a C-type lectin, hFcLec4, initiated β-integrin mediated phagocytosis after bacterial infection. Thus, knockdown of hFcLec4 and β-integrin was performed. The results showed that the translocation of MjRhoA from the cytoplasm to membrane was inhibited and the expression level of MjRhoA was decreased, suggesting that MjRhoA participated in hFcLec4-integrin mediated phagocytosis. Therefore, our study identified a new hFcLec4-integrin-RhoA dependent phagocytosis against bacterial infection in shrimp.

Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.

The Toll pathway is essential for inducing an immune response to defend against bacterial invasion in vertebrates and invertebrates. Although Toll receptors and the transcription factor Dorsal were identified in different shrimp, relatively little is known about how the Toll pathway is activated or the function of the pathway in shrimp antibacterial immunity. In this study, three Tolls (Toll1-3) and the Dorsal were identified in Marsupenaeus japonicus. The Toll pathway can be activated by gram-positive (G+) and gram-negative (Gˉ) bacterial infection. Unlike Toll binding to spatzle in Drosophila, shrimp Tolls could directly bind to pathogen associated molecular patterns (PAMPs) from G+ and Gˉ bacteria, resulting in Dorsal translocation into nucleus to regulate the expression of different antibacterial peptides (AMPs) in the clearance of infected bacteria. These findings suggest that shrimp Tolls are pattern recognition receptors and the Toll pathway in shrimp is different from the Drosophila Toll pathway but identical with the mammalian Toll-like receptor (TLR) pathway in its activation and antibacterial functions.

The Toll pathway is essential for inducing an immune response to defend against bacterial invasion in vertebrates and invertebrates. Although Toll receptors and the transcription factor Dorsal were identified in different shrimp, relatively little is known about how the Toll pathway is activated or the function of the pathway in shrimp antibacterial immunity. In this study, three Tolls (Toll1–3) and the Dorsal were identified in Marsupenaeus japonicus. The Toll pathway can be activated by Gram-positive (G+) and Gram-negative (G−) bacterial infection. Unlike Toll binding to Spätzle in Drosophila, shrimp Tolls could directly bind to pathogen-associated molecular patterns from G+ and G− bacteria, resulting in Dorsal translocation into nucleus to regulate the expression of different antibacterial peptides (AMPs) in the clearance of infected bacteria. These findings suggest that shrimp Tolls are pattern recognition receptors and the Toll pathway in shrimp is different from the Drosophila Toll pathway but identical with the mammalian Toll-like receptor pathway in its activation and antibacterial functions.