Abstract
Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei . LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrio alginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V . alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibrio penaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.
Highlights
Apoptosis is a genetically programmed process of controlled cell suicide that plays critical roles in organismal development, homeostasis, and the immune system through elimination of cells that are no longer useful [1]
In healthy shrimp, when normalized to mRNA expression in the hepatopancreas (1.00-fold), LvIAP1 was expressed at higher levels in the intestine (1.14-fold), epithelium (1.25-fold), hemocytes (1.34-fold), eyestalk (1.39-fold), gill (3.51-fold), heart (4.69-fold), pyloric cecum (7.64-fold), nerve (8.55-fold), stomach (17.42-fold), and muscle (30.34-fold) (Figure 3A), LvIAP2 was expressed at higher levels in the stomach (1.14fold), hemocytes (1.33-fold), eyestalk (1.70-fold), intestine (1.84-fold), pyloric cecum (2.09-fold), epithelium (2.27-fold), gill (3.34-fold), nerve (4.78-fold), heart (5.72-fold), and muscle (17.09-fold) (Figure 3B), LvIAP3 was expressed at higher levels in the intestine (1.32-fold), hemocytes (1.50-fold), stomach (1.99-fold), eyestalk (1.91-fold), epithelium (2.07-fold), pyloric cecum (3.11-fold), gill (4.12-fold), nerve (4.84-fold), heart (8.71-fold), and muscle (37.15-fold) (Figure 3C)
After white spot syndrome virus (WSSV) infection, LvIAP1 was upregulated in the gill, hemocytes, and intestine compared with the Phosphate-buffered saline (PBS)-injected group (Figure 4A–D); LvIAP2 was upregulated in the gill, hemocytes, and hepatopancreas (Figure 4F–H); but LvIAP3 was only slightly upregulated in the gill and intestine
Summary
Apoptosis is a genetically programmed process of controlled cell suicide that plays critical roles in organismal development, homeostasis, and the immune system through elimination of cells that are no longer useful [1]. Activation of the Toll and IMD pathways initiates an intracellular signaling cascade to activate the NF-κB family proteins Dorsal/Dif and Relish, respectively, promoting the expression of immune-related genes, such as antimicrobial peptide genes (AMPs) [9,10,11]. Knock-down of Drosophila IAP2 (DIAP2) in insect cells reduced the expression of AMPs induced by Gram-negative bacteria, suggesting a role of DIAP2 in the IMD pathway [15,16,17,18]. It was reported that Gram-negative bacterial infections induce binding of DIAP2 to the caspase homolog DREDD, targeting it for polyubiquitination in a RING finger-dependent manner for Relish processing and subsequent AMP expression [19,20]. We cloned three IAPs from the model crustacean Litopenaeus vannamei and investigated the roles of these proteins during WSSV infection and in regulation of shrimp AMP expression
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