Activation of Toll Pathway Is Different between Kuruma Shrimp and Drosophila

Abstract

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.

RNAi screening identifies a new Toll from shrimp that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides

“…The results showed that all the four types of pathogen could induce Dorsal activation with different degrees of nucleus translocation, but it seems to be not relevant in the context of the silencing of Toll4 (Figure 8). The prominent nuclear translocation of Dorsal in hemocytes after V. parahaemolyticus challenge is in good concurrence with previous reports that G - bacterial infection can induce the activation of Dorsal and its translocation (42, 46). These analyses with different pathogens strongly suggest that Toll4 mediated Dorsal activation is WSSV specific, which indicate that Toll4 could play a vital role in recognizing WSSV infection.…”

“…Besides, several Tolls from L. vananmei are able to detect some PAMPs directly, as shown by the fact that Toll1 and Toll3 can interact with CpG ODN 2395 in vitro (57). Surprisingly, another study demonstrates that three Tolls from M. japonicas , two of them homologous to the above Toll1 and Toll3 from L. vananmei (57), can directly bind to both PGN and LPS (46). These data suggest that one type of shrimp Toll could recognize more than one PAMP.…”

“…Before all experiment treatments, the shrimps (5% of total) were detected and confirmed to be free of common pathogens including white spot syndrome virus (WSSV), yellow head virus (YHV), taura syndrome virus (TSV), shrimp hemocyte iridescent virus (SHIV, also known as CQIV), infectious hypodemal and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus by PCR or RT-PCR methods according to standard operation procedures by Panichareon et al (71) and Qiu et al (72). Because many genes from the shrimp canonical Toll-Dorsal pathway can be activated by Gram-negative (G - ) bacteria (42, 46), V. parahaemolyticus thus was used here as a positive activator of the shrimp Toll-Dorsal pathway. The Gram-negative bacteria V. parahaemolyticus were cultured in Luria broth (LB) medium overnight at 37 °C, and the bacteria were harvested by centrifugation (5000 g, 10 min) and washed twice in phosphate buffer saline (PBS) to remove growth medium and finally resuspended in PBS to give 10 8 cells per ml.…”

“…Immunocytochemical staning was used to analysis shrimp Dorsal translocation in hemocyte recommended by a published method with a minor modification (46). In short, hemocytes were collected by centrifugation (1000 g , 5 min) at room temperature (RT) and deposited onto a glass slide, and then fixed immediately with 4% paraformaldehyde at RT for 5 min.…”

“…Fluorescence was visualized on a confocal laser scanning microscope (Leica TCS-SP5, Germany). WCIF ImageJ software was used to analyze the colocalization of Dorsal and Hochest-stained nuclei in hemocytes according to a previously published method (46).…”