Identification, Characterization, and Functional Analysis of Tube and Pelle Homologs in the Mud Crab Scylla paramamosain

Abstract

Tube and Pelle are essential components in Drosophila Toll signaling pathway. In this study, we characterized a pair of crustacean homologs of Tube and Pelle in Scylla paramamosain, namely, SpTube and SpPelle, and analyzed their immune functions. The full-length cDNA of SpTube had 2052 bp with a 1578 bp open reading frame (ORF) encoding a protein with 525 aa. A death domain (DD) and a kinase domain were predicted in the deduced protein. The full-length cDNA of SpPelle had 3825 bp with a 3420 bp ORF encoding a protein with 1140 aa. The protein contained a DD and a kinase domain. Two conserved repeat motifs previously called Tube repeat motifs present only in insect Tube or Tube-like sequences were found between these two domains. Alignments and structure predictions demonstrated that SpTubeDD and SpPelleDD significantly differed in sequence and 3D structure. Similar to TubeDD, SpTubeDD contained three common conserved residues (R, K, and R) on one surface that may mediate SpMyD88 binding and two common residues (A and A) on the other surface that may contribute to Pelle binding. By contrast, SpPelleDD lacked similar conservative residues. SpTube, insect Tube-like kinases, and human IRAK4 were found to be RD kinases with an RD dipeptide in the kinase domain. SpPelle, Pelle, insect Pelle-like kinases, and human IRAK1 were found to be non-RD kinases lacking an RD dipeptide. Both SpTube and SpPelle were highly expressed in hemocytes, gills, and hepatopancreas. Upon challenge, SpTube and SpPele were significantly increased in hemocytes by Gram-negative or Gram-positive bacteria, whereas only SpPelle was elevated by White Spot Syndrome Virus. The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle. These results suggest that SpTube, SpPelle, and SpMyD88 may form a trimeric complex involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria.

Identification, characterization, and functional studies of a Pelle gene in the Chinese mitten crab, Eriocheir sinensis

“…EsPelle mRNA could be detected in all examined tissues and was detected at high abundance in the gills, hepatopancreas, and hemocytes, as observed with the other two other crustacean Pelles (SpPelle and LvPelle) [26,27]. SpPelle is highly expressed in hemocytes, gills, and hepatopancreas [26].…”

“…SpPelle is highly expressed in hemocytes, gills, and hepatopancreas [26]. LvPelle mRNA is largely distributed in gills [27].…”

“…LvPelle (now identified as Tube isoforms) in the hepatopancreas is upregulated by LPS, Vibrio alginolyticus, and poly (I:C) [27]. SpPelle was significantly increased in hemocytes by Gram-negative Vibrio harveyi or Gram-positive bacteria S. aureus [26]. EsPelle is highly expressed in these immune tissues and is regulated by different microbes.…”

“…The SpPelle expression was not only significantly increased in hemocytes by Gram-negative or Gram-positive bacteria but also elevated by white spot syndrome virus (WSSV). The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle, which may suggest that SpTube, SpPelle, and SpMyD88 form a trimeric complex that is involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria [26]. Knockdowns of LvPelle using the RNAi strategy lead to higher shrimp mortality during Vibrio parahemolyticus infection and could reduce the genome copy number of WSSV in the infected muscle tissue.…”

“…To date, only two Pelles have been identified and characterized from crustaceans, Scylla paramamosain and Litopenaeus vannamei [26,27]. The SpPelle expression was not only significantly increased in hemocytes by Gram-negative or Gram-positive bacteria but also elevated by white spot syndrome virus (WSSV).…”