SpToll1 and SpToll2 modulate the expression of antimicrobial peptides in Scylla paramamosain.

Tolls and Toll-like receptors (TLRs) were the first pattern recognition receptors (PRRs) identified to play key roles in host innate immunity. However, relatively little is known about other types of Toll-like receptors in Scylla paramamosain, although a Toll-like receptor (SpToll1) has recently been cloned. In this study, we cloned and characterized another novel Toll-like receptor 2 (SpToll2) from S. paramamosain. The full-length cDNA of SpToll2 is 3391 bp with a 2646 bp open reading frame (ORF) encoding a putative protein of 881 amino acids, and predicted to contain six extracellular leucine-rich repeat (LRR) domains, a transmembrane domain and an intracellular Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis revealed that SpToll2 clustered with Drosophila Toll1, and shared high homology with PtToll4. Real-time qPCR analysis showed that SpToll2 was widely expressed in all tissues tested, with the highest level found in hemocytes and hepatopancreas while the lowest in heart and muscle. The transcript levels of both SpToll1 and SpToll2 in mud crabs hemocytes was induced following challenge with Vibrio parahaemolyticus, Staphylococcus aureus, Polyinosinic: polycytidylic acid (Poly I:C) and white spot syndrome virus (WSSV). In addition, recombinant SpToll1-LRR and SpToll2-LRR proteins could bind to V. parahaemolyticus, S. aureus, Escherichia coli, and Beta Streptococcus. In order to study the signaling pathway of AMPs' expression in mud crab, RNA interference were used to test the expression of SpAMPs after the challenges with V. parahaemolyticus or S. aureus. The data suggested that SpToll1and SpToll2 could regulate the transcripts of several AMPs and four immune related mediators (SpMyD88, SpTube, SpPelle and SpTRAF6) at different scale. While silencing of SpToll1 post pathogens challenge attenuated the expression of SpHistin, SpALF1 and SpALF5 in mud crab's hemocytes, depletion of SpToll2 post pathogens challenge inhibited the expression of SpALF1-6, SpGRP, SpArasin and SpHyastastin. Furthermore, the results of overexpression assay also showed SpToll1 and SpToll2 could enhance the promoter activities of SpALFs in mud crab. Taken together, these results indicated that SpToll1 and SpToll2 might play important roles in host defense against pathogen invasions in S. paramamosain.

Toll-like receptor 2 type 1 and type 2 polymorphisms in different chicken breeds.

Toll-like receptors mediate immune responses via recognition of pathogen-associated molecular patterns. Polymorphisms of toll-like receptors may affect their recognition of pathogen-associated molecular patterns, leading to varied host resistance to pathogenic infections. However, little is known about the polymorphisms of chicken toll-like receptors (ChTLR) among breeds. In this study, we cloned ChTLR2 type 1 and type 2 genes from 7 chicken breeds and analyzed their sequences. It was found that there were 10 amino acid polymorphism sites in ChTLR2 type 1 and type 2, among which 6 sites were in type 1 (5 sites in the extracellular domain and 1 site in the cytoplasmic domain) and 4 sites were in type 2 (all 4 sites in the extracellular domain). These results demonstrate that ChTLR2 type 1 and type 2 genes are polymorphic among chicken breeds, suggesting a varied resistance among breeds of chicken. We found that Laiwu Black chicken breeds have distinctive polymorphic sites at I699T in the type 1 and H561R in the type 2 ChTLR2 gene. Beijing Fatty chickens have distinctive sites at Q45R in type 1 and V66L in type 2. Nongda No.3 Chickens have distinctive sites at L115P, H232Y, and T494A in type 1. Hy-Line variety brown chickens have distinctive sites at I311V in type 2. Beijing White 939 chickens have distinctive sites at E284G in type 1 and A22V in type 2. These findings may provide some clues toward understanding the resistance of lighter chickens to salmonellosis.