Abstract
TLRs (Toll-like receptors) are essential in host defense against pathogens. There are two types of TLR5, namely, membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S), both of which perform a crucial role in flagellin response. TLR13 is a TLR that localizes to endosomes and recognizes nucleic acids released by internal microorganisms, including viruses, bacteria, and fungi. Here, the full-length coding sequence (CDS), protein structure, and immune response and subcellular localization of TLR5 (TLR5S) and TLR13 were characterized in large yellow croaker (Larimichthys crocea). These TLRs share high sequence homology with other ichthyic TLRs, while also having their own characters; qtPCR was determined and the results found that the three genes were constitutively expressed in all examined tissues: TLR5M was highly expressed in the spleen and liver; TLR13 expression was high in the kidney, liver, and spleen. And TLRs were upregulated following stimulation with Vibrio parahaemolyticus in the liver, spleen, and kidney. Immunofluorescence staining revealed that TLR5M were localized in the cytoplasm, while TLR5S and TLR13 were in the endosome. The evolutionary analysis has shown that TLR13 was clustered with TLR11, 19, 20, 21, and 22, while TLR5 and TLR3 were classified into a group; these results suggest that TLRs are vital in the defense of L. crocea against bacterial infection and further increase our understanding of TLR function in innate immunity in teleosts.
Highlights
TLRs (Toll-like receptors) were first discovered in Drosophila embryos and control the formation of the dorsal-ventral polarity of early embryos [1]
TLR5S lacks Toll/IL-1 receptor homology domain (TIR) and transmembrane domains compare to TLR5M, with an additional LRR_NT and 3 leucinerich repeat (LRR)
The extracellular region of TLR5M contained 18 β-sheets, 14 all located in the horseshoe-shaped concave surface, while the 7 α-helices were located on the horseshoe-shaped convex surface, and the rest were irregularly curled; the intracellular region was 5 α-helix surrounded by 4 parallel β fold
Summary
TLRs (Toll-like receptors) were first discovered in Drosophila embryos and control the formation of the dorsal-ventral polarity of early embryos [1]. TLR5-ligand interaction in mammals, TLR5, is primarily responsible for the detection of a kind of flagellin in bacterial flagella and the specific identification of constant functional domains that are relatively conserved between different bacteria [17]. To fish, they have two forms of TLR5, TLR5M (membrane form) and TLR5S (soluble form), which are responsible for identifying bacterial flagellin [18]. Studies have shown that the recombinant TLR5S of Sparus aurata can bind to flagellin of Vibrio anguillarum, and TLR5M activates the inflammatory response It is still controversial whether the exact function of TLR5S is to enhance the innate immune response or to avoid the excessive inflammatory response of TLR5M [19]. This study was focused on three genes, and the results will contribute to our understanding of the immune system in fish
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