Abstract
Toll-like receptors (TLRs), as important pattern recognition receptors, represent a significant component of fish immune systems and play an important role in resisting the invasion of pathogenic microorganisms. The TLR5 subfamily contains two types of TLR5, the membrane form of TLR5 (TLR5M) and the soluble form of TLR5 (TLR5S), whose detailed functions have not been completely elucidated. In the present study, we first identified two genes, TLR5M (ToTLR5M) and TLR5S (ToTLR5S), from golden pompano (Trachinotus ovatus). The full-length ToTLR5M and ToTLR5S cDNA are 3644 bp and 2329 bp, respectively, comprising an open reading frame (ORF) of 2673 bp, encoding 890 amino acids, and an ORF of 1935 bp, encoding 644 amino acids. Both the ToTLR5s possess representative TLR domains; however, only ToTLR5M has transmembrane and intracellular TIR domains. Moreover, the transcription of two ToTLR5s was significantly upregulated after stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and flagellin in both immune-related tissues (liver, intestine, blood, kidney, and skin) and nonimmune-related tissue (muscle). Furthermore, the results of bioinformatic and promoter analysis show that the transcription factors GATA-1 (GATA Binding Protein 1), C/EBPalpha (CCAAT Enhancer Binding Protein Alpha), and ICSBP (Interferon (IFN) consensus sequence binding protein) may play a positive role in moderating the expression of two ToTLR5s. Overexpression of ToTLR5M and ToTLR5S notably increases NF-κB (nuclear factor kappa-B) activity. Additionally, the binding assay revealed that two rToTLR5s can bind specifically to bacteria and pathogen-associated molecular patterns (PAMPs) containing Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Escherichia coli, Photobacterium damselae, Staphylococcus aureus, Aeromonas hydrophila, LPS, poly(I:C), flagellin, and peptidoglycan (PGN). In conclusion, the present study may help to elucidate the function of ToTLR5M/S and clarify their possible roles in the fish immune response to bacterial infection.
Highlights
Toll-like receptors (TLRs) play an important role in host defense in both invertebrates and vertebrates [1]
The cDNA sequence of ToTLR5S is 2329 bp, including 156 bp of the 5 untranslated region (5 -UTR); a 1935 bp open reading frame (ORF) encoding a polypeptide of 644 amino acids (GenBank accession number: MT596698; Figure 1B); and a 238 bp 3 -UTR, with a predicted molecular weight of 72.21 kDa and a theoretical isoelectric point of 8.84
In TLR5M, the result of amino acid sequence alignment of the Toll/IL-1 (Interleukin-1) receptor (TIR) domain showed that this region contains three conserved functional boxes (Box1, Box2, and Box3) (Figure 2B)
Summary
Toll-like receptors (TLRs) play an important role in host defense in both invertebrates and vertebrates [1]. The TLR family is regarded as a pivotal mediator that activates innate immunity and develops antigen-specific acquired immunity against invading microorganisms and pathogens, such as peptidoglycan, lipoprotein, lipopolysaccharide (LPS), flagellin, and polyinosinic:polycytidylic acid (poly(I:C)) [2,3,4]. As a TLR family member, TLR5 is a key PRR and can recognize PAMPs, such as the bacterial flagellum protein, by touching their flagellin [10,11,12,13]. Our results suggest that ToTLR5 may play an important role as a PRR in the immune response to pathogen infections and may be involved in the NF-кB activation pathway
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