Short communicationGenomic organization, evolution and functional characterization of soluble toll-like receptor 5 (TLR5S) in miiuy croaker (Miichthys miiuy)
Introduction
Vertebrates rely on the immune system to eliminate infectious pathogens, when they are suffering the invasion of microbes and pathogens [1], and the immune system consists of innate and adaptive immunity. The innate immune system, as the most universal form of host defense, is the first line of defense against invading pathogens [2]. Toll-like receptors (TLRs) family are a key pathogen recognition receptors (PRRs), highly conserved protein which widely exists in plants, invertebrates and vertebrate, mainly participate in the innate immune system, starting up the cell signal transduction. TLRs have exerted huge effect in the innate immunity and adaptive immunity. As a type I trans-membrane protein, the typical structure of TLRs is formed by three parts: extracellular domain, transmembrane domain and intracellular domain [3]. The extracellular domain is formed by a series of LRRs and participate in the identification of the pathogen-associated molecular patterns (PAMP) [4]. The intracellular domain has a highly conservative Toll/IL-1 receptor domain (TIR) [5], homology with IL-1R family intracellular domain, plays an important role in signal transduction. Since the first Toll was found in human in 1997, 13 types of TLRS were found in mammalian [1,6].
As a family member of TLRs, TLR5 is an important PRRs and plays a prominent role in protecting fish from disease [7]. TLR5 can recognize the PAMPs, like the bacterial flagellum protein [8,9]. TLR5 can activate the NF-κB through the MyD88-dependent pathway, and then produce the inflammatory factors [10]. Current study found two types of TLR5 in teleost: membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S). To date, TLR5S has not been found in mammalian. According to previous reports, TLR5S was found for the first time in Takifugu Rubripes [11]. Later, TLR5S was found in Onchorhynchus mykiss, Salmo salar, Ictalurus punctatus and Paralichthys olivaceus [[12], [13], [14], [15]]. TLR5M is comprised of TIR domain, trans-membrane region and LRR domain, but TLR5S has no TIR domain and trans-membrane region by comparison. Recent study showed that TLR5M identified flagellin, and NF-кB was activated to induce production of some immune response genes and TLR5S [12]. TLR5S was induced to recognize flagellin in fluid phase, and then binds to TLR5M to amplify the signal cascade [15]. To date, this unique amplifiable flagellin recognition system had only been found in fish.
Miiuy croaker (Miichthys miiuy) is a representative species in Sciaenidae family, is mainly distributed between the western Japan Sea and the East China Sea. Miiuy croaker is an economically important fish for its medicinal and nutritional value, but its aquaculture industry has been affected by the variety of pathogens, such as Vibrio harveyi [16]. As an immune-primitive animal, fishes mainly rely on their innate immune system to protect themselves from a variety of diseases by various PRRs [7]. As a family member of PRRs, TLR5 plays a very important role in protecting themselves against diseases [17]. In this study, we identified the TLR5S in miiuy croaker. The sequences analysis, syntenic relationship and phylogenetic analysis of mmiTLR5S enable us to better understand the characteristic of TLR5S. The preliminary function experiments of TLR5S on the expression pattern, and luciferase reporter assays were performed to test the role of TLR5S in immune response.
Section snippets
Sample treatment
Healthy miiuy croakers (average weight 750 g) were purchased from the Zhoushan Fisheries Research Institute (Zhejiang, China) and temporarily placed in the inflatable seawater tanks at laboratory temperature to adapt to the environment. After acclimatizing, only healthy fish can be used in the experiment as described in previous studies [18,19]. Fish samples were divided into two groups at random, control and injected groups. The injected group was injected with 1 ml Vibrio harveyi (1.5 × 108
Characteristics of mmiTLR5S gene
The full length mmiTLR5S gene was successfully obtained, which was 3335 bp, including 5′-untranslated region (UTR) of 138 bp and 3′UTR which has one RNA instability motif “ATTTA” sequence of 271 bp (Fig. 1A). A 1932 bp open reading frame (ORF) was used to predict encoding protein of 644 amino acids. MmiTLR5S was composed of two exons and one intron which followed the GT/AG rule (Fig. 1B). The SMART program predicted that mmiTLR5S only had LRRs domain (Fig. 1C). The molecular weight and the
Discussion
TLRs family is highly conservation in evolution, TLRs in most mammal can find the homologous in fish [4]. However the varied living environment of fish lead to TLRs family showed polymorphism in the evolutionary process [29]. Since the first TLR gene of teleost was identified in goldfish [30], at least 20 different TLR genes have been found in teleosts [31]. TLR5S, TLR14, TLR16, TLR18-23 and TLR25-28 were unique in teleosts [2,32]. TLR5S as a member of these genes perhaps has a very important
Acknowledgements
This study was supported by National Natural Science Foundation of China (31672682).
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2023, Developmental and Comparative ImmunologyCitation Excerpt :Typically, TNF receptor associated factor 6 (TRAF6) forms a complex with TAK (transforming growth factor-beta-activated kinase) and TAB (TAK-binding protein), and further activates downstream IKK-NF-κB and MAPK (mitogen-activated protein kinases) cascades through phosphorylation (Kawai and Akira, 2007; Min et al., 2018), eventually leads to the activation of transcription factors NF-κB and AP-1 and controls the expression of pro-inflammatory cytokines and other immune-related genes (An et al., 2010; Anthoney et al., 2018; Liao et al., 2017). Up to now, ten TLRs have been identified in humans, including TLR1-10 (Fore et al., 2021; Guan et al., 2010), more than 20 kinds of TLRs (TLR1, 2, 3, 4, 5M, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, 26) have been described in fish (Boudinot et al., 2014; Roach et al., 2005a; Xu et al., 2016; Huo et al., 2018), and TLR14 and TLR22 in fish are unique to the organism (Ishii et al., 2007). Many members in TLR family are common among different animals, but they have different ligands and functions (Jault et al., 2004).