Elsevier

Fish & Shellfish Immunology

Volume 80, September 2018, Pages 109-114
Fish & Shellfish Immunology

Short communication
Genomic organization, evolution and functional characterization of soluble toll-like receptor 5 (TLR5S) in miiuy croaker (Miichthys miiuy)

https://doi.org/10.1016/j.fsi.2018.05.048Get rights and content

Highlights

  • TLR5S was identified in miiuy croaker.

  • Miiuy croaker TLR5S highly expressed in liver.

  • Miiuy croaker TLR5S can activate NF-кB signaling pathway.

Abstract

Toll-like receptors (TLRs) play the key role in host defense of invasion of pathogens, not only in the innate immunity, but also in adaptive immunity. There are significant varieties and distinct features in fish TLRs, the TLR5 subfamily have two members (TLR5M and TLR5S). However, the exact role of TLR5 was lack of research in fish. In this study, a soluble form of TLR5 (TLR5S) was identified in miiuy croaker. The bioinformatics analysis showed that miiuy croaker TLR5S lacked the transmembrane domain and TIR domain. In other words, mmiTLR5S only has leucine-rich repeats (LRRs) domain, it is one of differences between TLR5M and TLR5S. Comparative genomic analysis showed that TLR5S might have happened an evolution between species. Expression analysis showed that mmiTLR5S was expressed in all tested miiuy croaker tissues and the mmiTLR5S expressions were significantly upregulated at 12 h in liver and kidney after Vibrio harveyi infection. Further functional experiments showed that NF-кB can be actived by mmiTLR5S, TLR5S might be an indispensable role in organism immune response. In short, the study of mmiTLR5S enriches the information of TLR5S and lays the foundation for future research on teleost TLRs system.

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).

References (34)

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