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Expression analysis of nine Toll-like receptors in yellow catfish (Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge
Accepted Manuscript Expression analysis of nine Toll-like receptors in yellow catfish (Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge Xiao-Ting Zhang, Gui-Rong Zhang, Ze-Chao Shi, Yu-Jie Yuan, Huan Zheng, Li Lin, Kai-Jian Wei, Wei Ji PII:
S1050-4648(17)30093-1
DOI:
10.1016/j.fsi.2017.02.021
Reference:
YFSIM 4443
To appear in:
Fish and Shellfish Immunology
Received Date: 5 November 2016 Revised Date:
16 January 2017
Accepted Date: 17 February 2017
Please cite this article as: Zhang X-T, Zhang G-R, Shi Z-C, Yuan Y-J, Zheng H, Lin L, Wei K-J, Ji W, Expression analysis of nine Toll-like receptors in yellow catfish (Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.02.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expression analysis of nine Toll-like receptors in yellow catfish
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(Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge
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Zhang Xiao-Ting1, 2, Zhang Gui-Rong1, 2, Shi Ze-Chao2, 3,
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Yuan Yu-Jie1, 2, Zheng Huan1, 2, Lin Li1, 2, Wei Kai-Jian1, 2*, Ji Wei1, 2*
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1 Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, College of
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Fisheries, Huazhong Agricultural University, Wuhan 430070, China
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2 Freshwater Aquaculture Collaborative Innovation Centre of Hubei Province, Wuhan
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430070, China
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3 Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture, Yangtze
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River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223,
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China
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* Corresponding author at: College of Fisheries, Huazhong Agricultural University, Wuhan
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430070, P. R. China
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E-mail addresses: [email protected] (W. Ji), [email protected] (K.-J. Wei)
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Abstract
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Toll-like receptors (TLRs) are important components of pattern recognition receptors (PRRs),
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which play significant roles in innate immunity to defense against pathogen invasion. Many
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TLRs have been found in teleosts, but there are no reports about cloning and expression of
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TLR genes in yellow catfish (Pelteobagrus fulvidraco). In this study, we analyzed the
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sequence characters and the relative mRNA expression levels of nine TLRs (TLR1, TLR2,
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TLR3, TLR4-1, TLR5, TLR7, TLR8-2, TLR9 and TLR22) in different tissues of yellow
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catfish. The results showed that all nine TLR genes are highly expressed in head kidney,
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trunk kidney, spleen and liver, all of which are related to host immunity. Subsequently we
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used Aeromonas hydrophila as a stimulating agent to detect the expression profiles of these
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nine TLRs in the liver, spleen, trunk kidney and head kidney of yellow catfish at different
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time points after injection with killed Aeromonas hydrophila. All nine TLRs responded to A.
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hydrophila challenge with tissue-specific patterns in different immune tissues. The kinetics
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of up- or down-regulation of these nine TLRs exhibited a similar trend, rising to an elevated
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level at first and then falling to the basal level, but the peak value differed at different time
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points in different tissues. The expression levels of the TLR3, TLR4-1, TLR9 and TLR22
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genes were significantly up-regulated after bacterial challenge in the liver, spleen, head
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kidney and trunk kidney. The relatively high expression of TLR genes in the immune tissues
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in response to the A. hydrophila challenge indicated that TLRs may play important roles in
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the innate immune response against gram-negative bacteria in yellow catfish.
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Keywords: Yellow catfish; Aeromonas hydrophila; Toll-like receptors; Gene expression;
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intraperitoneal injection; Innate immunity
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1. Introduction The innate immune system is the first and oldest line of host defense to the invasion of
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pathogens, and is regarded as the primary defense mechanism that exists in lower vertebrates
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such as fish [1]. Fish immune systems provide important comparative outgroups for an
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understanding of the evolution of immune systems, eventually leading to an increased
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understanding of general principles of immune system design [2]. Additionally, the aquatic
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environment is a unique medium in which microorganisms are constantly in contact with the
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potential host fish and their immune system. Failure of immunity is a major risk for
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commercial fish farming because of the unnaturally high rearing density of aquaculture
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species compare to wild species and the great cost of infections to aquaculture [3].
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The innate immune system recognizes pathogen-associated molecular patterns (PAMPs)
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that are structurally conserved among many microorganisms, via pattern recognition
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receptors (PRRs) encoded by the germ-line. The earliest characterized and the most
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extensively studied PRRs in both vertebrates and invertebrates are Toll-like receptors (TLRs)
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[4], which can recognize a wide range of PAMPs [5]. TLRs are expressed either on the cell
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surface or associated with intracellular vesicles. They are type I transmembrane
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glycoproteins, containing three important domains: an extracellular leucine-rich repeat (LRR)
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domain, a transmembrane domain and an intracellular Toll/interleukin-1 receptor (TIR)
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domain. TLRs recognize conserved PAMPs by the extracellular LRR domains that constitute
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a horse-shoe shape, and activate downstream signaling pathways through their TIR domain
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[6-8]. After recognizing PAMPs, TLRs transmit downstream signals into the cytosol and
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activate a cascade of adaptor molecules. Among these adaptor molecules, TIR domain
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containing adapter inducing IFN-
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differentiation factor 88 (MyD88) are two important adaptor proteins, which subsequently
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result in the induction of various cytokines such as TNF-α, IL-1β, IL-12, IL-8, IL-6 and IFN
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(TRIF, also known as TICAM1) and myeloid
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[9-11]. The Toll gene was first identified to control the establishment of the dorso-ventral
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pattern in fruit fly (Drosophila melanogaster) embryos [12]. Later, it was found to play an
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essential role in the fly’s immunity against fungal infection, associating the role of “Toll”
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with the synthesis of anti-microbial peptides [13]. Then a focus on immunity-related
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function of Toll started. To date, 13 TLRs (TLR1 - 13) have been identified in mammals and
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were classified into different families [14]. Huge diversified TLRs exist in fishes as
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compared to mammals due to the fact that the aquatic media not only allows transport but
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also the growth of microorganism [3]. At least 20 TLRs have been identified in more than a
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dozen teleost species [15]. Some TLRs are expressed on the host cell surface in order to
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recognize extracellular microbial structures, and others are expressed intracellularly on
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endocytic vesicles so as to specifically detect viral or bacterial nucleic acids [5]. Compared
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with mammals, fish lack some TLR members such as TLR6 and TLR10, whereas some
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TLRs are “fish-specific”, including TLR5s, TLR14, TLR19, TLR20, TLR21, TLR22 and
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TLR23 [16-18].
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At present, 27 TLRs have been identified in vertebrates. These have been divided into
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six major families, namely TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11 [19]. Selective
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pressure presumably for maintenance of specific PAMP recognition dominates the TLR1
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family (for lipopeptide), the TLR3 family (for dsRNA), the TLR4 family (for LPS), the
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TLR5 family (for flagellin), the TLR7 family (for nucleic acid and heme motifs) and the
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TLR11 family. TLRs within the same family recognize a general class of PAMPs [19].
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In recent years, scientists have intensively researched into the TLR signaling cascade
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and proved that some components of pathogens are the ligands of TLRs [20]. However, the
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direct evidence of ligand specificity has only been shown for a few TLRs [21]. Specifically,
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there is limited information on the roles of fish TLRs in the response to gram-negative 4
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bacteria challenge. Thus, which and how fish TLRs are involved in anti-bacterial immune
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defense is still largely unknown. Yellow catfish (Pelteobagrus fulvidraco), a teleost belonging to the family Bagridae, is
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extensively cultured in East and South Asia in semi-intensive and intensive systems. Due to
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its excellent meat quality, it is an important commercial freshwater fish in China [22]. In
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2014, the total production of yellow catfish reached 333,651 tonnes in China [23]. With the
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rapid development of yellow catfish pond culture, especially high density intensive
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aquaculture in China, some microbial diseases have become more serious and impeded
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further rapid development of the yellow catfish culture industry. Aeromonas hydrophila, a
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gram-negative bacterium, is the most common opportunistic pathogen that can infect yellow
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catfish and other aquatic animals. The infection of A. hydrophila spreads very rapidly and
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can result in bacterial septicemia and other serious damage and may result in death of
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aquatic animals [24]. Considering the current serious lag condition of fish immune research
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relative to aquaculture development in China, it is necessary to enhance the research of
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immune responses to bacterial diseases in fish. As important PRRs, TLRs can recognize the
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PAMPs of the microbes and defend against invading microbes. To illustrate the role of TLRs
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in teleosts, we investigated which fish TLRs play important roles in the immune response
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against Gram-negative bacteria pathogens by detecting the expression profiles of the nine
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yellow catfish TLRs at different time points after A. hydrophila challenge.
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This study aimed to (i) identify as many TLRs as possible in yellow catfish, and
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conduct a comparative analysis of yellow catfish TLRs against those identified from other
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species, and (ii) examine the mRNA expression changes of TLRs after bacterial challenge.
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Here we report phylogenies of nine yellow catfish TLR genes (TLR1, TLR2, TLR3, TLR4-1,
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TLR5, TLR7, TLR8-2, TLR9 and TLR22) and their expressions in healthy and bacterial
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challenge tissues. This study will enhance our understanding of the role of TLRs in the 5
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disease immune defense of teleosts and further help us to control fish microbial diseases.
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2. Materials and methods All experimental procedures were approved by the Institutional Animal Care and Use
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Committees (IACUC) of Huazhong Agricultural University (HZAU), Wuhan, China. No
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specific permits were required for the field studies described here. The study area is not
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privately-owned or protected in any way, and the field studies did not involve endangered or
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protected species.
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2.1. Animals, bacterial challenging and sampling
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The original strains of yellow catfish were provided by the Jingzhou fish farm in Hubei
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province, China, and were transported to the fish breeding base of HZAU. All fishes were
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raised at the same level of nutrition and management in our laboratory. They were
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temporarily kept in an indoor circulating water tank with fresh water at 24 °C and were fed
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to satiation with a commercial diet (Hubei Haid Feeds Company, Wuhan, China) twice a day
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(8:00 am and 4:00 pm).
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To clone the cDNA of TLR7 and investigate the expression profile of TLRs, five
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individuals (~ 100 g) were collected for sampling. The fish were sampled at 1:00 pm and
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then anesthetized with tricaine methanesulfonate (MS-222, 1,000 mg/L) before dissection.
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Eighteen different tissues (blood, ovary, brain, eye, gill, fin, skin, muscle, heart, liver, spleen,
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stomach, trunk kidney, head kidney, foregut, midgut, hindgut and swim bladder) were
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rapidly isolated for RNA extraction.
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We prepared 60 individuals (~100 g) to investigate the effect of bacterial challenge on
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the expressions of TLRs. To perform the bacterial challenge experiment, A. hydrophila were
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obtained from the microbiology laboratory of HZAU. Single colonies were picked out and
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inoculated into 200 mL Luria-Bertani (LB) liquid culture media, which were cultured in a 6
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4 °C with 0.2% formalin for 36 h, centrifuged at 4500 rpm and suspended twice using PBS
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(pH 7.2). Finally the killed bacteria were resuspended in sterile PBS and calculated under a
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microscope. After acclimation, five fish were randomly sampled as a control group, and the
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other fish in the experimental group were stimulated with formalin-killed A. hydrophila at a
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dose of 1.5×107 cells (suspended in 50 µL PBS with pH 7.2) per fish by intraperitoneal
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injection. Five fish were randomly sampled from the experimental group at 6 h, 12 h, 24 h,
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48 h and 72 h post-injection (hpi). The sampled fish were anesthetized with 300 mg/L
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MS-222, and then liver, spleen, head kidney and trunk kidney tissues were collected for
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RNA extraction.
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All tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C until
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RNA extraction.
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2.2. Extraction of RNA and the reverse transcription PCR (RT-PCR) RNA was isolated from different tissues and four immune tissues at different stages
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after bacterial challenge using the TRIZOL Reagent according to the manufacturer’s
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protocol. And the RNA samples were treated with DNAse to remove the potential genomic
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DNA. The integrity and purity of RNA were assayed by agarose gel electrophoresis and a
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Nanodrop ND-2000 spectrophotometer (Thermo Electrom Corporation, USA), respectively.
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cDNA was reverse transcribed from total RNA using a moloney murine leukemia virus
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(M-MLV) Reverse Transcriptase kit (Promega, USA) following the manufacturer’s protocol.
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Approximately 2 µg of total RNA was used and pre-denaturated at 70 °C for 5 min. PCR
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reactions were performed at 42 °C for 60 min.
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2.3. Data mining and phylogenetic analysis
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In our previous study, we sequenced the transcriptome of yellow catfish using Illumina
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sequencing technology [25]. All potential TLRs predicted by the transcriptome analysis were 7
ACCEPTED MANUSCRIPT pulled and identified using BLAST against NCBI. Nine nucleic acid sequences were chosen
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for this study: TLR1, TLR2, TLR3, TLR4-1, TLR5, TLR8-2, TLR9 and TLR22 with full
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length of cDNAs except TLR7 with only 370 bp. The retrieved sequences of TLR genes
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were translated using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Further, the
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predicted ORFs were verified by BLASTP against NCBI non-redundant protein sequence
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database. The Simple Modular Architecture Research Tool (SMART) was used to predict the
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conserved domains based on sequence homology and further confirmed by conserved
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domain prediction from BLAST. LRR finder 2.0 [26] was used to identify LRRs using their
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Toll-like receptor LRR database. TMHMM 2.0 was applied to predict the transmembrane
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domain in TLRs. Multiple sequence alignments were conducted using ClustalW
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(http://www.genome.jp/tools/clustalw/)
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(http://www.ch.embnet.org/software/BOX_form.html).
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conducted using MEGA5.0.3. GenBank accession numbers of sequences for amino acid
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multiple alignments and phylogenetic tree are shown in Table 1.
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2.4 Molecular cloning of TLR7
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and
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Based on the partial cDNA sequence and the TLR7 sequences of the other species,
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degenerate primers were designed by Primer Premier 5.0 (Table 2) to amplify the full-length
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cDNA sequence of TLR7. Using the cDNA prepared above as a template we amplified the
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TLR7 fragment under the following PCR conditions: initial incubation at 95 °C for 3 min,
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then 35 cycles were run - each consisting of 30 s at 95 °C, 40 s at 57 °C and 72 s at 72 °C.
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The final step was conducted at 72 °C for 10 min. The 20 µL PCR reaction mixture
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contained 2 µL of 10 × buffer, 2 µL of MgCl2 (25 mM), 1.5 µL of dNTP (2.5 mM), 1 U Taq
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DNA polymerase (Thermo, USA), 0.5 µL (10 µM) of each primer, 160 ng cDNA and 10.5
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µL ddH2O.
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The PCR product was purified with a centrifugal columnar agarose gel DNA extraction 8
ACCEPTED MANUSCRIPT kit and cloned into the pMD18-T easy vector (Takara, Japan). It was then transformed into
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the competent cells of Escherichia coli DH5α and plated on the LB-agar petri-dish. Positive
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colonies containing an insert of the expected size were screened by colony PCR. Three of
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them were sequenced by the Sangon Biotech Company (Shanghai, China).
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2.5. Quantitative real-time PCR (qRT-PCR) and statistical analysis
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QRT-PCR was conducted on the Roche LightCycler 480 Real-Time PCR System. The
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qRT-PCR primers for TLRs are listed in Table 2. The specificity of the primers for qRT-PCR
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was confirmed by agarose gel electrophoresis (data not shown). Yellow catfish β-actin was
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used as an internal control [27]. SYBR® Premix Ex TaqTM (Roche) was used following the
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manufacturer’s protocols. Each PCR was performed in a total volume of 20 µL, including 10
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µL of SYBR® Premix Ex TaqTM, 40 ng of cDNA, 0.2 µM of each sense and antisense primer,
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and 7 µL ddH2O. Reactions were performed by a three-step method, 95 °C for 1 min initially,
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followed by 40 cycles, 95 °C for 5 s, 60 °C for 20 s and 72 °C for 20 s. Melt curve analysis
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was carried out over a range from 55 °C to 99 °C at the end of each PCR run. For each
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sample, quantitative PCR was performed in triplicate. A negative control (template replaced
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by ddH2O) was used for each primer pair.
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Statistical analysis was performed using Graphpad software and calculated using the
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2-∆∆Ct method [27]. All data were expressed as the mean ± SE and checked for normality and
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homogeneity of variances before statistical analysis. A one-way analysis of variance
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(ANOVA) was conducted to compare the differences of expression levels among various
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samples. The Newman-Keuls test was used to perform the multiple comparisons. The level
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of statistical significance was set at P < 0.05 for all analyses.
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3. Results
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3.1. Identification and analysis of TLR genes 9
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The partial cDNA sequence of TLR7 that we cloned was 2915 bp encoding 971 aa. A
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total of nine putative TLR genes were identified in yellow catfish - TLR1, TLR2, TLR3,
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TLR4-1, TLR5, TLR7, TLR8-2, TLR9 and TLR22. TLR family members are characterized by possession of several structural features such
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as LRR, TM and TIR. The nine deduced TLR amino acid sequences in yellow catfish
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contained various numbers of LRR domains, and the LRR number in each TLR ranged from
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6 to 18: 6 (TLR1), 7 (TLR2), 18 (TLR3), 11 (TLR4-1), 12 (TLR5), 14 (TLR7), 8 (TLR8-2),
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12 (TLR9) and 14 (TLR22). All of the TLRs include a C-terminus LRR (LRR CT) and a TIR
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domain (Fig. 1). All TLRs mentioned in this study have the TM domain with the exception
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of TLR7.
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We used all available TLR amino acid sequences in selected species to perform
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sequence alignment. Phylogenetic analysis revealed that TLRs in yellow catfish and other
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species shared common characteristics, and the phylogenetic tree provided strong support for
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the identities of the yellow catfish TLRs (Fig. 2). All of the TLR families and all of the TLR
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genes within each family are about equally distant from the center of the tree.
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3.2. Expressions of TLR genes in different tissues
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Eighteen yellow catfish tissues were used to detect mRNA expressions of TLRs by
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qRT-PCR. The results showed that TLR genes were mostly constitutively expressed in all
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tested tissues, but with tissue preference (Fig. 3). TLR3, TLR4-1 and TLR5 were most
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highly expressed in the liver, whereas TLR2, TLR8-2, TLR9 and TLR22 were highly
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expressed in the spleen. Almost all TLR genes had relatively high expression levels in the
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liver, spleen, head kidney and trunk kidney, which are important in the immune system of
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fish. Interestingly, higher expression levels of TLR1, TLR3, TLR4-1, TLR7 and TLR8-2
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were detected in the brain tissue, indicating a role of these genes in central nervous system
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infections.
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3.3. Responses of TLR genes to bacterial challenge Expressions of the nine TLR genes in yellow catfish were analyzed in the liver, spleen,
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head kidney and trunk kidney tissues after bacterial challenge. All nine TLR genes
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responded to bacterial challenge but their responses were tissue-specific (Fig. 4). The
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kinetics of up- or down-regulation of the nine TLR genes exhibited a similarity of general
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trend, rising to a high level at first and then falling to the basal level, but the up- and
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down-regulation differed at different time points in different tissues.
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In the liver, almost all the TLR genes involved in this study were induced at particular
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time point after bacterial challenge except TLR2, for which no significant increase was
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observed. The expression levels of TLR1, TLR3, TLR7, TLR8-2 and TLR9 were
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significantly increased and reached a peak at 12 h post-injection (hpi) in the liver. The
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expression level of TLR22 was up-regulated at 6 hpi and no significant changes were
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observed at other time points compared to the control group. The expressions of TLR4-1 and
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TLR5 were significantly induced and peaked at 24 hpi.
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In the spleen, almost all the TLR expressions were induced at some time points except
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TLR8-2. No significant changes were observed for TLR8-2 after bacterial challenge in
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spleen. TLR1 expression was up-regulated significantly at 24 hpi and the expression level
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was maintained until 72 hpi. The expression of TLR2 was induced in spleen and peaked at
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24 hpi. The expression of TLR3 was significantly up-regulated at 6 hpi and maintained at a
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relatively high level until 24 hpi in spleen. The expression of TLR4-1 was similar to TLR5,
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with high levels at 12 hpi and 24 hpi. The expression level was up-regulated at 6 hpi for
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TLR7 and at 12 hpi for TLR22, and no significant changes were observed at other time
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points for both of these two genes. The expression of TLR9 was induced at all time points
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and peaked at 12 hpi.
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In the head kidney, all the nine TLR expressions were induced obviously after bacterial 11
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challenge, and the induction peaked at 24 hpi for TLR5 and at 12 hpi for other TLRs. The
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expression of TLR3 was significantly up-regulated at 6 hpi and maintained at a relatively
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high level until 24 hpi in head kidney. In the trunk kidney, similar expression trends were observed for TLR3, TLR4-1, TLR5,
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TLR7, TLR9 and TLR22, with obvious expression induction at 6 hpi and highest expression
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levels at 12 hpi. And then the expression levels decreased to the control level gradually. The
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expression levels of TLR2 and TLR8-2 were up-regulated at 24 hpi and maintained at a
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relatively high level until 48 hpi. The TLR1 expression was induced at 6 hpi and peaked at
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24 hpi, and its induction maintained at a relatively high level until 48 hpi.
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The expression levels of the nine TLR genes after A. hydrophila challenge can be
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characterized in the following general patterns: (1) All nine genes responded to bacterial
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challenge with obvious differences in different tissues; (2) The responses of TLR 3, 4-1, 9,
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22 to bacterial challenge presented a rapid over-expression at 6 hpi; (3) In the head kidney,
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expression levels of all nine TLR genes were up-regulated after bacterial challenge. Their
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expression levels and the corresponding time points of up-regulation varied with various
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genes, but in general the expression levels were upward within 24 hpi; (4) Expression levels
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of the nine TLRs in the spleen were significantly induced, except for that of TLR8-2.
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4. Discussion
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Whilst there is a remarkable increase in annotations documented in the literature or
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databases concerning the identities of various TLR genes and specific TLRs against various
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PAMPs of invasion [28-30], this increase does not cover fish, let alone yellow catfish.
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Herein, we cloned TLR7 and analyzed the gene sequence and phylogenetic status of nine
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TLRs (TLRs 1, 2, 3, 4-1, 5, 7, 8-2, 9, 22) in yellow catfish. In addition, the expression levels
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of these nine TLR genes were detected in different tissues of yellow catfish. Furthermore, we 12
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revealed the expression patterns of the nine TLR genes in four immune-relevant tissues in
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response to bacterial challenge to better understand the role of the nine TLR genes in the
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innate immune system. The deduced amino acid sequences of nine TLRs from yellow catfish showed greatest
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similarity to other vertebrates. Like most other vertebrates, almost all of the nine TLRs
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identified in this study have various numbers of LRR domains, a C-terminus LRR domain, a
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TM domain and a TIR domain except TLR7, which lacks the TM domain. We also predicted
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the amino acid sequences of TLR7 from human and house mouse, both of which also lack
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the TM domain, being consistent with the reports that TLR7 is located in the cytoplasm [31].
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Alignments and phylogenetic tree analysis demonstrated six major TLR families containing
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nearly all vertebrate TLRs, consistent with previous studies [32]. Significantly, it appears
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that vertebrate TLRs are not fast-evolving genes and that the discrepancies in molecular
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distances between species with shorter and longer generation times are relatively muted. As a
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consequence, TLRs in yellow catfish share highly conserved domains with other species,
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consistent with reports that TLRs are evolutionarily highly conserved, from the worm
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(Caenorhabditis elegans) to mammals [1].
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All nine TLRs examined in this study had a constitutive expression in all tissues of
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yellow catfish. A relatively high expression of TLRs was observed in the liver, spleen, trunk
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kidney and head kidney, which is very similar to that reported for most TLR genes in other
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species. For example, TLR1 and TLR2 were both constitutively expressed in a wide range of
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organs/tissues of fish and normally a higher level of TLR1 and TLR2 expression was
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observed in immune organs [17, 33]. Grass carp TLR22 is a non-maternal gene and
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prominently expressed in immune relevant tissues such as spleen and head kidney [30]. In
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addition, higher expression levels of TLR1, TLR3, TLR4-1, TLR7 and TLR8-2 were
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detected in the brain tissue of yellow catfish; this observation could be important in
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understanding the role of particular TLRs in pathological processes unique to brain tissue. Our investigation of the expression profiles of nine TLRs in four tissues of yellow
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catfish at different time points after A. hydrophila challenge has shown that all tissue types
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responded to the stimulation. Each TLR binds to its own set of preferential ligands such as
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lipoproteins (recognized by TLR1, TLR2, and TLR6), double-stranded (ds) RNA (TLR3),
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lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), single-stranded (ss) RNA (TLR7 and
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TLR8) and DNA (TLR9) [1, 34]. It is likely that more kinds of TLRs, as well as diverse
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PAMPs specific TLRs, exist due to a genome duplication event or to environmental
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adaptation in fish [35].
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TLR1 family members (TLRs 1, 2, 6, 10) specific for lipopeptide can primarily
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recognize PAMPs from bacteria and play pivotal roles in sensing microbial products [19, 33].
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TLR2 is best known for recognizing the conserved components of Gram-positive bacteria
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such as lipoteichoic acid (LTA), peptidoglycans (PGN), and lipoproteins [14]. But in our
336
study both TLR1 and TLR2 were significantly up-regulated after stimulation of A.
337
hydrophila with the exception of TLR2 in the liver. This indicates that TLR1 and TLR2 can
338
also respond to some components of Gram-negative bacteria.
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The expression levels of TLR3, TLR4-1, TLR7, TLR8-2 and TLR9 were significantly
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changed at most time points after bacterial (A. hydrophila) challenge in yellow catfish. But
341
in mammals, these TLRs are anti-viral receptors. RNA from RNA viruses is recognized by
342
TLR3 (double-stranded RNA), TLR7 and TLR8 (single-stranded RNA). DNA such as
343
unmethylated CpG DNA from DNA viruses and microbes is recognized by TLR9 [36]. Our
344
findings are not entirely consistent with previous results，which may be due to inherent
345
species disparity in the structural features of TLRs. Whilst most fish species lack TLR4,
346
experiments in zebrafish that have TLR4 showed that this fish Toll-like receptor cannot
347
recognize LPS, which is a typical ligand recognized by mammalian TLR4 [37, 38]. Our
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study may provide new information for the research of fish TLR4 and TLR4-1. Many fish species have two forms of TLR5, a membrane TLR5 and a soluble TLR5
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(TLR5s), and both of them can sense bacterial flagellin [15, 19, 39]. In this study, the
351
expression levels of TLR5 were up-regulated significantly at 6 hpi in all tissues except the
352
liver, in which the TLR5 expression was only induced at 24 hpi after A. hydrophila challenge.
353
We conclude that TLR5 in yellow catfish also recognizes the flagellin from A. hydrophila,
354
which has been reported in other fish. For example, TLR5 was involved in the immune
355
response against A. hydrophila infection in Cirrhinus mrigala [40] and it has also been
356
demonstrated that in rainbow trout the membrane-bound form was constitutively expressed,
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whereas the soluble form was induced after stimulation with flagellin [41].
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TLR22, one of the fish-specific TLRs that belongs to the TLR11 family, was
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significantly up-regulated in head kidney and trunk kidney after A. hydrophila challenge in
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this study. Some studies have suggested that TLR22 recognizes double-stranded RNA and is
361
mainly involved in antiviral protection [30, 42]. Based on mRNA/protein expression
362
profiling, later evidence indicates that TLR22 confers resistance against a wide range of viral,
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bacterial and lice infections [8, 18, 43]. According to the results of our study, we also predict
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that TLR22 is involved in the recognition of Gram-negative bacteria.
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There is now evidence that not all zebrafish TLRs have the same PAMP specificity or
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signaling activity as their mammalian counterparts [2]. Even the same TLR can express
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differently among various fish species after pathogen infection. In the spleen of large yellow
368
croaker (Pseudosciaena crocea), TLR1 and TLR3 were significantly up-regulated, while
369
TLR2 and TLR22 were down-regulated after A. hydrophila infection [44]. After the first
370
infection and the re-infection of A. hydrophila, TLR2, TLR3, TLR5, TLR21 were found to
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be significantly induced in anterior kidney (also called head kidney) of channel catfish
372
(Ictalurus punctatus) [45, 46]. The expression profiles of TLR 4, 5, 22 were also detected in
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and TLR22 were significantly up-regulated in Carassius auratus after infection of A.
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hydrophila, and the expression levels of TLR5 and TLR22 were the highest in spleen with
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52.56-fold and 28.14-fold increase, respectively [47].
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5. Conclusion
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In the present study, we have showed that nine TLRs (TLR1, TLR2, TLR3, TLR4-1,
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TLR5, TLR7, TLR8-2, TLR9 and TLR22) are constitutively expressed in different tissues of
380
yellow catfish and with high expression levels in four immune tissues (liver, spleen, head
381
kidney and trunk kidney). Expression levels of the nine TLRs were significantly
382
up-regulated in the four immune tissues in yellow catfish after stimulation with A.
383
hydrophila. Based on the results of our study and the results from other researchers, we
384
conclude that the TLRs might not recognize some specific ligands, and that they may
385
perform their roles together. Further studies will be necessary to illustrate whether and which
386
TLRs play a critical role in the immune defense against A. hydrophila challenge. In addition,
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further studies about which components of A. hydrophila can be recognized by TLRs in
388
yellow catfish will be particularly valuable as high-density aquaculture of this important fish
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expands in Asia.
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Acknowledgements
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We thank all the laboratory members for their help on the experiment. We also thank Prof.
393
Jonathan Gardner (Victoria University of Wellington) for his helpful edits on the manuscript.
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This study was supported by the Natural Science Foundation of China (Grant No. 31301122),
395
the Scientific Research Funds of Academic Restoration Stage for the Double-duty Cadres of
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Huazhong Agricultural University, and the Major Science and Technology Program for
397
Water Pollution Control and Treatment (Grant No. 2014ZX07203010-4).
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Author Contributions
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Zhang Xiao-Ting, Ji Wei and Wei Kai-Jian conceived and designed the experiments; Zhang
400
Xiao-Ting, Zhang Gui-Rong, and Zheng Huan performed the experiments; Zhang Xiao-Ting
401
and Ji Wei analyzed the data; Shi Ze-Chao, Yuan Yu-Jie and Zhang Gui-Rong contributed
402
reagents/materials/analysis tools; Zhang Xiao-Ting, Ji Wei and Wei Kai-Jian wrote the paper.
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All authors have read and approved the final manuscript.
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Tables
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Table 1 GenBank accession numbers of TLRs used in this study TLR2
TLR3
TLR4
TLR5
Homo sapiens
CAG38593.1
TLR1
AAY85647.1
ABC86910.1
AAI17423.1
AAI09119.1
Bos taurus
NP_001039969.1
NP_776622.1
Mus musculus
AAG37302.1
AAF28345.1
AAH99937.1
AAH29856.1
NP_058624.2
Gallus gallus
BAD67422.1
BAB16113.2
ADZ48550.1
ACR26315.1
TLR7
TLR8
TLR9
AAZ99026.1
AAZ95441.1
BAB19259.1
ACR26275.1
Xenopus laevis
AAK62677.1
AAK28488.1
ACR26243.1
NP_001088449.1 XP_002938702.3
Danio rerio
AAI63271.1
AAQ90474.1
XP_002934448.2 AAT37633.1
ACE74929.1 (TLR4a)
NP_001120883.1
XP_002933859.1
AAI63198.1 (TLR5b)
XP_003199309.2
XP_001340186.2
Ictalurus punctatus
AEI59662.1
AEI59663.1
AEI59664.1
AEI59665.1 (TLR4-1)
AEI59668.1 (TLR5-1)
AEI59670.1
AEI59672.1 (TLR8-2)
AEI59666.1 (TLR4-2)
AEI59669.1 (TLR5-2)
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Xenopus tropicalis
AAQ90475.1 (TLR4b)
Ctenopharyngodon
TLR22
ADZ17137.1
AAI32386.1
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ACT68332.1
ACT68333.1
ABI64155.1
idella
ACT68334.1 AEQ64878.1 (TLR4.2)
NP_001124066.1
NP_001122147.1
AEI59673.1
AEI59679.1
ADB96920.1
ADX97523.2
ACC93939.1
NP_001117884.1
(TLR8b)
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AEQ64879.1 (TLR4.3)
AEQ64880.1
ACV92063.1
CCK73195.1
ABE69177.1
AEQ64877.1
ABC86865.1
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(TLR4.4) Oncorhynchus mykiss
BAC65467.1
ACV41797.1
(TLR4.1)
Carassius auratus Cyprinus carpio Salmo salar Takifugu rubripes
Paralichthys olivaceus
AGO57934.1
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Carassius carassius
ACV41799.1 (TLR8a1) ACV41798.1 (TLR8a2)
AGL34631.1
AAW69374.1
BAJ19518.1
ADC45018.2
ADR66025.1
CCX35457.1
ABV59002.1
CAJ80696.1
AAW69377.1
BAF91187.1
AAW69375.1
AAW69376.1
AAW69378.1 (TLRS5) BAJ16368.1 (TLR5S)
Larimichthys crocea
BAE80691.1 ADK77870.1
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ACCEPTED MANUSCRIPT Table 2 Primer sequences and their designated application in this study Gene
Primer name
Sequence (5’→3’)
Amplicon length (nt) and primer information
TLR2
TLR3
TLR4
TLR4-1
TLR5
7-JR
TAYTGCTGYGCTTGAGGA
TLR7 clone
β-actin-F (forward)
TCCCTGTATGCCTCTGGTCGT
β-actin-R (reverse)
AAGCTGTAGCCTCTCTCGGTC
1-139F (forward)
TTTGCCACCTTCATTCTTGCT
1-275R (reverse)
TTCAGGGTCCAGATGCGATT
2-596F (forward)
TCAGGGTAGTTACACTCAGCC
138
2-733R (reverse)
GTATGGGTTCATTTGTCTTCAG
qRT-PCR
3-2100F (forward)
ACCCTTTAAGGCTGCGTAT
151
3-2250R (reverse)
CCTGTAGCGGGAGTCATT
qRT-PCR
4-368F (forward)
TGGACAACGACACGCTAAAAG
176
4-543R (reverse)
GTGGGCGAGGCGAGAAAA
qRT-PCR
41-1970F (forward)
GCTATGGCTGTATTCTGCT
243
41-2213R (reverse)
ACCACGATAACTTTACGACT
qRT-PCR
5-621F (forward)
ACGGATGTTTATGCCTGA
224
GGTTTTTGTGTTTCTCGC
qRT-PCR
7-11F (forward)
CGCTCCTCTTCAGATTCA
267
7-277R (reverse)
GACCGAGACAGGTTTAGTG
qRT-PCR
82-234F (forward)
ATTCACATCACTGCCTAACC
104
82-337R (reverse)
CAAGCACCTCCAACTCCT
qRT-PCR
9-2453F (forward)
CAGATGTTCGCTGTGGTT
130
9-2582R (reverse)
GTTGCTGCAATAGTAATAAGG
qRT-PCR
22-F(forward)
AAATGGGCTGTGGATAGT
22-R(reverse)
CAGGAAGGCAACGAATAG
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5-804R (reverse) TLR7
TLR8-2
TLR9
TLR22
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qRT-PCR 149
qRT-PCR
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TLR1
AACACCACCAACCTGACG
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β-actin
7-JF
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22
qRT-PCR
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Figure Legends
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Fig. 1 Comparison of TLR domain structures in P. fulvidraco
526
The domain organizations of TLRs were predicted using the SMART analysis. LRR:
527
leucine-rich repeat; TIR: Toll/IL-1 receptor; TM: transmembrane domain; CT: C-(carboxyl)
528
terminal.
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Fig. 2 The NJ phylogenetic tree based on amino acid sequences of TLRs
530
Phylogenetic tree of TLR amino acid sequences of P. fulvidraco and other organisms using
531
MEGA 5.03 by the neighbor-joining method. Bootstrap values were calculated from 1000
532
replicates. The number at the forks indicates the bootstrap and the length reflects the number
533
of substitutions along each branch. TLRs of P. fulvidraco are indicated by ●.
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Fig. 3 Relative expressions of TLR genes in different tissues of yellow catfish
535
qRT - PCR was used to analysis the distribution of TLR genes in 18 tissues of yellow catfish.
536
The data are presented as the relative expression ratios of the target genes in tissues, with
537
normalization to β-actin. Columns represent the means of 3 repeats for each treatment. Error
538
bars represent standard error of the means. The different bold lowercase letters above the
539
bars indicate significant differences (P < 0.05).
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Fig. 4 Relative expressions of TLR genes in yellow catfish after A. hydrophila challenge
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Expression profiles of the nine TLR genes in four tissues (liver, spleen, head kidney, and
542
trunk kidney) of yellow catfish after A. hydrophila challenge are presented as the relative
543
expression ratios of the target genes in tissues, with normalization to β-actin. Columns
544
represent the means of 3 repeats for each treatment. Error bars represent standard error of the
545
means. “*” indicates a significant difference in different time points compared to 0 h after
546
bacterial challenge in yellow catfish (P < 0.05).
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ACCEPTED MANUSCRIPT Highlights Nine TLR genes were identified in yellow catfish. Deduced amino acid sequences of nine TLRs were analyzed. All the Nine TLRs were mainly expressed in the liver, spleen, head kidney and trunk kidney.
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All the Nine TLRs responded to A. hydrophila challenge with different expression patterns in
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immune-related tissues.
S1050-4648(17)30093-1
DOI:
10.1016/j.fsi.2017.02.021
Reference:
YFSIM 4443
To appear in:
Fish and Shellfish Immunology
Received Date: 5 November 2016 Revised Date:
16 January 2017
Accepted Date: 17 February 2017
Please cite this article as: Zhang X-T, Zhang G-R, Shi Z-C, Yuan Y-J, Zheng H, Lin L, Wei K-J, Ji W, Expression analysis of nine Toll-like receptors in yellow catfish (Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.02.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expression analysis of nine Toll-like receptors in yellow catfish
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(Pelteobagrus fulvidraco) responding to Aeromonas hydrophila challenge
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Zhang Xiao-Ting1, 2, Zhang Gui-Rong1, 2, Shi Ze-Chao2, 3,
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Yuan Yu-Jie1, 2, Zheng Huan1, 2, Lin Li1, 2, Wei Kai-Jian1, 2*, Ji Wei1, 2*
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1 Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, College of
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Fisheries, Huazhong Agricultural University, Wuhan 430070, China
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2 Freshwater Aquaculture Collaborative Innovation Centre of Hubei Province, Wuhan
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430070, China
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3 Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture, Yangtze
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River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223,
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China
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* Corresponding author at: College of Fisheries, Huazhong Agricultural University, Wuhan
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430070, P. R. China
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E-mail addresses: [email protected] (W. Ji), [email protected] (K.-J. Wei)
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Abstract
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Toll-like receptors (TLRs) are important components of pattern recognition receptors (PRRs),
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which play significant roles in innate immunity to defense against pathogen invasion. Many
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TLRs have been found in teleosts, but there are no reports about cloning and expression of
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TLR genes in yellow catfish (Pelteobagrus fulvidraco). In this study, we analyzed the
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sequence characters and the relative mRNA expression levels of nine TLRs (TLR1, TLR2,
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TLR3, TLR4-1, TLR5, TLR7, TLR8-2, TLR9 and TLR22) in different tissues of yellow
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catfish. The results showed that all nine TLR genes are highly expressed in head kidney,
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trunk kidney, spleen and liver, all of which are related to host immunity. Subsequently we
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used Aeromonas hydrophila as a stimulating agent to detect the expression profiles of these
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nine TLRs in the liver, spleen, trunk kidney and head kidney of yellow catfish at different
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time points after injection with killed Aeromonas hydrophila. All nine TLRs responded to A.
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hydrophila challenge with tissue-specific patterns in different immune tissues. The kinetics
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of up- or down-regulation of these nine TLRs exhibited a similar trend, rising to an elevated
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level at first and then falling to the basal level, but the peak value differed at different time
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points in different tissues. The expression levels of the TLR3, TLR4-1, TLR9 and TLR22
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genes were significantly up-regulated after bacterial challenge in the liver, spleen, head
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kidney and trunk kidney. The relatively high expression of TLR genes in the immune tissues
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in response to the A. hydrophila challenge indicated that TLRs may play important roles in
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the innate immune response against gram-negative bacteria in yellow catfish.
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Keywords: Yellow catfish; Aeromonas hydrophila; Toll-like receptors; Gene expression;
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intraperitoneal injection; Innate immunity
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1. Introduction The innate immune system is the first and oldest line of host defense to the invasion of
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pathogens, and is regarded as the primary defense mechanism that exists in lower vertebrates
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such as fish [1]. Fish immune systems provide important comparative outgroups for an
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understanding of the evolution of immune systems, eventually leading to an increased
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understanding of general principles of immune system design [2]. Additionally, the aquatic
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environment is a unique medium in which microorganisms are constantly in contact with the
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potential host fish and their immune system. Failure of immunity is a major risk for
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commercial fish farming because of the unnaturally high rearing density of aquaculture
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species compare to wild species and the great cost of infections to aquaculture [3].
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The innate immune system recognizes pathogen-associated molecular patterns (PAMPs)
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that are structurally conserved among many microorganisms, via pattern recognition
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receptors (PRRs) encoded by the germ-line. The earliest characterized and the most
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extensively studied PRRs in both vertebrates and invertebrates are Toll-like receptors (TLRs)
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[4], which can recognize a wide range of PAMPs [5]. TLRs are expressed either on the cell
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surface or associated with intracellular vesicles. They are type I transmembrane
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glycoproteins, containing three important domains: an extracellular leucine-rich repeat (LRR)
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domain, a transmembrane domain and an intracellular Toll/interleukin-1 receptor (TIR)
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domain. TLRs recognize conserved PAMPs by the extracellular LRR domains that constitute
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a horse-shoe shape, and activate downstream signaling pathways through their TIR domain
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[6-8]. After recognizing PAMPs, TLRs transmit downstream signals into the cytosol and
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activate a cascade of adaptor molecules. Among these adaptor molecules, TIR domain
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containing adapter inducing IFN-
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differentiation factor 88 (MyD88) are two important adaptor proteins, which subsequently
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result in the induction of various cytokines such as TNF-α, IL-1β, IL-12, IL-8, IL-6 and IFN
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(TRIF, also known as TICAM1) and myeloid
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[9-11]. The Toll gene was first identified to control the establishment of the dorso-ventral
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pattern in fruit fly (Drosophila melanogaster) embryos [12]. Later, it was found to play an
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essential role in the fly’s immunity against fungal infection, associating the role of “Toll”
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with the synthesis of anti-microbial peptides [13]. Then a focus on immunity-related
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function of Toll started. To date, 13 TLRs (TLR1 - 13) have been identified in mammals and
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were classified into different families [14]. Huge diversified TLRs exist in fishes as
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compared to mammals due to the fact that the aquatic media not only allows transport but
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also the growth of microorganism [3]. At least 20 TLRs have been identified in more than a
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dozen teleost species [15]. Some TLRs are expressed on the host cell surface in order to
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recognize extracellular microbial structures, and others are expressed intracellularly on
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endocytic vesicles so as to specifically detect viral or bacterial nucleic acids [5]. Compared
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with mammals, fish lack some TLR members such as TLR6 and TLR10, whereas some
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TLRs are “fish-specific”, including TLR5s, TLR14, TLR19, TLR20, TLR21, TLR22 and
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TLR23 [16-18].
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At present, 27 TLRs have been identified in vertebrates. These have been divided into
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six major families, namely TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11 [19]. Selective
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pressure presumably for maintenance of specific PAMP recognition dominates the TLR1
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family (for lipopeptide), the TLR3 family (for dsRNA), the TLR4 family (for LPS), the
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TLR5 family (for flagellin), the TLR7 family (for nucleic acid and heme motifs) and the
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TLR11 family. TLRs within the same family recognize a general class of PAMPs [19].
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In recent years, scientists have intensively researched into the TLR signaling cascade
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and proved that some components of pathogens are the ligands of TLRs [20]. However, the
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direct evidence of ligand specificity has only been shown for a few TLRs [21]. Specifically,
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there is limited information on the roles of fish TLRs in the response to gram-negative 4
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bacteria challenge. Thus, which and how fish TLRs are involved in anti-bacterial immune
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defense is still largely unknown. Yellow catfish (Pelteobagrus fulvidraco), a teleost belonging to the family Bagridae, is
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extensively cultured in East and South Asia in semi-intensive and intensive systems. Due to
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its excellent meat quality, it is an important commercial freshwater fish in China [22]. In
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2014, the total production of yellow catfish reached 333,651 tonnes in China [23]. With the
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rapid development of yellow catfish pond culture, especially high density intensive
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aquaculture in China, some microbial diseases have become more serious and impeded
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further rapid development of the yellow catfish culture industry. Aeromonas hydrophila, a
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gram-negative bacterium, is the most common opportunistic pathogen that can infect yellow
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catfish and other aquatic animals. The infection of A. hydrophila spreads very rapidly and
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can result in bacterial septicemia and other serious damage and may result in death of
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aquatic animals [24]. Considering the current serious lag condition of fish immune research
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relative to aquaculture development in China, it is necessary to enhance the research of
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immune responses to bacterial diseases in fish. As important PRRs, TLRs can recognize the
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PAMPs of the microbes and defend against invading microbes. To illustrate the role of TLRs
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in teleosts, we investigated which fish TLRs play important roles in the immune response
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against Gram-negative bacteria pathogens by detecting the expression profiles of the nine
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yellow catfish TLRs at different time points after A. hydrophila challenge.
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This study aimed to (i) identify as many TLRs as possible in yellow catfish, and
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conduct a comparative analysis of yellow catfish TLRs against those identified from other
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species, and (ii) examine the mRNA expression changes of TLRs after bacterial challenge.
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Here we report phylogenies of nine yellow catfish TLR genes (TLR1, TLR2, TLR3, TLR4-1,
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TLR5, TLR7, TLR8-2, TLR9 and TLR22) and their expressions in healthy and bacterial
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challenge tissues. This study will enhance our understanding of the role of TLRs in the 5
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disease immune defense of teleosts and further help us to control fish microbial diseases.
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2. Materials and methods All experimental procedures were approved by the Institutional Animal Care and Use
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Committees (IACUC) of Huazhong Agricultural University (HZAU), Wuhan, China. No
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specific permits were required for the field studies described here. The study area is not
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privately-owned or protected in any way, and the field studies did not involve endangered or
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protected species.
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2.1. Animals, bacterial challenging and sampling
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The original strains of yellow catfish were provided by the Jingzhou fish farm in Hubei
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province, China, and were transported to the fish breeding base of HZAU. All fishes were
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raised at the same level of nutrition and management in our laboratory. They were
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temporarily kept in an indoor circulating water tank with fresh water at 24 °C and were fed
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to satiation with a commercial diet (Hubei Haid Feeds Company, Wuhan, China) twice a day
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(8:00 am and 4:00 pm).
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To clone the cDNA of TLR7 and investigate the expression profile of TLRs, five
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individuals (~ 100 g) were collected for sampling. The fish were sampled at 1:00 pm and
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then anesthetized with tricaine methanesulfonate (MS-222, 1,000 mg/L) before dissection.
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Eighteen different tissues (blood, ovary, brain, eye, gill, fin, skin, muscle, heart, liver, spleen,
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stomach, trunk kidney, head kidney, foregut, midgut, hindgut and swim bladder) were
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rapidly isolated for RNA extraction.
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We prepared 60 individuals (~100 g) to investigate the effect of bacterial challenge on
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the expressions of TLRs. To perform the bacterial challenge experiment, A. hydrophila were
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obtained from the microbiology laboratory of HZAU. Single colonies were picked out and
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inoculated into 200 mL Luria-Bertani (LB) liquid culture media, which were cultured in a 6
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4 °C with 0.2% formalin for 36 h, centrifuged at 4500 rpm and suspended twice using PBS
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(pH 7.2). Finally the killed bacteria were resuspended in sterile PBS and calculated under a
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microscope. After acclimation, five fish were randomly sampled as a control group, and the
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other fish in the experimental group were stimulated with formalin-killed A. hydrophila at a
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dose of 1.5×107 cells (suspended in 50 µL PBS with pH 7.2) per fish by intraperitoneal
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injection. Five fish were randomly sampled from the experimental group at 6 h, 12 h, 24 h,
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48 h and 72 h post-injection (hpi). The sampled fish were anesthetized with 300 mg/L
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MS-222, and then liver, spleen, head kidney and trunk kidney tissues were collected for
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RNA extraction.
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All tissue samples were immediately frozen in liquid nitrogen and stored at -80 °C until
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RNA extraction.
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2.2. Extraction of RNA and the reverse transcription PCR (RT-PCR) RNA was isolated from different tissues and four immune tissues at different stages
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after bacterial challenge using the TRIZOL Reagent according to the manufacturer’s
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protocol. And the RNA samples were treated with DNAse to remove the potential genomic
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DNA. The integrity and purity of RNA were assayed by agarose gel electrophoresis and a
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Nanodrop ND-2000 spectrophotometer (Thermo Electrom Corporation, USA), respectively.
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cDNA was reverse transcribed from total RNA using a moloney murine leukemia virus
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(M-MLV) Reverse Transcriptase kit (Promega, USA) following the manufacturer’s protocol.
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Approximately 2 µg of total RNA was used and pre-denaturated at 70 °C for 5 min. PCR
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reactions were performed at 42 °C for 60 min.
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2.3. Data mining and phylogenetic analysis
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In our previous study, we sequenced the transcriptome of yellow catfish using Illumina
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sequencing technology [25]. All potential TLRs predicted by the transcriptome analysis were 7
ACCEPTED MANUSCRIPT pulled and identified using BLAST against NCBI. Nine nucleic acid sequences were chosen
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for this study: TLR1, TLR2, TLR3, TLR4-1, TLR5, TLR8-2, TLR9 and TLR22 with full
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length of cDNAs except TLR7 with only 370 bp. The retrieved sequences of TLR genes
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were translated using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Further, the
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predicted ORFs were verified by BLASTP against NCBI non-redundant protein sequence
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database. The Simple Modular Architecture Research Tool (SMART) was used to predict the
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conserved domains based on sequence homology and further confirmed by conserved
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domain prediction from BLAST. LRR finder 2.0 [26] was used to identify LRRs using their
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Toll-like receptor LRR database. TMHMM 2.0 was applied to predict the transmembrane
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domain in TLRs. Multiple sequence alignments were conducted using ClustalW
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(http://www.genome.jp/tools/clustalw/)
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(http://www.ch.embnet.org/software/BOX_form.html).
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conducted using MEGA5.0.3. GenBank accession numbers of sequences for amino acid
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multiple alignments and phylogenetic tree are shown in Table 1.
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2.4 Molecular cloning of TLR7
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and
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Based on the partial cDNA sequence and the TLR7 sequences of the other species,
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degenerate primers were designed by Primer Premier 5.0 (Table 2) to amplify the full-length
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cDNA sequence of TLR7. Using the cDNA prepared above as a template we amplified the
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TLR7 fragment under the following PCR conditions: initial incubation at 95 °C for 3 min,
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then 35 cycles were run - each consisting of 30 s at 95 °C, 40 s at 57 °C and 72 s at 72 °C.
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The final step was conducted at 72 °C for 10 min. The 20 µL PCR reaction mixture
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contained 2 µL of 10 × buffer, 2 µL of MgCl2 (25 mM), 1.5 µL of dNTP (2.5 mM), 1 U Taq
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DNA polymerase (Thermo, USA), 0.5 µL (10 µM) of each primer, 160 ng cDNA and 10.5
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µL ddH2O.
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The PCR product was purified with a centrifugal columnar agarose gel DNA extraction 8
ACCEPTED MANUSCRIPT kit and cloned into the pMD18-T easy vector (Takara, Japan). It was then transformed into
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the competent cells of Escherichia coli DH5α and plated on the LB-agar petri-dish. Positive
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colonies containing an insert of the expected size were screened by colony PCR. Three of
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them were sequenced by the Sangon Biotech Company (Shanghai, China).
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2.5. Quantitative real-time PCR (qRT-PCR) and statistical analysis
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QRT-PCR was conducted on the Roche LightCycler 480 Real-Time PCR System. The
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qRT-PCR primers for TLRs are listed in Table 2. The specificity of the primers for qRT-PCR
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was confirmed by agarose gel electrophoresis (data not shown). Yellow catfish β-actin was
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used as an internal control [27]. SYBR® Premix Ex TaqTM (Roche) was used following the
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manufacturer’s protocols. Each PCR was performed in a total volume of 20 µL, including 10
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µL of SYBR® Premix Ex TaqTM, 40 ng of cDNA, 0.2 µM of each sense and antisense primer,
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and 7 µL ddH2O. Reactions were performed by a three-step method, 95 °C for 1 min initially,
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followed by 40 cycles, 95 °C for 5 s, 60 °C for 20 s and 72 °C for 20 s. Melt curve analysis
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was carried out over a range from 55 °C to 99 °C at the end of each PCR run. For each
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sample, quantitative PCR was performed in triplicate. A negative control (template replaced
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by ddH2O) was used for each primer pair.
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Statistical analysis was performed using Graphpad software and calculated using the
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2-∆∆Ct method [27]. All data were expressed as the mean ± SE and checked for normality and
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homogeneity of variances before statistical analysis. A one-way analysis of variance
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(ANOVA) was conducted to compare the differences of expression levels among various
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samples. The Newman-Keuls test was used to perform the multiple comparisons. The level
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of statistical significance was set at P < 0.05 for all analyses.
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3. Results
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3.1. Identification and analysis of TLR genes 9
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The partial cDNA sequence of TLR7 that we cloned was 2915 bp encoding 971 aa. A
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total of nine putative TLR genes were identified in yellow catfish - TLR1, TLR2, TLR3,
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TLR4-1, TLR5, TLR7, TLR8-2, TLR9 and TLR22. TLR family members are characterized by possession of several structural features such
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as LRR, TM and TIR. The nine deduced TLR amino acid sequences in yellow catfish
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contained various numbers of LRR domains, and the LRR number in each TLR ranged from
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6 to 18: 6 (TLR1), 7 (TLR2), 18 (TLR3), 11 (TLR4-1), 12 (TLR5), 14 (TLR7), 8 (TLR8-2),
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12 (TLR9) and 14 (TLR22). All of the TLRs include a C-terminus LRR (LRR CT) and a TIR
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domain (Fig. 1). All TLRs mentioned in this study have the TM domain with the exception
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of TLR7.
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We used all available TLR amino acid sequences in selected species to perform
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sequence alignment. Phylogenetic analysis revealed that TLRs in yellow catfish and other
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species shared common characteristics, and the phylogenetic tree provided strong support for
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the identities of the yellow catfish TLRs (Fig. 2). All of the TLR families and all of the TLR
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genes within each family are about equally distant from the center of the tree.
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3.2. Expressions of TLR genes in different tissues
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Eighteen yellow catfish tissues were used to detect mRNA expressions of TLRs by
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qRT-PCR. The results showed that TLR genes were mostly constitutively expressed in all
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tested tissues, but with tissue preference (Fig. 3). TLR3, TLR4-1 and TLR5 were most
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highly expressed in the liver, whereas TLR2, TLR8-2, TLR9 and TLR22 were highly
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expressed in the spleen. Almost all TLR genes had relatively high expression levels in the
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liver, spleen, head kidney and trunk kidney, which are important in the immune system of
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fish. Interestingly, higher expression levels of TLR1, TLR3, TLR4-1, TLR7 and TLR8-2
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were detected in the brain tissue, indicating a role of these genes in central nervous system
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infections.
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3.3. Responses of TLR genes to bacterial challenge Expressions of the nine TLR genes in yellow catfish were analyzed in the liver, spleen,
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head kidney and trunk kidney tissues after bacterial challenge. All nine TLR genes
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responded to bacterial challenge but their responses were tissue-specific (Fig. 4). The
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kinetics of up- or down-regulation of the nine TLR genes exhibited a similarity of general
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trend, rising to a high level at first and then falling to the basal level, but the up- and
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down-regulation differed at different time points in different tissues.
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In the liver, almost all the TLR genes involved in this study were induced at particular
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time point after bacterial challenge except TLR2, for which no significant increase was
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observed. The expression levels of TLR1, TLR3, TLR7, TLR8-2 and TLR9 were
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significantly increased and reached a peak at 12 h post-injection (hpi) in the liver. The
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expression level of TLR22 was up-regulated at 6 hpi and no significant changes were
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observed at other time points compared to the control group. The expressions of TLR4-1 and
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TLR5 were significantly induced and peaked at 24 hpi.
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In the spleen, almost all the TLR expressions were induced at some time points except
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TLR8-2. No significant changes were observed for TLR8-2 after bacterial challenge in
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spleen. TLR1 expression was up-regulated significantly at 24 hpi and the expression level
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was maintained until 72 hpi. The expression of TLR2 was induced in spleen and peaked at
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24 hpi. The expression of TLR3 was significantly up-regulated at 6 hpi and maintained at a
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relatively high level until 24 hpi in spleen. The expression of TLR4-1 was similar to TLR5,
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with high levels at 12 hpi and 24 hpi. The expression level was up-regulated at 6 hpi for
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TLR7 and at 12 hpi for TLR22, and no significant changes were observed at other time
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points for both of these two genes. The expression of TLR9 was induced at all time points
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and peaked at 12 hpi.
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In the head kidney, all the nine TLR expressions were induced obviously after bacterial 11
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challenge, and the induction peaked at 24 hpi for TLR5 and at 12 hpi for other TLRs. The
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expression of TLR3 was significantly up-regulated at 6 hpi and maintained at a relatively
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high level until 24 hpi in head kidney. In the trunk kidney, similar expression trends were observed for TLR3, TLR4-1, TLR5,
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TLR7, TLR9 and TLR22, with obvious expression induction at 6 hpi and highest expression
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levels at 12 hpi. And then the expression levels decreased to the control level gradually. The
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expression levels of TLR2 and TLR8-2 were up-regulated at 24 hpi and maintained at a
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relatively high level until 48 hpi. The TLR1 expression was induced at 6 hpi and peaked at
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24 hpi, and its induction maintained at a relatively high level until 48 hpi.
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The expression levels of the nine TLR genes after A. hydrophila challenge can be
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characterized in the following general patterns: (1) All nine genes responded to bacterial
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challenge with obvious differences in different tissues; (2) The responses of TLR 3, 4-1, 9,
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22 to bacterial challenge presented a rapid over-expression at 6 hpi; (3) In the head kidney,
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expression levels of all nine TLR genes were up-regulated after bacterial challenge. Their
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expression levels and the corresponding time points of up-regulation varied with various
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genes, but in general the expression levels were upward within 24 hpi; (4) Expression levels
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of the nine TLRs in the spleen were significantly induced, except for that of TLR8-2.
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4. Discussion
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Whilst there is a remarkable increase in annotations documented in the literature or
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databases concerning the identities of various TLR genes and specific TLRs against various
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PAMPs of invasion [28-30], this increase does not cover fish, let alone yellow catfish.
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Herein, we cloned TLR7 and analyzed the gene sequence and phylogenetic status of nine
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TLRs (TLRs 1, 2, 3, 4-1, 5, 7, 8-2, 9, 22) in yellow catfish. In addition, the expression levels
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of these nine TLR genes were detected in different tissues of yellow catfish. Furthermore, we 12
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revealed the expression patterns of the nine TLR genes in four immune-relevant tissues in
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response to bacterial challenge to better understand the role of the nine TLR genes in the
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innate immune system. The deduced amino acid sequences of nine TLRs from yellow catfish showed greatest
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similarity to other vertebrates. Like most other vertebrates, almost all of the nine TLRs
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identified in this study have various numbers of LRR domains, a C-terminus LRR domain, a
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TM domain and a TIR domain except TLR7, which lacks the TM domain. We also predicted
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the amino acid sequences of TLR7 from human and house mouse, both of which also lack
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the TM domain, being consistent with the reports that TLR7 is located in the cytoplasm [31].
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Alignments and phylogenetic tree analysis demonstrated six major TLR families containing
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nearly all vertebrate TLRs, consistent with previous studies [32]. Significantly, it appears
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that vertebrate TLRs are not fast-evolving genes and that the discrepancies in molecular
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distances between species with shorter and longer generation times are relatively muted. As a
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consequence, TLRs in yellow catfish share highly conserved domains with other species,
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consistent with reports that TLRs are evolutionarily highly conserved, from the worm
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(Caenorhabditis elegans) to mammals [1].
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All nine TLRs examined in this study had a constitutive expression in all tissues of
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yellow catfish. A relatively high expression of TLRs was observed in the liver, spleen, trunk
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kidney and head kidney, which is very similar to that reported for most TLR genes in other
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species. For example, TLR1 and TLR2 were both constitutively expressed in a wide range of
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organs/tissues of fish and normally a higher level of TLR1 and TLR2 expression was
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observed in immune organs [17, 33]. Grass carp TLR22 is a non-maternal gene and
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prominently expressed in immune relevant tissues such as spleen and head kidney [30]. In
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addition, higher expression levels of TLR1, TLR3, TLR4-1, TLR7 and TLR8-2 were
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detected in the brain tissue of yellow catfish; this observation could be important in
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understanding the role of particular TLRs in pathological processes unique to brain tissue. Our investigation of the expression profiles of nine TLRs in four tissues of yellow
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catfish at different time points after A. hydrophila challenge has shown that all tissue types
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responded to the stimulation. Each TLR binds to its own set of preferential ligands such as
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lipoproteins (recognized by TLR1, TLR2, and TLR6), double-stranded (ds) RNA (TLR3),
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lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), single-stranded (ss) RNA (TLR7 and
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TLR8) and DNA (TLR9) [1, 34]. It is likely that more kinds of TLRs, as well as diverse
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PAMPs specific TLRs, exist due to a genome duplication event or to environmental
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adaptation in fish [35].
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TLR1 family members (TLRs 1, 2, 6, 10) specific for lipopeptide can primarily
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recognize PAMPs from bacteria and play pivotal roles in sensing microbial products [19, 33].
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TLR2 is best known for recognizing the conserved components of Gram-positive bacteria
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such as lipoteichoic acid (LTA), peptidoglycans (PGN), and lipoproteins [14]. But in our
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study both TLR1 and TLR2 were significantly up-regulated after stimulation of A.
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hydrophila with the exception of TLR2 in the liver. This indicates that TLR1 and TLR2 can
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also respond to some components of Gram-negative bacteria.
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The expression levels of TLR3, TLR4-1, TLR7, TLR8-2 and TLR9 were significantly
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changed at most time points after bacterial (A. hydrophila) challenge in yellow catfish. But
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in mammals, these TLRs are anti-viral receptors. RNA from RNA viruses is recognized by
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TLR3 (double-stranded RNA), TLR7 and TLR8 (single-stranded RNA). DNA such as
343
unmethylated CpG DNA from DNA viruses and microbes is recognized by TLR9 [36]. Our
344
findings are not entirely consistent with previous results，which may be due to inherent
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species disparity in the structural features of TLRs. Whilst most fish species lack TLR4,
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experiments in zebrafish that have TLR4 showed that this fish Toll-like receptor cannot
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recognize LPS, which is a typical ligand recognized by mammalian TLR4 [37, 38]. Our
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study may provide new information for the research of fish TLR4 and TLR4-1. Many fish species have two forms of TLR5, a membrane TLR5 and a soluble TLR5
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(TLR5s), and both of them can sense bacterial flagellin [15, 19, 39]. In this study, the
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expression levels of TLR5 were up-regulated significantly at 6 hpi in all tissues except the
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liver, in which the TLR5 expression was only induced at 24 hpi after A. hydrophila challenge.
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We conclude that TLR5 in yellow catfish also recognizes the flagellin from A. hydrophila,
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which has been reported in other fish. For example, TLR5 was involved in the immune
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response against A. hydrophila infection in Cirrhinus mrigala [40] and it has also been
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demonstrated that in rainbow trout the membrane-bound form was constitutively expressed,
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whereas the soluble form was induced after stimulation with flagellin [41].
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TLR22, one of the fish-specific TLRs that belongs to the TLR11 family, was
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significantly up-regulated in head kidney and trunk kidney after A. hydrophila challenge in
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this study. Some studies have suggested that TLR22 recognizes double-stranded RNA and is
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mainly involved in antiviral protection [30, 42]. Based on mRNA/protein expression
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profiling, later evidence indicates that TLR22 confers resistance against a wide range of viral,
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bacterial and lice infections [8, 18, 43]. According to the results of our study, we also predict
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that TLR22 is involved in the recognition of Gram-negative bacteria.
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There is now evidence that not all zebrafish TLRs have the same PAMP specificity or
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signaling activity as their mammalian counterparts [2]. Even the same TLR can express
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differently among various fish species after pathogen infection. In the spleen of large yellow
368
croaker (Pseudosciaena crocea), TLR1 and TLR3 were significantly up-regulated, while
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TLR2 and TLR22 were down-regulated after A. hydrophila infection [44]. After the first
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infection and the re-infection of A. hydrophila, TLR2, TLR3, TLR5, TLR21 were found to
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be significantly induced in anterior kidney (also called head kidney) of channel catfish
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(Ictalurus punctatus) [45, 46]. The expression profiles of TLR 4, 5, 22 were also detected in
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and TLR22 were significantly up-regulated in Carassius auratus after infection of A.
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hydrophila, and the expression levels of TLR5 and TLR22 were the highest in spleen with
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52.56-fold and 28.14-fold increase, respectively [47].
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5. Conclusion
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In the present study, we have showed that nine TLRs (TLR1, TLR2, TLR3, TLR4-1,
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TLR5, TLR7, TLR8-2, TLR9 and TLR22) are constitutively expressed in different tissues of
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yellow catfish and with high expression levels in four immune tissues (liver, spleen, head
381
kidney and trunk kidney). Expression levels of the nine TLRs were significantly
382
up-regulated in the four immune tissues in yellow catfish after stimulation with A.
383
hydrophila. Based on the results of our study and the results from other researchers, we
384
conclude that the TLRs might not recognize some specific ligands, and that they may
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perform their roles together. Further studies will be necessary to illustrate whether and which
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TLRs play a critical role in the immune defense against A. hydrophila challenge. In addition,
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further studies about which components of A. hydrophila can be recognized by TLRs in
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yellow catfish will be particularly valuable as high-density aquaculture of this important fish
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expands in Asia.
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Acknowledgements
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We thank all the laboratory members for their help on the experiment. We also thank Prof.
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Jonathan Gardner (Victoria University of Wellington) for his helpful edits on the manuscript.
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This study was supported by the Natural Science Foundation of China (Grant No. 31301122),
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the Scientific Research Funds of Academic Restoration Stage for the Double-duty Cadres of
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Huazhong Agricultural University, and the Major Science and Technology Program for
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Water Pollution Control and Treatment (Grant No. 2014ZX07203010-4).
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Author Contributions
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Zhang Xiao-Ting, Ji Wei and Wei Kai-Jian conceived and designed the experiments; Zhang
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Xiao-Ting, Zhang Gui-Rong, and Zheng Huan performed the experiments; Zhang Xiao-Ting
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and Ji Wei analyzed the data; Shi Ze-Chao, Yuan Yu-Jie and Zhang Gui-Rong contributed
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reagents/materials/analysis tools; Zhang Xiao-Ting, Ji Wei and Wei Kai-Jian wrote the paper.
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All authors have read and approved the final manuscript.
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Tables
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Table 1 GenBank accession numbers of TLRs used in this study TLR2
TLR3
TLR4
TLR5
Homo sapiens
CAG38593.1
TLR1
AAY85647.1
ABC86910.1
AAI17423.1
AAI09119.1
Bos taurus
NP_001039969.1
NP_776622.1
Mus musculus
AAG37302.1
AAF28345.1
AAH99937.1
AAH29856.1
NP_058624.2
Gallus gallus
BAD67422.1
BAB16113.2
ADZ48550.1
ACR26315.1
TLR7
TLR8
TLR9
AAZ99026.1
AAZ95441.1
BAB19259.1
ACR26275.1
Xenopus laevis
AAK62677.1
AAK28488.1
ACR26243.1
NP_001088449.1 XP_002938702.3
Danio rerio
AAI63271.1
AAQ90474.1
XP_002934448.2 AAT37633.1
ACE74929.1 (TLR4a)
NP_001120883.1
XP_002933859.1
AAI63198.1 (TLR5b)
XP_003199309.2
XP_001340186.2
Ictalurus punctatus
AEI59662.1
AEI59663.1
AEI59664.1
AEI59665.1 (TLR4-1)
AEI59668.1 (TLR5-1)
AEI59670.1
AEI59672.1 (TLR8-2)
AEI59666.1 (TLR4-2)
AEI59669.1 (TLR5-2)
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Xenopus tropicalis
AAQ90475.1 (TLR4b)
Ctenopharyngodon
TLR22
ADZ17137.1
AAI32386.1
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ACT68332.1
ACT68333.1
ABI64155.1
idella
ACT68334.1 AEQ64878.1 (TLR4.2)
NP_001124066.1
NP_001122147.1
AEI59673.1
AEI59679.1
ADB96920.1
ADX97523.2
ACC93939.1
NP_001117884.1
(TLR8b)
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AEQ64879.1 (TLR4.3)
AEQ64880.1
ACV92063.1
CCK73195.1
ABE69177.1
AEQ64877.1
ABC86865.1
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(TLR4.4) Oncorhynchus mykiss
BAC65467.1
ACV41797.1
(TLR4.1)
Carassius auratus Cyprinus carpio Salmo salar Takifugu rubripes
Paralichthys olivaceus
AGO57934.1
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Carassius carassius
ACV41799.1 (TLR8a1) ACV41798.1 (TLR8a2)
AGL34631.1
AAW69374.1
BAJ19518.1
ADC45018.2
ADR66025.1
CCX35457.1
ABV59002.1
CAJ80696.1
AAW69377.1
BAF91187.1
AAW69375.1
AAW69376.1
AAW69378.1 (TLRS5) BAJ16368.1 (TLR5S)
Larimichthys crocea
BAE80691.1 ADK77870.1
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ACCEPTED MANUSCRIPT Table 2 Primer sequences and their designated application in this study Gene
Primer name
Sequence (5’→3’)
Amplicon length (nt) and primer information
TLR2
TLR3
TLR4
TLR4-1
TLR5
7-JR
TAYTGCTGYGCTTGAGGA
TLR7 clone
β-actin-F (forward)
TCCCTGTATGCCTCTGGTCGT
β-actin-R (reverse)
AAGCTGTAGCCTCTCTCGGTC
1-139F (forward)
TTTGCCACCTTCATTCTTGCT
1-275R (reverse)
TTCAGGGTCCAGATGCGATT
2-596F (forward)
TCAGGGTAGTTACACTCAGCC
138
2-733R (reverse)
GTATGGGTTCATTTGTCTTCAG
qRT-PCR
3-2100F (forward)
ACCCTTTAAGGCTGCGTAT
151
3-2250R (reverse)
CCTGTAGCGGGAGTCATT
qRT-PCR
4-368F (forward)
TGGACAACGACACGCTAAAAG
176
4-543R (reverse)
GTGGGCGAGGCGAGAAAA
qRT-PCR
41-1970F (forward)
GCTATGGCTGTATTCTGCT
243
41-2213R (reverse)
ACCACGATAACTTTACGACT
qRT-PCR
5-621F (forward)
ACGGATGTTTATGCCTGA
224
GGTTTTTGTGTTTCTCGC
qRT-PCR
7-11F (forward)
CGCTCCTCTTCAGATTCA
267
7-277R (reverse)
GACCGAGACAGGTTTAGTG
qRT-PCR
82-234F (forward)
ATTCACATCACTGCCTAACC
104
82-337R (reverse)
CAAGCACCTCCAACTCCT
qRT-PCR
9-2453F (forward)
CAGATGTTCGCTGTGGTT
130
9-2582R (reverse)
GTTGCTGCAATAGTAATAAGG
qRT-PCR
22-F(forward)
AAATGGGCTGTGGATAGT
22-R(reverse)
CAGGAAGGCAACGAATAG
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5-804R (reverse) TLR7
TLR8-2
TLR9
TLR22
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qRT-PCR 149
qRT-PCR
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TLR1
AACACCACCAACCTGACG
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7-JF
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22
qRT-PCR
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Figure Legends
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Fig. 1 Comparison of TLR domain structures in P. fulvidraco
526
The domain organizations of TLRs were predicted using the SMART analysis. LRR:
527
leucine-rich repeat; TIR: Toll/IL-1 receptor; TM: transmembrane domain; CT: C-(carboxyl)
528
terminal.
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Fig. 2 The NJ phylogenetic tree based on amino acid sequences of TLRs
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Phylogenetic tree of TLR amino acid sequences of P. fulvidraco and other organisms using
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MEGA 5.03 by the neighbor-joining method. Bootstrap values were calculated from 1000
532
replicates. The number at the forks indicates the bootstrap and the length reflects the number
533
of substitutions along each branch. TLRs of P. fulvidraco are indicated by ●.
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Fig. 3 Relative expressions of TLR genes in different tissues of yellow catfish
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qRT - PCR was used to analysis the distribution of TLR genes in 18 tissues of yellow catfish.
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The data are presented as the relative expression ratios of the target genes in tissues, with
537
normalization to β-actin. Columns represent the means of 3 repeats for each treatment. Error
538
bars represent standard error of the means. The different bold lowercase letters above the
539
bars indicate significant differences (P < 0.05).
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Fig. 4 Relative expressions of TLR genes in yellow catfish after A. hydrophila challenge
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Expression profiles of the nine TLR genes in four tissues (liver, spleen, head kidney, and
542
trunk kidney) of yellow catfish after A. hydrophila challenge are presented as the relative
543
expression ratios of the target genes in tissues, with normalization to β-actin. Columns
544
represent the means of 3 repeats for each treatment. Error bars represent standard error of the
545
means. “*” indicates a significant difference in different time points compared to 0 h after
546
bacterial challenge in yellow catfish (P < 0.05).
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ACCEPTED MANUSCRIPT Highlights Nine TLR genes were identified in yellow catfish. Deduced amino acid sequences of nine TLRs were analyzed. All the Nine TLRs were mainly expressed in the liver, spleen, head kidney and trunk kidney.
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All the Nine TLRs responded to A. hydrophila challenge with different expression patterns in
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immune-related tissues.