A C-type lectin that inhibits bacterial infection and facilitates viral invasion in black rockfish, Sebastes schlegelii

Accepted Manuscript A C-type lectin that inhibits bacterial infection and facilitates viral invasion in black rockfish, Sebastes schlegelii Yong Liu, Ning-qiu Li, Xin-peng Zhao, Bin Yue, Shu-wen He, Zhi-xin Gao, Shun Zhou, Min Zhang PII:

S1050-4648(16)30537-X

DOI:

10.1016/j.fsi.2016.08.053

Reference:

YFSIM 4154

To appear in:

Fish and Shellfish Immunology

Received Date: 8 June 2016 Revised Date:

18 August 2016

Accepted Date: 24 August 2016

Please cite this article as: Liu Y, Li N-q, Zhao X-p, Yue B, He S-w, Gao Z-x, Zhou S, Zhang M, A C-type lectin that inhibits bacterial infection and facilitates viral invasion in black rockfish, Sebastes schlegelii, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.08.053. 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.

ACCEPTED MANUSCRIPT

A C-type lectin that inhibits bacterial infection and facilitates viral invasion in black rockfish, Sebastes schlegelii Liu Yong1, Li Ning-qiu2, Zhao Xin-peng1, Yue Bin1, He Shu-wen1, Gao Zhi-xin1, Zhou Shun1, Zhang Min*, 1

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(1. Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, 266109,

*To whom correspondence should be addressed

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Mailing address:

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China; 2. Pearl River Fishery Research Institute, Chinese Academy of Fishery Sciences)

Marine Science and Engineering College Qingdao Agricultural University 700 Changcheng Road

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Qingdao 266109 PR China

Phone and Fax: 86-532-86080762

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E-mail: [email protected]

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Abstract

2 C-type lectins (CTLs) are important pattern recognition receptors (PRRs) that play vital roles in

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innate immunity. In teleosts, a number of CTLs have been reported, but their in vivo effects on

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host defense are still limited. In this study, a CTL homologue (SsLec1) was identified from black

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rockfish, Sebastes schlegelii, and its structure, expression and biological function was analyzed.

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The open reading frame of SsLec1 is 633 bp, with a 5’- untranslated region (UTR) of 36 bp and a

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3’- UTR of 117 bp. The deduced amino acid sequence of SsLec1 shares the highest overall

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identity (73.20%) with the CTL of Oplegnathus fasciatus. SsLec1 possesses conserved CTL

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features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine

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residues, the mannose-type carbohydrate-binding motif, the conserved calcium binding sites and

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a putative signal peptide. The expression of SsLec1 was highest in liver and could be induced by

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experimental infection with Listonella anguillarum. Recombinant SsLec1 (rSsLec1) purified

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from E. coli was able to bind and agglutinate the Gram-negative fish pathogens Vibrio

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ichthyoenteri and Vibrio vulnificus. The agglutinating ability of rSsLec1 was abolished in the

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presence of mannose or ethylenediaminetetraacetic acid. Further analysis showed that rSsLec1

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could enhance phagocytosis by macrophages. In vivo experiments indicated that rSsLec1 could

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inhibit bacterial infection and promote viral invasion. Taken together, these results suggest that

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SsLec1 is a novel CTL that possesses apparent immunoregulation property and plays a critical

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role in host defense against pathogens invasion.

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Keywords: Sebastes schlegelii; C-type lectin; bacterial agglutination; antibacterial; facilitate viral infection

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1. Introduction

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Lectins are non-enzyme protein that can bind with carbohydrates on the cell surface. They are widespread in living organisms, from microbes, plants, to animals [1]. Based on structural

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characteristics, lectins from animals are classified into several families [2], one of which is C-type

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lectins (CTLs). CTLs are a large and the most well-studied family, most of the CTLs are

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Ca2+-dependent, and a few of CTLs are Ca2+-independent [3]. The CTLs generally contain at least

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one carbohydrate recognition domain (CRD), which was consisted of 115~130 amino acids and

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folds into a double-loop spatial structure that is stabilized by the two disulfide bridges formed by

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four conserved cysteine residues in the CRD [3-4]. Based on carbohydrates binding specificity,

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CTLs are classified into two main categories, mannose-specific type and galactose-specific type,

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the corresponding conserved binding motif in CRD were Glu-Pro-Asn (EPN) or Gln-Pro-Asp

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(QPD), respectively [4].

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Since CTLs can recognize and bind the specific carbohydrate structure, which is designated as pathogen-associated molecular patterns (PAMPs), on the surface of microbes, therefore, they

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are considered as a major class of pattern recognition receptors (PRRs) and play critical roles in

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triggering innate immunity against invading pathogens. By interacting with pathogens, CTLs can

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stimulate a variety of immune responses, including pathogen recognition [5], cell adhesion [6],

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phagocytosis [7], production of reactive oxygen species, cytokine release [8-10], anti-bacterial,

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anti-fungal or anti-viral activity [11-13], and activating the complement system [14], etc.

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Furthermore, CTLs exist in two forms, either as secreted soluble proteins or as transmembrane

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proteins [15], the former including mannose-binding protein (MBP), a member of secreted CTLs,

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that can activate the complement system through the lectin pathway [16], the latter are known to

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function in dendritic cell activation and antigen uptake [17-18].

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In teleosts, a number of CTLs have been identified so far. Such as CTLs from ayu

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(Plecoglossus altivelis) [19], tongue sole (Cynoglossus semilaevis) [20], grass carp

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(Ctenopharyngodon idella) [21], orange-spotted grouper (Epinephelus coioides) [22], rainbow

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trout (Oncorhynchus mykiss) [23], Atlantic salmon (Salmo salar) [24], Japanese eels (Anguilla

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japonica) [25], common carp (Cyprinus carpio) [26-29], Japanese flounder (Paralichthys

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olivaceus) [30], and turbot (Scophthalmus maximus) [31], etc. Most of these CTLs are known to

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be able to interact with microorganisms or different PAMPs, and some of them could enhance

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phagocytosis, whereby playing important roles in innate immunity [19, 20, 31]. However, the in

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vivo effects of CTLs on fish defense against pathogens infection are still limited. Black rockfish (Sebastes schlegelii) is cultured widely in China, Korea and Japan as an

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economic fish species. However, the fish has long been suffered from serious diseases at present,

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and the immune mechanism of black rockfish responses to pathogens infection is very limited.

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In this study, a new CTL homolog from black rockfish (SsLec1) was identified and characterized.

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The tissue distribution and expression pattern of SsLec1 post-bacterial infection was examined.

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The agglutination activity and oposonization ability of recombinant SsLec1 (rSsLec1) was

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investigated. Moreover, the role of rSsLec1 in defense against bacterial and viral infection was

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analyzed. These results will be helpful to further understanding the biological functions of teleosts

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CTLs in innate immunity.

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2. Materials and methods

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Ethics statement

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The experiments involving live animals were conducted in accordance with the

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"Regulations for the Administration of Affairs Concerning Experimental Animals" promulgated

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by the State Science and Technology Commission of Shandong Province. The study was approved

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by the ethics committee of Qingdao Agricultural University.

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2.1. Fish

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Black rockfish (Sebastes schlegelii) (average 8.2±1.3 g) were purchased from a commercial

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fish farm in Shandong Province, China and maintained at 20°C in aerated seawater. Fish were

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acclimatized in the laboratory for two weeks before experimental manipulation. Before

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experiment, fish were randomly sampled and verified to be absent of pathogens in tissues as

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reported previously [32-33]. Before tissue collection, fish were euthanized with an overdose of

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tricaine methanesulfonate (Sigma, St. Louis, MO, USA) as reported previously [34].

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2.2. Bacterial and viral strains

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91 Listonella anguillarum C1 was kindly provided by Doctor Cheng of Qingdao Agricultural

University; fish megalocytivirus, infectious spleen and kidney necrosis virus (ISKNV), and Vibrio

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vulnificus PR1 were kindly provided by Doctor Li of Pearl River Fishery Research Institute,

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Chinese Academy of Fishery Sciences. Escherichia coli DH5α was purchased from Transgene

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(Beijing, China), Vibrio ichthyoenteri 1A00059 was purchased from Marine Culture Collection of

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China. Pseudomonas putida C1 and Streptococcus agalactiae G1 were preserved in the laboratory.

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S. agalactiae G1 was cultured in Brian Heart Infusion (BHI) medium, other strains were cultured

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in Luria-Bertani broth (LB) medium. S. agalactiae G1 and E. coli DH5α were cultured at 37°C , all other strains were cultured at 28°C.

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A cDNA library of black rockfish head kidney and spleen was constructed according to the

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method reported by Wang et al. [35]. Briefly, the bacterial fish pathogen L. anguillarum was

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cultured at 28°C to the mid-logarithmic phase, then washed and resuspended in

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phosphate-buffered saline (PBS). Five black rockfish (~450 g) as described above were

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randomly selected, and 5×107 colony forming units (CFU) (diluted in 1 ml PBS) of L.

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anguillarum was administered via intraperitoneal injection. At 24 h post-infection, tissues were

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collected under sterile conditions. Total RNA was isolated with an RNAprep Tissue Kit (Tiangen,

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Beijing, China), and a cDNA library was constructed with the Super SMART PCR (Polymerase

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Chain Reaction) cDNA synthesis kit (Clontech, Mountain View, CA, USA) according to the

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manufacturer’s instructions. The subsequent DNA sequence analysis showed that one of the

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clones contained the full-length cDNA of SsLec1, with 5’- and 3’-untranslated regions (UTRs).

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2.4. Sequence analysis

ACCEPTED MANUSCRIPT 117 The cDNA and amino acid sequence of SsLec1 were analyzed using the BLAST program at

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the National Center for Biotechnology Information (NCBI). Signal peptide search and domain

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search were performed with the simple modular architecture research tool (SMART) version 4.0.

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The molecular mass and theoretical isoelectric point (pI) were predicted using DNAman

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software package (Lynnon Biosoft, Quebec, Canada).

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2.5. Quantitative real time reverse transcription-PCR (qRT-PCR) analysis of SsLec1 expression

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2.5.1. SsLec1 expression in fish tissues under normal physiological conditions

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Blood, liver, gills, spleen, kidney, heart, muscle, brain and intestine were taken aseptically

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from five fishes and used for total RNA extraction with the RNAprep Tissue Kit (Tiangen, Beijing,

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China). One microgram of total RNA was used for cDNA synthesis with the Superscript II reverse

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transcriptase (Invitrogen, Carlsbad, CA, USA). qRT-PCR was carried out in a LightCycler 96

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system (Roche Applied Science, North Carolina, USA) using the SYBR ExScript qRT-PCR Kit

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(Takara, Dalian, China) as described previously [31]. The primers used to amplify SsLec1 are

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SsLec1RTF1 and SsLec1RTR1 (Table 1), Melting curve analysis was carried out at the end of each

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PCR to confirm the specificity of PCR products. The black rockfish elongation factor 1α (SsEF1A)

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gene was used as an internal control (GenBank accession no: KF430623), which was previously

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proved as an appropriate internal control for qRT-PCR normalization [36]. The expression level of

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SsLec1 was analyzed using comparative threshold cycle method (2−∆∆CT). The primers used to

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amplify SsEF1A are SsEF1AF1 and SsEF1AR1 (Table 1). All data are given in terms of relative

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mRNA levels to that of tissue in which SsLec1 expression was the lowest.

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2.5.2. SsLec1 expression in fish tissues in response to bacterial infection

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To examine SsLec1 expression in response to bacterial infection, L. anguillarum was cultured

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as above and resuspended in PBS to 1 × 107 colony forming units (CFU)/ml. Black rockfish were

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divided randomly into two groups (30 fishes/group), and injected intraperitoneally (i.p.) with 100

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µl of L. anguillarum or PBS (control). Fish were sacrificed at 4 h, 8 h, 12 h, 24 h, and 48 h

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post-infection, and kidney, liver and spleen were taken under aseptic conditions and used for

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qRT-PCR analysis as described above.

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Black rockfish head kidney (HK) macrophages were prepared as reported previously [37].

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Briefly, head kidneys of five fishes were removed under aseptic conditions, mixed and washed

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three times with PBS that contained 100 U of penicillin and streptomycin (Thermo Scientific

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HyClone, Beijing, China). The tissues were placed on a metal mesh and pressed through with 5

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ml of L15 medium (Thermo Scientific HyClone, Beijing, China) to create cell suspensions. The

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suspensions were collected, washed twice and resuspended in L-15 medium. HK macrophages

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were obtained from the cell suspensions by centrifugation in 34/51% Percoll at 400 ×g for 30

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min. The phagocytes appearing at the 34/51% interface were collected, washed twice with L-15,

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and resuspended in L-15 supplemented with 15% calf serum and 1% penicillin and streptomycin

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(L-15S). The viability of the cells was examined using the trypan blue dye exclusion method.

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The cells were adjusted to 2×106 cells/ml in L-15S and distributed into 96-well cell culture

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plates.

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2.5.4. SsLec1 expression in macrophages in response to bacterial infection

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Black rockfish HK macrophages were prepared as above. L. anguillarum was cultured as

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described above to OD600 of 0.8 and resuspended in L-15 medium to 107 colony forming unit

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(CFU)/ml. L. anguillarum suspension or PBS was added to cell culture at 100 µl/well. The plate

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was incubated at 28°C for 0.5 h, 4 h, 8 h, 12 h, 24 h and 48 h, respectively. The cells were

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collected at each time point and used for RNA extraction, cDNA synthesis and qRT-PCR analysis

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as described above.

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2.6. Plasmid construction

ACCEPTED MANUSCRIPT To construct the plasmid that expresses SsLec1, the coding sequence without signal peptide

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of SsLec1 was amplified with PCR primers SsLec1F2 and SsLec1R2 (Table 1). The PCR

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products were ligated with the T-A cloning vector pEASY-T1 (TransGen Biotech, Beijing,

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China). Then the recombinant plasmid was sequenced, and the correct recombinant plasmid was

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digested with EcoRV to retrieve the ~0.58 kb fragment, which was inserted into pET259 [38] at

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the SwaI site, resulting plasmid pETSsLec1.

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2.7. Expression and purification of recombinant SsLec1 from E. coli

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pETSsLec1 and the control vector pET259 were transformed into E. coli Transetta (DE3)

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(Trans, Beijing, China). The transformants were cultured in LB medium at 37°C to mid-log phase,

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and the expression of His-tagged recombinant SsLec1 (designated as rSsLec1) was induced by

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adding isopropyl-b-D-thiogalactopyranoside as described previously [39]. rSsLec1 was purified

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under denaturing conditions using nickel nitrilotriacetic acid columns (GE Healthcare, Piscataway,

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NJ, USA) according to the procedure recommended by the manufacturer. The purified protein

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were reconstituted as described previously [37]. The reconstituted proteins were dialyzed for 24 h

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against phosphate-buffered saline (PBS) at 4°C and concentrated using Amicon Ultra Centrifugal

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Filter Devices (Millipore, Billerica, MA, USA). A previously reported turbot protein, recombinant

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suppressor of cytokine signaling 3 (rSmSOCS3) [40], was purified and reconstituted under the

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same conditions as rSsLec1, then used as a negative control of rSsLec1. The purified protein was

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subjected to sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) and

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visualized by staining with Coomassie brilliant blue. The concentration of the protein were

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determined using the Bradford method with bovine serum albumin as a standard.

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2.8. Interaction of rSsLec1 with bacteria or virus

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Interaction of rSsLec1 with bacteria or virus was determined by whole cell enzyme-linked

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immunosorbent assay (ELISA) as reported previously [37] with a little modification. Briefly,

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bacterial cells were cultured in LB medium to mid-logarithmic phase and resuspended in PBS to 1

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×107 CFU/ml. ISKNV was prepared as reported previously [41] and resuspended in PBS to 1×

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ELISA plate (Sangon, Shanghai, China), and coated with 0.05% (w/v) poly-lysine. After fixing

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the cells with 0.05% (v/v) gluteraldehyde and blocking with 1% (w/v) bovine serum albumin, the

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cells were reacted with 0.25 mM rSsLec1, rSmSOCS or PBS (control) in TBS-Ca2+ buffer (50 mM

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Tris-Cl, 100 mM NaCl, 10 mM CaCl2, pH 7.5) at 4°C for 4 h and then treated with mouse anti-His

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antibody (Tiangen, Beijing, China) for 2 h at room temperature. After washing three times with

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PBS, horse-radish peroxidase-conjugated goat anti-mouse IgG (Tiangen, Beijing, China) was

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added to the plates. Color development was performed using the TMB Kit (Tiangen, Beijing,

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China). The plates were read at 450 nm with a Precision microplate reader (Molecular Devices,

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Toronto, Canada). Positive readings were defined as at least twice of that of the control.

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Agglutination assay was performed as described previously [31]. Briefly, Gram-negative bacteria E. coli, L. anguillarum, P. putida, V. ichthyoenteri and V. vulnificus, Gram-positive

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bactreia S. agalactiae were cultured as above and labeled with 0.2 mg/ml fluorescein

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isothiocyanate (FITC) (Bioss, Beijing, China) then resuspended in TBS-Ca2+ buffer to 2 ×109

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CFU/ml. Ten microliters of bacteria were mixed with 10 µl of native rSsLec1 (100 µg/ml) or equal

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rSmSOCS, followed by incubation at 25°C for 1 h. Agglutination was observed with a

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fluorescence microscope (Leica DM2500, Germany). To examine the effect of

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ethylenediaminetetraacetic acid (EDTA), FITC-labeled V. ichthyoenteri was incubated with

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rSsLec1 in TBS EDTA buffer (50 mMTris-CL,100 mM NaCl, 4 mM EDTA, pH 7.5) as described

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above. To determine the sugar-binding specificity of rSsLec1, 10 µl of rSsLec1 was mixed with an

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equal volume of 200 mM D-mannose, D-fructose, D-glucose, D-galactose,

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N-acetyl-Dgalactosamine, N-acetyl-D-glucosamine (Sangon, Shanghai, China), and sialic acid

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(Sigma, St. Louis, MO, USA), respectively, and incubated at 25°C for 1 h, followed by adding 10

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µl of FITC-labeled V. ichthyoenteri. The mixtures were incubated and observed for agglutination

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as described above.

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2.10. Effect of rSsLec1 on macrophages bactericidal activity

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vulnificus was cultured and incubated in TBS-Ca2+ buffer (1×105 CFU) in the presence or absence

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of rSsLec1 as described above. The incubation mixture was then added to the macrophage culture.

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The killing effect of macrophages was determined by the method described previously [31].

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Briefly, the plate was incubated at 25°C for 0 h or 5 h for killing to take place. The killing was

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stopped by adding 50 µl of 0.2% Tween 20 to each well, followed by adding of LB medium (100

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ml/well), the mixture were plated in triplicate on LB agar plates. The plates were incubated at

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28°C for 48 h, and the colonies that emerged on the plates were counted. Killing index, which

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reflects directly the killing effect of macrophages, was defined as follows: 1

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incubation/ colonies of 0 h incubation). Above experiment was repeated for five times.

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2.11. In vivo effects of rSsLec1 on pathogens invasion

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(colonies of 5 h

The in vivo effects of rSsLec1 on pathogens invasion were determined by the method previously reported [42]. Briefly, V. vulnificus and ISKNV was prepared as above and incubated

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in TBS-Ca2+ buffer (1×105 CFU) in the presence or absence of rSsLec1 as described above. Black

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rockfish as described above were randomly divided into six groups (15 fishes per group) and

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administered with above prepared mixture via intraperitoneal injection. Kidney was taken under

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aseptic conditions at 12 h and 24 h (for the group administered with V. vulnificus) or 3 d and 5 d

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(for the group administered with ISKNV) post-infection. To examine bacterial loads in the tissues,

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the tissues were homogenized in PBS, and the homogenates were diluted in PBS and plated in

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triplicate on LB agar plates. The plates were incubated at 28°C for 48 h, and the colonies that

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emerged on the plates were counted. To examine virus loads in the tissues, genomic DNA was

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extracted from the infected tissues and absolute quantitative real time PCR was carried out as

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reported previously to determine the virus copies in different tissues [33].

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2.12. Statistical analysis

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All statistical analyses were performed with SPSS 17.0 software (SPSS Inc., Chicago, IL,

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USA). Data were analyzed with analysis of variance (ANOVA), and statistical significance was

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defined as P < 0.05.

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3. Results

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3.1. Sequence characterization of SsLec1

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The sequence analysis showed that the 5’- UTR of SsLec1 full length cDNA (GenBank

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accession no: KX354356) is 36 bp, the open reading frame (ORF) is 633 bp and 3’-UTR is 117 bp,

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which contains a polyA tail of 107 bp. A putative polyadenylation signal, AATAAA, was found 13

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bp upstream of the poly-A tail (Fig. S1). Analyzed by DNAman software, the SsLec1 is consisted

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of 210 amino acid residues, its predicted molecular mass and theoretical isoelectric point were

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23.0 kDa and 4.5, respectively. Analyzed by SMART software, SsLec1 possesses a signal peptide

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composed of the N-terminal 28 residues, and was predicted to be an extracellular protein. In silico

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analysis revealed a conserved CRD domain of the CTL superfamily (residues 74-203) in SsLec1,

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which contains the mannose-type carbohydrate-binding motif, EPN; and conserved residues

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involved in Ca2+ binding. In addition, eleven cysteines were found in SsLec1, and four of which

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(C65, C138, C154, and C162) have the potential to form the disulfide bonds (Fig. 1). Sequence

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alignment showed that SsLec1 shares higher overall sequence identity with C-type lectin

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homologues from the fish Oplegnathus fasciatus (73.20%), Epinephelus akaara (65.83%) and

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Cyprinodon variegates (65.76%), respectively (GenBank accession nos: ACY66647, ACJ12598

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and XP_015226175).

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3.2. Expression of SsLec1 in fish tissues

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Under normal physiological conditions, qRT-PCR analysis showed that SsLec1 distributed

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in all the examined tissues, and with an increasing order, in brain, intestine, kidney, gill, blood,

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spleen, muscle, heart and liver (Fig. 2). The difference in expression level between liver and

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brain was 523.5 folds.

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3.3. Expression of SsLec1 in response to bacterial infection

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challenged with the fish pathogens L. anguillarum and SsLec1 expression in kidney, liver and

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spleen was analyzed by qRT-PCR at 4 h, 8 h, 12 h, 24 h and 48 h post-infection. The results

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showed that L. anguillarum induced SsLec1 expression in time dependent manners in kidney,

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liver and spleen (Fig. 3). However, the maximum inductions occurring at different times, in

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kidney and liver, SsLec1 expression reached peak at 12 h (the induction folds were 17.9- and

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14.3-, respectively), while in spleen, the maximum expression occurring at 8 h (the induction

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folds was 72.9-). Morever, the general induction patterns among different tissues were different.

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Following L. anguillarum infection, SsLec1 expression in kidney and spleen was significantly

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increased at early hours (4 h and 8 h) post-infection. In contrast, in liver, L. anguillarum

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infection induced no apparent SsLec1 expression until 8 h post infection.

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3.4. Expression of SsLec1 in macrophages in response to bacterial infection

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Rock blackfish HK macrophages were prepared and infected with the fish bacterial

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pathogen L. anguillarum. qRT-PCR analysis showed that, mRNA level of SsLec1 was

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significantly upregulated at 0.5 h, 4 h, 8h, 12 h, and 24 h post-infection, with maximum

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inductions occurring at 24 h (140.4- folds) (Fig. 4), followed by downregulation of mRNA level.

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3.5. Purification and reconstitution of rSsLec1 To investigate the biological effect of SsLec1, rSsLec1 was purified from E. coli as a

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denatured His-tagged protein. The purified protein was reconstituted and analyzed by

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SDS-PAGE, in which revealed a single band with an apparent molecular mass matching that

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predicted for rSsLec1 (20.5 kDa) (Fig. 5).

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3.6. Binding of rSsLec1 with different pathogens

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To examine the interaction of rSsLec1 wiht different pathogens, whole cell ELISA analysis

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was performed. The results showed that rSsLec1 could selectively interact with Gram-negtive

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bacteria V. vulnificus and V. ichthyoenteri, as well as fish virus ISKNV (Fig. 6).

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3.7. Effect of rSsLec1 on bacterial agglutination

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To investigate the effect of rSsLec1 on bacterial agglutination, rSsLec1 was incubated with different bacteria spieces. The results showed that rSsLec1 could strongly agglutinate V.

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ichthyoenteri, and agglutinate V. vulnificus at a middle level (Fig. 7 A and C). In contrast,

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rSmSOCS didn’t display any agglutinate ability (Fig. 7 B and D). The agglutinating ability of

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rSsLec1 against V. ichthyoenteri was completely lost at the presence of mannose or EDTA (Fig. 7

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E and F). However, treatment with galactose, glucose, fructose, N-acetyl-D-galactosamine,

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N-acetyl-D-glucosamine, or sialic acid, didn’t have any effect on the agglutinating ability of

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rSsLec1.

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3.8. Effect of rSsLec1 on macrophage bactericidal activity In order to examine whether rSsLec1 had any influence on HK macrophages bactericidal activity, V. vulnificus was treated with PBS (as control), rSmSOCS or rSsLec1 at different

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concentrations before adding into HK macrophages. After incubation, HK macrophages

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bactericidal activity was evaluated by using killing index. The results showed that compared with

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HK macrophages incubated with untreated V. vulnificus, killing index of HK macrophages

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incubated with rSsLec1 treated V. vulnificus was significantly increased (Fig. 8). In contrast, the

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killing index of HK macrophages incubated with rSmSOCS treated V. vulnificus was comparable

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to that of the HK macrophages incubated with untreated V. vulnificus. Moreover, rSsLec1

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treatment enhanced the killing index of HK macrophages in a dose-denpendent manner.

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3.9. In vivo effects of rSsLec1 on pathogens invasion

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To investigate in vivo effects of rSsLec1 on pathogens invasion, black rockfish were

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burdens or virus loads in the kidney of the infected fish were subsequently determined at

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different time post-infection. The results showed that at 12 h and 24 h post-infection, the

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numbers of V. vulnificus recovered from the kidney of control fish were 1.98- and 2.22-fold,

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respectively, of that recovered from rSsLec1-administered fish (Fig. 9 A); at 3 d and 5 d

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post-infection, the copies of ISKNV in the kidney of rSsLec1-administered fish were 4.5- and

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1.7-fold, respectively, of that in control fish (Fig. 9 B). In contrast, V. vulnificus or ISKNV in

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rSmSOCS-administered fish were comparable to those from the control fish.

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4. Discussion

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As a major class of PRRs, CTLs play critical roles in the teleost innate immunity through interaction with pathogens [5-7]. In this study, a new CTL, SsLec1, was identified and

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functionally analyzed from black rockfish. Structural analysis showed that SsLec1 possesses

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conserved structural characteristics of CTL, including a carbohydrate-recognition domain (CRD),

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four disulfide bond-forming cysteine residues used to stabilize the spatial structure of the CRD,

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the mannose-type carbohydrate-binding motif, the conserved calcium binding sites and a putative

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signal peptide. Sequence alignment indicated SsLec1 shares the highest overall identity (73.2%)

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with a CTL of O. fasciatus. The high sequence identity, together with the conserved CTL

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structural features, demonstrated SsLec1 is a new member of teleost CTLs family.

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Of the reported fish CTLs, a few of them had the highest mRNA expression in spleen, such as

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CTLs from rainbow trout, Atlantic salmon and carp [24, 28, 43]; or in gill, such as a CTL from

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large yellow croaker [44]. In addition, lots of CTLs were expressed predominately or exclusively

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in liver, a crucial organ for producing immune related genes [45-46], such as CTLs from grass

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carp, orange-spotted grouper, rainbow trout [11, 22-23], healthy catfish, Japanese flounder, turbot,

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tongue sole and roughskin sculpin [14, 20, 30, 38, 47], etc. Similarly, in the present study, SsLec1

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expression was found in many tissues and the highest expression was in liver. These results

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indicated SsLec1 mRNA was mainly synthesized in liver, and since SsLec1 possesses apparent

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signal peptide sequence, therefore, it could be secreted to other organs to exert multiple functions.

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Previous studies showed CTLs expression could be enhanced by LPS, bacterial, viral pathogens or

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parasites [19- 20, 31, 43, 46]. In this study, we found SsLec1 expression was significantly

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upregulated in the kidney, liver, spleen and macrophages post bacterial infection. These results

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implied SsLec1 may play an important role in host defense against bacterial infection. Many reports have shown fish or shellfish CTLs have the ability to react with bacterial or

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viral pathogens. For example, a CTL of Plecoglossus altivelis was able to bind Gram-positive and

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Gram-negative bacteria [19]; a CTL of Argopectens irradians could recognize and bind yeasts,

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Gram positive bacteria and Gram negative bacteria [48]. Similar with these reports, we found

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rSsLec1 could interact with Gram negative bacteria V. vulnificus and V. ichthyoenteri as well as

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fish megalocytivirus ISKNV. These results suggested SsLec1might have effects on pathogens

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invasion through interaction with the carbodrates on the surface.

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It is well demonstrated that fish CTLs could agglutinate bacteria cells through binding with

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carbohydrate patterns on the surface [19- 20, 31, 48]. In the case of SsLec1, we found rSsLec1

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was able to agglutinate Gram-negative bacteria V. ichthyoenteri and V. vulnificus, but not E. coli, L.

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Anguillarum, P. putida and Gram-positive bacteria S. agalactiae. These results were consistent

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with the results from microbes binding assays, and implied that SsLec1 is a selective CTL and

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non-substrate bacteria may lack the specific carbohydrates recognized by SsLec1. However, at the

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presence of EDTA or mannose, SsLec1 failed to agglutinate V. ichthyoenteri, suggesting SsLec1

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is a calcium dependent and mannose-binding CTL, moreover, these characteristics are in

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agreement with structural prediction and the presence of EPN motif.

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CTLs play critical roles in host defense against microbes. Several reports have shown fish

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CTLs have a positive influence on macrophage activity in turbot, tongue sole and ayu [19- 20, 31].

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Moreover, tongue sole CTL was found to be able to inhibit bacterial infection in vivo [20].

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Similarly in our study, the results of in vitro and in vivo experiments indicated that rSsLec1 treated

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bacteria were easy to be killed by macrophages and black rockfish, which implied an positive role

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of SsLec1 in host defense against bacterial invasion. Conversely, in vivo infection by ISKNV

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showed rSsLec1 treatment facilitated the invasion of ISKNV into the host, this result together with

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that rSsLec1 could bind with ISKNV, implied SsLec1 may act as a receptor of ISKNV, and

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facilitate the invasion process. Similar with this result, many reports have shown that lots of CTLs

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participated in virus infection and influenced their entry into host cells [49-51]. Wang et al

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reported a CTL from shrimp collaborated with calreticulin facilitates White Spot Syndrome Virus

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as a receptor of the CTL [52]. On the contrary, tongue sole CTL was found to be able to inhibit

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Rock Bream Iridovirus-C1 infection in vivo [20]. These results implied fish CTLs may have

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diverse functions in host defense against pathogens invasion and SsLec1 plays an important role in

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black rockfish innate immunity.

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In conclusion, we identified and characterized a CTL homolog (SsLec1) from black rockfish,

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S. schlegelii. mRNA expression of SsLec1 was abundant in liver, and could be induced by bacteria

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in kidney, liver, spleen and macrophages. The purified recombinant SsLec1 (rSsLec1) could

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selectively recgonize and agglutinate V. ichthyoenteri and V. vulnificus in a mannose-binding and

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calcium dependent manner. Furthermore, rSsLec1 could inhibit bacterial infection and facilitate

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viral infection in vivo. These observation add new insights to the in vivo functions of fish CTLs.

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Acknowledgements

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This work was supported by the National Natural Science Foundation of China (grant no. 31572652), the Open Fund of the Key Laboratory of Experimental Marine Biology, Chinese

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Academy of Sciences (grant no. FF2015N003), the Fishery Industry Innovation Team, Modern

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Agricultural Industry and Technology System of Shandong Province (grant no. SDAIT-12-06),

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and the Advanced Talents Foundation of Qingdao Agricultural University (grant no.

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6631114322).

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Tables Table 1 PCR primers used in this study Target genes EF1A SsLec1 SsLec1

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Underlined nucleotides are the enzyme restriction sites.

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Sequences (5’– 3’) 5’- AACCTGACCACTGAGGTGAAGTCTG-3’ 5’- TCCTTGACGGACACGTTCTTGATGTT-3’ 5’-AGGAGACTGGAGATGGGTGGA-3’ 5’-CGGACAGGTGGTTGTTTGG-3’ 5’- GATATCCTCATCGGCTTGTTGGC-3’ 5’- GATATCCTGGGGCAGGATGGC-3’

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Primers EF1AF1 EF1AR1 SsLec1RTF1 SsLec1RTR1 SsLec1F2 SsLec1R2

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Figure legends

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Fig. 1. Alignment of the CRD of SsLec1 with CRDs of other teleost CTLs. Sequence alignment was carried out using DNAman. The residues that are conserved among all the CRDs are in black, the residues that are ≥75% identical among the aligned sequences are in grey, the carbohydrate-specificity motifs are boxed, the conserved cysteine residues are indicated with “*”, and the conserved residues involved in Ca2+ binding are indicated with “ ”. The GenBank accession numbers of the aligned sequences are as follows: Oplegnathus fasciatus, ACY66647.1; Epinephelus akaara, ACJ38233; Cyprinodon variegates, XP_015226175; Epinephelus coioides, ACO06100; Oreochromis niloticus, XP_003450637; Austrofundulus limnaeus, XP_013868904.

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Fig. 2. SsLec1 expression in fish tissues under normal physiological conditions. SsLec1 expression in the brain, intestine, kidney, gill, blood, spleen, muscle, heart and liver of rock blackfish was determined by quantitative real time RT-PCR. The expression level of SsLec1 in kidney was set as 1. Values are shown as means±SE (N = 5). Fig. 3. SsLec1 expression in black rockfish in response to bacterial challenge. Black rockfish were infected with Listonella anguillarum or PBS (control). SsLec1 expression was determined by quantitative real time RT-PCR at various times post-challenge. Values are shown as means±SE (N = 5). Significance is indicated with asterisks. **P < 0.01.

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Fig. 4. SsLec1 expression in response to bacterial infection. HK macrophages of black rockfish were infected with Listonella anguillarum, and the control fish were administered with PBS. SsLec1 expression level was determined by quantitative real time RT-PCR at various time points. In each case, the expression level of the control fish was set as 1. Values are shown as means±SE

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(N = 5). Significances between PBS- and pathogen-infected fish are indicated with asterisks. ∗∗P < 0.01.

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Fig. 5. SDS-PAGE analysis of purified recombinant SsLec1. Recombinant SsLec1 (lane 1) was analyzed by SDS-PAGE (15%) and viewed after staining with Coommassie brilliant blue R-250. Lane 2, protein markers. Fig. 6. Binding of rSsLec1 to bacteria or virus. Vibrio vulnificus, Vibrio ichthyoenteri, and infectious spleen and kidney necrosis virus (ISKNV) were prepared as above. The relative binding ability of rSsLec1 to different microorganisms was determined by ELISA. Values are shown as means ±SE (N=5). **P < 0.01. Fig. 7. Agglutination of bacteria by rSsLec1. FITC-labeled Vibrio ichthyoenteri (A, B, E and F) or Vibrio vulnificus (C and D) was incubated with rSsLec1 (A, C, E and F) or rSmSOCS (B and D), and agglutination was observed with a fluorescence microscope. (E) FITC-labeled V. ichthyoenteri was incubated with rSsLec1 that had been pre-incubated with mannose. (F) FITC-labeled V. ichthyoenteri was incubated with rSsLec1 in the presence of EDTA.

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Fig. 9. In vivo Effect of rSsLec1 on bacteria or virus infection. Vibrio vulnificus or ISKNV was treated with PBS (control), rSmSOCS or rSsLec1 before being administrated into black rockfish. The amounts of pathogens in kidney were determined at 12 h and 24 h post-infection. The amounts of ISKNV in kidney were determined at 3 d and 5 d post-infection. Values are shown as means±SE (N = 5). ∗∗P < 0.01.

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Supplementary figures

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Fig. S1. DNA and amino acid sequences of SsLec1. The nucleotides and amino acids are numbered along the left margin. The translation start and stop codons are in bold, the signal peptide is underlined, and the polyadenylation signal is double underlined.

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Highlights

1. A C-type lectin homologue (SsLec1) was identified and characterized. 2. Expression of SsLec1 in tissues and macrophages could be induced by Listonella anguillarum.

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3. Recombinant SsLec1 purified from E. coli could selectively recgonize and agglutinate Vibrio ichthyoenteri and Vibrio vulnificus in a mannose-binding and calcium dependent manner.

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4. Recombinant SsLec1 could inhibit bacteria infection and facilitate viral infection in vivo..