- Email: [email protected]
CD94 of tongue sole Cynoglossus semilaevis binds a wide arrange of bacteria and possesses antibacterial activity
Accepted Manuscript CD94 of tongue sole Cynoglossus semilaevis binds a wide arrange of bacteria and possesses antibacterial activity Xue-peng Li, Yong-hua Hu PII:
S1050-4648(16)30637-4
DOI:
10.1016/j.fsi.2016.10.005
Reference:
YFSIM 4233
To appear in:
Fish and Shellfish Immunology
Received Date: 28 April 2016 Revised Date:
1 October 2016
Accepted Date: 3 October 2016
Please cite this article as: Li X-p, Hu Y-h, CD94 of tongue sole Cynoglossus semilaevis binds a wide arrange of bacteria and possesses antibacterial activity, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.10.005. 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
CD94 of tongue sole Cynoglossus semilaevis binds a wide arrange of bacteria and possesses antibacterial activity Xue-peng Lia,b,c, Yong-hua Hua,b* a
b
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Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China University of Chinese Academy of Sciences, Beijing, China
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*To whom correspondence should be addressed
Mailing address:
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Yong-hua Hu
Institute of Oceanology
Chinese Academy of Sciences 7 Nanhai Road
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Qingdao 266071, China Phone: +86-532-82891907
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Email: [email protected]
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Abstract
2 In this study, we examined the expression patterns and the functions of the tongue sole
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Cynoglossus semilaevis CD94, CsCD94. CsCD94 is composed of 209 amino acid residues and
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shares 43.0–50.2% overall identities with known teleost CD94 sequence. CsCD94 has a C-type
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lectin-like domain. Expression of CsCD94 occurred in multiple tissues and was upregulated
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during bacterial infection. Recombinant CsCD94 (rCsCD94) exhibited apparent binding and
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agglutinating activities against both Gram-positive and Gram-negative bacteria in a
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Ca2+-dependent manner. Treatment of bacteria with rCsCD94 enhanced phagocytosis of the
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bacteria by peripheral blood leukocytes. Furthermore, incubation of rCsCD94 with bacteria
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reduced the survival of the bacteria in vitro. Taken together, these results indicate that rCsCD94 is
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a key factor in the bactericidal and phagocytic effects of tongue sole, and reveal for the first time
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an essential role of fish CD94 in antibacterial immunity, thereby adding insight into the function
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of CD94.
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Keywords: CD94; Cynoglossus semilaevis; Agglutination; Phagocytosis; Antibacterial
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1. Introduction
24 As a major class of pattern-recognition receptors (PRRs), lectins are a group of
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carbohydrate-binding proteins that are neither antibodies nor enzymes, which were first
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discovered in plants, and subsequently found to exist ubiquitously in bacteria, invertebrates, and
28
vertebrates [1–4]. Based on their structural characteristics, lectins from animals are classified into
29
several families [5], including C-type lectins (CTLs). CTLs, the most diverse family of animal
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lectins, are divided into seven groups (I–VII) [6]. CTLs are involved in a broad range of biological
31
processes that include adhesion, endocytosis, and pathogen recognition and neutralization [7–9].
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All classical CTLs have a carbohydrate-recognition domain (CRD) that mediates specific
33
recognition and binding to oligosaccharides in the extracellular matrix and on solid surfaces such
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as microbes [10]. Ca2+ is thought to be involved in ligand binding and maintenance of the CRD
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structure [11,12]. In addition to the classical CTLs, many proteins with a C-type lectin-like
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domain (CTLD) have been identified, some of which, however, are Ca2+-independent and bind to
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proteins, rather than to sugars [13,14]. One such protein-binding molecule is cluster of
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differentiation 94 (CD94), which is part of several vertebrate natural killer (NK) lymphocyte
39
receptors that play important roles in innate immunity [15]. CD94 forms heterodimers with NK
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group 2 (NKG2) family molecules and either blocks or activates the cytotoxic activity of NK cells
41
toward target cells by interacting specifically with major histocompatibility complex class I
42
molecules [16,17]. CD94 belongs to group V of the CTL superfamily, which also includes CD69,
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lectin-like type II transmembrane disulfide-bonded homodimer Ly49, and natural killer
44
receptor-p1 (NKR-P1) [18].
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ACCEPTED MANUSCRIPT CD94 studies have thus far been focused nearly and exclusively on mammals, specifically
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humans and mice, although CD94 homologues have also been reported in teleosts. Sato et al
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isolated cDNA clones from the cichlid Paralabidochromis chilotes that, when translated into
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protein sequences, were found to be homologous to the mammalian C-type lectin CD94 [20].
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Genes homologous to CD94 cDNAs were also identified in another cichlid species, Oreochromis
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niloticus [21]. Simultaneously, cDNAs homologous to mammalian CD94 were identified in
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Botryllus schlosseri and Ciona intestinalis [22,23]. B. schlosseri CD94 (BsCD94) has been shown
52
to be expressed in a sub-population of blood cells that may be involved in colony allorecognition
53
[22]. C. intestinalis CD94-1 (CiCD94-1) was up-regulated in response to inflammation induced by
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lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating
55
blood [23]. Furthermore, anti-ciCD94-1 antibody was found to specifically inhibit the phagocytic
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activity of these cells [23].
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Half-smooth tongue sole (Cynoglossus semilaevis) is a demersal flatfish that inhabits mainly
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warm waters and is distributed in the Yellow Sea and the East China Sea. Although farming of
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tongue sole in China is relatively recent, due to its high economic value, tongue sole has become
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an important commercial marine fish species, with great potential for further expansion of farming.
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With the development of the tongue sole farming industry, diseases caused by bacterial pathogens,
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such as Vibrio and Edwardsiella spp. have become problematic, resulting in significant economic
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losses [24]. To date, information on half smooth tongue sole CD94 (CsCD94) is scarce. In this
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study, we investigated the expression patterns and antibacterial potential of CsCD94.
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2. Materials and methods
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2.1. Fish
69 Clinically healthy tongue sole (average weight, 12.6 g) were purchased from a commercial
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fish farm in Shandong Province, China, and maintained at 20 °C in aerated seawater. Before
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experimentation, the fish were acclimatized in the laboratory for 2 weeks and verified to be free of
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pathogens in the liver, kidney, and spleen, as reported previously [25]. For tissue collection, fish
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were euthanized with tricaine methanesulfonate (Sigma-Aldrich Corporation, St. Louis, MO,
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USA), as reported previously [26].
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2.2. Sequence analysis
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The cDNA and amino acid sequences of CsCD94 (GenBank accession no. XM_017031325)
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were analyzed using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi), hosted by the
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National Center for Biotechnology Information (NCBI) website. Domain searches were performed
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with
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(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The theoretical molecular mass and
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isoelectric point were predicted using EditSeq sequence editing software in the DNASTAR
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software package (DNASTAR, Inc., Madison, WI, USA). Multiple sequence alignments were
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created using the DNAMAN program (Lynnon Biosoft, Vaudreuil-Dorion, Quebec, Canada).
domain
search
program
of
the
NCBI
website
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conserved
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the
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2.3. Quantitative real time reverse transcription-PCR (qRT-PCR) analysis of CsCD94
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expression under normal conditions
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qRT-PCR analysis of CsCD94 expression under normal conditions was determined as
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follows. Total RNA from the spleen, liver, kidney, blood, intestine, muscle, gill, heart, and brain, 5
ACCEPTED MANUSCRIPT aseptically collected from five tongue sole, was extracted using the EZNA Total RNA Kit (Omega
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Bio-tek, Doraville, GA, USA). One microgram of total RNA was used for cDNA synthesis with
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the Superscript II reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA). qRT-PCR
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was performed using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the
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SYBR ExScript qRT-PCR Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China) [27]. The PCR
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reaction was performed in a 20 µl volume containing 10 µl SYBR® premix Ex Taq™(Tli
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RNaseH Plus), 0.2 µM each of specific forward primer (5’- CGGGGTTGTCAGTAGAGTGG -3’)
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and reverse primer (5’- TCGTACAAACCCATGGTCCG -3’), and 2.0 µl diluted cDNA (50
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ng/µl). The PCR conditions were 95 °C for 30 s, followed by 35 cycles of 95 °C for 15 s, 60 °C
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for 15 s, 72 °C for 20 s. The expression level of CsCD94 was analyzed using the comparative
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threshold cycle method (2−∆∆CT) with beta-actin as an internal reference [28]. The experiment was
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performed three times.
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2.4. qRT-PCR analysis of CsCD94 expression during bacterial infections
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qRT-PCR analysis of CsCD94 expression during bacterial infection was performed as
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reported previously [29]. Vibrio harveyi [30] was cultured in Luria-Bertani (LB) broth at 28 °C to
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an optical density at 600 nm (OD600) of 0.8. Then, the cells were washed with phosphate-buffered
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saline (PBS) and resuspended in PBS to a concentration of 1 × 106 CFU/ml. Tongue sole (as above)
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were divided randomly into two groups (20/group) and injected intraperitoneally with 50 µl V.
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harveyi or PBS. At 6, 12, 24, and 48 h post-infection, CsCD94 expression in the kidney, spleen,
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liver, and blood was determined by qRT-PCR, as described in section 2.3. The experiment was
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performed three times.
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2.5. Construction of plasmids
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To construct pEtCsCD94, which expresses His-tagged recombinant CsCD94 (rCsCD94), the coding
sequence
of
CsCD94
was
amplified 6
by
PCR
with
primers
F1
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(5’–GATATCATGGATCCACCAGAGAGCTTCAC– 3’, underlined sequence, EcoRV restriction
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endonuclease
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underlined sequence, EcoRV restriction endonuclease site). The PCR products were ligated with
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the T−A cloning vector T-Simple (TransGen Biotech, Beijing, China) and the recombinant
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plasmid was digested with EcoRV to retrieve the CsCD94-containing fragment, which was
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inserted into pET32a [31] at the EcoRV site, resulting in pETCsCD94.
and
R1
(5’–GATATCAAACTCCTTCGTACAAACCCATGGT–
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2.6 Purification of recombinant proteins
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site)
Recombinant proteins were expressed and purified as described previously [29,32]. Briefly,
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Escherichia coli BL21 (DE3) cells (Beijing TransGen Biotech Co., Ltd., Beijing, China) were
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transformed separately with pETCsCD94 and pET32a (Novagen, San Diego, CA, USA), the latter
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expressing rTrx (recombinant thioredoxin) [33]. Then, the transformants were cultured in LB
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medium at 37 °C to the mid-logarithmic phase and isopropyl-β-D-thiogalactopyranoside was
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added to the culture to a final concentration of 1 mM. After growing at 16 °C for an additional 10
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h, the cells were harvested by centrifugation (4200 g) and His-tagged proteins were purified using
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Ni-NTA Agarose (Qiagen, Inc., Valencia, CA, USA) as recommended by the manufacturer. The
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proteins were concentrated with PEG20000 (Beijing Solarbio Science & Technology Co., Ltd.,
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Beijing, China) and endotoxins were removed as reported previously [34]. The concentrated
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proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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(SDS-PAGE) and visualized after staining with Coomassie brilliant blue R-250. The
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concentrations of purified proteins were determined using the Bradford method with bovine serum
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albumin as a standard.
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2.7. Binding of rCsCD94 to bacteria
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The bacteria Edwardsiella tarda, Pseudomonas fluorescens, Vibrio anguillarum, V. harveyi, and Streptococcus iniae are fish pathogens described elsewhere [26,30,35–37]. Bacillus subtilis 7
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Center (Bejing, China) Bacterial cells were cultured in LB medium to OD600 0.8, washed and
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resuspended in TBS (50 mM Tris–HCl, 100 mM NaCl, pH 7.5) to 108 CFU/ml. Binding of
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rCsCD94 to bacteria was determined using an enzyme-linked immunosorbent assay (ELISA) [38].
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To determine the effect of sugars and lipopolysaccharides (LPS) on rCsCD94-bacteria interaction,
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bacteria were incubated with rCsCD94 as above in TBS-Ca2+ buffer containing 200 mM
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D-mannose, D-galactose, D-glucose, L-fucose, N-acetyl-D-glucosamine (Sangon, Shanghai,
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China), or LPS (30 µg/ml), which was prepared as reported previously [39]. The experiment was
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performed three times.
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2.8. Agglutination assay
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The agglutination assay was performed as reported previously [40]. Briefly, bacterial cells
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were cultured in LB medium as above, washed with TBS buffer, and resuspended in TBS, TBS
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containing
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ethylenediaminetetraacetic acid (EDTA) to 2 × 108 CFU/ml. rCsCD94 was added to the bacterial
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cells to a final concentration of 32 µg/ml. After incubation at 25 °C for 1 h, the bacterial cells were
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stained with 4,6-diamino-2-phenyl indole (Invitrogen Corporation), according to manufacturer’s
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instructions, and agglutination was observed with a fluorescence microscope (Nikon E800; Nikon
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Corporation, Tokyo, Japan). To determine the effect of sugars on agglutination, bacteria were
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incubated with rCsCD94 as above in TBS-Ca2+ buffer containing 200 mM D-mannose,
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D-galactose, D-glucose, or N-acetyl-D-glucosamine (Sangon, Shanghai, China). The experiment
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was performed three times.
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CaCl2
(TBS-Ca2+
buffer),
or
TBS-Ca2+
buffer
plus
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2.9. Effect of rCsCD94 on bacterial survival
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Bacteria were cultured in LB medium to OD600 of 0.8 and the cells were resuspended to 1 ×
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104 CFU/ml in TBS-Ca2+ buffer. Then, rCsCD94 or rTrx was added to the bacterial cell 8
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suspension to a final concentration of 32 µg/ml. Control cells were incubated in TBS-Ca2+ buffer
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at 25 °C for 1, 2, and 4 h, and bacterial survival was examined by plate count, as reported
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previously [41]. The experiment was performed three times.
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To prepare PBL, blood was collected from the caudal veins of half-smooth tongue sole (~850
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g) and diluted 1:1 with L-15 medium (Thermo Scientific HyClone, Beijing, China). The diluted
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blood was placed on top of 61% Percoll (Solarbio, Beijing, China) and centrifuged at 400 g for 10
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min. The white layer of PBL in the interface was recovered, washed twice and resuspended in
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L-15 medium.
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2.11. Fluorescence activated cell sorting (FACS)
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Phagocytosis determined by FACS was performed as reported previously [29,42]. Briefly, V.
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anguillarum was cultured as above and resuspended in 100 µg/ml of fluorescein isothiocyanate
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(FITC) (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China). After incubation at 37 °C for 2 h,
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the cells were collected by centrifugation and washed five times in TBS. rCsCD94 or rTrx were
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added to the cells to a concentration of 32 µg/ml. After incubation at 22 °C for 1 h, the cells were
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washed and resuspended in L-15 medium (Thermo Scientific HyClone, Beijing, China) to a
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concentration of 1 × 108 cells/ml. One milliliter of PBLs (~107 cells) was mixed with 100 µl of V.
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anguillarum, rCsCD94-treated V. anguillarum, rTrx-treated V. anguillarum, or L-15 medium
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(control), and incubated in the dark for 2 h. Then, and the cells were collected by centrifugation
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and washed with TBS three times. Extracellular fluorescence was quenched by adding 1 ml of
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0.125 % trypan blue in TBS, followed by incubation at 22 °C for 5 min. The cells were collected
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by centrifugation, suspended in 1 ml of TBS, and subjected to FACS analysis with a Partec
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CyFlow Counter (Sysmex Partec GmbH, Münster, Germany). The data were analyzed using
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WinMDI 2.9 software (The Scripps Research Institute, La Jolla, CA, USA). The experiment was
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repeated three times.
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2.12. Statistical analysis
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All experiments were performed at least three times and statistical analyses were performed
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using SPSS 17.0 software (IBM-SPSS Inc., Chicago, IL, USA). Data were analyzed with analysis
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of variance and statistical significance was defined as P < 0.05.
3. Results
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3.1. Sequence analysis of CsCD94
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CsCD94 is composed of 209 amino acid residues with a calculated molecular mass of 24.2
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kDa and an isoelectric point of 4.31. CsCD94 has a CTLD composed of residues 81–206. BLAST
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analysis showed that CsCD94 shares 43.0–50.2% overall sequence identities with the CD94
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homologues of Haplochromis burtoni, Maylandia zebra, Pundamilia nyererei, O. niloticus,
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Stegastes partitus, respectively (Fig. 1).
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3.2. Expression of CsCD94 in fish tissues
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Under normal physiological conditions, qRT-PCR showed that CsCD94 expression occurred,
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in increasing order, in the intestine, kidney, heart, liver, gill, blood, muscle, brain, and spleen of
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tongue sole (Fig. 2). After infection with the bacterial pathogen V. harveyi, CsCD94 expression
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was significantly upregulated at 6 h, 12 h and 24 h in kidney and blood, at 12 h and 24 h in spleen,
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and at 24 h in liver (Fig. 3). The maximum inductions in kidney, blood, spleen, and liver were
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59.4-fold, 11.7- fold, 10.7-fold, and 3.2-fold, respectively.
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3.3. Binding of rCsCD94 to bacteria
233 rCsCD94 was purified from E. coli (Fig. S1). When rCsCD94 was incubated with
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Gram-negative bacteria and Gram-positive bacteria, i.e., E. tarda, P. fluorescens, V. anguillarum,
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V. harveyi, B. subtilis, M. luteus, and S. iniae, rCsCD94 was able to bind to each in the presence of
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Ca2+, with the highest and lowest binding indexes observed with E. tarda and V. anguillarum,
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respectively (Fig. 4). Furthermore, the binding indexes increased with the dose of rCsCD94 (Fig.
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4). In contrast, rTrx, which was used as a control protein for rCsCD94, did not exhibit any binding
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to the bacteria (Fig. 4). The presence of D-mannose, D-galactose, D-glucose, L-fucose, and
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N-acetyl-D-glucosamine had no effect on the binding activity of rCsCD94 (data not shown).
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Pre-incubation of rCsCD94 with E. tarda LPS inhibited binding of the protein to the bacteria (Fig.
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S2).
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3.4. Effect of EDTA and monosaccharides on bacterial agglutination by rCsCD94
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The results of the agglutination assay showed that in TBS buffer plus calcium, rCsCD94
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caused agglutination of E. tarda, B. subtilis, M. luteus, V. harveyi, S. iniae, V. anguillarum, and P.
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fluorescens (Fig. 5). In contrast, in TBS buffer or in TBS buffer plus calcium and EDTA,
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rCsCD94 caused no apparent agglutination of any of the tested bacteria (Fig. 5). The presence of
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D-mannose, D-galactose, D-glucose, and N-acetyl-D-glucosamine had no effect on the
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agglutination activity of rCsCD94 (data not shown).
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3.5. Effect of rCsCD94 on bacterial survival
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To examine the effect of rCsCD94 on the survivability of the bound bacteria, E. tarda, P.
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fluorescens, V. anguillarum, V. harveyi, B. subtilis, M. luteus, and S. iniae were incubated with
258
rCsCD94 or rTrx for 1 h, 2 h, and 4 h, and the number of surviving bacterial was determined. The
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results showed that at 2 h and 4 h incubation, the numbers of rCsCD94-treated V. anguillarum, V. 11
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harveyi, B. subtilis, and M. luteus were significantly decreased, as compared to the control,
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whereas the numbers of these bacteria treated with rTrx were similar to that of the control (Fig.6).
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The numbers of rCsCD94-treated E. tarda, P. fluorescens, and S. iniae did not change
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significantly (data not shown).
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To examine the effect of rCsCD94 on phagocytosis, tongue sole PBLs were incubated with V.
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anguillarum treated with rCsCD94 or rTrx. FACS analysis showed that for the PBLs incubated
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with rCsCD94-treated V. anguillarum, the percentage of positive phagocytic cells (M1 = 64.3%)
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was significantly (P < 0.01) higher than that of the PBLs incubated with untreated V. anguillarum
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(M1 = 45.8%) (Fig. 7). Treatment with rTrx had no significant effect on phagocytosis.
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4. Discussion
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In this study, we examined the expression and biological property of CsCD94, a CD94
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homologue from tongue sole. CsCD94 possesses a typical CTLD and is relatively closely related
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to known CD94 proteins of teleost.
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In mammals, CD94 is expressed mainly on NK cells and a subset of CD8+ T cells [43–45].
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In C. intestinalis, ciCD94-1 was expressed in a subpopulation of blood cells and during larval
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development, as well as the early stages of metamorphosis in structures related to the nervous
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system [23]. In B. schlosseri, BsCD94-1 was also expressed on the surface of a subpopulation of
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blood cells [22]. However, in fish, the tissue-specific expression profiles of CD94 have not been
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documented. In our study, CsCD94 transcripts were detected ubiquitously in all examined tissues
284
of fish, which is in line with that observed with other tongue sole immune related lectins such as
285
CsCTL1 and CsBML [36,40]. The tissue-different expressions of CsCD94 suggests different
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levels of CsCD94 regulations in these tissues.
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expression of CD94 had previously been found to upregulate in association with human
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cytomegalovirus reactivation [47,48]. Furthermore, the upregulation of CD94 seemed to depend
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on the presence of interleukin-12 [49]. In C. intestinalis, ciCD94-1 was up-regulated in expression
291
by LPS-induced inflammation [23]. However, to the best of our knowledge, no report on the
292
induction of fish CD94 by bacterial pathogens has been documented. In this study, we found that
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CsCD94 expression was upregulated in a time-dependent manner during V. harveyi infection,
294
however, the expression patterns of CsCD94 differed in different tissues. In kidney and blood,
295
peak inductions of CsCD94 expression occurred at the early stage of infection, while in spleen and
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liver, peak inductions of CsCD94 expression occurred at relatively the late stage of infection.
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These results indicate that CsCD94 is involved in tissue immune response during bacterial
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invasion.
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Several studies have shown that lectins can bind and agglutinate bacteria. A fucose-binding
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lectin from tilapia (O. niloticus) agglutinated Aeromonas hydrophila and Enterococcus faecalis
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[50], a skin lectin HjCL from Japanese Bullhead Shark (Heterodontus japonicas) agglutinated E.
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tarda [51], a lactose-binding skin lectin from Japanese eel agglutinated E. coli [52], a serum lectin
303
from blue gourami (Trichogaster trichopterus) agglutinated A. hydrophila [53,54], and lectins of
304
Atlantic salmon were able to bind to V. anguillarum and A. salmonicida [55]. In tongue sole, a
305
C-type lectin exhibited agglutination and binding activities against V. anguillarum and M. luteus
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[36]. The C-type lectin-like receptors CD94 could not only bind to sLeX and α2,3-linked NeuAc
307
residues on multi-antennary complex-type N-glycans, but also bind to heparin and
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sulfate-containing polysaccharides [56]. Although CD94 belongs to group V of the C type lectins,
309
which lack most of the conserved Ca2+-binding residues [12,57], the glycan ligands for CD94 have
310
been found [56]. Although rat NKR-P1 is a membrane protein of NK cell, which belongs to group
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suggests that some of group V of the C type lectins may bind oligosaccharides in Ca2+-dependent
313
manner [58]. In the current study, we found that rCsCD94, in the presence of Ca2+, was able to
314
bind to both Gram-negative bacteria and Gram-positive bacteria. Furthermore, the binding of
315
rCsCD94 to bacteria was not affected by monosaccharides but was abolished by LPS, suggesting
316
that LPS is likely the target on bacterial cells that interacted with rCsCD94. In contrast to
317
rCsCD94, the control protein rTrx did not exhibit any binding to bacteria, indicating that the
318
bacterial interaction observed with rCsCD94 was specific. Agglutination analysis showed that in
319
the presence of calcium, rCsCD94 caused agglutination of all tested bacteria. The observation that
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EDTA, a chelating agent capable of binding to divalent metal ions such as Ca2+, abolished
321
agglutination caused by rCsCD94 suggests that rCsCD94 activity is calcium-dependent.
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Lectins with bacteriostatic and bactericidal effects have been reported in plants [59-61]. In
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fish, a mannose-binding lectin from cobia fish (Rahycentron canadum) displayed antibacterial
324
effects against E. coli [62]. In bighead carp (Aristichthys nobilis), the growth of V. harveyi was
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inhibited by GANL, a gill lectin [63]. In tongue sole, V. harveyi was killed by two lectins, i.e.,
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rCsBML1 and rCsBML2 [40]. V. anguillarum dissemination and colonization in the tissues of
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tongue sole were significantly reduced when the presence of rCsCTL1 [36]. In our study, we
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found that of the seven bacterial species that were bound and agglutinated by rCsCD94, only four
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(V. harveyi, V. anguillarum, B. subtilis, and M. luteus) were killed by rCsCD94. These results
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suggest that rCsCD94 has an ability to kill some species of bacteria, which indicate CsCD94
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involved in antibacterial immunity
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Lectins are thought to play a role in pathogen phagocytosis, with different modalities in 14
ACCEPTED MANUSCRIPT animals belonging to various phyla [64]. C-type lectin-like receptors such as the CD209
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(DC-SIGN) protein functions not only in adhesion but also as a phagocytic pathogen-recognition
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receptor [65]. In tongue sole, rCsCTL1 significantly enhanced HKMs activation as evidenced by
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the augmented phagocytic activity [36]. In C. intestinalis, anti-ciCD94-1 antibody specifically
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inhibited the phagocytic activity of granular amebocytes [23]. In this study, we found that in line
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with binding and agglutination, rCsCD94 promoted killing of bacteria. These observations,
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together with the fact that rCsCD94 enhanced PBL phagocytic activity, support the conclusion
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that CsCD94 is involved in tongue sole immunity.
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In conclusion, the results of this study showed that expression of CsCD94 is required for
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responding to bacterial infection. rCsCD94 exhibits apparent binding and agglutination activities
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against Gram-positive and Gram-negative bacteria in a Ca2+-dependent manner. rCsCD94 is a key
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factor in the bactericidal and phagocytic effect of tongue sole. These results reveal for the first
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time an essential role of teleost CD94 in antibacterial immunity and add insight into the function
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of CD94.
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Acknowledgments
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This work was supported by the grants from the National Basic Research Program of China
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(2012CB114406), the Scientific and Technological Innovation Project and the AoShan Talents
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Program Financially Supported by Qingdao National Laboratory for Marine Science and
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Technology (No.2015ASKJ02 and No. 2015ASTP), the Taishan Scholar Program of Shandong
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Province, and the Youth Innovation Promotion Association, CAS.
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Figure legends
Fig. 1. Sequence alignment of the CsCD94 homologues. Dots denote gaps introduced for maximum matching. Numbers in brackets indicate overall sequence identities between CsCD94
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and the compared sequences. Consensus residues are in black, and residues that are ≥75% identical among the aligned sequences are in blue. The GenBank accession numbers of the aligned sequences are as follows: Cynoglossus semilaevis, XP_016886814; Haplochromis burtoni,
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XP_005939744.1; Maylandia zebra, XP_004571228.1; Pundamilia nyererei, XP_005749836.1;
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Oreochromis niloticus, XP_005463899.1; Stegastes partitus, XP_008281066.1.
Fig. 2. CsCD94 expression in fish tissues. CsCD94 expression in the intestine, kidney, heart, liver, gill, blood, muscle, brain, and spleen of tongues sole was determined by quantitative real time RT-PCR. For convenience of comparison, the expression level in intestine was set as 1. Data are
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the means of three independent assays and presented as means ± SEM.
Fig. 3. CsCD94 expression in response to bacterial infection. Tongue sole were infected with or without (control) Vibrio harveyi, and CsCD94 expression in kidney, spleen, liver, and blood was
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determined by quantitative real time RT-PCR at 6 h, 12 h, 24 h, and 48 h after infection. In each case, the expression level of the control fish was set as 1. Data are the means of three independent
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assays and presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01.
Fig. 4. Binding of rCsCD94 to bacterial cells. Edwardsiella tarda, Vibrio anguillarum, Vibrio harveyi, Pseudomonas fluorescens, Bacillus subtilis, Micrococcus luteus, and Streptococcus iniae were incubated with or without different concentrations of rCsCD94 or rTrx, and binding of the protein to bacteria was determined by ELISA. Data are the means of three independent assays and presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01.
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were stained with DPAI and observed with a fluorescence microscope. Bar, 10 µm.
Fig. 6. Effect of rCsCD94 on bacterial survival. Vibrio harveyi, Vibrio anguillarum, Bacillus subtilis, and Micrococcus luteus were incubated with or without (control) rCsCD94 or rTrx for 1 h, 2 h, and 4 h, and the amount of bacteria was determined by plate count. Data are the means of
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Fig. 7. Effect of rCsCD94 on phagocytosis. Tongue sole peripheral blood leukocytes were incubated without bacteria (A), with FITC-labeled Vibrio anguillarum (B), or with FITC-labeled V. anguillarum that had been treated with rTrx (C) or rCsCD94 (D). The cells were then analyzed by fluorescence activated cell sorting. M1 represents the cellular population with enhanced fluorescence as a result of bacterial uptake. Values are shown as means ± SEM (N = 3). N, the
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Supplemental data Fig. S1. SDS-PAGE analysis of rCsCD94 and rTrx. Purified rCsCD94 and rTrx (lane 3 and 2, respectively) were analyzed by SDS-PAGE and viewed after staining with Coomassie brilliant
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blue R-250. Lane 1, protein markers.
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ACCEPTED MANUSCRIPT Fig. S2. Effect of LPS on the binding of rCsCD94 to Edwardsiella tarda. E. tarda was incubated with different concentrations of rTrx or rCsCD94 that had been pre-incubated with or without LPS. The control sample was E. tarda incubated without protein. Binding of protein to bacteria was determined by ELISA. Data are the means of three independent assays and presented as means ±
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Highlights ► CsCD94 has a C-type lectin-like domain. ► Expression of CsCD94 was upregulated during bacterial
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infection. ► rCsCD94 exhibited apparent binding and agglutinating activities against bacteria. ► Binding of rCsCD94 to bacteria promoted phagocytosis of peripheral blood leukocytes. ► rCsCD94 had the ability
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S1050-4648(16)30637-4
DOI:
10.1016/j.fsi.2016.10.005
Reference:
YFSIM 4233
To appear in:
Fish and Shellfish Immunology
Received Date: 28 April 2016 Revised Date:
1 October 2016
Accepted Date: 3 October 2016
Please cite this article as: Li X-p, Hu Y-h, CD94 of tongue sole Cynoglossus semilaevis binds a wide arrange of bacteria and possesses antibacterial activity, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2016.10.005. 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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CD94 of tongue sole Cynoglossus semilaevis binds a wide arrange of bacteria and possesses antibacterial activity Xue-peng Lia,b,c, Yong-hua Hua,b* a
b
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Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China University of Chinese Academy of Sciences, Beijing, China
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*To whom correspondence should be addressed
Mailing address:
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Yong-hua Hu
Institute of Oceanology
Chinese Academy of Sciences 7 Nanhai Road
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Qingdao 266071, China Phone: +86-532-82891907
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Email: [email protected]
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Abstract
2 In this study, we examined the expression patterns and the functions of the tongue sole
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Cynoglossus semilaevis CD94, CsCD94. CsCD94 is composed of 209 amino acid residues and
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shares 43.0–50.2% overall identities with known teleost CD94 sequence. CsCD94 has a C-type
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lectin-like domain. Expression of CsCD94 occurred in multiple tissues and was upregulated
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during bacterial infection. Recombinant CsCD94 (rCsCD94) exhibited apparent binding and
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agglutinating activities against both Gram-positive and Gram-negative bacteria in a
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Ca2+-dependent manner. Treatment of bacteria with rCsCD94 enhanced phagocytosis of the
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bacteria by peripheral blood leukocytes. Furthermore, incubation of rCsCD94 with bacteria
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reduced the survival of the bacteria in vitro. Taken together, these results indicate that rCsCD94 is
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a key factor in the bactericidal and phagocytic effects of tongue sole, and reveal for the first time
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an essential role of fish CD94 in antibacterial immunity, thereby adding insight into the function
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of CD94.
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Keywords: CD94; Cynoglossus semilaevis; Agglutination; Phagocytosis; Antibacterial
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1. Introduction
24 As a major class of pattern-recognition receptors (PRRs), lectins are a group of
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carbohydrate-binding proteins that are neither antibodies nor enzymes, which were first
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discovered in plants, and subsequently found to exist ubiquitously in bacteria, invertebrates, and
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vertebrates [1–4]. Based on their structural characteristics, lectins from animals are classified into
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several families [5], including C-type lectins (CTLs). CTLs, the most diverse family of animal
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lectins, are divided into seven groups (I–VII) [6]. CTLs are involved in a broad range of biological
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processes that include adhesion, endocytosis, and pathogen recognition and neutralization [7–9].
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All classical CTLs have a carbohydrate-recognition domain (CRD) that mediates specific
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recognition and binding to oligosaccharides in the extracellular matrix and on solid surfaces such
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as microbes [10]. Ca2+ is thought to be involved in ligand binding and maintenance of the CRD
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structure [11,12]. In addition to the classical CTLs, many proteins with a C-type lectin-like
36
domain (CTLD) have been identified, some of which, however, are Ca2+-independent and bind to
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proteins, rather than to sugars [13,14]. One such protein-binding molecule is cluster of
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differentiation 94 (CD94), which is part of several vertebrate natural killer (NK) lymphocyte
39
receptors that play important roles in innate immunity [15]. CD94 forms heterodimers with NK
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group 2 (NKG2) family molecules and either blocks or activates the cytotoxic activity of NK cells
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toward target cells by interacting specifically with major histocompatibility complex class I
42
molecules [16,17]. CD94 belongs to group V of the CTL superfamily, which also includes CD69,
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lectin-like type II transmembrane disulfide-bonded homodimer Ly49, and natural killer
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receptor-p1 (NKR-P1) [18].
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ACCEPTED MANUSCRIPT CD94 studies have thus far been focused nearly and exclusively on mammals, specifically
46
humans and mice, although CD94 homologues have also been reported in teleosts. Sato et al
47
isolated cDNA clones from the cichlid Paralabidochromis chilotes that, when translated into
48
protein sequences, were found to be homologous to the mammalian C-type lectin CD94 [20].
49
Genes homologous to CD94 cDNAs were also identified in another cichlid species, Oreochromis
50
niloticus [21]. Simultaneously, cDNAs homologous to mammalian CD94 were identified in
51
Botryllus schlosseri and Ciona intestinalis [22,23]. B. schlosseri CD94 (BsCD94) has been shown
52
to be expressed in a sub-population of blood cells that may be involved in colony allorecognition
53
[22]. C. intestinalis CD94-1 (CiCD94-1) was up-regulated in response to inflammation induced by
54
lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating
55
blood [23]. Furthermore, anti-ciCD94-1 antibody was found to specifically inhibit the phagocytic
56
activity of these cells [23].
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Half-smooth tongue sole (Cynoglossus semilaevis) is a demersal flatfish that inhabits mainly
58
warm waters and is distributed in the Yellow Sea and the East China Sea. Although farming of
59
tongue sole in China is relatively recent, due to its high economic value, tongue sole has become
60
an important commercial marine fish species, with great potential for further expansion of farming.
61
With the development of the tongue sole farming industry, diseases caused by bacterial pathogens,
62
such as Vibrio and Edwardsiella spp. have become problematic, resulting in significant economic
63
losses [24]. To date, information on half smooth tongue sole CD94 (CsCD94) is scarce. In this
64
study, we investigated the expression patterns and antibacterial potential of CsCD94.
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2. Materials and methods
67 68
2.1. Fish
69 Clinically healthy tongue sole (average weight, 12.6 g) were purchased from a commercial
71
fish farm in Shandong Province, China, and maintained at 20 °C in aerated seawater. Before
72
experimentation, the fish were acclimatized in the laboratory for 2 weeks and verified to be free of
73
pathogens in the liver, kidney, and spleen, as reported previously [25]. For tissue collection, fish
74
were euthanized with tricaine methanesulfonate (Sigma-Aldrich Corporation, St. Louis, MO,
75
USA), as reported previously [26].
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2.2. Sequence analysis
78
The cDNA and amino acid sequences of CsCD94 (GenBank accession no. XM_017031325)
80
were analyzed using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi), hosted by the
81
National Center for Biotechnology Information (NCBI) website. Domain searches were performed
82
with
83
(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The theoretical molecular mass and
84
isoelectric point were predicted using EditSeq sequence editing software in the DNASTAR
85
software package (DNASTAR, Inc., Madison, WI, USA). Multiple sequence alignments were
86
created using the DNAMAN program (Lynnon Biosoft, Vaudreuil-Dorion, Quebec, Canada).
domain
search
program
of
the
NCBI
website
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conserved
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the
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2.3. Quantitative real time reverse transcription-PCR (qRT-PCR) analysis of CsCD94
89
expression under normal conditions
90 91
qRT-PCR analysis of CsCD94 expression under normal conditions was determined as
92
follows. Total RNA from the spleen, liver, kidney, blood, intestine, muscle, gill, heart, and brain, 5
ACCEPTED MANUSCRIPT aseptically collected from five tongue sole, was extracted using the EZNA Total RNA Kit (Omega
94
Bio-tek, Doraville, GA, USA). One microgram of total RNA was used for cDNA synthesis with
95
the Superscript II reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA). qRT-PCR
96
was performed using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the
97
SYBR ExScript qRT-PCR Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China) [27]. The PCR
98
reaction was performed in a 20 µl volume containing 10 µl SYBR® premix Ex Taq™(Tli
99
RNaseH Plus), 0.2 µM each of specific forward primer (5’- CGGGGTTGTCAGTAGAGTGG -3’)
100
and reverse primer (5’- TCGTACAAACCCATGGTCCG -3’), and 2.0 µl diluted cDNA (50
101
ng/µl). The PCR conditions were 95 °C for 30 s, followed by 35 cycles of 95 °C for 15 s, 60 °C
102
for 15 s, 72 °C for 20 s. The expression level of CsCD94 was analyzed using the comparative
103
threshold cycle method (2−∆∆CT) with beta-actin as an internal reference [28]. The experiment was
104
performed three times.
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2.4. qRT-PCR analysis of CsCD94 expression during bacterial infections
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qRT-PCR analysis of CsCD94 expression during bacterial infection was performed as
109
reported previously [29]. Vibrio harveyi [30] was cultured in Luria-Bertani (LB) broth at 28 °C to
110
an optical density at 600 nm (OD600) of 0.8. Then, the cells were washed with phosphate-buffered
111
saline (PBS) and resuspended in PBS to a concentration of 1 × 106 CFU/ml. Tongue sole (as above)
112
were divided randomly into two groups (20/group) and injected intraperitoneally with 50 µl V.
113
harveyi or PBS. At 6, 12, 24, and 48 h post-infection, CsCD94 expression in the kidney, spleen,
114
liver, and blood was determined by qRT-PCR, as described in section 2.3. The experiment was
115
performed three times.
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2.5. Construction of plasmids
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To construct pEtCsCD94, which expresses His-tagged recombinant CsCD94 (rCsCD94), the coding
sequence
of
CsCD94
was
amplified 6
by
PCR
with
primers
F1
ACCEPTED MANUSCRIPT 121
(5’–GATATCATGGATCCACCAGAGAGCTTCAC– 3’, underlined sequence, EcoRV restriction
122
endonuclease
123
underlined sequence, EcoRV restriction endonuclease site). The PCR products were ligated with
124
the T−A cloning vector T-Simple (TransGen Biotech, Beijing, China) and the recombinant
125
plasmid was digested with EcoRV to retrieve the CsCD94-containing fragment, which was
126
inserted into pET32a [31] at the EcoRV site, resulting in pETCsCD94.
and
R1
(5’–GATATCAAACTCCTTCGTACAAACCCATGGT–
127 128
2.6 Purification of recombinant proteins
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3’,
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site)
Recombinant proteins were expressed and purified as described previously [29,32]. Briefly,
131
Escherichia coli BL21 (DE3) cells (Beijing TransGen Biotech Co., Ltd., Beijing, China) were
132
transformed separately with pETCsCD94 and pET32a (Novagen, San Diego, CA, USA), the latter
133
expressing rTrx (recombinant thioredoxin) [33]. Then, the transformants were cultured in LB
134
medium at 37 °C to the mid-logarithmic phase and isopropyl-β-D-thiogalactopyranoside was
135
added to the culture to a final concentration of 1 mM. After growing at 16 °C for an additional 10
136
h, the cells were harvested by centrifugation (4200 g) and His-tagged proteins were purified using
137
Ni-NTA Agarose (Qiagen, Inc., Valencia, CA, USA) as recommended by the manufacturer. The
138
proteins were concentrated with PEG20000 (Beijing Solarbio Science & Technology Co., Ltd.,
139
Beijing, China) and endotoxins were removed as reported previously [34]. The concentrated
140
proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
141
(SDS-PAGE) and visualized after staining with Coomassie brilliant blue R-250. The
142
concentrations of purified proteins were determined using the Bradford method with bovine serum
143
albumin as a standard.
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2.7. Binding of rCsCD94 to bacteria
146 147 148
The bacteria Edwardsiella tarda, Pseudomonas fluorescens, Vibrio anguillarum, V. harveyi, and Streptococcus iniae are fish pathogens described elsewhere [26,30,35–37]. Bacillus subtilis 7
ACCEPTED MANUSCRIPT and Micrococcus luteus were purchased from China General Microbiological Culture Collection
150
Center (Bejing, China) Bacterial cells were cultured in LB medium to OD600 0.8, washed and
151
resuspended in TBS (50 mM Tris–HCl, 100 mM NaCl, pH 7.5) to 108 CFU/ml. Binding of
152
rCsCD94 to bacteria was determined using an enzyme-linked immunosorbent assay (ELISA) [38].
153
To determine the effect of sugars and lipopolysaccharides (LPS) on rCsCD94-bacteria interaction,
154
bacteria were incubated with rCsCD94 as above in TBS-Ca2+ buffer containing 200 mM
155
D-mannose, D-galactose, D-glucose, L-fucose, N-acetyl-D-glucosamine (Sangon, Shanghai,
156
China), or LPS (30 µg/ml), which was prepared as reported previously [39]. The experiment was
157
performed three times.
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2.8. Agglutination assay
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The agglutination assay was performed as reported previously [40]. Briefly, bacterial cells
162
were cultured in LB medium as above, washed with TBS buffer, and resuspended in TBS, TBS
163
containing
164
ethylenediaminetetraacetic acid (EDTA) to 2 × 108 CFU/ml. rCsCD94 was added to the bacterial
165
cells to a final concentration of 32 µg/ml. After incubation at 25 °C for 1 h, the bacterial cells were
166
stained with 4,6-diamino-2-phenyl indole (Invitrogen Corporation), according to manufacturer’s
167
instructions, and agglutination was observed with a fluorescence microscope (Nikon E800; Nikon
168
Corporation, Tokyo, Japan). To determine the effect of sugars on agglutination, bacteria were
169
incubated with rCsCD94 as above in TBS-Ca2+ buffer containing 200 mM D-mannose,
170
D-galactose, D-glucose, or N-acetyl-D-glucosamine (Sangon, Shanghai, China). The experiment
171
was performed three times.
173
CaCl2
(TBS-Ca2+
buffer),
or
TBS-Ca2+
buffer
plus
4
mM
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2.9. Effect of rCsCD94 on bacterial survival
174 175
Bacteria were cultured in LB medium to OD600 of 0.8 and the cells were resuspended to 1 ×
176
104 CFU/ml in TBS-Ca2+ buffer. Then, rCsCD94 or rTrx was added to the bacterial cell 8
ACCEPTED MANUSCRIPT 177
suspension to a final concentration of 32 µg/ml. Control cells were incubated in TBS-Ca2+ buffer
178
at 25 °C for 1, 2, and 4 h, and bacterial survival was examined by plate count, as reported
179
previously [41]. The experiment was performed three times.
180 2.10. Preparation of peripheral blood leukocytes (PBL)
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To prepare PBL, blood was collected from the caudal veins of half-smooth tongue sole (~850
184
g) and diluted 1:1 with L-15 medium (Thermo Scientific HyClone, Beijing, China). The diluted
185
blood was placed on top of 61% Percoll (Solarbio, Beijing, China) and centrifuged at 400 g for 10
186
min. The white layer of PBL in the interface was recovered, washed twice and resuspended in
187
L-15 medium.
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2.11. Fluorescence activated cell sorting (FACS)
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Phagocytosis determined by FACS was performed as reported previously [29,42]. Briefly, V.
192
anguillarum was cultured as above and resuspended in 100 µg/ml of fluorescein isothiocyanate
193
(FITC) (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China). After incubation at 37 °C for 2 h,
194
the cells were collected by centrifugation and washed five times in TBS. rCsCD94 or rTrx were
195
added to the cells to a concentration of 32 µg/ml. After incubation at 22 °C for 1 h, the cells were
196
washed and resuspended in L-15 medium (Thermo Scientific HyClone, Beijing, China) to a
197
concentration of 1 × 108 cells/ml. One milliliter of PBLs (~107 cells) was mixed with 100 µl of V.
198
anguillarum, rCsCD94-treated V. anguillarum, rTrx-treated V. anguillarum, or L-15 medium
199
(control), and incubated in the dark for 2 h. Then, and the cells were collected by centrifugation
200
and washed with TBS three times. Extracellular fluorescence was quenched by adding 1 ml of
201
0.125 % trypan blue in TBS, followed by incubation at 22 °C for 5 min. The cells were collected
202
by centrifugation, suspended in 1 ml of TBS, and subjected to FACS analysis with a Partec
203
CyFlow Counter (Sysmex Partec GmbH, Münster, Germany). The data were analyzed using
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WinMDI 2.9 software (The Scripps Research Institute, La Jolla, CA, USA). The experiment was
205
repeated three times.
206 207
2.12. Statistical analysis
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All experiments were performed at least three times and statistical analyses were performed
210
using SPSS 17.0 software (IBM-SPSS Inc., Chicago, IL, USA). Data were analyzed with analysis
211
of variance and statistical significance was defined as P < 0.05.
3. Results
214 215
3.1. Sequence analysis of CsCD94
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CsCD94 is composed of 209 amino acid residues with a calculated molecular mass of 24.2
218
kDa and an isoelectric point of 4.31. CsCD94 has a CTLD composed of residues 81–206. BLAST
219
analysis showed that CsCD94 shares 43.0–50.2% overall sequence identities with the CD94
220
homologues of Haplochromis burtoni, Maylandia zebra, Pundamilia nyererei, O. niloticus,
221
Stegastes partitus, respectively (Fig. 1).
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3.2. Expression of CsCD94 in fish tissues
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Under normal physiological conditions, qRT-PCR showed that CsCD94 expression occurred,
226
in increasing order, in the intestine, kidney, heart, liver, gill, blood, muscle, brain, and spleen of
227
tongue sole (Fig. 2). After infection with the bacterial pathogen V. harveyi, CsCD94 expression
228
was significantly upregulated at 6 h, 12 h and 24 h in kidney and blood, at 12 h and 24 h in spleen,
229
and at 24 h in liver (Fig. 3). The maximum inductions in kidney, blood, spleen, and liver were
230
59.4-fold, 11.7- fold, 10.7-fold, and 3.2-fold, respectively.
231 10
ACCEPTED MANUSCRIPT 232
3.3. Binding of rCsCD94 to bacteria
233 rCsCD94 was purified from E. coli (Fig. S1). When rCsCD94 was incubated with
235
Gram-negative bacteria and Gram-positive bacteria, i.e., E. tarda, P. fluorescens, V. anguillarum,
236
V. harveyi, B. subtilis, M. luteus, and S. iniae, rCsCD94 was able to bind to each in the presence of
237
Ca2+, with the highest and lowest binding indexes observed with E. tarda and V. anguillarum,
238
respectively (Fig. 4). Furthermore, the binding indexes increased with the dose of rCsCD94 (Fig.
239
4). In contrast, rTrx, which was used as a control protein for rCsCD94, did not exhibit any binding
240
to the bacteria (Fig. 4). The presence of D-mannose, D-galactose, D-glucose, L-fucose, and
241
N-acetyl-D-glucosamine had no effect on the binding activity of rCsCD94 (data not shown).
242
Pre-incubation of rCsCD94 with E. tarda LPS inhibited binding of the protein to the bacteria (Fig.
243
S2).
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244 245
3.4. Effect of EDTA and monosaccharides on bacterial agglutination by rCsCD94
246
The results of the agglutination assay showed that in TBS buffer plus calcium, rCsCD94
248
caused agglutination of E. tarda, B. subtilis, M. luteus, V. harveyi, S. iniae, V. anguillarum, and P.
249
fluorescens (Fig. 5). In contrast, in TBS buffer or in TBS buffer plus calcium and EDTA,
250
rCsCD94 caused no apparent agglutination of any of the tested bacteria (Fig. 5). The presence of
251
D-mannose, D-galactose, D-glucose, and N-acetyl-D-glucosamine had no effect on the
252
agglutination activity of rCsCD94 (data not shown).
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3.5. Effect of rCsCD94 on bacterial survival
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To examine the effect of rCsCD94 on the survivability of the bound bacteria, E. tarda, P.
257
fluorescens, V. anguillarum, V. harveyi, B. subtilis, M. luteus, and S. iniae were incubated with
258
rCsCD94 or rTrx for 1 h, 2 h, and 4 h, and the number of surviving bacterial was determined. The
259
results showed that at 2 h and 4 h incubation, the numbers of rCsCD94-treated V. anguillarum, V. 11
ACCEPTED MANUSCRIPT 260
harveyi, B. subtilis, and M. luteus were significantly decreased, as compared to the control,
261
whereas the numbers of these bacteria treated with rTrx were similar to that of the control (Fig.6).
262
The numbers of rCsCD94-treated E. tarda, P. fluorescens, and S. iniae did not change
263
significantly (data not shown).
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266
To examine the effect of rCsCD94 on phagocytosis, tongue sole PBLs were incubated with V.
268
anguillarum treated with rCsCD94 or rTrx. FACS analysis showed that for the PBLs incubated
269
with rCsCD94-treated V. anguillarum, the percentage of positive phagocytic cells (M1 = 64.3%)
270
was significantly (P < 0.01) higher than that of the PBLs incubated with untreated V. anguillarum
271
(M1 = 45.8%) (Fig. 7). Treatment with rTrx had no significant effect on phagocytosis.
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4. Discussion
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In this study, we examined the expression and biological property of CsCD94, a CD94
276
homologue from tongue sole. CsCD94 possesses a typical CTLD and is relatively closely related
277
to known CD94 proteins of teleost.
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In mammals, CD94 is expressed mainly on NK cells and a subset of CD8+ T cells [43–45].
279
In C. intestinalis, ciCD94-1 was expressed in a subpopulation of blood cells and during larval
280
development, as well as the early stages of metamorphosis in structures related to the nervous
281
system [23]. In B. schlosseri, BsCD94-1 was also expressed on the surface of a subpopulation of
282
blood cells [22]. However, in fish, the tissue-specific expression profiles of CD94 have not been
283
documented. In our study, CsCD94 transcripts were detected ubiquitously in all examined tissues
284
of fish, which is in line with that observed with other tongue sole immune related lectins such as
285
CsCTL1 and CsBML [36,40]. The tissue-different expressions of CsCD94 suggests different
286
levels of CsCD94 regulations in these tissues.
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288
expression of CD94 had previously been found to upregulate in association with human
289
cytomegalovirus reactivation [47,48]. Furthermore, the upregulation of CD94 seemed to depend
290
on the presence of interleukin-12 [49]. In C. intestinalis, ciCD94-1 was up-regulated in expression
291
by LPS-induced inflammation [23]. However, to the best of our knowledge, no report on the
292
induction of fish CD94 by bacterial pathogens has been documented. In this study, we found that
293
CsCD94 expression was upregulated in a time-dependent manner during V. harveyi infection,
294
however, the expression patterns of CsCD94 differed in different tissues. In kidney and blood,
295
peak inductions of CsCD94 expression occurred at the early stage of infection, while in spleen and
296
liver, peak inductions of CsCD94 expression occurred at relatively the late stage of infection.
297
These results indicate that CsCD94 is involved in tissue immune response during bacterial
298
invasion.
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Several studies have shown that lectins can bind and agglutinate bacteria. A fucose-binding
300
lectin from tilapia (O. niloticus) agglutinated Aeromonas hydrophila and Enterococcus faecalis
301
[50], a skin lectin HjCL from Japanese Bullhead Shark (Heterodontus japonicas) agglutinated E.
302
tarda [51], a lactose-binding skin lectin from Japanese eel agglutinated E. coli [52], a serum lectin
303
from blue gourami (Trichogaster trichopterus) agglutinated A. hydrophila [53,54], and lectins of
304
Atlantic salmon were able to bind to V. anguillarum and A. salmonicida [55]. In tongue sole, a
305
C-type lectin exhibited agglutination and binding activities against V. anguillarum and M. luteus
306
[36]. The C-type lectin-like receptors CD94 could not only bind to sLeX and α2,3-linked NeuAc
307
residues on multi-antennary complex-type N-glycans, but also bind to heparin and
308
sulfate-containing polysaccharides [56]. Although CD94 belongs to group V of the C type lectins,
309
which lack most of the conserved Ca2+-binding residues [12,57], the glycan ligands for CD94 have
310
been found [56]. Although rat NKR-P1 is a membrane protein of NK cell, which belongs to group
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312
suggests that some of group V of the C type lectins may bind oligosaccharides in Ca2+-dependent
313
manner [58]. In the current study, we found that rCsCD94, in the presence of Ca2+, was able to
314
bind to both Gram-negative bacteria and Gram-positive bacteria. Furthermore, the binding of
315
rCsCD94 to bacteria was not affected by monosaccharides but was abolished by LPS, suggesting
316
that LPS is likely the target on bacterial cells that interacted with rCsCD94. In contrast to
317
rCsCD94, the control protein rTrx did not exhibit any binding to bacteria, indicating that the
318
bacterial interaction observed with rCsCD94 was specific. Agglutination analysis showed that in
319
the presence of calcium, rCsCD94 caused agglutination of all tested bacteria. The observation that
320
EDTA, a chelating agent capable of binding to divalent metal ions such as Ca2+, abolished
321
agglutination caused by rCsCD94 suggests that rCsCD94 activity is calcium-dependent.
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Lectins with bacteriostatic and bactericidal effects have been reported in plants [59-61]. In
323
fish, a mannose-binding lectin from cobia fish (Rahycentron canadum) displayed antibacterial
324
effects against E. coli [62]. In bighead carp (Aristichthys nobilis), the growth of V. harveyi was
325
inhibited by GANL, a gill lectin [63]. In tongue sole, V. harveyi was killed by two lectins, i.e.,
326
rCsBML1 and rCsBML2 [40]. V. anguillarum dissemination and colonization in the tissues of
327
tongue sole were significantly reduced when the presence of rCsCTL1 [36]. In our study, we
328
found that of the seven bacterial species that were bound and agglutinated by rCsCD94, only four
329
(V. harveyi, V. anguillarum, B. subtilis, and M. luteus) were killed by rCsCD94. These results
330
suggest that rCsCD94 has an ability to kill some species of bacteria, which indicate CsCD94
331
involved in antibacterial immunity
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Lectins are thought to play a role in pathogen phagocytosis, with different modalities in 14
ACCEPTED MANUSCRIPT animals belonging to various phyla [64]. C-type lectin-like receptors such as the CD209
334
(DC-SIGN) protein functions not only in adhesion but also as a phagocytic pathogen-recognition
335
receptor [65]. In tongue sole, rCsCTL1 significantly enhanced HKMs activation as evidenced by
336
the augmented phagocytic activity [36]. In C. intestinalis, anti-ciCD94-1 antibody specifically
337
inhibited the phagocytic activity of granular amebocytes [23]. In this study, we found that in line
338
with binding and agglutination, rCsCD94 promoted killing of bacteria. These observations,
339
together with the fact that rCsCD94 enhanced PBL phagocytic activity, support the conclusion
340
that CsCD94 is involved in tongue sole immunity.
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In conclusion, the results of this study showed that expression of CsCD94 is required for
342
responding to bacterial infection. rCsCD94 exhibits apparent binding and agglutination activities
343
against Gram-positive and Gram-negative bacteria in a Ca2+-dependent manner. rCsCD94 is a key
344
factor in the bactericidal and phagocytic effect of tongue sole. These results reveal for the first
345
time an essential role of teleost CD94 in antibacterial immunity and add insight into the function
346
of CD94.
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Acknowledgments
349
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This work was supported by the grants from the National Basic Research Program of China
351
(2012CB114406), the Scientific and Technological Innovation Project and the AoShan Talents
352
Program Financially Supported by Qingdao National Laboratory for Marine Science and
353
Technology (No.2015ASKJ02 and No. 2015ASTP), the Taishan Scholar Program of Shandong
354
Province, and the Youth Innovation Promotion Association, CAS.
15
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Figure legends
Fig. 1. Sequence alignment of the CsCD94 homologues. Dots denote gaps introduced for maximum matching. Numbers in brackets indicate overall sequence identities between CsCD94
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and the compared sequences. Consensus residues are in black, and residues that are ≥75% identical among the aligned sequences are in blue. The GenBank accession numbers of the aligned sequences are as follows: Cynoglossus semilaevis, XP_016886814; Haplochromis burtoni,
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XP_005939744.1; Maylandia zebra, XP_004571228.1; Pundamilia nyererei, XP_005749836.1;
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Oreochromis niloticus, XP_005463899.1; Stegastes partitus, XP_008281066.1.
Fig. 2. CsCD94 expression in fish tissues. CsCD94 expression in the intestine, kidney, heart, liver, gill, blood, muscle, brain, and spleen of tongues sole was determined by quantitative real time RT-PCR. For convenience of comparison, the expression level in intestine was set as 1. Data are
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the means of three independent assays and presented as means ± SEM.
Fig. 3. CsCD94 expression in response to bacterial infection. Tongue sole were infected with or without (control) Vibrio harveyi, and CsCD94 expression in kidney, spleen, liver, and blood was
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determined by quantitative real time RT-PCR at 6 h, 12 h, 24 h, and 48 h after infection. In each case, the expression level of the control fish was set as 1. Data are the means of three independent
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Fig. 4. Binding of rCsCD94 to bacterial cells. Edwardsiella tarda, Vibrio anguillarum, Vibrio harveyi, Pseudomonas fluorescens, Bacillus subtilis, Micrococcus luteus, and Streptococcus iniae were incubated with or without different concentrations of rCsCD94 or rTrx, and binding of the protein to bacteria was determined by ELISA. Data are the means of three independent assays and presented as means ± SEM. ∗P < 0.05, ∗∗P < 0.01.
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were stained with DPAI and observed with a fluorescence microscope. Bar, 10 µm.
Fig. 6. Effect of rCsCD94 on bacterial survival. Vibrio harveyi, Vibrio anguillarum, Bacillus subtilis, and Micrococcus luteus were incubated with or without (control) rCsCD94 or rTrx for 1 h, 2 h, and 4 h, and the amount of bacteria was determined by plate count. Data are the means of
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Fig. 7. Effect of rCsCD94 on phagocytosis. Tongue sole peripheral blood leukocytes were incubated without bacteria (A), with FITC-labeled Vibrio anguillarum (B), or with FITC-labeled V. anguillarum that had been treated with rTrx (C) or rCsCD94 (D). The cells were then analyzed by fluorescence activated cell sorting. M1 represents the cellular population with enhanced fluorescence as a result of bacterial uptake. Values are shown as means ± SEM (N = 3). N, the
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Supplemental data Fig. S1. SDS-PAGE analysis of rCsCD94 and rTrx. Purified rCsCD94 and rTrx (lane 3 and 2, respectively) were analyzed by SDS-PAGE and viewed after staining with Coomassie brilliant
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ACCEPTED MANUSCRIPT Fig. S2. Effect of LPS on the binding of rCsCD94 to Edwardsiella tarda. E. tarda was incubated with different concentrations of rTrx or rCsCD94 that had been pre-incubated with or without LPS. The control sample was E. tarda incubated without protein. Binding of protein to bacteria was determined by ELISA. Data are the means of three independent assays and presented as means ±
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Highlights ► CsCD94 has a C-type lectin-like domain. ► Expression of CsCD94 was upregulated during bacterial
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infection. ► rCsCD94 exhibited apparent binding and agglutinating activities against bacteria. ► Binding of rCsCD94 to bacteria promoted phagocytosis of peripheral blood leukocytes. ► rCsCD94 had the ability
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