Ayu C-reactive protein/serum amyloid P agglutinates bacteria and inhibits complement-mediated opsonophagocytosis by monocytes/macrophages

doi:10.1016/j.fsi.2018.02.038

Fish & Shellfish Immunology

Volume 76, May 2018, Pages 58-67

https://doi.org/10.1016/j.fsi.2018.02.038 Get rights and content

Highlights

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We characterized a short-chain PTX (PaCRP/SAP) gene from ayu.
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PaCRP/SAP is a positive APP in ayu responding to Vibrio anguillarum infection.
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PaCRP/SAP agglutinated bacteria and exhibited high affinity to PGN and LPS.
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PaCRP/SAP inhibited complement-mediated opsonophagocytosis by ayu MO/MΦ.

Abstract

The short-chain pentraxins (PTXs), including C-reactive protein (CRP) and serum amyloid P (SAP), are soluble pattern recognition molecules (PRMs) that exhibit calcium-dependent binding to bacterial surface molecules. They opsonize pathogens or other particles by phagocytic clearance. However, the detailed functions of short-chain PTXs in teleosts remained unclear. In this study, we identified a short-chain PTX gene from ayu, Plecoglossus altivelis, and tentatively named as PaCRP/SAP. Sequence analysis revealed that PaCRP/SAP has typical characteristics of fish CRP/SAP and is mostly closely related to rainbow smelt (Osmerus mordax) SAP. PaCRP/SAP transcripts were detected in all tested tissues, with the highest level in the liver, and its expression significantly upregulated following Vibrio anguillarum infection. The active recombinant mature PaCRP/SAP (rPaCRP/SAPm) agglutinated Gram-negative bacteria (Escherichia coli, V. anguillarum, Aeromonas hydrophila, and Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes) in a calcium-dependent manner in vitro, and it correspondingly bound peptidoglycan and lipopolysaccharide in a dose-dependent manner. The binding of rPaCRP/SAPm to E. coli and S. aureus resulted in a clear inhibition of the deposition of ayu complement 3 (PaC3) on the bacteria. Furthermore, rPaCRP/SAPm decreased phagocytosis of rPaCRP/SAPm-bound E. coli and S. aureus cells by ayu monocytes/macrophages (MO/MΦ) in a complement-dependent way. However, rPaCRP/SAPm alone had no significant influence on phagocytosis. These results provided the first evidence that PaCRP/SAP might function in ayu immune responses via agglutinating bacteria and inhibiting complement-mediated opsonophagocytosis by MO/MΦ.

Introduction

The innate immune system recognizes microorganisms via a limited number of germline-encoded pattern-recognition receptors (PRRs). Based on cellular localization and function, PRRs are classified into two major groups: (1) cell-associated receptors, which are localized in different cellular compartments [1], and (2) soluble PRRs such as pentraxins (PTXs), which represent the functional ancestors of antibodies and are involved in pathogen opsonization, complement activation, and self versus modified self-discrimination [2]. PTXs, which belong to a family of animal lectins [3,4], are distinctively characterized by the presence of a highly conserved motif of known as PTX signature (HxCxS/TWxS) in their carboxy-terminal regions [5]. They are a key component of the humoral arm of innate immunity, processing functions in various pathophysiological conditions [6]. PTXs can be divided into two subclasses, the classical short-chain PTXs including C-reactive protein (CRP) and serum amyloid P (SAP), and the long-chain PTXs, which have an unrelated amino-terminal region coupled to a carboxy-terminal PTX-like domain [7].

In mammals, the short-chain PTXs are well characterized, both structurally and functionally. CRP and SAP are similarly composed of five identical subunits in a disc-like configuration and share extensive sequence homology, indicating that they evolved from a common ancestor [8]. They are involved in acute-phase response to trauma, injury or infection [9]. Furthermore, they can bind to various ligands in a characteristic calcium-dependent manner, such as DNA, chromatin, fibronectin, glycosaminoglycans, heparin, and the structures of microbial surfaces including lipopolysaccharide (LPS), peptidoglycan (PGN), and phosphorylcholine, etc [8,[10], [11], [12]]. Hence, CRP and SAP can also bind to a range of microbes and other particles such as apoptotic cells and dead cells to opsonize pathogens or cells by phagocytic clearance [9,13]. Previous reports have revealed that short-chain PTXs have affect the opsonophagocytosis of pathogens in two ways, by interacting directly with cell-surface Fcγ receptors (FcγR) or by affecting the classical complement pathway through several complement components (C1q, factor H, C4b-binding protein, C5b6 complex, etc.) [9,[14], [15], [16], [17]]. Mammalian CRP are reported to enhance opsonization, whereas the ultimate impacts of SAP are inconsistent, some show the induction of the opsonophagocytosis, and others show the inhibition [7,10,11,[18], [19], [20], [21]].

In fish, some CRP and SAP were isolated from serum like plaice (Pleuronectes platessa L.), dogfish (Mustelus cannis), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and the striped catfish (Pangasianodon hypophthalmus) by using affinity chromatography and named based on their binding properties compared with mammalian homologues [[22], [23], [24], [25], [26], [27], [28], [29]]. Recently, more cDNA sequences of CRP and SAP had been characterized in teleost, such as common carp (Cyprinus carpio), snapper (Pagrus auratuse), black rockfish (Sebastes schlegelii), rock bream (Oplegnathus fasciatus), half-smooth tongue sole (Cynoglossus semilaevis) and Atlantic salmon [[30], [31], [32], [33], [34], [35], [36], [37]]. Fish encode several PTX-like molecules, but no clear separation of CRP and SAP by sequence homology and phylogenetic tree analysis is observed [37]. Hence, such short-chain PTXs in fish are tentatively named as CRP/SAP molecules [37].

Fish CRP/SAP are widely expressed in the tissues and cells of the innate immune system. Upon infection via a pathogen, their expressions increase dramatically and immediately [22,27,[34], [35], [36], [38],38]. Fish CRP/SAP can bind and agglutinate many bacteria, exhibiting calcium-dependent lectin activity [29,33,34]. However, detailed immunological functions of fish CRP/SAP were rarely reported. Only rainbow trout CRP and half-smooth tongue sole SAP are reported to be involved in the phagocytosis and host resistance to pathogen infections [28,36,39]. However, the exact mechanism of CRP/SAP in fish immune responses is still not clear.

Ayu (Plecoglossus altivelis) is an economically important fish in East Asia [40]. Pathogen infections have resulted in both production and animal welfare problems [41]. It is therefore important to study immune modulation of ayu against pathogens. In this study, we characterized a CRP/SAP gene from ayu (PaCRP/SAP), and analyzed its expression profile upon Vibrio anguillarum infection. In addition, its agglutination activity to bacteria and effects on opsonophagocytosis by monocytes/macrophages (MO/MΦ) were investigated.

Section snippets

Fish rearing

Healthy ayu, weighing 40–50 g and without any pathological signs, were obtained from a fishery in Ninghai County, Ningbo City, China and healthy. Fish were kept in fresh-water tanks in a recirculating system at 20–22 °C for two-week acclimation prior to the experiments commencing. All experiments were performed according to the Experimental Animal Management Law of China and approved by the Animal Ethics Committee of Ningbo University.

Molecular cloning of PaCRP/SAP cDNA

The cDNA sequence of PaCRP/SAP gene was obtained from a

Sequence analysis of PaCRP/SAP gene

The isolated cDNA, 1146 bp in length, contains an open reading frame of 675 bp encoding for a polypeptide of 225 amino acids containing a 16-residue signal peptide. The polypeptide is 25.2 kDa in size and produces a 23.4 kDa mature protein with the theoretical pI of 6.47. BLAST search revealed that this sequence is homologous to fish CRP and SAP, thus tentatively named it as PaCRP/SAP. Multiple alignment with other known sequences of PTX family revealed that PaCRP/SAP has the similar

Discussion

Mammalian short-chain PTXs (CRP and SAP) recognize a wide range of exogenous pathogenic substances and mediate through FcγR or the complementary pathway leading to pathogen clearance [7,17,20]. No clear separation of CRP and SAP in fish by sequence homology and phylogenetic tree analysis is observed, and they are tentatively named as CRP/SAP [37]. However, the information on fish CRP/SAP functions is limited. In the present work, we studied a CRP/SAP like gene, PaCRP/SAP, from ayu. PaCRP/SAP

Acknowledgments

We thank Dr. Mark Whitehead, Institute of Integrative Biology, University of Liverpool, UK for his expertise and critical reading of the manuscript. The project was supported by the Program for the Natural Science Foundation of China (31402323; 31772876; 31372555), the Natural Science Foundation of Zhejiang Province (LY14C190007; LZ18C190001), the Scientific Innovation Team Project of Ningbo (2015C110018), the Natural Science Foundation of Ningbo City of China (2017A610284), Ningbo Science and

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Citation Excerpt :
On the other hand, expression of TrSAP/CRP gene was observed in the skin as well as liver in juvenile at 57 dph (Fig. 6). In contrast to mammalian SAPs and CRPs, fish PTXs are expressed in various tissues including the skin (e.g., in common skate (Tsutsui et al., 2009), ayu (Shi et al., 2018), mudskipper (Cai et al., 2019)), although the highest levels are often found in the liver. In addition, two isoforms of PTX in cod fish were detected in the skin of larvae at 3 days post hatching by immunohistochemistry, and they were detected in the skin mucus of the adults by western blotting (Magnadóttir et al., 2018).
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Full length articleAyu C-reactive protein/serum amyloid P agglutinates bacteria and inhibits complement-mediated opsonophagocytosis by monocytes/macrophages

Highlights

Abstract

Introduction

Section snippets

Fish rearing

Molecular cloning of PaCRP/SAP cDNA

Sequence analysis of PaCRP/SAP gene

Discussion

Acknowledgments

Cell

Vet. Immunol. Immunopathol.

Vasc. Pharmacol.

Mol. Immunol.

Trends Immunol.

J. Autoimmun.

Comp. Biochem. Physiol. C

J. Biol. Chem.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Dev. Comp. Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Dev. Comp. Immunol.

Fish Shellfish Immunol.

Dev. Comp. Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Fish Shellfish Immunol.

Mol. Immunol.

Full length article
Ayu C-reactive protein/serum amyloid P agglutinates bacteria and inhibits complement-mediated opsonophagocytosis by monocytes/macrophages