An l-rhamnose-binding lectin from Nile tilapia (Oreochromis niloticus) possesses agglutination activity and regulates inflammation, phagocytosis and respiratory burst of monocytes/macrophages

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Highlights

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    The RBL-like gene was identified in Nile tilapia (OnRBL-L).

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    OnRBL-L expression was significantly up-regulated following challenges with bacteria.

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    Recombinant OnRBL-L possessed binding and agglutination bacteria ability.

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    Recombinant OnRBL-L could participate in the regulation of inflammation, phagocytosis and respiratory burst.

Abstract

Rhamnose-binding lectins (RBLs), a Ca2+-independent lectin family, are widely present in vertebrates and invertebrates, which involve in the innate immune response. However, the functional characterization and related regulation mechanisms of RBLs remain unclear in teleost fish. In this study, an l-rhamnose-binding lectin-like (OnRBL-L) was identified and functionally characterized from Nile tilapia (Oreochromis niloticus). The open reading frame of OnRBL-L is 678 bp encoding 225 aa. The sequence of OnRBL-L has relatively conservative characteristic peptide motifs, including YGR, DPC, and KYL-motif. Expression analysis showed that OnRBL-L was abundantly distributed in intestine tissue, and widely existed in all detected tissues. Meanwhile, the expression of OnRBL-L increased significantly in vivo (liver, spleen, head kidney, intestine, gills and peripheral blood) and in vitro (monocytes/macrophages) following challenges with two important tilapia pathogenic bacteria Streptococcus agalactiae and Aeromonas hydrophila. In addition, the recombinant OnRBL-L was found to bind and agglutinate S. agalactiae and A. hydrophila. Furthermore, OnRBL-L could participate in non-specific cellular immune defense, including reducing the expression of pro-inflammatory factors (IL-6IL-8 and TNF-α), and enhancement of the phagocytosis and respiratory burst of MO/MФ. Overall, our results provide new insights into the understanding of RBL as an important pattern recognition molecule and regulator in non-specific cell immunity in an early vertebrate.

Introduction

The innate immune system is an evolutionarily ancient defense system, which is essential for the first line of defense against pathogenic microorganisms (Kumar et al., 2011). In general, the innate immunity can be activated rapidly upon recognition of foreign threat, causing a series of effector immune responses, such as immobilization, inflammation, chemotaxis, opsonization, phagocytosis and killing (Turvey and Broide, 2010; Vasta et al., 2011). These reactions are associated with many receptor molecules including toll-like receptors and lectins, which are responsible for recognizing the presence of foreign microorganisms (Janeway and Medzhitov, 2002; Vasta et al., 2011). Among them, lectins, as a group of carbohydrate-binding proteins, are the key components of innate immunity and play multiple functions in host defense (Ogawa et al., 2011; Vasta et al., 2011). By recognizing and binding to the carbohydrate of potential pathogenic microorganisms, lectins can actively participate in a series of immune responses, including agglutination, regulation of inflammation, complement activation, opsonophagocytosis, and apoptotic cell clearance, thereby reducing infection and maintaining host homeostasis (Eddie Ip et al., 2009; Ogawa et al., 2011; Vasta et al., 2011; Hansen et al., 2016). According to their structures and specific binding motifs, lectins can be classified into C-type lectins, galectins, F-type lectins, pufflectins, X-type lectins/intelectins, pentraxins, L-type lectins and rhamnose-binding lectins (Zelensky and Gready, 2005; Ogawa et al., 2011; Vasta et al., 2011).

The rhamnose-binding lectins (RBLs) are Ca2+-independent lectins, which show the specific binding activities to rhamnose and galactose (Ogawa et al., 2011). Most RBLs are composed of one or multiple characteristic carbohydrate-recognition domains (CRDs), which can recognize and bind to various microbes, including bacteria, fungi, viruses, and parasites, triggering related immune responses, and then play an important role in host defense (Ogawa et al., 2011; Vasta et al., 2011). Generally, RBLs are predominantly existed in oocytes and hepatocytes, and widely distributed in the immune-related tissues including spleen, skin, intestine, and kidney (Vasta et al., 2011; Thongda et al., 2014). RBLs were first isolated and identified from sea urchins (Anthocidaris crassispina) (Ozeki et al., 1991). In recent years, RBLs has also been mainly found and reported in teleost fishes and invertebrates, such as steelhead trout (Oncorhynchus mykiss), chum salmon (Oncorhynchus keta), ponyfish (Leiognathus nuchalis), ayu (Plecoglossus altivelis), channel catfish (Ictalurus punctatus), sea bass (Dicentrarchus labrax), turbot (Scophthalmus maximus), and ascidian (Botryllus schlosseri) (Tateno et al., 2001; Shiina et al., 2002; Okamoto et al., 2005; Gasparini et al., 2008; Watanabe et al., 2008; Cammarata et al., 2014; Thongda et al., 2014; Gao et al., 2018). These studies mainly focused on the identification of RBLs, tissue distribution and the expression of RBLs in immune tissues (such as intestine, skin and gills) after pathogenic infection, and suggested that RBLs play an important role in the innate immune response of teleost fish. Moreover, the related immune mechanism still need to be further studied.

Aquaculture has been well accepted as the present and future most important supply of aquatic protein source for human consumption, and is more integrated into the global food system, playing an important role in global food security (Kayansamruaj et al., 2020; Naylor et al., 2021). Nile tilapia (Oreochromis niloticus), major economically cultured fish in the world, suffers from widespread disease outbreaks due to various pathogens, including Streptococcus agalactiae and Aeromonas hydrophila, resulted in tremendous economic losses to the aquaculture industry (Yardimci and Aydin, 2011; Wang et al., 2017; Kayansamruaj et al., 2020). Therefore, it is urgent to understand the immune defense mechanisms of tilapia to prevent the outbreak of disease. Many efforts have been made in the identification of immune-related molecules and their related activities in regulating the innate immune response during infection. In Nile tilapia, four RBL genes were identified by Auburn University, and analyzed the changes of RBLs expression in mucosal tissues after pathogen infection (Zhang et al., 2018). However, the study of RBLs functional characterization in the host defense against pathogen infection was limited, especially in non-specific cellular defense.

Here we reported the functional characterization of an l-rhamnose-binding lectin-like from O. niloticus, provisionally named to as OnRBL-L. The OnRBL-L was successfully cloned and identified from the O. niloticus. The expression level of OnRBL-L was investigated in immune-related tissues and monocytes/macrophages (MO/MФ) upon bacterial infection (S. agalactiae and A. hydrophila). Further, the functional characterization of recombinant OnRBL-L protein was performed using bacterial binding and agglutination assays. Moreover, the effects of (r)OnRBL-L on non-specific cell immunity including inflammation, phagocytosis and respiratory burst were demonstrated. These findings indicate that OnRBL-L possesses the ability of bacterial binding and agglutination, which is helpful to understand the potential regulation role of RBL in non-specific cell immunity in an early vertebrate.

Section snippets

Animals, bacterial challenge and samples preparation

O. niloticus were procured from Guangdong tilapia breeding farm (Guangzhou, China), about 80 ± 10 g. Before the experiment, fish were fed into the culture system at 28 ± 2 °C for 2–3 weeks, as in previous descriptions (Mu et al., 2017; Yin et al., 2018). All animal protocols were reviewed and approved by the University Animal Care and Use Committee of the South China Normal University (an approval reference number SCNU-SLS-2021-012).

Ten tissues including liver, head kidney, hind kidney, muscle,

Sequence analysis of OnRBL-L

An l-rhamnose-binding lectin-like gene was cloned from O. niloticus and provisionally named OnRBL-L. The ORF of OnRBL-L was 678 bp and encoded 225 aa. The predicted molecular mass was 24.69 kDa, with a theoretical isoelectric point of 6.30. Multiple sequence alignment result indicated that OnRBL-L was homologous to other known and predicted RBLs (Fig. 1A). In addition, the OnRBL-L was 93.33%, 71.24%, 64.91%, 54.82%, 36.56% and 32.20% identical to Haplochromis burtoni, large yellow croaker (

Discussion

As an important component of innate immune system, lectins play an important role in the first line of host defense (Ogawa et al., 2011; Vasta et al., 2011). Lectins have genomic and structural diversity in vertebrates and invertebrates, which greatly expand the range of carbohydrate ligands and further enhance their diversity in function exertion (Vasta et al., 2011). In this study, we identified and functionally characterized a RBL-like gene from O. niloticus, which indicated that OnRBL-L

Acknowledgements

This project was supported by National Natural Science Foundation of China (31902396, 31972818), Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation) (2019A1515012065), China Postdoctoral Science Foundation (2019M652942), and Open Foundation of Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals (PBEA2020YB02).

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