|Other Abstract||Shellfish such as Ruditapes philippinarum usually live in a water environment rich in microorganisms and are often attacked by a variety of pathogens. Most of the previous studies on shellfish immunology focused on humoral immunity based on antimicrobial peptides and cellular immunity based on phagocytosis. As an important part of cellular immunity, extracellular traps (ETs) are a kind of cellular immune defense response discovered in recent years to resist the invasion of pathogens. In the process of resisting pathogen invasion, ETs take intracellular nucleic acid as the skeleton, and carry immune polypeptides such as elastase, cathepsin, histone and defensins to form a fibrous network structure, which can encapsulate, restrict and kill pathogens. ETs can inhibit the invasion of pathogens by concentrating diverse active polypeptides to increase the concentration of local antimicrobial peptides. At present, few studies about ETs have been identified in marine shellfish, and there is still a lack of systematic and in-depth studies on the production characteristics and immune function of ETs. In this study, we selected the hemocytes of R. philippinarum as the research object, explored the characteristics and immune activity of ETs, analyzed the antibacterial components and antibacterial mechanisms of ETs, and constructed the microweb of ETs in vitro. The results can provide a theoretical basis for in-depth understanding the innate immunity of bivalve mollusks, as well as a theoretical reference for the development of marine drugs. The results of the study are as follows:
(1) Identification and function of ETs from R. philippinarum
The hemocytes of R. philippinarum were induced by zymosan, which is a typical inducer of ETs, and the morphological characteristics of ETs in hemocytes of R. philippinarum was observed. The changes of mitochondrial parameters and intracellular reactive oxygen species (ROS) were detected to analyze the characteristics of ETs induced by zymosan in hemocytes of R. philippinarum. The bacteriostatic function of ETs was evaluated by the expression of antimicrobial related genes and the inhibitory effect on pathogens. The results showed that zymosan could induce the formation of ETs, and the optimal concentration and induction time were 0.5 μg/mL and 30 minutes. Under the induction condition, the production of intracellular ROS and myeloperoxidase increased and the expression of ROS-related genes (PI3K, AKT and HIF) was up-regulated. As the main source of ROS, mitochondria migrated to the cell surface. In response to the ROS burst, the mitochondrial membrane potential decreased, and the mitochondrial permeability transition pore opened. Mitochondrial ROS inhibitor (Mito-TEMPO) significantly inhibited the formation of ETs, suggesting that mitochondrial ROS were necessary for the formation of ETs. NADPH oxidase is a major regulatory protein of ROS. Inhibition of NADPH oxidase (DPI) also inhibited ET formation, demonstrating a positive correlation between the formation of ETs and the activity of NADPH oxidase. In addition, we found that zymosan-induced ETs exhibited antibacterial activities against gram-negative bacteria (Vibrio anguillarum, Vibrio harveyi and Escherichia coli) and gram-positive bacteria (Micrococcus luteus), suggesting that ETs may be an antimicrobial mode in R. philippinarum and play an important role in immune defense responses.
(2) Response of ETs to pathogen invasion in R. philippinarum
Marine Vibrio is the main pathogen of bivalve shellfish such as R. philippinarum. We selected V. anguillarum to study the role of ETs in cellular immune response. ETs were involved in the immune response to V. anguillarum invasion. During the process, the production of ROS and myeloperoxidase increased, and the formation of ETs was inhibited by the addition of NADPH oxidase inhibitors (DPI) and myeloperoxidase inhibitors (ABAH), confirming that ROS and myeloperoxidase were involved in ETs.In terms of energy, the expression of glycolysis related genes and the activities of glycolysis related enzymes (pyruvate kinase and hexokinase) were significantly enhanced during the process of ETs, suggesting that glycolysis was involved in the V. anguillarum-induced ETs and may be one of the energy sources of ETs. In terms of bactericidal activity, ETs could entrap and kill the invading V. anguillarum and had a significant killing effect on V. anguillarum.
To explore the main components of V. anguillarum that induced ETs, we selected four bacterial cell wall components (lipopolysaccharide, lipid A, teichoic acid and acetylmuramic acid) to induce ETs, and the results showed that lipopolysaccharide induced the most significant effect. The ET formation induced by lipopolysaccharide was accompanied by increased production of intracellular ROS. However, NADPH oxidase inhibitors (DPI) cannot inhibit the formation of ETs, demonstrating that
lipopolysaccharide induced ETs was independent of NADPH oxidase. In this process, the nuclear membrane bulges, tended to separate from chromatin. In addition, the mitochondrial membrane potential decreased, the mitochondrial permeability transition pore opened, and the increased mitochondrial ROS released into the extracellular. Mitochondrial ROS inhibitor (Mito-TEMPO) also significantly inhibited the formation
of ETs, suggesting that mitochondrial ROS were required in the formation of ETs induced by lipopolysaccharide. These results suggested that ETs induced by V. anguillarum may be mainly caused by cell walls such as lipopolysaccharide. Lipopolysaccharide induced-ETs were independent of NADPH oxidase, which was different from the mechanism of zymosan-induced ETs, suggesting that there were multiple induction mechanisms for ETs in R. philippinarum.
(3) Discovery and function of immune polypeptides from ETs in R. philippinarum
The effect of ETs is often accompanied by the production and action of antimicrobial peptides. In the early stage, the research group had made some progress in antimicrobial peptides from R. philippinarum. On this basis, the types, functions and bactericidal mechanisms of antimicrobial peptides involved in ETs were explored. Defensin (Rpdef1α), Macins (RpMacin-1 and RpMacin-2) and ubiquitin (RpUbi) were
involved in the immune response of hemocyte ETs induced by lipopolysaccharide, which preliminarily indicated that the antibacterial function of ETs may mainly come from the immunoactive peptides. Subsequently, the bacteriostatic experiments in vitro of recombined protein of defensin (rRpdef1α), Macin (rRpmacin-1 and rRpmacin-2) and ubiquitin (rRpUbi) showed that they had killing effect on both gram-negative and gram-positive bacteria. Among them, rRpdef1α not only exhibited broad-spectrum antimicrobial activity against Vibrio species, but also inhibited the formation of bacterial biofilms. In the study of bactericidal mechanism, rRpdef1α could increase the bacterial membrane permeabilization, leading to the overflow of bacterial contents and cause bacterial death. These results suggested that Rpdef1α was a potent antimicrobial agent, participating in the resistance of ETs to pathogen invasion. For Macin proteins, rRpMacin-1 and rRpMacin-2 killed E. coli within 400 and 1000 min at a minimum inhibitory concentration, respectively. It was worth noting that rRpMacin-2 had a stronger inhibition effect on the growth and biofilm formation of gram-negative bacteria such as marine Vibrio than that of rRpMacin-1. In addition, rRpMacin-1 and rRpMacin-2 had a strong damage effect on bacterial membrane. These results suggested that the stronger antimicrobial activity of rRpMacin-2 may be related to the stronger destruction effect of bacterial membrane. Moreover, the same type of antimicrobial peptides with different activities simultaneously participated in the process of ETs, which can synergistically improve the immune activity of ETs. For ubiquitin protein, rRpUbi still showed intense antibacterial activity, which was mainly through binding to bacterial DNA. These results indicated that antimicrobial peptides with different bactericidal modes participated in the formation of ETs, which can improve the bactericidal activity and efficiency of ETs, and maintain the health and stability of organisms.
(4) Construction and activity of ETs in vitro from R. philippinarum
The structure of ETs was isolated, and the antibacterial function of the separated ETs was evaluated. Furthermore, DNA and solid phase synthetic defensins were mixed to prepare the microweb structure of ETs to evaluate the feasibility of constructing ETs structure in vitro. The results showed that the structure of ETs isolated in vitro was still
intact, and it could capture Vibrio parahaemolyticus and destroy the bacterial membrane system. The microweb structure composed of equal mass ratio of DNA (negative charge) and solid phase synthetic defensins (positive charge) was positive charged and had similar structure to the ETs isolated in vitro. The bactericidal activity of the constructed ETs showed that the microweb structure had a strong killing effect on E. coli and V. anguillarum. These results indicated that ETs could be prepared in vitro, and the constructed microweb structure obtained had good antibacterial activity. In summary, this study identified the production of ETs in hemocytes of ETs in R. philippinarum, discussed the response of ETs to invasion of pathogenic microorganisms, preliminarily studied the antibacterial components and antibacterial mechanism of ETs, and constructed microweb structure similar to ETs in vitro. The results comprehensively revealed the role of ETs in cellular immunity of R. philippinarum, and provided a theoretical basis for the development of cell immunity of mollusks and the application of nucleic acid-polypeptide.|