Cellular immune responses such as phagocytosis and apoptosis
Cellular immune responses, such as phagocytosis and apoptosis, are very important processes to eradicate the invading pathogens and reduce concomitant host damage. And all of these processes are fine-tuned by the ubiquitin system (Li et al., 2016). Phagocytosis is a major mechanism of cellular immune reaction to recognize and ingest non-self molecules and cell debris. This immune process includes a series of physiological changes, such as phagocytic receptors recognition, signaling pathway activation, and phagosomal maturation (Rosales, 2011; Zhang et al., 2014; Song et al., 2015). Among them, phagosomal maturation is important for host immune responses, which can be regulated by the ubiquitin system during pathogen infection (Li et al., 2016). In the present study, when CgUbe2g1 was interfered by RNAi, the phagocytic rate of oyster hemocyte decreased dramatically after LPS stimulation, suggesting that CgUbe2g1 might involve in the signal transduction and phagosomal maturation during oyster haemocyte phagocytosis (Nakatogawa, 2013; Li et al., 2016). Apoptosis is a vital cell suicide modality for defensing parasites and pathogens in innate immunity (Riedl and Shi, 2004; Elmore, 2007; Estrada et al., 2014). In the present study, the apoptosis rate of hemocyte from CgUbe2g1 interfered oysters increased significantly after LPS stimulation, which was similar as the reports in human (Kim et al., 2005; Li et al., 2016). The activation of NF-κB and inflammatory responses in human were also reported to be inhibited after Ub-conjugating enzyme UbcH5 was interfered, and apoptosis was eventually triggered (Kim et al., 2005). Two major apoptotic pathways, the intrinsic and extrinsic apoptotic signaling pathways, have been reported in oyster (Kiss, 2010; Sokolova, 2009; Zhang et al., 2011). Although it was still not clear which apoptotic pathway was regulated by ubiquitin system, the results of in vivo RNAi experiments clearly demonstrated the involvement of CgUbe2g1 in the apoptosis pathway of C. gigas.
Because the ubiquitin system is indispensable for immune responses, it has become an important target for pathogens to escape the host immune defenses (Kim et al., 2005; Li et al., 2016). For example, Chlorogenic acid OspG of Shigella flexneri could bind many ubiquitinated E2 proteins, such as UbcH5 and Ubch7, to prevent the ubiquitination and inhibit NF-κB signaling pathway (Kim et al., 2005; Pruneda et al., 2014). OspI in S. flexneri could inactivate Ubc13 to inhibit TRAF6 polyubiquitination (Sanada et al., 2012). In the present study, the ROS level was found to be significantly decreased after CgUbe2g1 RNAi. ROSs are produced during normal metabolism of aerobic organisms (Aguirre et al., 2005), and they are highly induced when the organism is attacked by invaders or exposing to contaminant (Aguirre et al., 2005; Winterbourn, 2008). Oysters have evolved antioxidant defense system to rapidly and efficiently remove ROS to protect cells from damaging effect (Wang et al., 2018). The lower ROS level might affect the generation of antioxidant enzymes, which could be utilized by pathogens to escape the immune system (Severo et al., 2013; Li et al., 2016; Wang et al., 2018). This result demonstrated that E2s (such as CgUbe2g1) and the ubiquitin system were critical for immune responses and pathogens elimination in oysters.
In conclusion, an ubiquitin-conjugating enzyme E2, CgUbe2g1, was identified from Pacific oyster C. gigas. It was mainly expressed in hemocyte, mantle, hepatopancreas, and male gonad. The rCgUbe2g1 protein was found to induce the ubiquitination in vitro via binding E1-activated and un-activated Ub. CgUbe2g1 could play vital roles in innate immune system of C. gigas, including activation of inflammatory response and regulation of cellular immune response after pathogen stimulation.
Acknowledgments This research was supported by a grant (No. U1706204) from National Science Foundation of China, Aoshan Talent Cultivation Program (No. 2017ASTCP-OS13) supported by Qingdao National Laboratory for Marine Science and Technology, Dalian High Level Talent Innovation Support Program (2015R020), Earmarked Fund (CARS-49) from Modern Agro-industry Technology Research System, Outstanding Talents and Innovative Teams of Agricultural Scientific Research in the Ministry of Agriculture, and the Research Foundation for Distinguished Professor in Liaoning (to L. S.) and Talented Scholars in Dalian Ocean University (to L. W.).