Tumor necrosis factor receptor-associated factors (TRAFs) are a family of cytoplasmic adapters that play important roles in innate and acquired immunity, promoting intracellular signal transduction via receptors, such as tumor necrosis factor receptor (TNFR), toll-like receptors-interleukin-1binding receptors (TLR/IL-1R), the Epstein–Barr virus protein LMP1, and the RIG-I–like receptor family, potentiating the recruitment of signal transduction proteins (Arch et al., 1998; Evans et al., 2018; Guo et al., 2020). Seven members of the TRAF family, TRAF1-7, have been identified and participated in a host of signaling pathways relative to immunity. TRAF2, a prototypical TRAF, combines with TNFR1, TNFR2, CD40, and LMP-1 to induce downstream signaling factors, participating in cell survival and apoptosis, immunity, and infection process (Han et al., 2003). TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 can be recruited by CD40, whose cytoplasmic tail has two TRAF-interacting motifs; TRAF2 and TRAF3 bind to PxQxT motif to positively regulate the downstream factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), while TRAF6 binds to another motif to activate NF-κB (Bishop, 2004; Hauer et al., 2005). In addition, TRAF4 modulated mitogen-activated protein kinase, c-Jun N-terminal kinase, Wnt/β-catenin, and NF-κB signaling pathways via interacting with other molecules in mammals subsequently exerting functions in immunity (Xu et al., 2002; Wang et al., 2014; Lalani et al., 2018).

Interacting with receptors to modulate pathways, TRAFs can function as a regulatory factor in physiological processes. TRAF1 can regulate pathways that can be mediated by TRAF2, and TRAF3 can increase TRAF6-mediated NF-κB transcriptional activity while inhibiting the TRAF2/5-mediated NF-κB signal pathway (Hauer et al., 2005; Xie et al., 2006; Foight and Keating, 2016). These findings have revealed that different members of the TRAFs’ family interactions with others can result in different effects. TRAF members can interact to form homo- and hetero-dimers with distinct regulatory functions involved in signal transduction (Arron et al., 2002). In the CD40 signal pathway, TRAF1, TRAF2, TRAF3, and TRAF6 can form homo-dimers and bind CD40 receptors. TRAF3 and TRAF5 can form hetero-oligomers (Arron et al., 2002; Xie et al., 2006). These findings indicate that TRAFs may regulate immune-related pathways through their interactions. However, at present, the specific mechanism of the mutual regulation and binding of TRAFs is still unclear.

In recent years, most TRAFs from bivalves in marine mollusks have been identified and studied. Four Chlamys nobilis TRAF genes were identified, and their messenger RNA (mRNA) expression levels in hemolymph were significantly increased by exposure to Vibrio parahaemolyticus compared to the control group, which indicates that TRAFs are involved in host immunity (Zhang et al., 2021). HcTRAF6 expression level was significantly up-regulated in gills and hemocytes in response to immune stimulation (Aeromonas hydrophila and lipopolysaccharide (LPS) stimulation) in Hyriopsis cumingii (Huang et al., 2018). In the oyster, TRAF2 and TRAF6 from Crassostrea gigas and TRAF6 from Mizuhopecten yessoensis were induced to express upon bacterial infection (He et al., 2013; Huang et al., 2016; Mao et al., 2017). Although TRAFs can have an impact in response to immune-related stimulation, their functions and molecular mechanisms in different bivalves still need to be explored.

The oyster Pinctada fucata martensii, a type of seawater pearl oyster, relies primarily on innate immunity to defend against invading pathogens (Zhang et al., 2022). In previous studies, the TRAF3, TRAF6, and TRAF7 genes from the pearl oyster were identified (Huang et al., 2012; Jiao et al., 2014; Lei et al., 2016); Huang et al. and Jiao et al. have observed that TRAF3 and TRAF6 are involved in the innate immune response to V. alginolyticus and LPS stimulation, respectively. However, less is well understood about two P. f. martensii TRAFs, TRAF2 (PmTRAF2) and TRAF4 (PmTRAF4), and the role of these genes in the immune response remains obscure. In this study, PmTRAF2 and PmTRAF4 were identified in P. f. martensii hemocyte transcriptome, and their expression levels were analyzed in different tissues. The expression levels of PmTRAF2, PmTRAF3, PmTRAF4, and PmTRAF6 were analyzed in the hepatopancreas in response to LPS and polyinosinic acid (PolyI:C) stimulation, and nucleus insertion surgery. In addition, we used yeast two-hybrid to verify the interacting proteins. Our study offers new insights to further study the function of TRAF and the mechanism of shellfish immunity.

Worsening coastal water quality has brought various diseases to P. f. martensii, which lacks a specific immune system. Thus, studying the role of immune-related genes in pathogen response may improve the survival of P. f. martensii (He et al., 2019b). TRAFs are major signal transducers of the TNF receptor superfamily and other receptors, which can regulate various cellular activities and innate immune responses, including cell survival, proliferation, and death (Jiao et al., 2014). In the present study, we cloned two novel TRAFs, PmTRAF2, and PmTRAF4, in P. f. martensii. The protein domain of PmTRAF2 and orthologs of other species contain the typical RING finger domain, TRAF-type zinc finger domain, TRAF_BIRC3_bd domain, and MATH domain (TRAF-C) ( Figure 3B ). The MATH domain is crucial for homo- and hetero-oligomerization of TRAFs and the interaction of upstream and downstream factors, while the RING finger domain is involved in the activation of downstream signaling pathways (Zapata et al., 2007; Ha et al., 2009), which helps to investigate the immune-related signaling pathways involved in PmTRAF2. The PmTRAF4 TRAF-type zinc finger domains and MATH domain and their numbers are similar to those of other mollusk and vertebrate TRAF4. However, PmTRAF4 lacks the RING finger domain, and its deletion may affect the transmission of downstream signaling proteins. The results of the phylogenetic analysis revealed that TRAF2 and TRAF4 were highly conserved across all species, and PmTRAF2 and PmTRAF4 were closer to bivalves’ homologs, which suggests that PmTRAF2 and PmTRAF4 may have similar functions to other homologs.

TRAFs genes were detected and expressed at high levels in various immune-related tissues (gill, hemocytes, and hepatopancreas) in mollusks, suggesting their involvement in the host immune response. For instance, the mRNA of Patinopecten yessoensis’s intact TRAFs (TRAF2, TRAF3, TRAF4, TRAF6, and TRAF7) were mainly abundant in gills and hemocytes (Wang et al., 2015). In C. nobilis, high expression levels of CnTRAF2, CnTRAF3, and CnTRAF4 were observed in hemocytes, whereas CnTRAF6 was highly expressed in gills (Zhang et al., 2021). C. gigas TRAF3-S and TRAF3-L transcripts were up-regulated in hemolymph and muscle relative to the gill of the internal reference sample (Huang et al., 2014). Consistent with the above results, the mRNA transcripts of PmTRAF2 and PmTRAF4 were highly expressed in hepatopancreas and gills, indicating that they operated in immune defense. TRAF2 is one of the major recruitment targets for TRADD and TNFR5 proteins, and all three genes from P. f. martensii share high expression in gills and hepatopancreas (Hsu et al., 1996; He et al., 2019a; Wu et al., 2020). It can be speculated that PmTRADD and PmTNFR5 can recruit PmTRAF2 and play a role in apoptosis and other immune responses.

To investigate the function of TRAF family (PmTRAF2, PmTRAF3, PmTRAF4 and PmTRAF6) in P.f. martensii’s immune response, their expression levels were examined in the hepatopancreas after three immune stimulation, including nuclear insertion surgery, LPS challenge, and PolyI:C challenge. Several reports have indicated that the TNF–TNFR system (TNFR, TRAF, and NF-κB family members) responded to the nuclear insertion surgery. These genes may be involved in allograft immunity in pearl oysters (Wu et al., 2020). Here, in hepatopancreas tissues, PmTRAF3 and PmTRAF4 transcripts with significant up-regulation at different times after nuclear insertion surgery, and PmTRAF2 and PmTRAF6 with down-regulation at all times, indicating that four TRAFs might participate in the immune defense at different time. The expression pattern of PmTRAF2 was similar to PmTRAF6, which suggests that they may have the same regulation in response to nuclear insertion surgery. PmTRAF3 and PmTRAF4 transcripts were rapidly increased at 6 h, which illustrates that PmTRAF3 and PmTRAF4 may quickly involve in immune response after nuclear insertion surgery. In addition, four TRAFs mRNA in hepatopancreas were decreased at a short time (24 h and 48 h). However, in a longer period of time (5 d to 30 d), PmTRAF2 transcripts in hemocytes were significantly upregulated on 5 d, 15 d, and 30 d after nuclear insertion surgery, while PmTRAF3 was significantly upregulated on 15 d (Wu et al., 2020). The difference between the two results indicates that the downregulation of PmTRAFs mRNA in the hepatopancreas may be caused by time or that different tissues have different immune responses, which requires more experiments to verify.

Pathogen-associated molecular patterns (PAMPs) are highly conserved molecular structures that are common in pathogenic microorganisms and serve as ligand receptors for pattern recognition receptor (PRR) binding, and LPS and PolyI:C were typical PRR moleculars (Shen et al., 2022). PmTRAF4 transcript was downregulated within 96 h exposure to PolyI:C. Contrary to this result, the transcript level of Larimichthys crocea TRAF4 in gill was markedly upregulated at 6 h post PolyI:C (Chen et al., 2022). In Patinopecten yessoensis, TRAF4 was upregulated in the hemocytes post-bacterial infection (Micrococcus luteus and Vibrio anguillarum) (Wang et al., 2015). The above different phenomena suggest that PmTRAF4 may have different expression patterns in innate defenses in response to pathogens. Furthermore, TRAF4 can inhibit innate immune signaling pathways, such as TRAF4 inhibits nucleotide-binding oligomerization domain 2-induced NF-κB activation (Marinis et al., 2011); TRAF4 inhibits the toll-like receptors signaling pathway by associating with p47phox and interacting with TRAF6 and toll-IL-1 receptor domain-containing adaptor-inducing IFN-β (Takeshita et al., 2005). Whether TRAF4 can inhibit the immune response to pathogens is worth exploring. PmTRAF2, PmTRAF3, and PmTRAF6 were significantly induced to express at multiple time points within 96 h under LPS and PolyI:C, which suggests that their involvement in the innate immune system may be highly sensitive to the invasion of pathogenic microorganisms. Post LPS and PolyI:C infection, the expression levels of PmTRAF3 and PmTRAF6 increased sharply at 12 h, whereas PmTRAF2 at 24 h, which may indicate that PmTRAF3 and PmTRAF6 were more sensitive to LPS and PolyI:C model pathogen invasion than PmTRAF2. However, PmTRAF2 was responsive to LPS challenge in the gill tissue of P. f. martensii, with expression significantly up-regulated at 6 h and 12 h relative to the control group, which may be caused by the ability of gills to recognize and defend against LPS model pathogens rapidly (Wu et al., 2020; Zhang et al., 2022). Our results speculate that PmTRAFs (PmTRAF2, PmTRAF3, PmTRAF4 and PmTRAF6) may be the important molecule in the host’s immune defense.

Based on our studies, PmTRAF2, PmTRAF3, and PmTRAF6 may exert a similar action at the transcript level. Therefore, these may have a coordinative effect on signaling pathways. TRAF2, TRAF3, or TRAF6 have been found to mediate CD40-induced activation of NF-κB (Lu et al., 2003). Thus studying the relationship between them is important. Furthermore, in a previous report, TRAFs members can participate in signal transduction by interacting to form homo- and hetero-dimers with distinct regulatory functions (Arron et al., 2002). The TRAF-C structural domain of TRAF3 interacts with the TRAF-N structural domain and zinc fingers 4 and 5 of TRAF2 to form multimers, which inhibits the capability of TRAF2 to induce the NF-κB activation (He et al., 2004). Using RNA interference and immunoblotting, the researchers found for the first time in nonhemopoietic cells that TRAF6 can regulate CD40 signaling by associating with TRAF2 (Davies et al., 2005). Therefore, yeast two-hybrid assays were performed to understand the relationship between TRAF2 and TRAF3, and TRAF6 in the present study, which is crucial for subsequent exploration of the downstream signaling. The yeast two-hybrid assays have revealed that PmTRAF2 interacts with PmTRAF6 while not interacting with PmTRAF3. The TRAF domain is crucial for homo- and hetero-oligomerization of TRAFs. However, TRAF6 forms a heterodimer by binding to the RING domain of TRAF2 via its RING domain (Das et al., 2021). How PmTRAF2 and PmTRAF6 form a heterodimer and their molecular mechanism for downstream signaling still need to be explored.

The novel PmTRAF2 and PmTRAF4 were identified from P. f. martensii and had highly conserved regions of amino acid sequences. PmTRAF2 and PmTRAF4 with abundant expression levels in hepatopancreas and gill. Transcripts of PmTRAF2, PmTRAF3, PmTRAF6, and PmTRAF4 were markedly changed post- LPS injection, PolyI:C injection, and nuclear insertion surgery, indicating that these genes may play a role in P. f. martensii innate immunity. PmTRAF2 interacts with PmTRAF6 instead of PmTRAF3, potentially affecting downstream immune signaling pathways, such as NF-κB signaling pathways. The above results provide new perspectives for further investigation of TRAF’s immune mechanisms in bivalves.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

MZ: Investigation and designed the study, Analyzed all data, Writing - Original draft preparation; CS: Analyzed all sequencing data; HL: Conceptualization, Methodology, Writing- Reviewing and Editing; YW: Investigation, Analyzed all sequencing data; BL: Prepared the samples, Conclusion. All authors contributed to the article and approved the submitted version.