IGFBP2 And IGFBP5 Of Golden Pompano (Trachinotus Ovatus) Are Involved In Antimicrobial Immune Response

Trachinotus ovatus,an important economic fish in the southern coastal area of China,is becoming a pillar culture species in deep-water net cages,with rapid development and a promising future.However,in recent years,frequent strikes of infectious diseases have severely hampered their healthy development.Studies have shown that insulin-like growth factor binding proteins（IGFBPs）play an important biological role in the regulation of apoptosis,growth,cell differentiation,immune cell functions,and inflammation.However,present studies mostly emphasize the importance of IGFBPs in higher vertebrates such as mammals,and IGFBPs in teleosts are still poorly studied.Based on the transcriptome of T.ovatus,we cloned and identified four of T.ovatus IGFBPs homologous genes:TroIGFBP2a,TroIGFBP2b,TroIGFBP5a,and TroIGFBP5b.Then,we analyzed their potential roles in resistance to bacterial infection.We further discussed the antibacterial immune mechanism of TroIGFBP5b from various aspects.The main results are as follows.（Ⅰ）Cloning and expression analysis of TroIGFBP2a/2b and TroIGFBP5a/5bThe open reading frame（ORF）lengths of TroIGFBP2a/2b are 861 bp and 810 bp,encoding 286 and 269 amino acids,respectively.The ORF lengths of TroIGFBP5a/5b are 810 bp and 801 bp,encoding 269 and 266 amino acids,respectively.Sequence analysis showed that TroIGFBP2a/2b and TroIGFBP5a/5b are composed of three segments:a signal peptide（SP）,an insulin-like growth factor-binding domain（IBD）and a thyroglobulin type I repeats domain（TYD）,each of which is highly conserved during vertebrate evolution.The phylogenetic tree indicated that TroIGFBP2a/2b and TroIGFBP5a/5b cluster with IGFBP2a/2b and IGFBP5a/5b respectively,in teleosts.Homology analysis of the amino acid sequences showed that TroIGFBP5b is the most conserved among the four IGFBP genes studied,and its homology with other vertebrates（mammals,reptiles,amphibians,and birds）ranges from 51.66%to 58.74%,while with other teleosts,it shows up to 70.22%to 97.38%homology.In addition,the results of fluorescence quantitative PCR（q RT-PCR）showed that TroIGFBP2a/2b and TroIGFBP5a/5b could be expressed in various tissues of healthy T.ovatus,but with a discrepancy in distribution.TroIGFBP2a/2b and TroIGFBP5b had the highest expression in the liver,but TroIGFBP5a was expressed in the intestine.After being stimulated by Vibrio harveyi,the m RNA expression of TroIGFBP2a/2b and TroIGFBP5a/5b increased substantially in the liver,spleen,head kidney,and intestine,taking on a first upwards and then downwards trend,while the expression level of TroIGFBP5b was the highest.Based on these findings,we hypothesized that TroIGFBP2a/2b and TroIGFBP5a/5b may participate in the body’s anti-bacterial immune response,with TroIGFBP5b exhibiting the strongest response.（Ⅱ）In vivo and in vitro antibacterial activity of TroIGFBP5bBased on the aforementioned results,the anti-bacterial immunity of TroIGFBP5b was further studied.A prokaryotic expression vector was constructed to obtain the active recombinant protein r TroIGFBP5b.In vitro experiments indicated that r TroIGFBP5b contributed to advancing HKLs proliferation and boosting HKMs phagocytosis.Subcellular localization showed that TroIGFBP5b was present in both nucleus and cytoplasm,but only mature TroIGFBP5b（without signal peptide）could enter the nucleus.Overexpression and RNAi experiments with TroIGFBP5b revealed that overexpression increases the capacity to eliminate bacteria in vivo,while RNAi does the opposite.All these results suggest that TroIGFBP5b can significantly activate immunocytes and engage in the process of antibacterial immunity in T.ovatus.（Ⅲ）Key motifs in the antibacterial immune process of TroIGFBP5bTo explore the mechanism of the antibacterial function of TroIGFBP5b,we cloned three mutants of TroIGFBP5b:⁶⁵GVY⁶⁷-deletant,²³⁵CWCV²³⁸-deletant,and HBM(Heparin-binding motif,²²⁰KRKQCK²²⁵)-deletant,namely,TroIGFBP5b-ΔGVY,TroIGFBP5b-ΔCWCV,and TroIGFBP5-ΔHBM,respectively.In vivo experiments showed that compared with the TroIGFBP5b overexpression group,the antibacterial performance of the TroIGFBP5-ΔHBM overexpression group is severely weakened,and the levels of immune-related cytokines（IL-8,IL-10,IL-1β,and TNF-α）were also severely decreased.However,there was no palpable undulation in the expression level of the TroIGFBP5b-ΔGVY overexpression group and the TroIGFBP5b-ΔCWCV overexpression group,suggesting that HBM is essential for the antibacterial immunity function of TroIGFBP5b.In vitro,we conducted functional experiments and determined that r TroIGFBP5b-ΔHBM is incapable of boosting HKLs proliferation and HKMs phagocytosis.Subcellular localization studies confirmed that TroIGFBP5b and these three deletion mutants were all localized in the cytoplasm of golden pompano snout（GPS）cells.After stimulation by V.harveyi or LPS,some of the green fluorescence in the TroIGFBP5b-transfected group was transferred from the cytoplasm to the nucleus,while such transfer did not occur in the TroIGFBP5b-△HBM-transfected group.Based on the above results,we speculate that the antibacterial effect of TroIGFBP5b may be required to enter the nucleus to exert its antibacterial effects,and the absence of HBM prevents its entry from cytoplasm into the nucleus.Moreover,the absence of HBM deprived TroIGFBP5b of its function to activate immune cells.Therefore,HBM is the key motif in the antibacterial and immune processes of TroIGFBP5b.（Ⅳ）The HBM motif of the TroIGFBP5b gene is involved in the activation of the NF-κB signalling pathwayTo determine whether the antibacterial function of TroIGFBP5b involves the NF-κB signalling pathway,and to fully understand the role that HBM plays in this process,a dual luciferase reporter system was conducted.The results showed that TroIGFBP5b activates NF-κB in a dose-dependent manner.However,the HBM deletion mutant cannot activate the NF-κB pathway.On this basis,immunofluorescence experiments further verified that overexpression of TroIGFBP5b can promote the translocation of the p65 protein into the nucleus.However,overexpression of TroIGFBP5b-ΔHBM cannot promote this transfer.Moreover,overexpression of TroIGFBP5b in vivo upregulated the expression of NF-κB pathway-related genes（p65 and i KB-α）in the liver,spleen,and head kidney.However,overexpressed TroIGFBP5b-ΔHBM cannot upregulate these genes in vivo.All these results suggest that TroIGFBP5b participates in the NF-κB immune signalling pathway in T.ovatus,in which HBM features prominently.（Ⅴ）Antibacterial and immunomodulatory effects of a TroIGFBP5b-derived peptideTo investigate the mechanism by which TroIGFBP5b participates in antibacterial immunity,we synthesized the derived peptide TroIGFBP5b-22（SLYLPNCDRKGFFKRKQCKPSR）from the cationic core region of TroIGFBP5b containing HBM by chemical methods.Software analysis showed that TroIGFBP5b-22is an amphiphilic cationic polypeptide with a small molecular weight.In vitro,antibacterial tests showed that TroIGFBP5b-22 had obvious antibacterial activity against Staphylococcus aureus and V.alginolyticus.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）against S.aureus and V.alginolyticus were 32μM and 64μM,respectively,and inhibition of both occurred in a dose-dependent manner.Further studies have discovered that TroIGFBP5b-22 can significantly inhibit the proliferation of murine cancer cells（Hepa1-6）and promote the growth of GPS cells.As mentioned,it is suggested that TroIGFBP5b-22,a peptide derivative of TroIGFBP5b,has certain antibacterial activity as well as immunomodulatory effects,which provides basis for deeper research into the role of TroIGFBP5b’s in the antibacterial immunity of T.ovatus.In conclusion,we have confirmed the participation of TroIGFBP2a/2b and TroIGFBP5a/5b in the antibacterial immunity of T.ovatus.The mechanisms behind the functions of TroIGFBP5b are also explored.These studies not only enrich our knowledge about the antibacterial immunity function of IGFBPs in teleosts but also have great significance for future explorations of innate immune mechanisms.