Exosomes, secreted by most cells, are critical antimicrobial immune factors in animals. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to regulation of innate immune signaling. However, the relationship between exosomes and Rab GTPases, resulting in antimicrobial activity in vertebrates and invertebrates during pathogenic infection, has not been addressed. In this study, SpRab11a was reported to have a protective effect on the survival rate of mud crabs Scylla paramamosain after Vibrio parahaemolyticus challenge through the stimulation of exosome secretion and modulation of anti-LPS factor (ALF) expression. Furthermore, Sp14-3-3 was confirmed to be densely packaged in exosomes after V. parahaemolyticus infection, which could recruit the MyD88 and TLR by binding the Toll/IL-1R domain to the plasma membrane, promoting the translocation of Dorsal from the cytoplasm into the nucleus, and thereby regulating ALFs expression in the hemocytes of mud crab in response to the bacterial infection. The findings therefore provide, to our knowledge, a novel mechanism that underlies the cross-talk between SpRab11a-regulated exosome formation and ALFs expression in innate immune response in invertebrates, with a crustacean species, mud crab S. paramamosain, as a model study.
Rab GTPases constitute the largest family of small GTPases, functioning as molecular switches that alternate between the two conformational states of the GTP-bound “on” and “off” forms (1). Rab GTPases regulate vesicle trafficking in cells (2), and their effectors coordinate consecutive stages of transport, such as the formation and motility of vesicles and organelles, as well as the tethering of vesicles with target membranes (3). The evidence has demonstrated the essential role of Rab GTPases in innate immunity through modulating the transport of innate immune receptors and adaptors (4). Previous studies have shown the involvement of Rab proteins (including Rab GTPases) in crustacean’s innate immunity: Rab9 played a key role in antivirus protection through regulating autophagy in shrimp (5). Rab7 interacts with the envelope proteins of white spot syndrome virus to block multiple viral infection processes, thereby protecting the host against white spot syndrome virus in shrimp (6). In addition, Rab GTPases play different roles in the exosomal pathway and mediate exosome secretion (7, 8). However, the underlying mechanisms are entirely unclear.
Several types of membrane vesicles of endosomal (exosomes) and plasma (microvesicles) membrane origin are released by the cells into the extracellular environment (9). Exosomes are common membrane-bound and saucer-shaped vesicles (30–100 nm in diameter) that are delimited by a lipid bilayer and float at a density of 1.13–1.19 g/ml in sucrose gradients (10, 11). Exosomes can be formed in the endosomes, carrying a selection of components from the cell and displaying various functional properties (12). The importance of exosomes in intercellular communication via the transfer of proteins, lipids, and nucleic acids has been affirmed in numerous studies (13). Exosomes are packaged up by parent cells and sent over to their neighbors to transfer biological signals (14, 15). Exosomes have been implicated in controlling normal and pathogenic processes (16). During viral infection, host exosome pathways can be hijacked by viruses, and virally modified exosomes contribute to the virus spread and immune evasion (17–19). The infected cells can excrete exosomes containing functional biomolecules to stimulate the antiviral immune response in neighboring cells in mud crab Scylla paramamosain (20). Exosomes also display strong antibacterial effects, which suggest a crucial role of exosomes during pathogenic infection (21–23). However, very little is known about how exosomes regulate immune responses in crustaceans.
Most invertebrates mainly rely on innate immunity (24). The 14-3-3 protein can effectively induce innate immune responses (25, 26). Members of the evolutionary conserved 14-3-3 family of proteins have previously been implicated in the regulation of multiple signaling pathways for apoptosis, cell trafficking, regulation of cytoskeletal dynamics, and neuronal plasticity (27). In Drosophila, 14-3-3ε, one of the two homologs (14-3-3ε and 14-3-3ζ) of 14-3-3, has been known to be a regulator of antimicrobial peptide secretion and be important in the innate immunity and resistance to bacterial infection (28, 29). Recently, reports revealed that 14-3-3 proteins could be transported by exosome, and that exosomal 14-3-3 has potential functions for the stimulation of immune signaling pathways (30–32). However, the effects of 14-3-3 proteins in the immunity in invertebrates, especially in regulating immune-related signaling pathways through exosomes, have been rarely reported. In our previous studies, the exosomal microRNAs have functions in the antiviral process and maintain the homeostasis of hemolymph microbiota on V. parahaemolyticus infection in mud crab S. paramamosain (20, 21).
Hence in this study, the effect of Rab protein on the innate immunity of invertebrates through the stimulation of exosome secretion and modulation of anti-LPS factor (ALF) expression was investigated for the first time in mud crab. The results found that the enrichment of SpRab11a in the hemocytes of mud crab (S. paramamosain) is induced on bacterial stimulation, and the transport of Sp14-3-3 protein into infected hemocytes is carried out through the secretion of SpRab11a-regulated exosomes. Furthermore, exosomal Sp14-3-3 protein is found to be an important promoter of the expression of ALFs. These findings provided new insights into how SpRab11a tightly regulates the immune response in resistance to the pathogenic infection in invertebrates.
Materials and Methods
Animals used in this study did not include endangered or protected species. The animals were processed according to the “Regulations for the Administration of Affairs Concerning Experimental Animals” established by the Guangdong Provincial Department of Science and Technology on the Use and Care of Animals.
Bacterial challenge and sample collection
Healthy mud crabs (∼50 g each) were taken from a local crab farm (Niutianyang, Shantou, Guangdong, China) and acclimated to laboratory conditions (salinity: 8‰; temperature: 25°C) for a week before further processes. A volume of 200 μl of V. parahaemolyticus (1 × 106 CFUs/ml) or sterile PBS (200 μl) was injected into the base of the fourth leg of each crab in the experimental or control groups, respectively. Hemocytes were collected from at least three mud crabs from each group. For the hemocyte collection, the hemolymph was extracted with a syringe preloaded with 1 ml of anticoagulant buffer (0.45 M NaCl, 10 mM KCl, 10 mM EDTA, and 10 mM HEPES [pH 7.45]) and immediately centrifuged at 800 × g for 15 min at 4°C, and the hemocytes were suspended in PBS. The hemocytes were used for RNA or protein extraction. Total RNA was extracted using the TRIzol reagent (Cwbio, Beijing, China). Cytoplasmic and nuclear protein were separately extracted by NE-PER (Thermo Fisher Scientific).
RNA interference assay
Based on the sequence of SpRab24, SpRab32b, SpRab11a, and Sp14-3-3 (the sequence is available at GenBank [https://www.ncbi.nlm.nih.gov/genbank/], accession numbers OL321174, OL321173, OL321175, and JQ218935.1), the small interfering RNA (siRNA) specifically targeting gene was designed (Supplemental Table I). The siRNA was synthesized using the In vitro Transcription T7 Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Then 25 μg of SpRab24-siRNA, SpRab32b-siRNA, SpRab11a-siRNA, and Sp14-3-3-siRNA were injected into each mud crab, respectively. To enhance the RNA interference (RNAi) efficiency, we performed a second injection 24 h after the first injection with siGFP as the control. The hemocytes were collected from the mud crab at 24 h after the second injection, and total RNA was extracted. The expressions of these genes were detected by quantitative RT-PCR (RT-qPCR) using primers (RT-F and RT-R) (Supplemental Table I) to check the RNAi efficiency. All experiments were repeated three times.
Bacteria clearance and survival rate assays
After knockdown of SpRab24, SpRab32b, SpRab11a, or Sp14-3-3 by RNAi, 200 µl of exosome solution (1 × 108 vesicles/ml) or V. parahaemolyticus (1 × 106 CFUs/ml) was injected into the base of the fourth leg of each crab, respectively. Then the hemolymph was extracted at 48 h after the second V. parahaemolyticus injection. The hemolymph was diluted, spread on 2216E agar plates, and incubated overnight at 37°C, and the number of bacterial colonies was counted. The cumulative mortality of mud crabs was examined daily for up to 72 h. All experiments were performed in triplicate. The data were used for statistical analyses.
Isolation and analysis of exosomes
For exosome isolation, hemolymph of mud crabs was separated, after centrifuging at 800 × g for 15 min, to collect the supernatants. Next, supernatants were subjected to ultracentrifugation, followed by sucrose density-gradient centrifugation. The solution between 1.3 and 0.95 M sucrose solution was obtained and filtrated through 0.22-μm filters. Exosome preparations were verified by transmission electron microscopy. Exosomes were dissolved in PBS buffer, dropped into a carbon-coated copper grid, and then stained with 2% uranyl acetate. Images of the sample were captured using Philips CM120 BioTwin transmission electron microscope (FEI Company). Particle size and concentration were measured with a NanoSight LM20 (NanoSight, Malvern, U.K.). Concentrations were reported in particles per milliliter and adjusted to 1 ml of serum. Exosome markers TSG101/CD81 were used to reflect exosome concentration by Western blot or immunocytochemical staining.
The hemolymph was collected from three mud crabs and immediately centrifuged at 800 × g for 15 min to obtain the hemocytes. The hemocytes were washed with PBS and fixed by adding 4% paraformaldehyde. After incubation in 0.1% Triton X-100 (15 min), hemocytes were blocked with 5% BSA for 40 min. Hemocytes were later incubated overnight with primary Abs purchased from Abcam (Cambridge, U.K.) (1:100 dilution in 1% BSA). After washing with PBS, the hemocytes were then incubated with the second Ab, goat anti-rabbit Alexa Fluor 488 (1:1000 dilution in 1% BSA). The reaction was kept in the dark for 1 h and then washed with PBS. The hemocyte nucleus was stained with DAPI for 10 min at room temperature and washed again. At last, hemocytes were observed under a fluorescence microscope.
Purified GST-tagged Sp14-3-3 (200 μg) was incubated with the glutathione resin (1:1) and incubated at 4°C for 2 h with slight rotation. Then the natural proteins from mud crab with 1 mM PMSF were added and incubated at 4°C overnight. The mixture (resin and binding proteins) was washed three times by PBS to remove the unbound proteins. Elution buffer (10 mM reduced glutathione, 50 mM Tris–HCl [pH 8.0]) was added to wash out the bound proteins. SDS-PAGE was conducted to analyze the proteins. GST was used as a control. The proteins in the gel were stained with silver. Finally, the protein that specifically bound to Sp14-3-3 was identified by mass spectrometry.
Western blot analysis
Protein concentrations were quantified using a Pierce BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Haimen, China). The proteins were then separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Before incubation with antiserum against the proteins of interest (1:1000 dilution in blocking milk solution) at 4°C overnight, the membrane was blocked with 5% nonfat milk diluted in TBST buffer (150 mM NaCl, 3 mM EDTA, 0.1% Tween 20, 50 mM Tris–HCl [pH 8.0]) for 1 h at room temperature. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary Ab (Bio-Rad) for 2 h at room temperature and subsequently detected by ECL substrate (Thermo Scientific). The references and catalog numbers of Abs used in this paper are in Supplemental Fig. 3, and the specificity of the Abs developed in the context of this study against recombinant proteins was tested by Western blot in Supplemental Fig. 3.
Coimmunoprecipitation (Co-IP) analysis was conducted according to Dynabeads Co-Immunoprecipitation Kit (Thermo Scientific). A total of 50 μg anti–Sp14-3-3, anti-MyD88, anti-Pelle, or anti-IgG, all obtained from Abcam (Cambridge, U.K.), was used for Ab immobilization and incubated with 50 μg of total hemocyte extract.
Bacterial strains and sample collection
V. parahaemolyticus was isolated from mud crab (S. paramamosain) samples collected from a local crab farm (Niutianyang, Shantou, Guangdong, China). After detection and identification, V. parahaemolyticus was stored at −80°C in the cryogenic vials for further experiments.
Recombinant expression, purification, and antiserum production
PCR fragments representing SpRab11a and Sp14-3-3 were amplified using specific primers (Supplemental Table I). Subsequently, the PCR products were purified and digested with restriction enzymes (EcoRI and XhoI) for ligation of the final DNA fragments into the pGEX-6P-1 (+) vector (Madison, WI). Escherichia coli BL21 (DE3) cells (TransGen Biotech, Beijing, China) were transformed with the recombinant plasmid pGEX-6P-1 (+)-SpRab11a or pGEX-6P-1 (+)-Sp14-3-3 to express the recombinant SpRab11a or Sp14-3-3 protein. Fusion protein expression was induced under four different conditions: 1 mM IPTG for 3 h at 37°C, 0.25 mM IPTG for 3 h at 37°C, 1 mM IPTG for 3 h at 16°C, and 0.25 mM IPTG for 3 h at 16°C. The fusion protein was purified with GST resin (Merck KGaA, Darmstadt, Germany) in accordance with the manufacturer’s instructions. Subsequently, mouse antiserum against SpRab11a or Sp14-3-3 was prepared according to the following method: the purified recombinant protein SpRab11a or Sp14-3-3 was mixed with the complete adjuvant (1:1) and injected into the peritoneum (abdominal cavity) of healthy 6-wk-old mice (KM murine) (100 μg recombinant protein/mouse) weekly in a 4-wk-long experiment after shaken for 2 h. One week after the last injection, the mice were anesthetized with ether, and the serum Abs were obtained after standing overnight at 4°C. Finally, the specificity of serum Abs was assayed by Western blot.
All data were subjected to one-way ANOVA using Origin Pro8.0, with p < 0.05 considered as statistically significant. All experiments were carried out in triplicates and repeated for three biological replicates. All data were representative of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).
SpRab11a drives bacterial resistance
The Rab GTPase emerged as a critical regulator of the host defense pathway that can eliminate bacterial pathogens. In this study, SpRab24, SpRab32b, and SpRab11a were expressed in the hemocytes of both healthy and V. parahaemolyticus–injected mud crabs. The results showed that the expression of SpRab24, SpRab32b, and SpRab11a rapidly increased in the hemocytes of mud crab in response to V. parahaemolyticus infection at both mRNA and protein levels (Fig. 1A–E). The results indicated that SpRab24, SpRab32b, and SpRab11a may be important in mud crab S. paramamosain to defend V. parahaemolyticus infection.
Knockdown of the target genes was performed to investigate the function of SpRab24, SpRab32b, and SpRab11a in bacterial infection (Fig. 1F), and the number of bacteria present in the mud crab hemolymph was counted (Fig. 1G). The result showed that the total hemolymph bacteria number for the SpRab11a-knockdown mud crabs was significantly increased as compared with that for the controls. However, there was no significant difference in total hemolymph bacteria number for the SpRab24- or SpRab32b-knockdown mud crabs from the controls. The SpRab11a-knockdown mud crabs were then used for a challenge with V. parahaemolyticus, and the results found that the survival rate of SpRab11a-siRNA–injected mud crabs was significantly decreased when compared with the control (GFP-siRNA–injected) individuals (Fig. 1H).
Expressions of SpRab11a in mud crabs injected with graded concentrations of SpRab11a-siRNA were detected at the mRNA and protein levels (Fig. 1I, 1J). Total hemolymph bacteria number and the survival rate of SpRab11a-siRNA–injected mud crabs were determined. The results showed that the SpRab11a expression had a negative correlation with the number of total hemolymph bacteria and a positive correlation with the survival rate of mud crabs (Fig. 1K, 1L). Collectively, the results indicated that SpRab11a contributes to resistance to bacterial infection in mud crab.
SpRab11a is related to exosome secretion
Rab proteins are small GTPases that regulate the trafficking of membrane components during endocytosis and exocytosis, including the release of extracellular vesicles. To analyze whether the cellular mechanism of SpRab11a in bacterial clearance is related to exosomes, the subcellular localization of SpRab11a and colocalization between SpRab11a and the exosomal biomarker TSG101 were assessed in mud crab after the V. parahaemolyticus infection. Immunofluorescence microscopic observations revealed that more SpRab11a localizes to the cytoplasm in response to V. parahaemolyticus infection (Fig. 2A). Intriguingly, a striking colocalization was observed between SpRab11a and TSG101 (Fig. 2B). The hemocytes of V. parahaemolyticus–infected mud crab contained more TSG101-positive structures than those of V. parahaemolyticus mixed with SpRab11a-siRNA–injected individuals, indicating that the SpRab11a inhibition could decrease exosome formation (Fig. 2C).
To ascertain the role of SpRab11a in exosome biogenesis in mud crab, we isolated exosomes from the hemolymph of V. parahaemolyticus–injected mud crabs (namely, exosome-Vp) and PBS-injected control exosomes (exosome-PBS). Both exosome-PBS and exosome-Vp have a typical exosome morphology (Fig. 2D), with similar size (Fig. 2F), and are immune positive for exosome markers (CD81 and TSG101) (Fig. 2E). The results indicated that exosomes were successfully isolated from mud crabs challenged with either V. parahaemolyticus or PBS, and their natural characteristics are almost similar between the two groups. Furthermore, the results of Western blot and NanoSight particle tracking analysis revealed that the number of exosomes isolated from the hemolymph of V. parahaemolyticus–challenged mud crab was higher than that isolated from the controls (Fig. 2E, 2F). Knockdown of SpRab11a resulted in reduced exosome generation in the hemolymph of mud crabs (Fig. 2G, 2H). Collectively, these results indicated that the small GTPase SpRab11a contributed to the secretion of exosomes.
The bacterial clearance ability is related to the exosomes-regulated ALFs
Exosomes (exosome-Vp or exosome-PBS) and V. parahaemolyticus or SpRab11a-siRNA, respectively, were coinjected into mud crabs to monitor the total bacteria in vivo and the mortality rate, with healthy mud crabs (wild type) serving as the control groups. The results showed that exosome-Vp can inhibit the proliferation of bacteria in the hemolymph and enhance the mortality rate of mud crabs (Fig. 3A, 3B), which can compensate for the damage caused by SpRab11a RNAi. To confirm that exosome-Vp has critical roles in the antibacterial ability of mud crabs, we analyzed the number of bacteria present in the mud crab hemolymph and the survival rate of mud crab after injecting with V. parahaemolyticus along with exosomes at gradient concentrations. The results showed that the volume of exosome-Vp was negatively correlated with total hemolymph bacteria number (Fig. 3C), but positively correlated with the survival rate of mud crabs (Fig. 3D). The data suggested that the SpRab11a could promote hemocytes to produce exosomes to prevent pathogenic invasions.
Because exosomes can remove bacteria from hemolymph, the possibility that mud crab–derived exosomes could have direct bacteriostatic or bactericidal activity was examined. Exposure of bacterial cultures to intact exosome-Vp at increasing volume ranging from 200 to 1000 µl showed no differences in bacterial growth with control cultures (Fig. 3E). In light of these results, it should be investigated whether the antibacterial activity of exosomes could be derived from bioactive substances in exosomes that are taken up by neighboring or distant cells and subsequently modulate recipient cells. For this, the ability of the isolated exosomes to be internalized by mud crab hemocytes was analyzed by labeling the isolated exosomes with 3,3′-dioctadecyloxacarbocyanine perchlorate (green) before injection. When hemocytes from the injected crabs were collected and labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (red) before examination with a confocal laser scanning microscope, the results showed that the isolated exosomes could be internalized in hemocytes (Fig. 3F). It has been reported that Rab7 can regulate the expression of antimicrobial peptides via the Toll signaling pathway in the Chinese mitten crab (33). And in our previous study, exosomes were found to maintain the homeostasis of hemolymph microbiota on V. parahaemolyticus infection in mud crab S. paramamosain (21). Therefore, SpRab11a was suspected to regulate the expression of antimicrobial peptides through exosomes. Then the expression of six ALFs was first evaluated in mud crab hemocytes after V. parahaemolyticus challenge. The results showed that the expression of ALF-1 to ALF-5 was significantly upregulated (Fig. 3G). The expressions of ALF-1 to ALF-6 on V. parahaemolyticus challenge after injection with exosome-Vp, exosome-PBS, or exosome-Vp mixed with SpRab11a-siRNA were analyzed. The result showed that exosome-Vp can stimulate the regulation of ALF-1, ALF-4, and ALF-5 and can compensate for the downregulation of ALF-1, ALF-4, and ALF-5 caused by SpRab11a-siRNA (Fig. 3H). All the earlier results indicated that SpRab11a specifically upregulated the expression of three different ALFs (ALF-1, -4, and -5) by exosomes on pathogenic infection.
Exosome induces Dorsal translocation into the nucleus
The signaling pathways regulated by exosomes that allow the expressions of ALFs were investigated. In Drosophila, antimicrobial peptide expression is mainly regulated by the Toll and Immune Deficiency pathways (34). Furthermore, the cytokine-activated JAK/STAT is a key pathway for antiviral responses in both Drosophila and mammals (35). Therefore, the potential roles of exosomes in the activation of the Toll, Immune Deficiency, and JAK/STAT pathways were examined. The translocation of the transcription factors, including Relish, STAT, and Dorsal, from the cytoplasm to the nucleus on injection of V. parahaemolyticus mixed with either exosome-PBS or exosome-Vp was investigated, and V. parahaemolyticus challenge was set up as a positive control. Results of the immunocytochemical analysis showed that exosome-Vp can only increase the translocation of Dorsal (Fig. 4A, 4Ca), but not STAT (Fig. 4B, 4Cb) and Relish (Fig. 4C, 4Cc), from the cytoplasm into the nucleus. Western blot analysis using proteins extracted from cytoplasm and nucleus of hemocytes revealed similar results obtained from the immunocytochemical analysis (Fig. 4D, 4E).
SpRab11a-regulated exosomes promote the expression of ALFs through the Dorsal translocation
To further investigate the relationship between the exosome and Dorsal translocation, we excluded the influence of SpRab11a on the Dorsal translocation. The r-SpRab11a and SpRab11a-siRNA or GST and GFP-siRNA were coincubated with the hemocytes, respectively. The results showed that Dorsal was not translocated from the cytoplasm into the nucleus in the r-SpRab11a- and SpRab11a-siRNA–coincubated hemocytes as compared with GST and GFP-siRNA–coincubated counterparts (Fig. 5A, 5Aa, 5C, 5Cc). Western blot analysis showed similar results (Fig. 5B, 5Bb, 5D, 5Dd).
To determine the role of exosomes in the translocation of Dorsal from the cytoplasm into the nucleus of hemocytes, we used the exosome-PBS or exosome-Vp simultaneously mixed with SpRab11a-siRNA for injecting into mud crabs after the injection with V. parahaemolyticus. The results showed that exosome-Vp, but not exosome-PBS, could induce the translocation of Dorsal from the cytoplasm into the nucleus (Fig. 5E). Although the earlier results found that SpRab11a-siRNA could reduce Dorsal translocation in the hemocytes, the supplementation of exosomes can compensate for this reduction. The same results were obtained from Western blot analysis using proteins extracted from both cytoplasm and nucleus of hemocytes (Fig. 5F). Taken together, the results suggested that Dorsal was translocated from the cytoplasm into the nucleus by exosomes, which was not related to the SpRab11a.
Exosomal Sp14-3-3 modulates expressions of ALFs
From the earlier-obtained results, exosomes can be easily absorbed by hemocytes to enhance the immune responses, and the exosome-Vp can promote the translocation of Dorsal from the cytoplasm into the nucleus of hemocytes. It is hypothesized that exosomes produced by V. parahaemolyticus–infected mud crabs themselves contain immune-regulatory molecules. Therefore, the proteomic profiles of exosome-Vp and exosome-PBS were studied. Among the differentially expressed exosomal proteins, Sp14-3-3 was the most significantly expressed in the exosome-Vp group (Supplemental Fig. 1), and the protein was selected to investigate its potential roles in the immune response of mud crabs against V. parahaemolyticus infection.
The results of Western blot showed the significantly high expression of Sp14-3-3 in exosome-Vp when compared with the exosome-PBS (Fig. 6A). To ascertain whether exosome-Vp is involved in the regulation of ALFs via exosomal Sp14-3-3, we determined the relative expressions of Sp14-3-3 in the mud crabs injected with either exosome-Vp or exosome-PBS. The results revealed a significant upregulation of Sp14-3-3 in the exosome-Vp–injected mud crabs, compared with the exosome-PBS–injected individuals (Fig. 6B), indicating that exosome-Vp could lead to the Sp14-3-3 accumulation in the recipient cells. The immunocytochemical analysis also confirmed the findings of Western blot (Fig. 6C). To determine whether Sp14-3-3 protein is mediated to the nuclear translocation of Dorsal, we used siRNA to knock down the Sp14-3-3 expression at both mRNA and protein levels (Fig. 6D, 6E). Furthermore, immunostaining and Western blot were performed to analyze Dorsal translocation in Sp14-3-3-siRNA–injected mud crabs. The nuclear translocation of Dorsal in Vp-infected mud crabs was further enhanced by exosome-Vp, which also increased the Dorsal translocation in infected crabs that were treated with Sp14-3-3-siRNA to decrease endogenous Sp14-3-3 protein levels (Fig. 6F, 6G). Sp14-3-3 packaged in the exosome-Vp stimulated the expression of ALF-1, ALF-4, and ALF-5 (Fig. 6H).
Sp14-3-3 interacts with MyD88 and TLR to affect Toll signaling
Components of bacteria and viruses can activate TLRs in vertebrate’s cells, which trigger the formation of the Myddosome and a signaling network that culminates in the production of the inflammatory mediators required to combat the infection. To understand the regulatory mechanisms of Sp14-3-3 in the Toll pathway, we examined Myddosome. The Ab against MyD88 was coupled to CNBr-activated Sepharose 4B for binding to Pelle from Sp14-3-3-siRNA–, exosome-Vp–, and V. parahaemolyticus–challenged mud crabs. The interactions of MyD88 with Pelle were analyzed by Co-IP assays. The amount of Myddosome in the hemocytes of exosome-Vp and Sp14-3-3-siRNA coinjected mud crabs was significantly lower than that in the exosome-Vp–injected mud crabs (Fig. 7A). A similar trend was observed in the Co-IP assays that Pelle bound to MyD88 from Sp14-3-3-siRNA–, exosome-Vp–, and V. parahaemolyticus–challenged mud crabs (Fig. 7B). The results suggested that Sp14-3-3 could promote the formation of Myddosome in the Toll pathway.
Pulldown and mass spectrometry assays were performed to determine the interaction between Sp14-3-3 and proteins in the Toll pathway using recombinant proteins of Sp14-3-3. The results showed that Sp14-3-3 could interact with MyD88 and TLR in the Toll pathway (Fig. 7C, Supplemental Fig. 2). The mass spectrometry analysis was further confirmed by the immunoprecipitation experiment (Fig. 7D–7F). The structural domains of MyD88 and TLR were analyzed (Fig. 7G). The domains of MyD88 (GST-MyD88-TIR [Toll/IL-1R] and GST-MyD88-DEATH) and TLR (GST-TLR-TIR) were used for pulldown assays. The results showed that the TIR domain (of both MyD88 and TLR) was responsible for the interaction with Sp14-3-3 (Fig. 7H).
Sp14-3-3 facilitates MyD88 recruitment to TLR
To further explore the mechanism that Sp14-3-3 interacts with MyD88 and TLR to regulate the production of ALFs, we detected the expressions of MyD88 and TLR at both mRNA and protein levels. The results showed that exosome-Vp could significantly enhance the expression of MyD88 and TLR in the mud crab hemocytes in both normal conditions and knockdown of Sp14-3-3 or SpRab11a conditions (Fig. 8A, 8B). This indicated that exosomes participate in the TLR-MyD88-Dorsal pathway through Sp14-3-3. Then the localization of MyD88 in mud crab hemocytes was analyzed, and the results found that MyD88 was localized in the plasma membrane after challenge with V. parahaemolyticus. The membrane aggregation of MyD88 increased by exosome-Vp stimulation but decreased by Sp14-3-3 or SpRab11a knockdown (Fig. 8C).
The colocalization of MyD88 and TLR was detected to address the possible role of Sp14-3-3 in MyD88 recruitment. The results revealed that exosome-Vp stimulates the accumulation of MyD88-TLR near to the plasma membrane, and both Sp14-3-3-siRNA and SpRab11a-siRNA showed a negative effect of the exosome-Vp on MyD88 recruitment (Fig. 8D). When MyD88 Ab was used for the Co-IP assay, high levels of TLR were detected in the V. parahaemolyticus– or exosome-Vp–injected mud crabs as compared with the Sp14-3-3-siRNA– or SpRab11a-siRNA–injected mud crabs (Fig. 8E). In the case of using TLR Ab, a higher level of MyD88 in the V. parahaemolyticus– or exosome-Vp–injected mud crabs was found when compared with those in the Sp14-3-3-siRNA– or SpRab11a-siRNA–injected mud crabs (Fig. 8F). The data indicated that Sp14-3-3 present in the exosome-Vp plays an important role in the stimulation of MyD88 expression and the activation of the Toll pathway (Fig. 9).
Rab GTPases serve as central integrators of endocytic processes, vesicle formation, trafficking maturation, and recycling and degradation (36). It has been shown that the intracellular trafficking and the immune function of cells are linked in multiple ways, and this coordination is critical for dynamic and specialized immune defenses (37). The previous study has reported that Rab GTPases can modulate immune responses by regulating the transport of immune receptors (38), the secretion of chemokines and cytokines (39), and the critical immune surveillance processes of endocytosis and phagocytosis (40). It seems that Rab GTPases may be crucially important for exosome secretion. However, the role of Rab GTPases in controlling pathogens by regulating exosomes is still poorly defined. In this study, SpRab11a was found to be involved in antibacterial responses of mud crab through the promotion of exosome secretion. In our study, the role of Rab GTPases by promoting exosome secretion in the innate immunity was first explored in crustacean mud crab.
Exosomes are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates (41). The components delivered by exosomes into recipient cells effectively alter their biological responses (42). Our previous study revealed that miR-137 and miR-7847 were less packaged in the exosomes of mud crabs challenged with white spot syndrome virus to help the suppression of viral infection (20), whereas miR-224 was densely packaged in those of mud crab challenged with V. parahaemolyticus to maintain the hemolymph microbiota homeostasis and had a protective effect on the survival rates after the pathogen infection in mud crab (21). Exosomes from a tick cell line serve as vehicles for the transmission of viral RNA and proteins to human cells (43). Interest increased around exosomes, because they appeared to have multiple functions in the immune system of the host (44). However, molecular determinants and mechanisms of exosome transmission to the recipient cell, especially exosomal protein, are poorly understood.
14-3-3 is a multifunction protein involved in various biological processes, such as cell-cycle regulation, apoptosis, and autophagy (45). Sp14-3-3 has been reported to be associated with the regulation of antimicrobial peptides, but the specific mechanism has not been explored (46). In this study, Sp14-3-3 was detected gathering exosomes released from V. parahaemolyticus–infected mud crabs. Furthermore, the exosomal Sp14-3-3 was found to be involved in the regulation of exosome-induced ALFs in the recipient hemocytes. Interestingly, the 14-3-3 proteins are a family of adaptor proteins playing important roles in cell signaling, but the functions of exosomal 14-3-3 in immunity are rarely reported.
By binding with hundreds of ligands, 14-3-3 proteins can participate in multiple cellular biological functions, such as cell-cycle, apoptosis, autophagy, signal transduction, and other cellular activities (47). The participation of 14-3-3 proteins in the regulation of NF-κB, JAK/STAT, and PPAR signaling pathways has been previously reported (48). In Drosophila, 14-3-3ε has a crucial role in antimicrobial peptide secretion and innate immunity (29). The secretion of antimicrobial peptides is essentially important in innate immunity (49), but the molecular mechanism that controls the release of them from immune challenge cells is still limited. Also, the information about the association between 14-3-3 proteins and innate immunity is unknown. In this study, for the first time (to our knowledge), Sp14-3-3 was found to be essential for the expression of ALFs after V. parahaemolyticus infection and the recruitment of MyD88 to TLR through its TIR domain on the plasma membrane for signal transduction in crustacean mud crab.
Exosomes can trigger bioactive substances accumulating in the targeted cells to influence immune responses. Tumor cell–derived exosomes inhibit T cell activation by binding programmed death ligand 1 to the receptor programmed death 1 expressed on activated T cells, leading to immune escape to promote tumor cell growth (50). The 14-3-3ζ expression could be upregulated in hepatocellular carcinoma cells and be transmitted from hepatocellular carcinoma cells to T cells through exosomes. 14-3-3ζ overexpression inhibited the activity and proliferation of peripheral blood CD3+ T cells and deviated the differentiation of naive T cells from effector T cells to regulatory T cells (31). It is similar to the findings in this study that, after being stimulated by pathogens, the intracellular Sp14-3-3 can be increased through exosomes, which is essential for its antibacterial immunity, but the ratio of ALFs expression by exosomal 14-3-3 activation and by cellular 14-3-3 regulation of the recipient cells remains to be further investigated.
In summary, it was revealed that SpRab11a could lead to the secretion of exosomes containing Sp14-3-3 protein after bacterial challenge in this study. Furthermore, the critical role of exosomal Sp14-3-3 in the recruitment of MyD88 and TLR to the plasma membrane, promoting Dorsal translocation, and thereby regulating ALFs expression in the hemocytes on the bacterial infection, was determined (Fig. 9). For the first time, to our knowledge, the findings in this study demonstrate that SpRab11a positively regulates the expression of ALFs by promoting the secretion of exosomes containing Sp14-3-3 in invertebrates, which is not reported in vertebrates so far. The results presented in this article would shed some light on the function of exosomal protein in invertebrates.
This work was supported by the Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (Grant GML2019ZD0606), the National Natural Science Foundation of China (Grant 42076125), a 2020 Li Ka Shing Foundation Cross-Disciplinary Research grant (2020LKSFG01E), and the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Teams (Grant 2019KJ141).
The online version of this article contains supplemental material.
Q.S., S. Lin, and M.Z. performed the experiments. Q.S. and S. Li designed the experiments and analyzed the data. H.M. and Y.Z. provided technical support. Q.S., Y.G., N.T.T., and S. Li wrote the manuscript. All authors read and approved the contents of the manuscript and its publication.
The sequences presented in this article have been submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers OL321174, OL321173, and OL321175.
The authors have no financial conflicts of interest.