Fig 1.
Exosomes secreted from Vibrio parahaemolyticus-infected mud crab participate in anti-bacterial regulation.
(A-B) Exosomes isolated from mud crabs injected with PBS and V. parahaemolyticus were detected by electron microscopy (A) and Nanosight particle tracking analysis (B). Scale bar, 200 nm. (C) Western blot analysis of exosomal protein markers (Flotillin-1 and TSG101) and cytoplasmic marker (Negative control) Calnexin in cell lysate and exosomes. (D) The delivery of exosomes to mud crab hemocytes. The indicated exosomes (Dio-labeled, green) were injected into mud crabs for 6 h, after which hemocytes (DiI-labeled, red) were isolated and analyzed by confocal microscopy. Scale bar, 20 μm. (E) Effects of exosomes on mud crab mortality. The specific treatments are shown on the top and the mortality was examined daily. (F) Effects of exosomes on bacteria number in mud crab hemolymph. Hemolymph bacteria number for the different treatments were counted using a fluorescence microscope at 100× magnification. (Vp means V. parahaemolyticus, exosome-Vp or exosome-PBS means exosomes isolated from the hemolymph of crabs challenged with V. parahaemolyticus or PBS). Significant statistical differences between treatments are indicated with asterisks (**, p<0.01).
Fig 2.
Exosomes regulate hemolymph microbiota homeostasis through activation of ROS and ALFs.
(A-B) The effects of the indicated exosomes on ROS production during V. parahaemolyticus infection in mud crabs. The level of ROS was measured by fluorescence microscopy. Scale bar, 50 μm (A) and microplate reader (B). (C) The effect of exosomes on the mRNA levels of ALF1-ALF6, and β-actin was used as internal reference, each treatment contains 5 crabs and three independent experiments were performed. (D) The effects of the indicated exosomes on hemolymph microbiota diversity. Mud crabs were co-injected with exosomes and V. parahaemolyticus for 48 h, after which hemolymph was collected and subjected to 16S rDNA sequencing. (E-F) The effects of the indicated exosomes on the composition of hemolymph microbiota at phylum (Top 10) (E) and genera (Top 35) (F) levels. Data represent mean ± s.d. of triplicate assays (*, p<0.05; **, p<0.01).
Fig 3.
Exosomal miR-224 modulates hemolymph microbiota homeostasis in mud crabs.
(A) miRNA sequencing analysis for exosome-V.p and exosome-PBS is presented as a heatmap. The top three up- and downregulated miRNAs in the indicated exosomes are listed. (B-C) The effects of the indicated miRNAs on ALF1 expression in mud crabs. Mimics (B) or AMOs (C) of the indicated miRNAs were co-injected with V. parahaemolyticus into mud crabs for 48 h, followed by the analysis of ALF1 expression using qPCR. Data were presented relative to the value of Vp+ mimic-NC or Vp+ AMO-NC group, which were treated as standard “1”. mimic-NC and AMO-NC stand for disordered nucleic acids which serve as the negative controls. (D) The expression levels of miR-224 in mud crabs challenged with different exosomes, U6 was used as an internal reference. (E-F) The participation of miR-224 in exosome-mediated ROS production. The indicated exosomes, AMO-miR-224 and V. parahaemolyticus were co-injected into mud crabs, followed by the detection of ROS using fluorescence microscopy, Scale bar, 50 μm (E) and microplate reader (F). (G) The effect of miR-224 silencing on exosome-mediated ALFs regulation. (H-I) The involvement of miR-224 in exosome-mediated hemolymph microbiota homeostasis. Hemolymph was collected from mud crabs with the indicated treatments, following by determining the bacterial cell count (H) and species (I) analysis. The data of Vp+Exosome-PBS and Vp+Exosome-Vp groups were from Fig 2D. Each experiment was performed in triplicate and data are presented as mean ± s.d. (*, p<0.05; **, p<0.01).
Fig 4.
HSP70 is the direct downstream target for miR-224 in mud crab.
(A) Target gene prediction of miR-224 using Targetscan and miRanda softwares. (B) Cloning of wild-type and mutated 3’UTRs of HSP70 into the pIZ-V5-EGFP plasmid. The sequences targeted by miR-224 are underlined. (C-D) The direct interactions between miR-224 and HSP70 in insect cells. Drosophila S2 cells were co-transfected with miR-224 and/or the indicated plasmids for 48 h, followed by analysis of the relative fluorescence intensities. Data were presented relative to the value of miR-224+ EGFP-HSP70-3’UTR group, which was treated as standard “1”. (E-F) The effect of miR-224 silencing on the expression level of HSP70 in mud crab post-injection with AMO-miR-224. The mRNA (E) and protein (F) levels were examined at 48 h post-injection. Gray-scale value quantification was conducted using Image J software. (G-H) The effect of miR-224 overexpression on the mRNA and protein levels of HSP70 in mud crabs. (I) The co-localization of miR-224 and HSP70 mRNA in mud crab hemocytes. miR-224 and HSP70 mRNA were determined with FAM-labeled miR-224 probe (red) and Cy3-labeled HSP70 mRNA probe (green), Cy3-labeled Tubulin mRNA probe (green) was used as a negative control, the arrows indicated co-localization. Experiments were performed in triplicates, with the data shown representing the mean ± s.d. (**, p<0.01).
Fig 5.
Role of HSP70 in exosomal miR-224-mediated hemolymph microbiota homeostasis.
(A) The participation of HSP70 in miR-224-mediated ALFs regulation in mud crabs. AMO-miR-224 was co-injected with HSP70-siRNA into V. parahaemolyticus-challenged mud crabs, followed by analysis of the expression levels of ALFs using qPCR. Data were presented relative to the value of Vp group, which was treated as standard “1”. (B-C) The involvement of HSP70 in miR-224-mediated ROS production. The level of ROS in mud crab hemocytes was determined using fluorescence microscopy, Scale bar, 50 μm (B) and microplate reader (Data were presented relative to the value of Vp group, which was treated as standard “1”) (C). (D) The effect of the indicated exosomes on HSP70 expression. Isolated exosomes from mud crabs treated with PBS and V. parahaemolyticus were injected into mud crabs for 48 h, followed by determination of HSP70 protein level using Western blot analysis, tubulin was used as an internal reference. (E-F) The effect of HSP70 silencing on exosome-mediated hemolymph microbiota homeostasis. Hemolymph was collected from mud crabs with the indicated treatments and subjected to 16S rDNA sequencing, then the bacterial cell count (E) and species (F) were analyzed. The data of Vp+Exosome-PBS and Vp+Exosome-Vp groups were from Fig 2D. All the data are the average from at least three independent experiments, mean ± s.d. (*, p<0.05; **, p<0.01).
Fig 6.
miR-224-mediated suppression of HSP70 results in disruption of the HSP70-TRAF6 complex and TRAF6-Ecsit complex formation.
(A) Identification of proteins that bind to HSP70. Mud crab hemocytes lysates were subjected to Co-immunoprecipitation (Co-IP) assay using anti-HSP70 IgG, followed by separation using SDS-PAGE and identification of the proteins by mass spectrometry. (B) Interactions between HSP70 and TRAF6 in mud crab, the cell lysates were analyzed using Co-IP with anti-HSP70 IgG, then the IP products were subjected to Western blot assay to detect TRAF6. (C) The effect of TRAF6 silencing on ALFs regulation. Mud crabs were injected with TRAF6-siRNA or GFP-siRNA for 48 h, followed by analysis of ALFs expression using qPCR. Data were presented relative to the value of Vp group, which was treated as standard “1”. (D-E) Effect of TRAF6 silencing on ROS production in mud crabs. The level of ROS in mud crab hemocytes was analyzed using fluorescence microscopy, Scale bar, 50 μm (D) and microplate reader (Data were presented relative to the value of Vp group, which was treated as standard “1”) (E). (F) Identification of proteins that bind to TRAF6. Mud crab hemocytes lysates were subjected to Co-IP assay using anti-TRAF6 IgG, followed by separation using SDS-PAGE and identification of the proteins by mass spectrometry. (G) The interaction between TRAF6 and Ecsit in mud crabs. The cell lysates were analyzed using Co-IP with anti-TRAF6 IgG, then the IP products were subjected to Western blot assay to detect Ecsit. (H) The interaction between HSP70 and Ecsit in mud crabs. Cell lysates were subjected to Co-IP analysis with anti-HSP70 IgG and anti-Ecsit IgG, followed by Western blot analysis using the indicated antibodies. (I) The interactions between HSP70 and TRAF6, TRAF6 and Ecsit in mud crabs after the indicated treatments. Hemocytes lysates were collected from mud crabs challenged with Vp, Vp+ Exosome-PBS and Vp+ Exosome-Vp, followed by Co-IP assay using anti-TRAF6 IgG, then the IP products were subjected to Western blot assay to detect HSP70 and Ecsit. Data shown represent the mean ± s.d. for triplicate assays (**, p<0.01).
Fig 7.
TRAF6-Ecsit complex mediates hemolymph microbiota homeostasis.
(A) The effect of the indicated exosomes on the protein level of TRAF6 in mitochondria. Mud crabs were treated with either Exosome-PBS or Exosome-Vp for 48 h, then the mitochondria were isolated and subjected to Western blot assay to detect TRAF6. Tubulin and HSP60 were used as negative indicator and positive indicator to evaluate the purity of the isolated mitochondria, respectively. (B-C) The effect of the indicated exosomes on mROS production. The mROS level in mud crab hemocytes was determined using fluorescence microscopy, Scale bar, 50 μm (B) and microplate reader (Data were presented relative to the value of WT group, which was treated as standard “1”) (C). (D) The effect of TRAF6 silencing on the expression and ubiquitination levels of Ecsit. Mud crabs were treated with TRAF6-siRNA for 48 h, followed by the detection of total and ubiquitinated Ecsit through Western blot assay. (E) The effect of the indicated exosomes on the expression and ubiquitination levels of Ecsit. Mud crabs were treated with Exosome-PBS or Exosome-Vp for 48 h, followed by the detection of total and ubiquitinated Ecsit through Western blot assay. (F) The protein level of Ecsit in mud crab hemocytes nuclei after treatment with the indicated exosomes was determined by Western blot analysis. Tubulin and Histone H3 were used to evaluate the purity of the isolated nuclei. (G) The localization of Ecsit in mud crab hemocytes after treated with the indicated exosomes was determined using immunofluorescence assay with mouse anti-Ecsit antibody, Scale bar, 10 μm. (H) The effect of Ecsit overexpression on the transcription of ALFs. pGL3-Basic and renilla luciferase activities serve as internal reference, Data were presented relative to the value of pAc 5.1-Empty vector group, which was treated as standard “1”. (I-J) The effects of TRAF6 or Ecsit silencing on exosome-mediated hemolymph microbiota homeostasis. Hemolymph was collected from mud crabs after the indicated treatments and was used to determine bacteria number (I) and species (J). The data of Vp+Exosome-PBS and Vp+Exosome-Vp groups were from Fig 2D. (K) Effects of TRAF6 or Ecsit silencing on exosome-VP-mediated anti-bacterial function in mud crab. The specific treatments are shown on the top and the mortality was examined daily. Data shown represent that of three independent experiments (*, p<0.05; **, p<0.01).
Fig 8.
Proposed schematic diagram for exosomal miR-224-mediated hemolymph microbiota homeostasis during V. parahaemolyticus infection in mud crabs.