The ARM repeat domain of hemocyanin interacts with MKK4 to modulate antimicrobial peptides expression

Abstract

The mitogen-activated protein kinase (MAPK) intracellular signaling pathway mediates numerous biological processes, including antimicrobial immune response by inducing antimicrobial peptides (AMPs) production. Although MAPK signaling cascade proteins have been identified in penaeid shrimp, their modulation via the MKK4-p38-c-Jun cascade and effect on AMPs production is unknown. Here, we show that hemocyanin (PvHMC), antimicrobial peptides (anti-lipopolysaccharide factor, crustin, and penaeidins), and MKK4-p38-c-Jun cascade proteins are simultaneously induced by pathogens (Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus) in Penaeus vannamei. Intriguingly, knockdown of PvHMC with or without pathogen challenge attenuated the expression of MKK4-p38-c-Jun cascade proteins and their phosphorylation level, which consequently decreased AMPs expression. Further analysis revealed that PvHMC interacts via its armadillo (ARM) repeat domain with PvMKK4 to modulate the p38 MAPK signaling pathway. Thus, the ARM repeat domain enables penaeid shrimp hemocyanin to modulate AMPs expression during antimicrobial response by activating the p38 MAPK signaling pathway.

Keywords: Cell biology; Immunology.

Graphical abstract

Figure 1

Microbial pathogens induce both hemocyanin and antimicrobial peptides expression (A–C) PvHMC mRNA levels, (D–F) ALF mRNA levels, (G–I) CRU mRNA levels, and (J–L) PEN mRNA levels in Penaeus vannamei hepatopancreas after challenge with Vibrio parahaemolyticus, Staphylococcus aureus, and WSSV, respectively. mRNA levels of the indicated genes were quantified by qRT-PCR, and normalized to those of EF1α mRNA. Results reported as mean ± SEM (n = 3). ∗p <0.05, ∗∗p <0.01 vs. control (PBS). PvHMC; hemocyanin; ALF, anti-lipopolysaccharide factor; CRU, crustin; PEN, penaedin; WSSV, white spot syndrome virus.

Figure 2

Hemocyanin modulates antimicrobial peptides expression Relative mRNA expression levels of (A) PvHMC, (B) ALF, (C) CRU, and (D) PEN in Penaeus vannamei hepatopancreas after RNAi-mediated knockdown of PvHMC gene. Relative mRNA expression levels of (E) PvHMC, (F) ALF, (G) CRU, and (H) PEN in Penaeus vannamei hepatopancreas after PvHMC gene knockdown followed by Vibrio parahaemolyticus challenge. mRNA levels of the indicated genes were quantified by qRT-PCR, and normalized to those of EF1α mRNA. Results reported as mean ± SEM (n = 3). ∗p <0.05, ∗∗p <0.01 vs. control. PvHMC; hemocyanin; ALF, anti-lipopolysaccharide factor; CRU, crustin; PEN, penaedin.

Figure 3

Microbial pathogens induce the expression of PvHMC and p38 MAPK cascade genes Relative mRNA expression levels of (A–C) PvHMC, (D–F), PvMKK4, (G–I) Pvp38, and (J–L) Pvc-Jun in Penaeus vannamei hepatopancreas at the indicated time points post challenge with Vibrio parahaemolyticus, Staphylococcus aureus, and WSSV, respectively. mRNA levels of the indicated genes were quantified by qRT-PCR, and normalized to those of EF1α mRNA. Results reported as mean ± SEM (n = 3). ∗p <0.05, ∗∗p <0.01 vs. control. (M−P) Western blot analysis of PvHMC, PvMKK4, Pvp38, and tubulin proteins and phosphorylation levels in Penaeus vannamei hepatopancreas after injection with PBS (control), Vibrio parahaemolyticus, Staphylococcus aureus, and WSSV, respectively. Protein and phosphorylation levels were determined by western blot. The immunoblots shown are representative of at least two independent experiments. PvHMC; hemocyanin; PvMKK4, mitogen-activated protein kinase kinase four; Pvp38, p38 mitogen-activated protein kinase; Pvc-Jun, Penaeus vannamei transcription factor c-Jun; WSSV, white spot syndrome virus.

Figure 4

Hemocyanin modulates p38 MAPK signaling to induce antimicrobial peptides expression in shrimp hepatopancreas (A) PvHMC mRNA and protein levels, (B) PvMKK4 mRNA, protein, and phosphorylation levels, (C) Pvp38 mRNA, protein, and phosphorylation levels, (D) PvATF2 mRNA, protein, and phosphorylation levels, and (E) Pvc-Jun mRNA levels, after shrimp were injected with dsControl or dsPvHMC. (F) PvHMC mRNA and protein levels, (G) PvMKK4 mRNA, protein, and phosphorylation levels, (H) Pvp38 mRNA, protein, and phosphorylation levels, and (I) Pvc-Jun mRNA levels, after shrimp were injected with dsControl or dsPvHMC followed by Vibrio parahaemolyticus challenge. Relative mRNA expression levels of (J) PvMKK4, (K) ALF, (L) CRU, and (M) PEN genes in shrimp hepatopancreas after PvMKK4 knockdown. Relative mRNA expression levels of (N) Pvp38, (O) ALF, (P) CRU, and (Q) PEN genes in shrimp hepatopancreas after Pvp38 knockdown. mRNA levels of the indicated genes were quantified by qRT-PCR, and normalized to those of EF1α mRNA, whereas protein and phosphorylation levels were determined by Western blot. Results reported as mean ± SEM (n = 3). ∗p <0.05, ∗∗p <0.01 vs. control. The immunoblots shown are representative of at least two independent experiments.

Figure 5

Hemocyanin interacts with p38 MAPK signaling proteins (A and B) Immunoblots of protein-protein interaction analysis between GST-PvHMC and His-PvMKK4 or GST-PvHMC and His-Pvp38. (C and D) Co-immunoprecipitation and immunoblot analysis of the interaction between PvHMC-FLAG and PvMKK4-V5 or PvHMC-FLAG and Pvp38-V5. (E) Protein domain structure of Penaeus vannamei hemocyanin showing the N-domain, M-domain (with the ARM repeat domain motif), and C-domain. (F) Immunoblots of protein-protein interaction analysis between GST (control), GST-PvHMC-N, GST-PvHMC-M, GST-PvHMC-M-ΔARM, and GST-PvHMC-C with His-PvMKK4. Results reported are representative of at least two independent experiments.

Figure 6

Diagrammatic summary of proposed mechanism by which hemocyanin (PvHMC) modulates p38 MAPK signaling during antimicrobial response in penaeid shrimp Microbial pathogens induce PvHMC expression, promoting binding via its armadillo (ARM) repeat domain with MKK4 to activate (phosphorylate) p38 and c-Jun, which allows phosphorylated c-Jun to enter the nucleus to promote the transcription of antimicrobial peptide genes. PvHMC can also undergo proteolytic degradation in response to microbial pathogens to generate peptides with antimicrobial activity.