Aedes aegypti CLIPB9 activates prophenoloxidase-3 in the presence of CLIPA14 after fungal infection

Abstract

Melanization is an integral part of the insect defense system and is often induced by pathogen invasion. Phenoloxidases (POs) are critical enzymes that catalyze melanin formation. PO3 is associated with the antifungal response of the mosquito, Aedes aegypti, but the molecular mechanism of the prophenoloxidase-3 (PPO3) activation is unclear. Here we report that PPO3 cleavage activation is mediated by a clip-domain serine protease, CLIPB9. We purified recombinant CLIPB9 and found that it cleaved PPO3 and increased PO activity in the hemolymph. We then identified CLIPA14 (a serine protease homolog) by co-immunoprecipitation using anti-CLIPB9 antibody. After being cleaved by CLIPB9, Ae. aegypti CLIPA14 acted as a cofactor for PPO3 activation. In addition, dsRNA co-silencing of CLIPB9 and CLIPA14 genes reduced melanization after infection with the entomopathogen, Beauveria bassiana, making the adult mosquitoes more sensitive to fungal infection. These results illustrate the roles of CLIPB9 and CLIPA14 in the PPO activation pathway and revealed the complexity of the upstream serine protease network controlling melanization.

Keywords: antifungal response; immune melanization; prophenoloxidase; serine protease; serine protease homolog.

Figure 1

B. bassiana induced melanin synthesis is mediated by Ae. aegypti CLIPB9. (A) Mosquitoes appeared melanization after infected with B. bassiana. The melanotic masses were found in mosquitoes infected with B. bassiana but not in control PBS treatment. Abdominal images were observed under an OLYMPUS SZX16 microscope. The red arrows indicate the melanotic masses. Scale bars, 200 μm. (B) PPO in hemolymph was activated by B. bassiana conidia. PO activity was detected after 10-fold dilution of fresh hemolymph. The hemolymph with the addition of 1 × 10³ conidia was significantly activated compared to the hemolymph without the addition of conidia. The bars represent the mean ± SEM (n = 3). **P < 0.01. (C) Immunoblotting analysis of CLIPB9 expression in hemolymph after B. bassiana infection. H, mosquito hemolymph was uninfected. H + B, mosquito hemolymph infected with B. bassiana. Anti-LPII antibody was used as the loading control.

Figure 2

Purification and activation of recombinant proCLIPB9 and proCLIPB9_Xa. (A) Purified recombinant proCLIPB9 and proCLIPB9_Xa were analyzed by SDS-PAGE and immunoblotting. Left, CBB staining; Right, immunoblotting assays using anti-CLIPB9 antibody (CLIPB9 Ab). (B) The cleavage of purified proCLIPB9_Xa by Factor Xa was detected by immunoblotting. After incubation of Factor Xa with proCLIPB9 (100 ng) or proCLIPB9_Xa (100 ng), immunoblotting was performed using anti-CLIPB9 antibody. (C) Amidase activity assay of CLIPB9_Xa. The catalytic activity of CLIPB9_Xa cleaved by Factor Xa was detected using IEARpNa as a substrate. The bars represent the mean ± SEM (n = 3). **P < 0.01; ns, P > 0.05.

Figure 3

Cleavage activation of Ae. aegypti PPO3 by Factor Xa-treated proCLIPB9_Xa. (A) PPO3 was activated by CLIPB9. Recombinant PPO3 was incubated with buffer, Factor Xa, proCLIPB9, proCLIPB9_Xa, or Factor Xa and proCLIPB9/proCLIPB9_Xa. The mixtures containing 300 ng PPO3 were used in the PO activity assay (upper panel), and the mixtures containing 100 ng PPO3 were analyzed by immunoblotting using PPO3 antibodies (bottom panel). (B) CLIPB9 activates PPO in hemolymph. The 1:10 diluted hemolymph samples were incubated with proCLIPB9 or Factor Xa-treated proCLIPB9Xa. PO activities were measured using dopamine as a substrate and plotted as mean ± SEM (n = 3). **P < 0.01; *P < 0.05; ns, P > 0.05.

Figure 4

A potential interacting protein of CLIPB9. Proteomic analysis of CLIPB9 (A) and CLIPA14 (B) associated proteins. Co-IP experiments were performed on Bb-infected and control cell-free hemolymph using anti-CLIPB9 or anti-CLIPA14 antibody. Proteins eluted from the immunoaffinity columns were treated with trypsin for LC-MS/MS analysis. Peak processing was performed using Proteome Discoverer 2.4.1.15 (Thermo Fisher Scientific) software. (C) Immunoblotting analysis of CLIPA14 expression in hemolymph after B. bassiana infection. H, mosquito hemolymph was uninfected. H + B, mosquito hemolymph infected with B. bassiana. Anti-LPII antibody was used as the loading control. (D) Purified recombinant proCLIPA14 and proCLIPA14_Xa analyzed by SDS-PAGE and immunoblotting. Left, CBB staining. Right, immunoblotting using anti-CLIPA14 antibody.

Figure 5

Ae. aegypti CLIPA14 treatment significantly increased the PO activity. (A) Enhancement of PPO3 activation by CLIPA14. proCLIPB9_Xa or its mixture with proCLIPA14_Xa was treated with Factor Xa and then incubated with 300 ng PPO3 prior to PO activity measurement. (B) The cleavage of proCLIPA14 by activated CLIPB9_Xa was detected by immunoblotting. Factor Xa-treated CLIPB9_Xa was incubated with proCLIPA14 (100 ng) and the mixture and controls were analyzed by immunoblotting using anti-V5 antibody. (C) The cleavage of purified proCLIPA14_Xa by Factor Xa was detected by immunoblotting. After incubation of Factor Xa with proCLIPA14 (100 ng) or proCLIPA14_Xa (100 ng), the mixture was immunoblotted with anti-CLIPA14 antibody. (D) The CLIPA14_Xa-mutant can also act as a cofactor to increase PO activity. ProCLIPB9_Xa, proCLIPA14_Xa, and proCLIPA14_Xa-mutant were activated by Factor Xa and then incubated with PPO3. Using dopamine as the substrate to measure PO activity. CLIPA14_Xa-mut, CLIPA14_Xa-mutant. The bars represent the mean ± SEM (n = 3). **P < 0.01; ns, P > 0.05.

Figure 6

The roles of CLIPB9 and CLIPA14 in mosquito antifungal defense. (A) Effects of silencing of CLIPB9 and CLIPA14 on the survival rate of mosquitoes after B. bassiana (Bb) infection. Depletion of CLIPB9 or CLIPA14 reduced mosquito resistance to B. bassiana compared to the control group (iEGFP_Bb), and concomitant depletion of CLIPB9 and CLIPA14 further reduced the resistance of mosquitoes to B. bassiana. Three biological repeats were conducted. The significance between different survival curves was compared using the log-rank (Mantel-Cox) test. *, P < 0.05; **P < 0.01. (B) Effects of silencing CLIPB9 and CLIPA14 on melanization in mosquitoes after B. bassiana infection. Compared to the control group (iEGFP_Bb), the melanotic masses on the abdomen of mosquitoes were reduced by varying degrees in the CLIPB9-depleted group, the CLIPA14-depleted group, and the co-silenced group. Abdominal images were observed under an OLYMPUS SZX16 microscope. The red arrows indicate the melanotic masses. Scale bars, 200 μm.