Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes

Abstract

Melanization is an innate immune response in arthropods that encapsulates and kills invading pathogens. One of its rate-limiting steps is the activation of prophenoloxidase (PPO), which is controlled by an extracellular proteinase cascade and serpin inhibitors. The molecular composition of this system is largely unknown in mosquitoes with the exception of serpin-2 (SRPN2), which was previously identified as a key negative regulator of melanization. Using reverse genetic and biochemical techniques, we identified the Anopheles gambiae clip-serine proteinase CLIPB9 as a PPO-activating proteinase, which is inhibited by SRPN2. Double knockdown of SRPN2 and CLIPB9 reversed the pleiotrophic phenotype induced by SRPN2 silencing. This study identifies the first inhibitory serpin-serine proteinase pair in mosquitoes and defines a regulatory unit of melanization. Additionally, the interaction of CLIPB9 and SRPN2 affects the life span of adult female mosquitoes and therefore constitutes a well-defined potential molecular target for novel late-life acting insecticides.

Fig. 1

Activation of purified recombinant proCLIPB9_Xa by factor Xa. Activation of the proteinase zymogen was analyzed by Western blot using mouse anti-His antibodies (a) and spectrophotometric assay using IEARpNA as a substrate (b). The bars represent mean ± SD (n = 3). Statistically significant differences are indicated with different letters (one-way ANOVA followed by Newman-Keuls test, P < 0.05). Circle, proCLIPB9_Xa zymogen; square, catalytic domain of proCLIPB9_Xa

Fig. 2

An. gambiae SRPN2 binds and inhibits CLIPB9. a, b Western blot analysis of SRPN2-CLIPB9 complex. CLIPB9_Xa was incubated with recombinant SRPN2 (a) or An. gambiae hemolymph (b). Western blot analysis was performed using mouse anti-His (left) or rabbit anti-SRPN2 (right) antibodies. Triangles, non-complexed SRPN2; circles, proCLIPB9_Xa zymogen; squares, catalytic domain of proCLIPB9_Xa; asterisks, SRPN2-CLIPB9 complex. (c) Inhibition of CLIPB9 activity by recombinant SRPN2. Purified recombinant SRPN2 was incubated with CLIPB9_Xa. The residual IEARase activities of CLIPB9_Xa were plotted as mean ± SD (n = 3) against the corresponding molar ratios of SRPN2 and CLIPB9_Xa. (d) Kinetic analysis of the inhibition of CLIPB9 by SRPN2. CLIPB9_Xa (2.8 pmol) was added to a mixture of 300 μM IEARpNa and SRPN2 at 0, 12.3, 30.8, 61.5, 123, 307.5, 615, and 922.5 nM. The progress of enzyme inactivation at each concentration of SRPN2 was followed by measuring the ΔA₄₀₅ of the reaction every 49 s (inset). The rate of complex formation was calculated as described in [31]

Fig. 3

CLIPB9 cleaves and activates M. sexta PPO. CLIPB9_Xa cleaves M. sexta PPO in plasma (a) and as purified protein (b), causing a significant increase in PO activity (c, d). Arrows mark the doublet bands representing PPO (white) and PO (black). Bars represent mean ± 1 SD (n = 3); statistically significantly differences are indicated by different letters (one-way ANOVA followed by Newman-Keuls test, P < 0.05)

Fig. 4

CLIPB9 knockdown partially reverts the SRPN2-depletion phenotype. a Images of abdominal wall preparations 12 days post injection; tumors are marked by white arrows. b The total melanotic area per abdomen (arbitrary unit) is significantly reduced in dsCLIPB9/dsSRPN2-treated compared to SRPN2-depleted mosquitoes (U test, P < 0.0001). No melanotic tumors were observed in dsGFP or dsCLIPB9-treated mosquitoes; thus, they were excluded from the analysis. cCLIPB9-KD alone did not affect survival, but partially rescued the mortality effect induced by SRPN2-KD. The combined data set of four independent biological repeats is shown; for individual results, see Fig. S2. Statistically significant differences between survival curves are indicated by letters (log rank test, df = 3, χ² = 128.8, P < 0.0001; pairwise comparisons: dsGFP vs. dsCLIPB9, df = 1, χ² = 0.351, P = 0.554; dsGFP vs. dsSRPN2, df = 1, χ² = 99.8, P < 0.0001; dsGFP vs. dsCLIPB9/dsSRPN2, df = 1, χ² = 19.45, P < 0.0001; dsSRPN2 vs. dsCLIPB9/dsSRPN2, df = 1, χ² = 30.14, P < 0.0001). d Mean daily percent mortality rate from a above, calculated according to [5]