Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response

Abstract

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

Keywords: Anopheles gambiae; Clip domain serine protease cascades; Melanization; Mosquito innate immunity.

Fig. 1.

CLIPB8 and CLIPB10 hemolymph proteins respond to microbial infection. CLIPB8 is consumed in the hemolymph in response to (A) B. bassiana and (B) S. aureus, but not (C) E. coli infection. CLIPB10 is cleaved at 24 hours after (E) S. aureus but not (D) B. bassiana nor (F) E. coli infections. Western blots of hemolymph extracts obtained at the indicated time points from (A,D) B. bassiana (2000 spores/mosquito), (B,E) S. aureus and (C,F) E. coli injected mosquitoes and probed with αCLIPB8. Western blots were probed with (A-C) αCLIPB8 and (D-F) αCLIPB10, respectively. All hemolymph samples were extracted from 40 mosquitoes, quantified using Bradford protein quantification assay, and equal amounts of protein were loaded into each well. Membranes were reprobed with αAPOII (without stripping) to control for loading. The figures shown are representative of 3 independent biological experiments. C, control naïve mosquitoes.

Fig. 2.

CLIPB4 and CLIPB17 act upstream of CLIPB8 and CLIPB10 in the serine protease cascade regulating melanization. Western blots showing (A) CLIPB8 protein levels and (B) CLIPB10 cleavage profile in the hemolymph of mosquitoes treated with the indicated gene-specific double-stranded (ds) RNA at 10 and 24 hrs after S. aureus infection, respectively. Two representative trials are shown per experiment. All hemolymph samples were extracted from 40 mosquitoes, quantified using Bradford protein quantification assay, and equal protein amounts were loaded into each well. Membranes were reprobed with αAPOII (without stripping) as loading control. (C) Schematic diagram showing the hierarchical organization of SPCLIP1 with respect to the catalytic CLIPBs in the melanization response to systemic bacterial infections. CLIPB4 and CLIPB17 are shown in a rectangular box because their respective hierarchies have not been determined. Dashed lines indicate that the enzymatic steps are not yet fully characterized.

Fig. 3.

TEP1 and the core cSPH module control the activation of CLIPB8 and CLIPB10. Western blots showing (A) CLIPB10 cleavage profile and (B) CLIPB8 protein levels in the hemolymph of mosquitoes treated with the indicated gene-specific double-stranded (ds) RNAs at 10 and 24 hrs after S. aureus infection, respectively. In (A), low (left) and extended (right) exposures of the same membrane are shown in order to reveal the CLIPB10-C band that is often very weak. All hemolymph samples were extracted from 40 mosquitoes, quantified using Bradford protein quantification assay, and equal protein amounts were loaded into each well. Membranes were reprobed with αAPOII (without stripping) as loading control. The figures shown are representative of 3 independent biological experiments. (C) Schematic diagram showing the hierarchical organization of TEP1, the core cSPH module and the catalytic CLIPBs and CLIPC9 in the melanization response to systemic bacterial infections. Dashed lines indicate that the enzymatic steps are not yet fully characterized.

Fig. 4.

Recombinant proCLIPA8 is cleaved by B4, B9 and B10 in vitro. Recombinant proCLIPA8-V5-His is cleaved by (A) Factor Xa-activated B4_Xa-His, (B) B9_Xa-His, and (C) B10_Xa-His at the predicted site, examined by Western blot analyses using anti-His and anti-V5 antibodies. Zymogens of CLIPB members were activated by Factor Xa, and incubated with wild-type proCLIPA8 for (A,B) 1 hour at RT or (C) 30 min on ice. The activation of corresponding CLIPB members by Factor Xa was included for each Western blot analysis. A positive control was included with Factor Xa-cleavage of proCLIPA8_Xa for 2 hours at RT. Both anti-His and anti-V5 antibodies detected the cleaved fragment of wild-type proCLIPA8 at a similar size as that of Factor Xa-cleaved proCLIPA8_Xa. Solid and hollow arrows indicate the full-length and activated forms of CLIPB members, respectively; for CLIPA8_Xa, solid and hollow stars indicate the full-length and cleaved forms, respectively.

Fig. 5.

CLIPB4 and CLIPB17 are central players in the melanization response to bacterial infections. MelASA assay conducted on the indicated mosquito genotypes at 12 hrs post (A) E. coli, (B) S. aureus and 18 hrs post (C) M. luteus infections. The amount of melanin deposits present in infected mosquito excreta was measured from Whatman papers placed at the bottom of the paper cups housing 35 (for E. coli and S. aureus) or 50 (for M. luteus) mosquitoes per sample. Statistical analysis was done using unpaired t-tests to compare two treatment groups, and One-Way ANOVA with Dunnett’s post-test to compare multiple treatment groups. Means are shown in red lines. Data shown are from at least 6 independent trials. *, P<0.05; ***, P<0.001; ****, P<0.0001.

Fig. 6.

CLIPB14 is required for mosquito tolerance but not resistance to bacterial infections. (A-D) Survival analysis of the indicated mosquito genotypes challenged with (A and C) E. coli, and (B and D) S. aureus. A representative experiment of at least three independent trials is shown. The Kaplan-Meier survival test was used to calculate the percent survival. Statistical significance of the observed differences was calculated using the Log-rank test. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. (E, F) Bacterial proliferation assays conducted on the indicated mosquito genotypes injected with (E) E. coli (OD₆₀₀=0.8) and (F) S. aureus (OD₆₀₀=0.8). Batches of 8 whole mosquitoes each were homogenized in LB medium at 24 hours post-infection and colony forming units (CFUs) were scored on LB plates supplemented with the appropriate antibiotic. Each point on the scatter plot represents the mean CFU per mosquito per batch. Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s multiple comparisons test, with P-values less than 0.05 considered significant. Means are in red. P-values are shown only for samples that significantly differ from the control (dsLacZ). Data presented are from 4 independent experiments.