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

Division of Biology, Kansas State University, 271 Chalmers Hall, Manhattan, KS 66506, USA.
Cellular and Molecular Life Sciences CMLS (Impact Factor: 5.81). 10/2010; 68(11):1929-39. DOI: 10.1007/s00018-010-0543-z
Source: PubMed


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.

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Available from: Kristin Michel, Oct 14, 2015
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    • "The data presented herein go further by assessing the effect of thermolysin on the cleavage of plasma proteins across the animal kingdom, and more specifically, by showing how thermolysin kills insects through the activation of the PPO-based melanization cascade. Insect PPOs have been studied for over a century, and thus, the biochemistry of the melanization pathway is relatively well understood (Fig. S1; Ashida and Brey, 1998; Cerenius et al., 2008; Kanost and Gorman, 2008; Hillyer, 2010; Jiang et al., 2011). In this study, changes to three key proteins belonging to the PPO activation pathway were conclusively detected both in vivo and in vitro. "
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    ABSTRACT: Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.
    Archives of Insect Biochemistry and Physiology 01/2015; 88(1). DOI:10.1002/arch.21178 · 1.02 Impact Factor
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    • "PCR product from these reactions was precipitated using 1 volume of isopropanol and resuspended in deionized water. dsGFP (An et al., 2010) and dsCsIAP1 were synthesized as described previously (An et al., 2010) using 1 μg of product from the second round of PCR as template in a total reaction volume of 20 μl. Purified dsRNA was resuspended in RNase-free water at a final concentration of 3 μg/μl. "
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    ABSTRACT: Biting midges in the genus Culicoides are important vectors of arboviral diseases, including epizootic haemorrhagic disease, bluetongue and most likely Schmallenberg, which cause significant economic burdens worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the difficulty of consistent culturing of certain species and the absence of molecular techniques such as RNA interference (RNAi). Here, we report the establishment of RNAi as a research tool for the adult midge, Culicoides sonorensis. Based on previous research and transcriptome analysis, which revealed putative small interfering RNA pathway member orthologues, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to perform RNA silencing. Injection of control double-stranded RNA targeting green fluorescent protein (dsGFP), into the haemocoel of 2–3-day-old adult female midges resulted in survival curves that support virus transmission. dsRNA injection targeting the newly identified C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) orthologue resulted in a 40% decrease of transcript levels and 73% shorter median survivals as compared with dsGFP-injected controls. These results reveal the conserved function of IAP1. Importantly, they also demonstrate the feasibility of RNAi by dsRNA injection in adult midges, which will greatly facilitate studies of the underlying mechanisms of vector competence in C. sonorensis.
    Insect Molecular Biology 11/2014; 24(1). DOI:10.1111/imb.12139 · 2.59 Impact Factor
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    • "SRPN-2 is a key component in negatively regulating melanization, and knock down of SRPN-2 induced melanotic pseudotumors in adults. Melanization induces the production of cytotoxic semiquinones and reactive oxygen intermediates, which likely contribute to reducing longevity (An et al., 2011). However, double knock-down of SRPN-2 and CLIPB9 rescues the phenotype induced by SRPN2 silencing (An et al., 2011). "
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    ABSTRACT: Insect prophenoloxidase (PPO) is an important innate immunity protein due to its involvement in cellular and humoral defense. It belongs to a group of type-3 copper-containing proteins that occurs in almost all organisms. Insect PPO has been studied for over a century, and the PPO activation cascade is becoming clearer. The insect PPO activation pathway incorporates several important proteins, including pattern-recognition receptors (PGRP, β GRP, and C-type lectins), serine proteases, and serine protease inhibitors (serpins). Due to their complexity, PPO activation mechanisms vary among insect species. Activated phenoloxidase (PO) oxidizes phenolic molecules to produce melanin around invading pathogens and wounds. The crystal structure of Manduca sexta PPO shows that a conserved amino acid, phenylalanine (F), can block the active site pocket. During activation, this blocker must be dislodged or even cleaved at the N-terminal sequence to expose the active site pockets and allow substrates to enter. Thanks to the crystal structure of M. sexta PPO, some domains and specific amino acids that affect PPO activities have been identified. Further studies of the relationship between PPO structure and enzyme activities will provide an opportunity to examine other type-3 copper proteins, and trace when and why their various physiological functions evolved. Recent researches show that insect PPO has a relationship with neuron activity, longevity, feces melanization (phytophagous insects) and development, which suggests that it is time for us to look back on insect PPO beyond the view of immunity in this review.
    Frontiers in Physiology 07/2014; 5:252. DOI:10.3389/fphys.2014.00252 · 3.53 Impact Factor
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