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: 5.86). 10/2010; 68(11):1929-39. DOI: 10.1007/s00018-010-0543-z
Source: PubMed

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.

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Available from: Kristin Michel, Aug 04, 2015
    • "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 · 1.16 Impact Factor
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    • "For membrane feeding assays, hyper-reticulocytosis was induced in mice by injection of 6 mg/mL of phenylhydrazine 3 days prior to infection with 10 7 parasitized red blood cells (pRBCs). Three days after infection, exflagellation of male gametocytes was checked as described previously (Blagborough and Sinden, 2009). Blood containing infectious P. berghei gametocytes was then collected by cardiac puncture, split into two equal aliquots and mixed with pooled serum from mice immunized with either AgSRPN2 or GFP (each n = 8) at a dilution of 1:5 serum:blood and fed to A. stephensi mosquitoes through plastic membrane feeders. "
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    ABSTRACT: The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2 (AgSRPN2). Antibodies against AgSRPN2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines.
    International journal for parasitology 07/2013; 43(11). DOI:10.1016/j.ijpara.2013.06.004 · 3.40 Impact Factor
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    • "AgCLIPB9, the first PAP to be identified in mosquitoes, is inhibited by AgSRPN2. Double knockdown of AgSRPN2 and AgCLIPB9 partially reversed the phenotypes induced by AgSRPN2 silencing (An et al., 2010). One interpretation of the partial reversion of the AgSRPN2 depletion phenotype is that AgSRPN2 has additional PAP proteinase targets, which are currently being identified. "
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    ABSTRACT: In vector-borne diseases, the complex interplay between pathogen and its vector's immune system determines the outcome of infection and therefore disease transmission. Serpins have been shown in many animals to be key regulators of innate immune reactions. Their control over regulatory proteolytic cascades ultimately decides whether the recognition of a pathogen will lead to an appropriate immune response. In mosquitoes, serpins (SRPNs) regulate the activation of prophenoloxidase and thus melanization, contribute to malaria parasite lysis, and likely Toll pathway activation. Additionally, in culicine mosquitoes, SRPNs are able to regulate hemostasis in the vertebrate host, suggesting a crucial role during bloodfeeding. This review summarizes the annotation, transcriptional regulation, and current knowledge of SRPN function in the three mosquito species for which the complete genome sequence is available. Additionally, we give a brief overview of how SRPNs may be used to prevent transmission of vector-borne diseases.
    Journal of insect physiology 09/2012; DOI:10.1016/j.jinsphys.2012.08.015 · 2.50 Impact Factor
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