The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.

The arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core o...

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Main Authors: Gregory L Sousa, Ritika Bishnoi, Richard H G Baxter, Michael Povelones
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2020-10-01
Series:PLoS Pathogens
Online Access:https://doi.org/10.1371/journal.ppat.1008985
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spelling doaj-d3c8fe171bcf49bf92a7b8cbbe63d1b22021-06-09T04:33:58ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742020-10-011610e100898510.1371/journal.ppat.1008985The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.Gregory L SousaRitika BishnoiRichard H G BaxterMichael PovelonesThe arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core of a hierarchical cascade downstream of mosquito complement that is required for microbial melanization. However, our understanding of the regulatory relationship of the CLIP-SPH cascade with the catalytic CLIP-SPs driving melanization is incomplete. Here, we report on the development of a novel screen to identify melanization pathway components based on the quantitation of melanotic mosquito excreta, eliminating the need for microdissections or hemolymph enzymatic assays. Using this screen, we identified CLIPC9 and subsequent functional analyses established that this protease is essential for the melanization of both Escherichia coli and the rodent malaria parasite Plasmodium berghei. Mechanistically, septic infection with E. coli promotes CLIPC9 cleavage and both full-length and cleaved CLIPC9 localize to this bacterium in a CLIPA8-dependent manner. The steady state level of CLIPC9 in the hemolymph is regulated by thioester-containing protein 1 (TEP1), suggesting it functions downstream of mosquito complement. In support, CLIPC9 cleavage is inhibited following SPCLIP1, CLIPA8, and CLIPA28 knockdown positioning it downstream of the CLIP-SPH cascade. Moreover, like CLIPA8 and CLIPA28, CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2). This report demonstrates how our novel excretion-based approach can be utilized to dissect the complex protease networks regulating mosquito melanization. Collectively, our findings establish that CLIPC9 is required for microbial melanization in An. gambiae and shed light on how the CLIP-SPH cascade regulates this potent immune response.https://doi.org/10.1371/journal.ppat.1008985
collection DOAJ
language English
format Article
sources DOAJ
author Gregory L Sousa
Ritika Bishnoi
Richard H G Baxter
Michael Povelones
spellingShingle Gregory L Sousa
Ritika Bishnoi
Richard H G Baxter
Michael Povelones
The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.
PLoS Pathogens
author_facet Gregory L Sousa
Ritika Bishnoi
Richard H G Baxter
Michael Povelones
author_sort Gregory L Sousa
title The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.
title_short The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.
title_full The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.
title_fullStr The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.
title_full_unstemmed The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae.
title_sort clip-domain serine protease clipc9 regulates melanization downstream of spclip1, clipa8, and clipa28 in the malaria vector anopheles gambiae.
publisher Public Library of Science (PLoS)
series PLoS Pathogens
issn 1553-7366
1553-7374
publishDate 2020-10-01
description The arthropod melanization immune response is activated by extracellular protease cascades predominantly comprised of CLIP-domain serine proteases (CLIP-SPs) and serine protease homologs (CLIP-SPHs). In the malaria vector, Anopheles gambiae, the CLIP-SPHs SPCLIP1, CLIPA8, and CLIPA28 form the core of a hierarchical cascade downstream of mosquito complement that is required for microbial melanization. However, our understanding of the regulatory relationship of the CLIP-SPH cascade with the catalytic CLIP-SPs driving melanization is incomplete. Here, we report on the development of a novel screen to identify melanization pathway components based on the quantitation of melanotic mosquito excreta, eliminating the need for microdissections or hemolymph enzymatic assays. Using this screen, we identified CLIPC9 and subsequent functional analyses established that this protease is essential for the melanization of both Escherichia coli and the rodent malaria parasite Plasmodium berghei. Mechanistically, septic infection with E. coli promotes CLIPC9 cleavage and both full-length and cleaved CLIPC9 localize to this bacterium in a CLIPA8-dependent manner. The steady state level of CLIPC9 in the hemolymph is regulated by thioester-containing protein 1 (TEP1), suggesting it functions downstream of mosquito complement. In support, CLIPC9 cleavage is inhibited following SPCLIP1, CLIPA8, and CLIPA28 knockdown positioning it downstream of the CLIP-SPH cascade. Moreover, like CLIPA8 and CLIPA28, CLIPC9 processing is negatively regulated by serine protease inhibitor 2 (SRPN2). This report demonstrates how our novel excretion-based approach can be utilized to dissect the complex protease networks regulating mosquito melanization. Collectively, our findings establish that CLIPC9 is required for microbial melanization in An. gambiae and shed light on how the CLIP-SPH cascade regulates this potent immune response.
url https://doi.org/10.1371/journal.ppat.1008985
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