The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs

English version

Autoři: Layla El Moussawi ^aff001; Johnny Nakhleh ^aff001; Layla Kamareddine ^aff002; Mike A. Osta ^aff001
Působiště autorů: Department of Biology, American University of Beirut, Beirut, Lebanon ^aff001; Department of Biomedical Sciences, Qatar University, Doha, Qatar ^aff002
Vyšlo v časopise: The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs. PLoS Pathog 15(11): e32767. doi:10.1371/journal.ppat.1008194
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008194

Souhrn

Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. The activation of cSPHs requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network.

Klíčová slova:

Immune response – Malarial parasites – Mosquitoes – Parasitic diseases – Plasmodium – Regulator genes – Serine proteases – Staphylococcus aureus

Zdroje

1. Veillard F, Troxler L, Reichhart JM. Drosophila melanogaster clip-domain serine proteases: Structure, function and regulation. Biochimie. 2016; 122: 255–269. doi: 10.1016/j.biochi.2015.10.007 26453810

2. Cerenius L, Kawabata S, Lee BL, Nonaka M, Soderhall K. Proteolytic cascades and their involvement in invertebrate immunity. Trends Biochem Sci. 2010; 35: 575–583. doi: 10.1016/j.tibs.2010.04.006 20541942

3. Kanost MR, Jiang H. Clip-domain serine proteases as immune factors in insect hemolymph. Curr Opin Insect Sci. 2015; 11: 47–55. doi: 10.1016/j.cois.2015.09.003 26688791

4. Nakhleh J, El Moussawi L, Osta MA. The melanization response in insect immunity. In: Jurenka R, editor. Advances in Insect Physiology. Cambridge, Academic Press, USA; 2017. pp 2–20.

5. Povelones M, Osta MA, Christophides GK. The complement system of malaria vector mosquitoes. In: Jurenka R, editor. Advances in Insect Physiology. Cambridge, Academic Press, USA; 2016. pp 223–242.

6. Cerenius L, Soderhall K. The prophenoloxidase-activating system in invertebrates. Immunol Rev. 2004; 198: 116–126. doi: 10.1111/j.0105-2896.2004.00116.x 15199959

7. Christensen BM, Li J, Chen CC, Nappi AJ. Melanization immune responses in mosquito vectors. Trends Parasitol. 2005; 21: 192–199. doi: 10.1016/j.pt.2005.02.007 15780842

8. Vavricka CJ, Christensen BM, Li J. Melanization in living organisms: a perspective of species evolution. Protein Cell. 2010; 1: 830–841. doi: 10.1007/s13238-010-0109-8 21203925

9. Cao X, Gulati M, Jiang H. Serine protease-related proteins in the malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol. 2017; 88: 48–62. doi: 10.1016/j.ibmb.2017.07.008 28780069

10. Waterhouse RM, Kriventseva EV, Meister S, Xi Z, Alvarez KS, Bartholomay LC, et al. Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Science. 2007; 316: 1738–1743. doi: 10.1126/science.1139862 17588928

11. Gupta S, Wang Y, Jiang H. Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously. Insect Biochem Mol Biol. 2005; 35: 241–248. doi: 10.1016/j.ibmb.2004.12.003 15705503

12. Wang Y, Lu Z, Jiang H. Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs. Insect Biochem Mol Biol. 2014; 50: 82–91. doi: 10.1016/j.ibmb.2014.04.005 24768974

13. Yu XQ, Jiang H, Wang Y, Kanost MR. Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol. 2003; 33: 197–208. doi: 10.1016/s0965-1748(02)00191-1 12535678

14. Lu Z, Jiang H. Expression of Manduca sexta serine proteinase homolog precursors in insect cells and their proteolytic activation. Insect Biochem Mol Biol. 2008; 38: 89–98. doi: 10.1016/j.ibmb.2007.09.011 18070668

15. Wang Y, Jiang H. A positive feedback mechanism in the Manduca sexta prophenoloxidase activation system. Insect Biochem Mol Biol. 2008; 38: 763–769. doi: 10.1016/j.ibmb.2008.04.008 18625399

16. Lee KY, Zhang R, Kim MS, Park JW, Park HY, Kawabata S, et al. A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae. Eur J Biochem. 2002; 269: 4375–4383. doi: 10.1046/j.1432-1033.2002.03155.x 12199717

17. Kim MS, Baek MJ, Lee MH, Park JW, Lee SY, Soderhall K, et al. A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae. J Biol Chem. 2002; 277: 39999–40004. doi: 10.1074/jbc.M205508200 12185078

18. Piao S, Song YL, Kim JH, Park SY, Park JW, Lee BL, et al. Crystal structure of a clip-domain serine protease and functional roles of the clip domains. EMBO J. 2005; 24: 4404–4414. doi: 10.1038/sj.emboj.7600891 16362048

19. Povelones M, Bhagavatula L, Yassine H, Tan LA, Upton LM, Osta MA, et al. The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae. PLoS Pathog. 2013; 9: e1003623. doi: 10.1371/journal.ppat.1003623 24039584

20. Yassine H, Kamareddine L, Chamat S, Christophides GK, Osta MA. A serine protease homolog negatively regulates TEP1 consumption in systemic infections of the malaria vector Anopheles gambiae. J Innate Immun. 2014; 6: 806–818. doi: 10.1159/000363296 25012124

21. Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, et al. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell. 2004; 116: 661–670. doi: 10.1016/s0092-8674(04)00173-4 15006349

22. Nakhleh J, Christophides GK, Osta MA. The serine protease homolog CLIPA14 modulates the intensity of the immune response in the mosquito Anopheles gambiae. J Biol Chem. 2017; 292: 18217–18226. doi: 10.1074/jbc.M117.797787 28928218

23. Povelones M, Upton LM, Sala KA, Christophides GK. Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1. PLoS Pathog. 2011; 7: e1002023. doi: 10.1371/journal.ppat.1002023 21533217

24. Oliveira Gde A, Lieberman J, Barillas-Mury C. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science. 2012; 335: 856–859. doi: 10.1126/science.1209678 22282475

25. Castillo JC, Ferreira ABB, Trisnadi N, Barillas-Mury C. Activation of mosquito complement antiplasmodial response requires cellular immunity. Sci Immunol. 2017; 2.

26. Fraiture M, Baxter RH, Steinert S, Chelliah Y, Frolet C, Quispe-Tintaya W, et al. Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium. Cell Host Microbe. 2009; 5: 273–284. doi: 10.1016/j.chom.2009.01.005 19286136

27. Povelones M, Waterhouse RM, Kafatos FC, Christophides GK. Leucine-rich repeat protein complex activates mosquito complement in defense against Plasmodium parasites. Science. 2009; 324: 258–261. doi: 10.1126/science.1171400 19264986

28. Schnitger AK, Kafatos FC, Osta MA. The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections. J Biol Chem. 2007; 282: 21884–21888. doi: 10.1074/jbc.M701635200 17537726

29. An C, Budd A, Kanost MR, Michel K. Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes. Cell Mol Life Sci. 2011; 68: 1929–1939. doi: 10.1007/s00018-010-0543-z 20953892

30. Volz J, Muller HM, Zdanowicz A, Kafatos FC, Osta MA. A genetic module regulates the melanization response of Anopheles to Plasmodium. Cell Microbiol. 2006; 8: 1392–1405. doi: 10.1111/j.1462-5822.2006.00718.x 16922859

31. Volz J, Osta MA, Kafatos FC, Muller HM. The roles of two clip domain serine proteases in innate immune responses of the malaria vector Anopheles gambiae. J Biol Chem. 2005; 280: 40161–40168. doi: 10.1074/jbc.M506191200 16188883

32. Zhang X, An C, Sprigg K, Michel K. CLIPB8 is part of the prophenoloxidase activation system in Anopheles gambiae mosquitoes. Insect Biochem Mol Biol. 2016; 71: 106–115. doi: 10.1016/j.ibmb.2016.02.008 26926112

33. Osta MA, Christophides GK, Kafatos FC. Effects of mosquito genes on Plasmodium development. Science. 2004; 303: 2030–2032. doi: 10.1126/science.1091789 15044804

34. Koutsos AC, Blass C, Meister S, Schmidt S, MacCallum RM, Soares MB, et al. Life cycle transcriptome of the malaria mosquito Anopheles gambiae and comparison with the fruitfly Drosophila melanogaster. Proc Natl Acad Sci U S A. 2007; 104: 11304–11309. doi: 10.1073/pnas.0703988104 17563388

35. Simoes ML, Mlambo G, Tripathi A, Dong Y, Dimopoulos G. Immune regulation of Plasmodium is Anopheles species specific and infection intensity dependent. MBio. 2017; 8.

36. Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G. Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog. 2006; 2: e52. doi: 10.1371/journal.ppat.0020052 16789837

37. Mendes AM, Awono-Ambene PH, Nsango SE, Cohuet A, Fontenille D, Kafatos FC, et al. Infection intensity-dependent responses of Anopheles gambiae to the African malaria parasite Plasmodium falciparum. Infect Immun. 2011; 79: 4708–4715. doi: 10.1128/IAI.05647-11 21844236

38. Cohuet A, Osta MA, Morlais I, Awono-Ambene PH, Michel K, Simard F, et al. Anopheles and Plasmodium: from laboratory models to natural systems in the field. EMBO Rep. 2006; 7: 1285–1289. doi: 10.1038/sj.embor.7400831 17099691

39. Yassine H, Kamareddine L, Osta MA. The mosquito melanization response is implicated in defense against the entomopathogenic fungus Beauveria bassiana. PLoS Pathog. 2012; 8: e1003029. doi: 10.1371/journal.ppat.1003029 23166497

40. Kurata S, Ariki S, Kawabata S. Recognition of pathogens and activation of immune responses in Drosophila and horseshoe crab innate immunity. Immunobiology. 2006; 211: 237–249. doi: 10.1016/j.imbio.2005.10.016 16697917

41. An C, Ishibashi J, Ragan EJ, Jiang H, Kanost MR. Functions of Manduca sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways. J Biol Chem. 2009; 284: 19716–19726. doi: 10.1074/jbc.M109.007112 19487692

42. Kan H, Kim CH, Kwon HM, Park JW, Roh KB, Lee H, et al. Molecular control of phenoloxidase-induced melanin synthesis in an insect. J Biol Chem. 2008; 283: 25316–25323. doi: 10.1074/jbc.M804364200 18628205

43. Kim CH, Kim SJ, Kan H, Kwon HM, Roh KB, Jiang R, et al. A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect. J Biol Chem. 2008; 283: 7599–7607. doi: 10.1074/jbc.M710216200 18195005

44. Rhodes VL, Thomas MB, Michel K. The interplay between dose and immune system activation determines fungal infection outcome in the African malaria mosquito, Anopheles gambiae. Dev Comp Immunol. 2018; 85: 125–133. doi: 10.1016/j.dci.2018.04.008 29649553

45. Shin SW, Kokoza V, Bian G, Cheon HM, Kim YJ, Raikhel AS. REL1, a homologue of Drosophila dorsal, regulates toll antifungal immune pathway in the female mosquito Aedes aegypti. J Biol Chem. 2005; 280: 16499–16507. doi: 10.1074/jbc.M500711200 15722339

46. Michel K, Budd A, Pinto S, Gibson TJ, Kafatos FC. Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei. EMBO Rep. 2005; 6: 891–897. doi: 10.1038/sj.embor.7400478 16113656

47. De Gregorio E, Han SJ, Lee WJ, Baek MJ, Osaki T, Kawabata S, et al. An immune-responsive Serpin regulates the melanization cascade in Drosophila. Dev Cell. 2002; 3: 581–592. doi: 10.1016/s1534-5807(02)00267-8 12408809

48. Ligoxygakis P, Pelte N, Ji C, Leclerc V, Duvic B, Belvin M, et al. A serpin mutant links Toll activation to melanization in the host defence of Drosophila. EMBO J. 2002; 21: 6330–6337. doi: 10.1093/emboj/cdf661 12456640

49. Reichhart JM, Gubb D, Leclerc V. The Drosophila serpins: multiple functions in immunity and morphogenesis. Methods Enzymol. 2011; 499: 205–225. doi: 10.1016/B978-0-12-386471-0.00011-0 21683256

50. Soukup SF, Culi J, Gubb D. Uptake of the necrotic serpin in Drosophila melanogaster via the lipophorin receptor-1. PLoS Genet. 2009; 5: e1000532. doi: 10.1371/journal.pgen.1000532 19557185

51. Tong Y, Jiang H, Kanost MR. Identification of plasma proteases inhibited by Manduca sexta serpin-4 and serpin-5 and their association with components of the prophenol oxidase activation pathway. J Biol Chem. 2005; 280: 14932–14942. doi: 10.1074/jbc.M500532200 15695806

52. Dudzic JP, Hanson MA, Iatsenko I, Kondo S, Lemaitre B. More Than Black or White: Melanization and Toll share regulatory serine proteases in Drosophila. Cell Rep. 2019; 27: 1050–1061 e1053. doi: 10.1016/j.celrep.2019.03.101 31018123

53. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, van der Keur M, van der Linden R, et al. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Mol Biochem Parasitol. 2004; 137: 23–33. doi: 10.1016/j.molbiopara.2004.04.007 15279948

54. Blandin S, Moita LF, Kocher T, Wilm M, Kafatos FC, Levashina EA. Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene. EMBO Rep. 2002; 3: 852–856. doi: 10.1093/embo-reports/kvf180 12189180

55. Kamareddine L, Nakhleh J, Osta MA. Functional interaction between Apolipophorins and Complement regulate the mosquito immune response to systemic infections. J Innate Immun. 2016; 8: 314–326. doi: 10.1159/000443883 26950600

56. Labrousse A, Chauvet S, Couillault C, Kurz CL, Ewbank JJ. Caenorhabditis elegans is a model host for Salmonella typhimurium. Curr Biol. 2000; 10: 1543–1545. doi: 10.1016/s0960-9822(00)00833-2 11114526

57. Kamareddine L, Fan Y, Osta MA, Keyhani NO. Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito. Parasit Vectors. 2013; 6: 22. doi: 10.1186/1756-3305-6-22 23336669

58. Osta MA, Rizk ZJ, Labbe P, Weill M, Knio K. Insecticide resistance to organophosphates in Culex pipiens complex from Lebanon. Parasit Vectors. 2012; 5: 132. doi: 10.1186/1756-3305-5-132 22759898

59. Bell AS, Blanford S, Jenkins N, Thomas MB, Read AF. Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: technique validation and first applications. J Invertebr Pathol. 2009; 100: 160–168. doi: 10.1016/j.jip.2009.01.006 19320043

60. Garver LS, de Almeida Oliveira G, Barillas-Mury C. The JNK pathway is a key mediator of Anopheles gambiae antiplasmodial immunity. PLoS Pathog. 2013; 9: e1003622. doi: 10.1371/journal.ppat.1003622 24039583

61. Muller HM, Dimopoulos G, Blass C, Kafatos FC. A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes. J Biol Chem. 1999; 274: 11727–11735. doi: 10.1074/jbc.274.17.11727 10206988