Skip to main content

Advertisement

SpringerLink
Log in

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

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

dKD:

Double knockdown

IEARpNA:

Acetyl-Ile-Glu-Ala-Arg-p-nitroanilide

KD:

Knockdown

KS:

Kolmogorov–Smirnov

LLA:

Late-life acting

MS:

Mass spectrometry

PPO:

Prophenoloxidase

References

  1. Enayati A, Hemingway J (2010) Malaria management: past, present, and future. Annu Rev Entomol 55:569–591

    Article  PubMed  CAS  Google Scholar 

  2. Kelly-Hope L, Ranson H, Hemingway J (2008) Lessons from the past: managing insecticide resistance in malaria control and eradication programmes. Lancet Infect Dis 8:387–389

    Article  PubMed  Google Scholar 

  3. Hemingway J, Ranson H (2000) Insecticide resistance in insect vectors of human disease. Annu Rev Entomol 45:371–391

    Article  PubMed  CAS  Google Scholar 

  4. Read AF, Lynch PA, Thomas MB (2009) How to make evolution-proof insecticides for malaria control. PLoS Biol 7:e1000058

    Article  PubMed  Google Scholar 

  5. Blanford S, Chan BH, Jenkins N, Sim D, Turner RJ, Read AF, Thomas MB (2005) Fungal pathogen reduces potential for malaria transmission. Science 308:1638–1641

    Article  PubMed  CAS  Google Scholar 

  6. Michel K, Budd A, Pinto S, Gibson TJ, Kafatos FC (2005) Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei. EMBO Rep 6:891–897

    Article  PubMed  CAS  Google Scholar 

  7. Kanost M, Gorman MJ (2008) In: Beckage NE (ed.) Insect immunology. Academic Press/Elsevier, San Diego, pp 69–96

  8. Satoh D, Horii A, Ochiai M, Ashida M (1999) Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning. J Biol Chem 274:7441–7453

    Article  PubMed  CAS  Google Scholar 

  9. Kan H, Kim CH, Kwon HM, Park JW, Roh KB, Lee H, Park BJ, Zhang R, Zhang J, Soderhall K, Ha NC, Lee BL (2008) Molecular control of phenoloxidase-induced melanin synthesis in an insect. J Biol Chem 283:25316

    Article  PubMed  CAS  Google Scholar 

  10. Tang H, Kambris Z, Lemaitre B, Hashimoto C (2006) Two proteases defining a melanization cascade in the immune system of Drosophila. J Biol Chem 281:28097

    Article  PubMed  CAS  Google Scholar 

  11. Nappi A, Poirie M, Carton Y (2009) The role of melanization and cytotoxic by-products in the cellular immune responses of Drosophila against parasitic wasps. Adv Parasitol 70:99–121

    Article  PubMed  CAS  Google Scholar 

  12. Zhu Y, Wang Y, Gorman MJ, Jiang H, Kanost MR (2003) Manduca sexta serpin-3 regulates prophenoloxidase activation in response to infection by inhibiting prophenoloxidase-activating proteinases. J Biol Chem 278:46556–46564

    Article  PubMed  CAS  Google Scholar 

  13. De Gregorio E, Han SJ, Lee WJ, Baek MJ, Osaki T, Kawabata S, Lee BL, Iwanaga S, Lemaitre B, Brey PT (2002) An immune-responsive serpin regulates the melanization cascade in Drosophila. Dev Cell 3:581–592

    Article  PubMed  Google Scholar 

  14. Ligoxygakis P, Pelte N, Ji C, Leclerc V, Duvic B, Belvin M, Jiang H, Hoffmann JA, Reichhart JM (2002) A serpin mutant links Toll activation to melanization in the host defence of Drosophila. EMBO J 21:6330–6337

    Article  PubMed  CAS  Google Scholar 

  15. Zou Z, Shin SW, Alvarez KS, Kokoza V, Raikhel AS (2010) Distinct melanization pathways in the mosquito Aedes aegypti. Immunity 32:41–53

    Article  PubMed  CAS  Google Scholar 

  16. Gettins PG (2002) Serpin structure, mechanism, and function. Chem Rev 102:4751–4804

    Article  PubMed  CAS  Google Scholar 

  17. Michel K, Suwanchaichinda C, Morlais I, Lambrechts L, Cohuet A, Awono-Ambene PH, Simard F, Fontenille D, Kanost MR, Kafatos FC (2006) Increased melanizing activity in Anopheles gambiae does not affect development of Plasmodium falciparum. Proc Natl Acad Sci USA 103:16858–16863

    Article  PubMed  CAS  Google Scholar 

  18. Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, Brey PT, Collins FH, Danielli A, Dimopoulos G, Hetru C, Hoa NT, Hoffmann JA, Kanzok SM, Letunic I, Levashina EA, Loukeris TG, Lycett G, Meister S, Michel K, Moita LF, Muller HM, Osta MA, Paskewitz SM, Reichhart JM, Rzhetsky A, Troxler L, Vernick KD, Vlachou D, Volz J, von Mering C, Xu J, Zheng L, Bork P, Kafatos FC (2002) Immunity-related genes and gene families in Anopheles gambiae. Science 298:159–165

    Article  PubMed  CAS  Google Scholar 

  19. Volz J, Muller HM, Zdanowicz A, Kafatos FC, Osta MA (2006) A genetic module regulates the melanization response of Anopheles to Plasmodium. Cellular microbiology 8:1392

    Article  PubMed  CAS  Google Scholar 

  20. Volz J, Osta MA, Kafatos FC, Muller HM (2005) The roles of two clip domain serine proteases in innate immune responses of the malaria vector Anopheles gambiae. J Biol Chem 280:40161–40168

    Article  PubMed  CAS  Google Scholar 

  21. Paskewitz SM, Andreev O, Shi L (2006) Gene silencing of serine proteases affects melanization of Sephadex beads in Anopheles gambiae. Insect Biochem Mol Biol 36:701–711

    Article  PubMed  CAS  Google Scholar 

  22. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113

    Article  PubMed  Google Scholar 

  23. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Article  PubMed  CAS  Google Scholar 

  24. Clamp M, Cuff J, Searle SM, Barton GJ (2004) The Jalview Java alignment editor. Bioinformatics 20:426–427

    Article  PubMed  CAS  Google Scholar 

  25. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191

    Article  PubMed  CAS  Google Scholar 

  26. Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21:2104–2105

    Article  PubMed  CAS  Google Scholar 

  27. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690

    Article  PubMed  CAS  Google Scholar 

  28. Jiang H, Mulnix AB, Kanost MR (1995) Expression and characterization of recombinant Manduca sexta serpin-1B and site-directed mutants that change its inhibitory selectivity. Insect Biochem Mol Biol 25:1093–1100

    Article  PubMed  CAS  Google Scholar 

  29. Jiang H, Wang Y, Korochkina SE, Benes H, Kanost MR (1997) Molecular cloning of cDNAs for two pro-phenol oxidase subunits from the malaria vector, Anopheles gambiae. Insect Biochem Mol Biol 27:693–699

    Article  PubMed  CAS  Google Scholar 

  30. An C, Jiang H, Kanost MR (2010) Proteolytic activation and function of the cytokine Spatzle in the innate immune response of a lepidopteran insect, Manduca sexta. FEBS J 277:148

    Article  PubMed  CAS  Google Scholar 

  31. Schick C, Kamachi Y, Bartuski AJ, Cataltepe S, Schechter NM, Pemberton PA, Silverman GA (1997) Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase. J Biol Chem 272:1849

    Article  PubMed  CAS  Google Scholar 

  32. Blandin S, Moita LF, Kocher T, Wilm M, Kafatos FC, Levashina EA (2002) Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene. EMBO Rep 3:852–856

    Article  PubMed  CAS  Google Scholar 

  33. Wilkinson M, McInerney JO, Hirt RP, Foster PG, Embley TM (2007) Of clades and clans: terms for phylogenetic relationships in unrooted trees. Trends Ecol Evol 22:114–115

    Article  PubMed  Google Scholar 

  34. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704

    Article  PubMed  Google Scholar 

  35. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574

    Article  PubMed  CAS  Google Scholar 

  36. Li JS, Ruyl Kim S, Christensen BM, Li J (2005) Purification and primary structural characterization of prophenoloxidases from Aedes aegypti larvae. Insect Biochem Mol Biol 35:1269–1283

    Article  PubMed  CAS  Google Scholar 

  37. Jiang H, Wang Y, Ma C, Kanost MR (1997) Subunit composition of pro-phenol oxidase from Manduca sexta: molecular cloning of subunit ProPO-P1. Insect Biochem Mol Biol 27:835–850

    Article  PubMed  CAS  Google Scholar 

  38. Huang L, Sternberg PW. In: Community TCeR (ed.) WormBook

  39. Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, Collins WE, Campbell CC, Gwadz RW (1986) Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 234:607–610

    Article  PubMed  CAS  Google Scholar 

  40. Lambrechts L, Morlais I, Awono-Ambene PH, Cohuet A, Simard F, Jacques JC, Bourgouin C, Koella JC (2007) Effect of infection by Plasmodium falciparum on the melanization immune response of Anopheles gambiae. Am J Trop Med Hyg 76:475–480

    PubMed  Google Scholar 

  41. Riehle MM, Markianos K, Niare O, Xu J, Li J, Toure AM, Podiougou B, Oduol F, Diawara S, Diallo M, Coulibaly B, Ouatara A, Kruglyak L, Traore SF, Vernick KD (2006) Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region. Science 312:577–579

    Article  PubMed  CAS  Google Scholar 

  42. Muller HM, Dimopoulos G, Blass C, Kafatos FC (1999) A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes. J Biol Chem 274:11727–11735

    Article  PubMed  CAS  Google Scholar 

  43. Pinto SB, Lombardo F, Koutsos AC, Waterhouse RM, McKay K, An C, Ramakrishnan C, Kafatos FC, Michel K (2009) Discovery of Plasmodium modulators by genome-wide analysis of circulating hemocytes in Anopheles gambiae. Proc Natl Acad Sci USA 106:21270–21275

    Article  PubMed  CAS  Google Scholar 

  44. Jiang H, Wang Y, Yu XQ, Zhu Y, Kanost M (2003) Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1 J and serine proteinase homologs. Insect Biochem Mol Biol 33:1049–1060

    Article  PubMed  CAS  Google Scholar 

  45. Pike RN, Buckle AM, le Bonniec BF, Church FC (2005) Control of the coagulation system by serpins. Getting by with a little help from glycosaminoglycans. FEBS J 272:4842–4851

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. M. Gorman for purified M. sexta PPO and T. Graves, K. Kjos, R. Woolsey, and G. Hammon for mosquito rearing. Thanks go to Drs. J. Tomich and Y. Hiromasa at the KSU Proteomics Core Laboratory for protein digestion and mass spectrometry. Real-time PCR analyses were performed at the COBRE Core I, KSU. This work was supported by NIH grants 3P20RR017708-07S1 and P20RR017686 subawards to K.M. and GM41247 to M.K. This is contribution 10-368-J from the Kansas Agricultural Experiment Station.

Author information

Authors and Affiliations

  1. Division of Biology, Kansas State University, 271 Chalmers Hall, Manhattan, KS, 66506, USA

    Chunju An

  2. Division of Biology, Kansas State University, 267 Chalmers Hall, Manhattan, KS, 66506, USA

    Kristin Michel

  3. European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany

    Aidan Budd

  4. Department of Biochemistry, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA

    Michael R. Kanost

Authors
  1. Chunju An
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aidan Budd
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael R. Kanost
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kristin Michel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kristin Michel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

An, C., Budd, A., Kanost, M.R. et al. Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes. Cell. Mol. Life Sci. 68, 1929–1939 (2011). https://doi.org/10.1007/s00018-010-0543-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-010-0543-z

Keywords

Search

Navigation