Reference

An immune-responsive Serpin regulates the melanization cascade in Drosophila.

De Gregorio E Han SJ Lee WJ Baek MJ Osaki T Kawabata S Lee BL Iwanaga S Lemaitre B Brey PT
Developmental cell. 2002.

Abstract

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.

Main claims

  1. Spn27A is a negative regulator of the melanization cascade required to prevent excessive melanization in response to injury or infection. [v1]
    Supported using null mutant of Spn27A by (Ligoxygakis et al., 2002) and (Bidla et al., 2009)

    Last change on 2022-06-21 14:03 by Hannah Westlake Orcid 0009-0000-0013-8980

Major claims

  1. Spn27A is an acute immune-responsive gene regulated by the Toll pathway [v1]
    Previously shown by same authors (De Gregorio et al., 2002), confirmed by (Ligoxygakis et al., 2002)

    Last change on 2022-06-21 14:03 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. • Northern blot showed that Spn27A is maximally induced 3h following mixed bacterial or fungal infection [v1]

      To be assessed

      Last change on 2022-06-21 14:13 by Hannah Westlake Orcid 0009-0000-0013-8980

    2. • Expression of Spn27A is abolished in Spz mutants [v1]

      To be assessed

      Last change on 2022-06-21 14:13 by Hannah Westlake Orcid 0009-0000-0013-8980

    3. • MALDI showed that Spn27A is post-translationally modified to produce a 66.59 kDa protein [v1]

      To be assessed

      Last change on 2022-06-21 14:14 by Hannah Westlake Orcid 0009-0000-0013-8980

    4. • Spn27A was found in culture medium of S2 cells, suggesting it is secreted [v1]

      To be assessed

      Last change on 2022-06-21 14:14 by Hannah Westlake Orcid 0009-0000-0013-8980

  2. Serpin-27A regulates the melanization cascade through the specific inhibition of PPO processing by the terminal serine protease PPAE (prophenoloxidase activating enzyme). [v1]
    The target SP of Serpin 27A is not known; evidence indicates that Spn27A directly targets Sp7(MP2/PAE1) but probably also other serine proteases (An et al., 2013).

    Last change on 2022-06-21 14:07 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. • The C-terminal region of Spn27A is homologous to the region around the conserved cleavage site of insect PPOs [v1]

      To be assessed

      Last change on 2022-06-21 14:12 by Hannah Westlake Orcid 0009-0000-0013-8980

    2. • Addition of rSpn27A to Drosophila pupal homogenates completely blocked PO activity [v1]

      To be assessed

      Last change on 2022-06-21 14:12 by Hannah Westlake Orcid 0009-0000-0013-8980

    3. • Addition of rSpn27A to hemolymph plasma of Bombyx mori and Galleria mellonella significantly blocked PPO activation in both species, while Spn43Ac did not [v1]

      To be assessed

      Last change on 2022-06-21 14:12 by Hannah Westlake Orcid 0009-0000-0013-8980

    4. • Mutation of a key Lys at the cleavage site of Spn27A to Ala completely removed its inhibitory effect on PPO activation [v1]

      To be assessed

      Last change on 2022-06-21 14:12 by Hannah Westlake Orcid 0009-0000-0013-8980

    5. • Enzymatic activity of PPAE from Holotrichia diomphalia was strongly inhibited in the presence of rSpn27A, but not a mutated version [v1]

      To be assessed

      Last change on 2022-06-21 14:12 by Hannah Westlake Orcid 0009-0000-0013-8980

    6. • Western blots showed that rSpn27A but not an inactive version inhibited cleavage of PPO [v1]

      To be assessed

      Last change on 2022-06-21 14:12 by Hannah Westlake Orcid 0009-0000-0013-8980

    7. • The Black cells mutation suppresses the Spn27A phenotype; all phenotypes except for female sterility were suppressed in a Bc mutant background [v1]

      Supported by same authors (Binggeli et al., 2014)

      Last change on 2022-06-21 14:12 by Hannah Westlake Orcid 0009-0000-0013-8980

  3. Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing excessive melanization. [v1]
    Supported using null mutant (Bidla et al., 2009; Ligoxygakis et al., 2002).

    Last change on 2022-06-21 14:08 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. • Spn27A1 mutant adults had constitutive melanization of the wings and cuticle, and larvae sometimes had melanotic tumors and melanization of the cuticle and internal organs [v1]

      To be assessed

      Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

    2. • Injury with a needle of Spn27A1 mutant larvae led to uncontrolled hemocoelic melanization within 2h of pricking, leading to high death rates and complete blackening of the larva [v1]

      To be assessed

      Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

    3. • Injury of Spn27A1 adults led to more intense melanization than wild-type, but the phenotype was not as significant as in larvae and only weakly reduced survival [v1]

      To be assessed

      Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

    4. • Injection of rSpn27A at the wound site inhibited melanization in Spn27A1 mutants [v1]

      To be assessed

      Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

    5. • Wasp-parasitization of Spn27A1 larvae induced a strong systemic hemocoelic melanization reaction, but it was not restricted to the encapsulation process as in wild-type larvae [v1]

      To be assessed

      Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

Minor claims

  1. Maternal spn27A expression is required during embryogenesis to prevent sterility of female offspring. [v1]
    Supported by results of (Hashimoto et al., 2003) and (Ligoxygakis et al., 2003) Spn27A is required to inhibit inappropriate cleavage of Easter during development.

    Last change on 2022-06-21 14:08 by Hannah Westlake Orcid 0009-0000-0013-8980

  2. Spn27A may have a role in metamorphosis [v1]
    According to FlyBase modENCODE, Spn27A is very highly expressed during the pupal stage. The decreased viability of these larvae is likely due to the escape of bacteria from the gut during metamorphosis triggering an immune response that is uncontrolled due to the loss of Spn27A, leading to constitutive melanization and death. Similar pupal lethality is also observed in animals lacking hemocytes and is rescued by raising flies in axenic conditions (Arefin et al., 2015; Charroux and Royet, 2009; Defaye et al., 2009). Similarly, environmental microbes can induce melanization in the absence of melanization cascade inhibitors (Tang et al., 2008).

    Last change on 2022-06-21 14:09 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. • Spn27A1 mutant larvae were viable and reached pupal stage, but only 30% successfully eclosed [v1]

      To be assessed

      Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

  3. Spn27A is not required to regulate AMP expression [v1]
    Confirmed by (Ligoxygakis et al., 2002), although these authors saw a weak constitutive expression of Dpt in the Spn27A mutant. AMPs are commonly activated as an indirect result of melanization (e.g (Seisenbacher et al., 2011))

    Last change on 2022-06-21 14:09 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. • The Spn27A1 mutation did not affect Drs or Dpt expression in challenged or unchallenged conditions. [v1]

      To be assessed

      Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

  4. Melanization is required to resist B. bassiana infection [v1]
    (Binggeli et al., 2014) extended these results; note that loss of melanization typically has a less marked effect on survival to pathogens than loss of Imd/Toll (Leclerc et al., 2006), and its contribution becomes more apparent when combined with other immune deficiencies (Tang et al., 2006).

    Last change on 2022-06-21 14:09 by Hannah Westlake Orcid 0009-0000-0013-8980

    1. • Spn27A1 adults were resistant as wild-type to M. luteus, E. coli and A. fumigatus, but were susceptible to B. bassiana (less so than Spz mutants) [v1]

      To be assessed

      Last change on 2022-06-21 14:10 by Hannah Westlake Orcid 0009-0000-0013-8980

    2. • Bc and Bc, Spn27A1 double mutants had similar survival as Spn27A1 mutants [v1]

      To be assessed

      Last change on 2022-06-21 14:10 by Hannah Westlake Orcid 0009-0000-0013-8980

Methods

  1. Monitoring of enzyme activity using chromogenic substrates, gel mobility shift assays, Western blots to monitor cleavage of PPO. Generation of Spn27A1 null mutant by P-element mobilization. Survivals and assessment of AMP expression. [v1]

    Last change on 2022-06-21 14:11 by Hannah Westlake Orcid 0009-0000-0013-8980

Additional context

Not annotated yet

Additional files

Not annotated yet

References

  1. An C, Zhang M, Chu Y, Zhao Z. 2013. Serine Protease MP2 Activates Prophenoloxidase in the Melanization Immune Response of Drosophila melanogaster. PLOS ONE 8:e79533. [DOI link]


    Arefin B, Kucerova L, Krautz R, Kranenburg H, Parvin F, Theopold U. 2015. Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State. PLOS ONE 10:e0136593. [DOI link]


    Bidla G, Hauling T, Dushay MS, Theopold U. 2009. Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors. J Innate Immun 1:301–308. [DOI link]


    Binggeli O, Neyen C, Poidevin M, Lemaitre B. 2014. Prophenoloxidase activation is required for survival to microbial infections in Drosophila. PLoS Pathog 10:e1004067. [DOI link]


    Charroux B, Royet J. 2009. Elimination of plasmatocytes by targeted apoptosis reveals their role in multiple aspects of the Drosophila immune response. Proc Natl Acad Sci U S A 106:9797–9802. [DOI link]


    De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B. 2002. The Toll and Imd pathways are the major regulators of the immune response in Drosophila. EMBO J 21:2568–2579. [DOI link]


    Defaye A, Evans I, Crozatier M, Wood W, Lemaitre B, Leulier F. 2009. Genetic ablation of Drosophila phagocytes reveals their contribution to both development and resistance to bacterial infection. J Innate Immun 1:322–334. [DOI link]


    Hashimoto C, Kim DR, Weiss LA, Miller JW, Morisato D. 2003. Spatial regulation of developmental signaling by a serpin. Dev Cell 5:945–950. [DOI link]


    Leclerc V, Pelte N, El Chamy L, Martinelli C, Ligoxygakis P, Hoffmann JA, Reichhart J-M. 2006. Prophenoloxidase activation is not required for survival to microbial infections in Drosophila. EMBO Rep 7:231–235. [DOI link]


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


    Ligoxygakis P, Roth S, Reichhart J-M. 2003. A Serpin Regulates Dorsal-Ventral Axis Formation in the Drosophila Embryo. Curr Biol 13:2097–2102. [DOI link]


    Seisenbacher G, Hafen E, Stocker H. 2011. MK2-Dependent p38b Signalling Protects Drosophila Hindgut Enterocytes against JNK-Induced Apoptosis under Chronic Stress. PLOS Genet 7:e1002168. [DOI link]


    Tang H, Kambris Z, Lemaitre B, Hashimoto C. 2008. A serpin that regulates immune melanization in the respiratory system of Drosophila. Dev Cell 15:617–626. [DOI link]


    Tang H, Kambris Z, Lemaitre B, Hashimoto C. 2006. Two proteases defining a melanization cascade in the immune system of Drosophila. J Biol Chem 281:28097–28104. [DOI link]

    [v1]

    Last change on 2022-06-21 14:16 by Hannah Westlake Orcid 0009-0000-0013-8980