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Drosophila innate immunity: regional and functional specialization of prophenoloxidases.

Dudzic JP, Kondo S, Ueda R, Bergman CM, Lemaitre B - BMC Biol. (2015)

Bottom Line: Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation.Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps.We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions.

View Article: PubMed Central - PubMed

Affiliation: Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 19, 1015, Lausanne, Switzerland. jan.dudzic@epfl.ch.

ABSTRACT

Background: The diversification of immune systems during evolution involves the expansion of particular gene families in given phyla. A better understanding of the metazoan immune system requires an analysis of the logic underlying such immune gene amplification. This analysis is now within reach due to the ease with which we can generate multiple mutations in an organism. In this paper, we analyze the contribution of the three Drosophila prophenoloxidases (PPOs) to host defense by generating single, double and triple mutants. PPOs are enzymes that catalyze the production of melanin at the site of infection and around parasites. They are the rate-limiting enzymes that contribute to the melanization reaction, a major immune mechanism of arthropods. The number of PPO-encoding genes is variable among insects, ranging from one in the bee to ten in the mosquito.

Results: By analyzing mutations alone and in combination, we ascribe a specific function to each of the three PPOs of Drosophila. Our study confirms that two PPOs produced by crystal cells, PPO1 and PPO2, contribute to the bulk of melanization in the hemolymph, upon septic or clean injury. In contrast, PPO3, a PPO restricted to the D. melanogaster group, is expressed in lamellocytes and contributes to melanization during the encapsulation process. Interestingly, another overlapping set of PPOs, PPO2 and PPO3, achieve melanization of the capsule upon parasitoid wasp infection.

Conclusions: The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish fully a particular function. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation. Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps. We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions.

No MeSH data available.


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Both PPO1 and PPO2 but not PPO3 contribute to injury-related melanization in adults and larvae. Melanization of adults (a) and larvae (b) after clean injury is abolished only in the simultaneous absence of PPO1 and PPO2. A normal melanization spot was observed in the two PPO3 mutants. Arrows indicate the pricking site. Adults and larvae were wounded with a tungsten needle and blackening of the wound was recorded 1 h later in larvae and 16 h later in adults. A representative picture is shown for each genotype
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Fig2: Both PPO1 and PPO2 but not PPO3 contribute to injury-related melanization in adults and larvae. Melanization of adults (a) and larvae (b) after clean injury is abolished only in the simultaneous absence of PPO1 and PPO2. A normal melanization spot was observed in the two PPO3 mutants. Arrows indicate the pricking site. Adults and larvae were wounded with a tungsten needle and blackening of the wound was recorded 1 h later in larvae and 16 h later in adults. A representative picture is shown for each genotype

Mentions: Using a needle to injure wild-type larvae or adults induces a melanization spot at the wound site, the extent of which is usually proportional to the injury size. This blackening reaction results from de novo melanin synthesis catalyzed by PO and is further enhanced by the presence of microbial products [22]. Our previous study attributed all hemolymphatic PO activity to PPO1 and PPO2 due to the absence of melanization in the PPO1Δ,2Δ,(31) stocks, while single PPO1 or PPO2 mutants showed only reduced or almost normal melanization, respectively. Consistent with this finding, no melanization spot on the cuticle of injured PPO1Δ,2Δ and PPO1Δ,2Δ,31 mutants was observed (Fig. 2a, b). In contrast, PPO3 mutants show a wild-type level of melanization in injured larvae and adults. We next measured PO activity via the L-DOPA assay in hemolymph samples extracted from larvae. We found no significant difference in PO activity between PPO31 and wild-type hemolymph samples. In contrast, hemolymph samples of PPO1,2 deficient larvae show no PO activity, indicating that PPO3 alone is not sufficient to produce PO activity in hemolymph (Additional file 1: Figure S1). This confirms that PPO1 and PPO2 together produce all injury-mediated melanization in larvae and adults [2]. Survival analyses had shown that PO is required for resistance to microbial infection, notably to Gram-positive bacteria and fungi [2]. Survival analyses using the new fly lines now show that the strict PPO1Δ,2Δ double mutation recapitulates all the phenotypes previously described using PPO1Δ,2Δ,(31): PPO1Δ,2Δ flies are more susceptible to septic injury with the Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis and exhibit a mild susceptibility to natural infection with the fungus Beauveria bassiana (Fig. 3). In contrast, PPO3SK3 flies exhibit a wild-type level of resistance upon challenge with the same microbes. These experiments confirm the important role of melanization in fighting infection by Gram-positive bacteria and fungi, and are consistent with our previous results indicating that PPO1 and 2 are the sole sources for hemolymphatic PO. Additional data (Fig. 4a, b) show that PPO31 does not markedly affect the Toll and Imd pathways in adults as revealed by the wild-type inducibility of Diptericin and Drosomycin, their respective read-out genes, in this mutant.Fig. 2


Drosophila innate immunity: regional and functional specialization of prophenoloxidases.

Dudzic JP, Kondo S, Ueda R, Bergman CM, Lemaitre B - BMC Biol. (2015)

Both PPO1 and PPO2 but not PPO3 contribute to injury-related melanization in adults and larvae. Melanization of adults (a) and larvae (b) after clean injury is abolished only in the simultaneous absence of PPO1 and PPO2. A normal melanization spot was observed in the two PPO3 mutants. Arrows indicate the pricking site. Adults and larvae were wounded with a tungsten needle and blackening of the wound was recorded 1 h later in larvae and 16 h later in adults. A representative picture is shown for each genotype
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595066&req=5

Fig2: Both PPO1 and PPO2 but not PPO3 contribute to injury-related melanization in adults and larvae. Melanization of adults (a) and larvae (b) after clean injury is abolished only in the simultaneous absence of PPO1 and PPO2. A normal melanization spot was observed in the two PPO3 mutants. Arrows indicate the pricking site. Adults and larvae were wounded with a tungsten needle and blackening of the wound was recorded 1 h later in larvae and 16 h later in adults. A representative picture is shown for each genotype
Mentions: Using a needle to injure wild-type larvae or adults induces a melanization spot at the wound site, the extent of which is usually proportional to the injury size. This blackening reaction results from de novo melanin synthesis catalyzed by PO and is further enhanced by the presence of microbial products [22]. Our previous study attributed all hemolymphatic PO activity to PPO1 and PPO2 due to the absence of melanization in the PPO1Δ,2Δ,(31) stocks, while single PPO1 or PPO2 mutants showed only reduced or almost normal melanization, respectively. Consistent with this finding, no melanization spot on the cuticle of injured PPO1Δ,2Δ and PPO1Δ,2Δ,31 mutants was observed (Fig. 2a, b). In contrast, PPO3 mutants show a wild-type level of melanization in injured larvae and adults. We next measured PO activity via the L-DOPA assay in hemolymph samples extracted from larvae. We found no significant difference in PO activity between PPO31 and wild-type hemolymph samples. In contrast, hemolymph samples of PPO1,2 deficient larvae show no PO activity, indicating that PPO3 alone is not sufficient to produce PO activity in hemolymph (Additional file 1: Figure S1). This confirms that PPO1 and PPO2 together produce all injury-mediated melanization in larvae and adults [2]. Survival analyses had shown that PO is required for resistance to microbial infection, notably to Gram-positive bacteria and fungi [2]. Survival analyses using the new fly lines now show that the strict PPO1Δ,2Δ double mutation recapitulates all the phenotypes previously described using PPO1Δ,2Δ,(31): PPO1Δ,2Δ flies are more susceptible to septic injury with the Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis and exhibit a mild susceptibility to natural infection with the fungus Beauveria bassiana (Fig. 3). In contrast, PPO3SK3 flies exhibit a wild-type level of resistance upon challenge with the same microbes. These experiments confirm the important role of melanization in fighting infection by Gram-positive bacteria and fungi, and are consistent with our previous results indicating that PPO1 and 2 are the sole sources for hemolymphatic PO. Additional data (Fig. 4a, b) show that PPO31 does not markedly affect the Toll and Imd pathways in adults as revealed by the wild-type inducibility of Diptericin and Drosomycin, their respective read-out genes, in this mutant.Fig. 2

Bottom Line: Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation.Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps.We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions.

View Article: PubMed Central - PubMed

Affiliation: Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 19, 1015, Lausanne, Switzerland. jan.dudzic@epfl.ch.

ABSTRACT

Background: The diversification of immune systems during evolution involves the expansion of particular gene families in given phyla. A better understanding of the metazoan immune system requires an analysis of the logic underlying such immune gene amplification. This analysis is now within reach due to the ease with which we can generate multiple mutations in an organism. In this paper, we analyze the contribution of the three Drosophila prophenoloxidases (PPOs) to host defense by generating single, double and triple mutants. PPOs are enzymes that catalyze the production of melanin at the site of infection and around parasites. They are the rate-limiting enzymes that contribute to the melanization reaction, a major immune mechanism of arthropods. The number of PPO-encoding genes is variable among insects, ranging from one in the bee to ten in the mosquito.

Results: By analyzing mutations alone and in combination, we ascribe a specific function to each of the three PPOs of Drosophila. Our study confirms that two PPOs produced by crystal cells, PPO1 and PPO2, contribute to the bulk of melanization in the hemolymph, upon septic or clean injury. In contrast, PPO3, a PPO restricted to the D. melanogaster group, is expressed in lamellocytes and contributes to melanization during the encapsulation process. Interestingly, another overlapping set of PPOs, PPO2 and PPO3, achieve melanization of the capsule upon parasitoid wasp infection.

Conclusions: The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish fully a particular function. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation. Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps. We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions.

No MeSH data available.


Related in: MedlinePlus