Figure 1.
Generation and phenotypic characterization of PPO1 and PPO2 deletion mutants.
(A) Schematic representation of PPO1Δ and PPO2Δ deletions. The gene map was adapted from FlyBase and includes PPO1 (top) and PPO2 with its neighboring gene CG13743 (bottom). The PPO2 mutant was generated after the mobilization of the transposable element Mi{ET1}PPO2 MB05593 inserted in the 3′ end of PPO2. The imprecise excision deleted a fragment of 5.2 kb including the 3′ non-coding sequence of the neighboring gene CG13743. PPO1Δ mutant flies were generated by homologous recombination of PPO1 with the w gene. The flanking sequences used for recombination (dotted lines) are indicated. (B) Lifespan analysis of unchallenged flies reveals an increased mortality of PPO1Δ, PPO2Δ double mutants (***, p<0.0005). Each survival curve corresponds to one experiment of 3 samples with 20 flies each. p values were calculated using the Log-rank test.
Figure 2.
PPO2 forms the crystals of crystal cells.
(A) Bright field (BF) and fluorescence micrographs of crystal cells of L3 larvae expressing a Lz-Gal4, UAS-GFP construct combined with the PPO mutations (top panel). Arrows indicate crystals within crystal cells. Immunostaining with antibodies against PPO1 (red), or GFP (green) reveals that PPO1 antibody does not detect PPO1 in the crystals of crystal cells (medium and bottom panels). (B) Immunostaining with antibodies against PPO2 (red), or GFP (green) shows that PPO2 composes the crystals of crystal cells. Specificity of the antibody was confirmed by the absence of signal in crystal cells from PPO2Δ and PPO1Δ, PPO2Δ mutant larvae (medium and bottom panels). Arrows indicate crystals within crystal cells. (C) No melanization of crystal cells was observed in PPO2Δ and PPO1Δ, PPO2Δ double mutant larvae after spontaneous activation of the PPO by incubating larvae at 65°C for 10 minutes. (D) Number of melanized crystal cells found in the posterior region of L3 larvae after heating. No melanized crystal cells were found in PPO2Δ and PPO1Δ, PPO2Δ double mutant larvae. Data were analyzed by t test and values represent the mean ± s.e. of at least 10 different larvae per genotype. (E) PPO1and PPO2 are not detected upon crystal cell depletion. Hemolymph samples were derived from unchallenged F1 progeny of Lz-Gal4, UAS-GFP crossed with UAS-Bax/CyO-GFP larvae. A representative Western Blot analysis using Drosophila anti-PO1 and anti-PO2 antibodies is shown.
Figure 3.
Both PPO1 and PPO2 contribute to injury related melanization in larvae.
(A) Melanization of larvae after clean injury is abolished in the simultaneous absence of PPO1 and PPO2. A reduced melanization spot was observed in PPO1Δ mutants. PPO2Δ mutant larvae displayed a more intense melanization at the wound site when compared to wild-type. Arrows indicate the pricking site. Larvae were wounded with a tungsten needle and blackening of the wound was recorded 30 minutes later. A representative picture is shown for each genotype. (B) PPO1Δ larvae have a defect in melanization rate upon pricking compared to wild-type and PPO2 mutant larvae. CantonsS larvae were used as an additional wild-type control. Larvae were wounded with a tungsten needle and the presence of a blackening wound was recorded every 5 minutes up to 30 minutes. Data were analyzed using the Log rank test and values represent the mean±s.e. of at least three independent experiments of 10 larvae each.
Figure 4.
Both PPO1 and PPO2 contribute to melanization of wasp capsule.
Top panels: representative photos showing infected eggs of A. tabida parasitoid wasp were not melanized in the PPO1Δ, PPO2Δ mutant larvae. Note that A. tabida larvae escape the capsule and develop better in the double-mutant compared to single mutants and wild-type (the position of the larva is indicated with dashed dots). Lower panels: Light micrographs showing melanin deposited on the surface of the eggs of A. tabida removed from the hemocoel of wild-type and PPO mutant larvae at 6 days post-infection. Phalloidin staining reveals the presence of lamellocytes around the egg of wild-type and PPO mutant larvae. Bars = 300 µm.
Figure 5.
Increase of PO activity in absence of PPO2.
(A) Hemolymph PO activity in unchallenged and wounded adult flies. Spontaneous PO activity is abolished in PPO1Δ, PPO2Δ double mutant flies (***, p<0.0005) and strongly reduced in PPO1Δ mutants (**, p<0.005) compared to wild-type. In PPO2Δ deficient flies, a stronger PO activity is observed even in the absence of wounding (*, p<0.05). Adult flies were punctured and 3 h later their hemolymph was examined for PO activity. Hemolymph of wounded Bc flies and unchallenged Spn27A1 flies was used as control. Data were analyzed by t test and values represent the mean±s.e. of three independent experiments. (B) A representative Western Blot analysis using Drosophila anti-PPO1 (top) and anti-PPO2 (bottom) antibodies. Hemolymph samples were collected from adult flies subjected to injury contaminated with a mixture of Gram-positive Micrococcus luteus and Gram-negative Erwinia carotovora carotovora 15 (E. carotovora) bacteria. Absence of PPO1 and PPO2 in the respective single and double mutant flies is confirmed. Of note, higher levels of the mature form of PPO1 were observed in PPO2Δ mutants consistent with the notion that PPO2 negatively impacts PPO1. Hemolymph was extracted from flies 3 h post wounding.
Figure 6.
Epistatic relationships between Serpin 27A and PPOs.
Pictures of larvae (left panel), pupae (center panel) and adults (right panel) were taken in the absence of wounding. Spn27A1 mutants exhibit spontaneous melanization (black arrows) which is suppressed in a PPO1Δ PPO2Δ double mutant background. This uncontrolled melanization is not completely abolished in PPO1Δ, Spn27A1 mutant flies (note the melanized spot indicated by the arrow). A distinctive pattern of melanized spots under the epidermis was observed in Spn27A1, PPO1Δ larvae and pupae. The Spn27A1 melanization was stronger in the absence of PPO2 with pupae turning black and adults presenting black spots and wing defects (not shown).
Figure 7.
Melanization is not required for Toll and Imd pathway activities.
(A) Expression of Diptericin in PPO mutant flies. Total RNA was extracted from animals either uninfected or collected 6 h and 12 h after septic injury with Gram-negative bacteria E. carotovora. Shown are the relative expression levels of Dpt in relation to RpL32. Single and double PPO mutants as well as Bc flies have wild-type Dpt expression levels. The Imd pathway mutant Relish was used as a negative control. (B) Expression of Drosomycin in PPO mutant flies 24 h after septic injury with Gram-positive bacteria M. luteus show that PPO1Δ, PPO2Δ mutant flies (*,p<0.05) have an enhanced Toll pathway activity compared to wild-type. (C) PPO1Δ (*, p<0.05), PPO2Δ (**, p<0.005), and double PPO1Δ, PPO2Δ mutant flies (***, p<0.0005) have an enhanced Toll pathway activity compared to wild- type flies upon infection to heat-inactivated M. luteus. Toll pathway mutant spätzlerm7 was used as a negative control. Shown are the relative expression levels of Drs in relation to RpL32. 100% corresponds to Drs expression level of wild-types flies 24 h after septic injury with the Gram-positive bacteria M. luteus. Data were analyzed using one-way ANOVA and post-test and values represent the mean±s.e. of at least three independent experiments.
Figure 8.
Contribution of PPO1 and PPO2 to wound healing and to host defense.
(A, B) Survival rate of flies following injury with clean needle show that concomitant deletion of PPO1 and PPO2 impairs the capacity of flies to survive severe wounding (p<0.0001) compared to wild-type flies. (C, D) PPO1 and PPO2 are not required to survive septic injury with Gram-negative bacteria (S. typhimurium and E. cloacae). (E, F) PPO1Δ, PPO2Δ mutant flies are less resistant to infections with Gram-positive DAP-type bacteria L. monocytogenes (p = 0.0002) and B. subtilis (p<0.0001) compared to wild-type animals. (G, H) PPO1Δ, PPO2Δ mutant flies are severely affected in their capacity to resist infection with Gram-positive Lys-type bacteria E. faecalis and S. aureus (p<0.0001). PPO1Δ and PPO1Δ, PPO2Δ flies are even more susceptible to S. aureus than spzrm7 flies p<0.0001. PPO1Δ mutant flies also exhibit a reduced survival to S. aureus infection (p<0.0001). (I, J) PPO1Δ, PPO2Δ mutant flies exhibit a moderate susceptibility upon septic injury with the yeast C. albicans (p = 0.0101) and a strong susceptibility to injection of A. fumigatus spores (p<0.0001). PPO2Δ mutant flies also die significantly faster from A. fumigatus infection (p = 0.0018 (K, L) Natural infection with entomopathogenic fungi B. bassiana and M. anisopliae reveals that PPO1Δ, PPO2Δ flies have a reduced survival rate compared to wild-type flies (p<0.0001); so did PPO1Δ single mutant flies (p = 0.0084). Black cells (A, B), Relish E20 (C, D) and spätzlerm7 (E, L), respectively, were used as controls. x-axis: Time post-infection in days; y-axis: Percentage of living flies. Data were analyzed by Log-rank test and values are pooled from three independent experiments. Survival to E. faecalis, S. aureus and B. bassiana have been repeated using fly lines carrying the PPO mutations in an OregonR background (Fig. S4C–E).
Figure 9.
Microbial persistence in wild-type and PPO1Δ, PPO2Δ double mutant flies.
Persistence of E. carotovora (A), S. typhimurium (B), L. monocytogenesis (C), E. faecalis (D), S. aureus (E) and C. albicans (F) were evaluated at different times post- infection in wild-type and PPO1Δ, PPO2Δ mutant flies (referred to as PPOΔ). Statistical analysis reveals no bacterial persistence difference in PPO1Δ, PPO2Δ double mutants compared to wild-type flies upon E. carotovora, S. typhimurium, L. monocytogenesis, E. faecalis and C. albicans infections. However, we noticed an increased counts of S. aureus in PPO1Δ, PPO2Δ double mutants compared to wild-type flies (1 day: p = 0.04113; 2 days: p = 0.002165; 3 days: p = 0.004922; 4 days: p = 0.009524). Bars = median. The number of colony forming units (CFU) is expressed per fly and in a logarithmic scale. Data were analyzed by Wilcoxon test and values are pooled from three independent experiments.
Table 1.
Contribution of melanization to survival and microbial infection.