Layers of immunity: Deconstructing the Drosophila effector response
- Global Health Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
- Centre for Ecology and Conservation, University of Exeter, United Kingdom
Figures
Figure 1 with 1 supplement
Each immune module functions largely independently from other immune modules.
(A) Activation of the Imd pathway, as revealed by DptA expression following infection with a mixture of heat-killed E. coli and M. luteus, is broadly wild-type in single-module mutants other than Imd. In the case of DPhag flies, we observed a lesser induction at 6 hr compared to wild-type. (B) Activation of the Toll pathway, as revealed by the expression of Drs, is broadly wild-type in single-module mutants other than DTOLL flies. In the case of DMel, we observed a slightly greater Toll pathway activity. (C) The ability of plasmatocytes to phagocytose bacterial particles is not negatively affected in single-module mutants except DPhag (also see Figure 1—figure supplement 1). (D) Cuticle blackening after clean injury is not impaired except in DMel flies. (E) Activation of the Imd pathway remains strongly inducible in compound mutants except when deficient for the Imd pathway. (F) Activation of the Toll pathway remains strongly inducible in compound mutants except when flies were deficient of the Toll pathway. Note that Drs receives a minor input from Imd signaling (Leulier et al., 2000), explaining minor induction of Drs in DToll flies at early time points. (G) The ability of plasmatocytes to phagocytose bacterial particles is not significantly affected in compound mutants except those including DPhag. (H) Cuticle blackening after clean injury is not impaired in compound mutants except in DMel flies.
Figure 1—figure supplement 1
We used DPhag mutants in the genetic background of Melcarne et al., 2019a (+; DPhag) in some experiments, and confirm here that these mutants are equally deficient in phagocytic capacity to DPhag flies from the iso DrosDel genetic background (DPhag).
Figure 2 with 1 supplement
Lifespans of mutants used in this study in unchallenged flies (A, B) and upon clean injury (C, D) conditions at 25°C.
See Figure 2—figure supplement 1 for 29°C comparisons.
Figure 2—figure supplement 1
Lifespans of mutants used in this study in unchallenged flies(A, B) and upon clean injury (C, D) conditions at 29°C.
Figure 3 with 1 supplement
Heatmap of lifespans of immune module-deficient flies upon infection by various pathogens.
Darker blue indicates lower survival, while white indicates maximum survival. Experiments used either 7 or 10 days as a maximum lifespan/time course, and the heatmap is adjusted accordingly per row to have white fill for the maximum possible lifespan of that row. Heatmap colors: blue (low survival), white (high survival). Survival curves are presented in Supplementary file 2, and a summary of susceptibilities is provided in Figure 3—figure supplement 1. Small numbers beside mean lifespans indicate total sample size per genotype per treatment.
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Figure 3—source data 1
Editable version of Figure 3.
- https://cdn.elifesciences.org/articles/107030/elife-107030-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Summary table of susceptibilities per the three conditions outlined in section ‘Systemic infections and survival’.
A ✓ indicates the pathway contributes to defense against a given pathogen. In the event that a single-module mutant is not more susceptible to the infection, but contributes to increased mortality in a double mutant line, a ✓ is given and the co-deleted relevant pathways highlighted by their first letter. Black boxes indicate no difference to wild-type or to clean injury.
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Figure 3—figure supplement 1—source data 1
Editable version of Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/107030/elife-107030-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Growth kinetics of Pr. rettgeri (A), S. aureus (B), and C. albicans (C) in wild-type, single-module mutant and DITPM flies.
Survival curves underlying data in Figure 3 are shown for comparison. Each data point reflects a pooled sample of five flies. Error bars reflect 1 standard deviation from the mean.
Figure 4—figure supplement 1
Survival to infection of females against P. rettgeri, S. aureus, and C. albicans.
Survival trends largely parallel the rank order of males (Figure 4).
Figure 5
Measurement of Pathogen Load Upon Death (PLUD) upon infection with Pr. rettgeri, S. aureus, and C. albicans.
(A) The PLUD of Pr. rettgeri in individual module mutant flies is not significantly different from wild-type. The PLUD of DITPM flies was not significantly different, although censoring of a single low-PLUD outlier in the wild-type would result in a significant difference between wild-type and DITPM flies (p < 0.01). (B) The PLUD of S. aureus-infected flies is not different across any genotype. (C) The PLUD of C. albicans-infected flies was significantly lower in DPhag flies with a notably lower mean PLUD. This difference was robust to use of different DPhag genetic backgrounds (merged data shown here). DMel flies and DITPM flies also had significantly lower PLUD. Error bars reflect 1 standard deviation from the mean. * = P < .05, ** = P < .01, *** = P < .001.
Author response image 1
Figure 3 with 2 decimals places of rounding for mean lifespans.
The 7-day clean injury mean lifespan of WT is 6.74 days, and of ΔMel is 6.37 days. Due to rounding, in the 1 decimal Figure 3 this difference appears as if it is only 0.3 days, but it closer to 0.4 days. Regardless, this level of difference, which appears rather clearly in a survival curve, is well below the level of difference we have chosen to highlight in our study.
Tables
Table 1
List of mutants used in this study.
‘-’ indicates the deleted immune module.
| Deficient pathway | ||||||
|---|---|---|---|---|---|---|
| Drosophila lines | Reference | Name | IMD | Toll | Phagocytosis | Melanization |
| iso; iso; RelE20 | Hedengren et al., 1999 | ∆IMD | - | + | + | + |
| iso; iso; spzrm7 | Lemaitre et al., 1996 | ∆TOLL | + | - | + | + |
| iso; NimC11; Eater1 | Melcarne et al., 2019b | ∆Phag | + | + | - | + |
| iso; PPO1∆, PPO2∆; iso | Binggeli et al., 2014 | ∆Mel | + | + | + | - |
| +; +; RelE20, spzrm7 | This paper | ∆IMD, ∆TOLL | - | - | + | + |
| iso; PPO1∆, PPO2∆; NimC11,Eater1 | This paper | ∆Phag, ∆Mel | + | + | - | - |
| iso; NimC11; Eater1, RelE20 | This paper | ∆IMD, ∆Phag | - | + | - | + |
| iso; PPO1∆ PPO2∆; RelE20 | This paper | ∆IMD, ∆Mel | - | + | + | - |
| iso; PPO1∆ PPO2∆; spzrm7 | This paper | ∆TOLL, ∆MelPPS | + | - | + | - |
| iso Hay-pshDef; iso; iso | Dudzic et al., 2019 | ∆TOLL, ∆MelHP | + | - | + | - |
| iso Hay-pshDef; NimC11; Eater1 RelE20 | This paper | ∆ITPM | - | - | - | - |
Additional files
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Supplementary file 1
Microbe characteristics and growth conditions.
- https://cdn.elifesciences.org/articles/107030/elife-107030-supp1-v1.xlsx
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Supplementary file 2
Survival curves for data summarized in Figure 3.
- https://cdn.elifesciences.org/articles/107030/elife-107030-supp2-v1.zip
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Supplementary file 3
Survival analysis outputs for data summarized in Figure 3.
- https://cdn.elifesciences.org/articles/107030/elife-107030-supp3-v1.zip
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Supplementary file 4
Supplemental discussion of fly genetics and considerations for the use of genotypes described in this study.
- https://cdn.elifesciences.org/articles/107030/elife-107030-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107030/elife-107030-mdarchecklist1-v1.docx
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Layers of immunity: Deconstructing the Drosophila effector response
eLife 14:RP107030.
https://doi.org/10.7554/eLife.107030.3
