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. 2017 Sep 5;15(1):79.
doi: 10.1186/s12915-017-0408-0.

Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps

Anna Dostálová  1 , Samuel Rommelaere  2 , Mickael Poidevin  3 , Bruno Lemaitre  4
Affiliations

Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps

Anna Dostálová et al. BMC Biol. .

Abstract

Background: Members of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. In this study, we address the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPq Δ , lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4.

Results: Survival analyses with TEPq Δ flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. Our results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, we show that TEPq Δ flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation.

Conclusions: Collectively, our study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation.

Keywords: Beauveria; Complement; Drosophila; Entomopathogenic fungus; Innate immunity; Insect; Parasitoid wasp; Phagocytosis.

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Figures

Fig. 1
Fig. 1
Flies devoid of inducible TEPs are viable and do not show increased susceptibility to wounding. a Left panel: eGFP-TEP2 proteins produced by an endogenously eGFP-tagged TEP2 locus were detected in haemolymph samples using an anti-GFP antibody. UC unchallenged, Bb septic injury with B. bassiana, Ctrl haemolymph from control y,w flies (not expressing a GFP). Three bands corresponding in size to the full-length tagged protein and two products of proteolytic cleavage were observed (highlighted with *). Right panel: eGFP-TEP4 proteins produced in flies overexpressing a TEP4-GFP fusion using a fat body driver (genotype UAS-TEP4-GFP/+;; Lpp-Gal4/+). A major band with the expected size corresponding to eGFP-TEP4 was observed as well as many smaller bands. A shorter cleaved product was observed at 48 h post-infection. b Genomic location of the four genes encoding secreted TEPs with the position of the transposon insertions causing mutation. c Lifespan of unchallenged male and female flies at 25 °C. TEPq Δ flies have a shorter lifespan than the w 1118 controls (log-rank test, P < 0.001). d Survival to clean injury. Males were pricked in the thorax with a clean needle and kept at 25 °C. TEPq Δ flies are as resistant as the wild-type (log-rank test, P > 0.05). e Survival to oxidative stress. Flies were fed on 1.5% H2O2 in standard food and flipped on fresh medium every 2 days. TEPq Δ flies are as resistant as the wild-type (log-rank test, P > 0.05). c, d, e Shown are representative survival experiments of a minimum of two independent repeats. Forty flies minimum were used for each genotype per repeat
Fig. 2
Fig. 2
Survival to systemic bacterial infection. Male flies were pricked in the thorax with a needle dipped in a concentrated bacterial culture. Data were analyzed by log-rank test. Shown are representative experiments of a minimum of two independent repeats (three where a difference from the control flies was observed). x-axis: time post-infection in days; y-axis: percentage of living flies. a–d Survival to septic injury with Gram-negative bacteria (E. carotovora, S. typhimurium and E. cloacae) and the Gram-positive bacterium S. aureus. No statistically significant difference was observed between TEPq Δ and wild-type (w 1118) flies. e, f Survival to septic injury with Gram-positive bacteria E. faecalis and L. innocua. Statistically significant differences were observed between TEPq Δ and wild-type (w 1118) flies (P < 0.001 for E. faecalis and P = 0.00172 for L. innocua)
Fig. 3
Fig. 3
Survival to fungal infection. Male flies were either covered with spores (a, b, labelled as natural infection) or pricked in the thorax with a needle dipped in a concentrated fungal spore suspension (c–f). Data were analyzed by log-rank test. Shown are representative experiments of a minimum of two independent repeats (three where a difference from the control flies was observed). x-axis: time post-infection in days; y-axis: percentage of living flies. a–c Statistically significant differences were observed between TEPq Δ and wild-type (w 1118) flies (P < 0.001 for B. bassiana, both natural infection and pricking; P = 0.0039 for M. anisopliae). d–f No statistically significant difference was observed between the TEPq Δ and the wild-type (w 1118) flies in the case of N. crassa, A. fumigatus and C. albicans infection by septic injury. g Quantification of B. bassiana DNA 3 days post-infection normalised to the host RpL32 DNA. Values represent the mean ± standard error (SE) of three independent experiments and were analysed using Mann-Whitney test (two-sided). The quantity of fungal DNA is significantly elevated in the TEPq Δ flies as compared to the control w 1118 line (P < 0.001). Overactivation of the Toll pathway in TEPq Δ flies by overexpressing ModSP rescues the increased fungal growth caused by the absence of TEPq Δ (P = 0.005)
Fig. 4
Fig. 4
Survival to parasitoid wasps. Second instar larvae were exposed to female parasitoid wasps, and the emergence of wasps (black boxes) and flies (white boxes) was monitored. Of note, a significant fraction of infested animals die as larvae and pupae (dashed boxes). We observed a significant difference in the outcome of infection between the TEPq Δ and the wild-type flies (a A. tabida, chi-square = 97.59, df = 3, P < 0.001; b L. boulardi, chi-square = 14.81, df = 3, P = 0.02). In the case of L. boulardi infections, flies carrying a lamellocyte marker (misshapen-Gal4, UAS-mCherry) were used to monitor lamellocyte differentiation. Results are represented as a sum of a minimum of three independent experiments. Statistical significance was calculated using Pearson’s chi-square test
Fig. 5
Fig. 5
Phagocytosis of bacteria. a The number of haemocytes in prepupae in the TEPq Δ and wild-type w 1118 line. No statistically significant difference was observed (Mann-Whitney test, two-sided). b Phagocytosis of bacterial particles assessed by ex vivo and in vivo phagocytosis assays. Significantly lower rates of phagocytosis of the Gram-positive bacteria E. faecalis (P = 0.032) and S. aureus (P = 0.015) were detected in the TEPq Δ prepupae in the in vivo assay. No statistically significant differences were observed in phagocytosis of E. coli in vivo or E. faecalis or S. aureus ex vivo. Data were pooled from at least four independent experiments and analysed by Mann-Whitney test (two-sided). c Representative images of haemocytes of third instar larvae with internalised spores of M. anisopliae 2575-RFP after incubation in vitro. Scale bar represents 5 μm. d Flies with reduced number of plasmatocytes (hmlΔ-Gal4 > UAS-Bax) do not show an increased susceptibility to septic injury with B. bassiana. Data were analyzed by log-rank test. Shown is a representative experiment of two independent repeats
Fig. 6
Fig. 6
Toll and Imd pathway induction in the TEPq Δ mutant. Expression of antimicrobial peptide genes normalised to ribosomal protein gene RpL32 after septic injury. a Induction of Diptericin (Dpt, Imd pathway read-out) in response to septic injury with E. carotovora. TEPq Δ flies show a wild-type level of induction of Diptericin. b Induction of Drosomycin (Drs; Toll pathway read-out) in response to septic injury with M. luteus. TEPq Δ flies show a significantly lower level of Drs expression 24 h post-infection (P = 0.013). c TEPq Δ flies show a significantly lower level of Drs expression 24 h post-infection with E. faecalis (P < 0.001 at 48 h post-infection). d TEPq Δ flies show a reduced Drs expression after septic injury with B. bassiana (P = 0.005 at 24 h post-infection and P < 0.001 at 48 h post-infection). e TEPq Δ flies show reduced Drs expression in response to the injection of purified E. faecalis peptidoglycan (PG; measured 16 h post-injection, P = 0.0286, Mann-Whitney test, two-sided) and heat inactivated spores of B. bassiana (Heat inactivated; measured 16 h post-injection, P = 0.0079, Mann-Whitney test, two-sided). f Drosomycin expression 24 h after challenge with B. bassiana is enhanced in TEP4-GFP overexpressing flies (P = 0.0079). UC unchallenged; Sec-GFP, flies overexpressing a secreted form of GFP were used as control. Data were analysed using t test comparing the values in TEPq Δ flies to wild-type w 1118 flies. Values represent the mean ± SE of at least two independent experiments
Fig. 7
Fig. 7
Role of TEPs in regulation of the Toll pathway. a–c Male flies were pricked in the thorax with a needle dipped in a concentrated suspension of B. bassiana spores. Data were analyzed by log-rank test. Each panel shows a representative experiment of a minimum of three independent repeats. x-axis: time post-infection in days; y-axis: percentage of living flies. a TEPq Δ , GNBP3 hades double mutant flies are highly susceptible to B. bassiana as compared to the w 1118 control line (P < 0.001) with a survival curve comparable to that of spz rm7 flies. TEPq Δ or GNBP3 hades single mutant flies show a milder phenotype (P = 0.004 and P < 0.001, respectively, as compared to the double mutants). b TEPq Δ , ModSP 1 double mutant flies are highly susceptible to B. bassiana as compared to the w 1118 control line (P < 0.001) with a survival curve comparable to that of spz rm7 flies. TEPq Δ or ModSP 1 simple mutant flies show a milder phenotype (P < 0.001 for both as compared to the double mutant). c psh 1 , TEPq Δ double mutant flies show an increased susceptibility to B. bassiana compared to the w 1118 wild-type flies (P < 0.001). The survival curve is comparable to that of spz rm7 flies. TEPq Δ or psh 1 single mutant flies show a milder phenotype (P = 0.002 and P = 0.005, respectively), as compared to the double mutants. d Female flies were pricked in the thorax with a needle dipped in a concentrated suspension of B. bassiana spores, and the expression of Drs was measured 24 h post-infection. Values represent the mean ± SE of three independent experiments and were analysed using the Mann-Whitney test (two-sided). Drs expression for all the genotypes tested except for GNBP3 Δ was significantly lower than in the wild-type w 1118 flies, as indicated by asterisks in the chart (0.01 < P < 0.03 for all cases). There were no statistically significant differences between TEPq Δ flies and other compound knockouts (i.e. TEPq Δ , GNBP3 hades or TEPq Δ , ModSP 1 or psh 1 , TEPq Δ.) e Drs expression in response to injection of purified proteases of B. subtilis. No statistically significant differences were observed between the TEPq Δ and the wild type w 1118. f Drs expression in unchallenged male flies of the indicated genotypes. Values represent the mean ± SE of three independent experiments and were analysed using the Mann-Whitney test (two-sided). Overexpression of ModSP induces Drs expression to the same levels in the presence or absence of TEPs
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