Prevalence of local immune response against oral infection in a Drosophila/Pseudomonas infection model

Abstract

Pathogens have developed multiple strategies that allow them to exploit host resources and resist the immune response. To study how Drosophila flies deal with infectious diseases in a natural context, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses, indicating that this bacterium has developed specific strategies to escape the fly immune response. Using a combined genetic approach on both host and pathogen, we showed that P. entomophila virulence is multi-factorial with a clear differentiation between factors that trigger the immune response and those that promote pathogenicity. We demonstrate that AprA, an abundant secreted metalloprotease produced by P. entomophila, is an important virulence factor. Inactivation of aprA attenuated both the capacity to persist in the host and pathogenicity. Interestingly, aprA mutants were able to survive to wild-type levels in immune-deficient Relish flies, indicating that the protease plays an important role in protection against the Drosophila immune response. Our study also reveals that the major contribution to the fly defense against P. entomophila is provided by the local, rather than the systemic immune response. More precisely, our data points to an important role for the antimicrobial peptide Diptericin against orally infectious Gram-negative bacteria, emphasizing the critical role of local antimicrobial peptide expression against food-borne pathogens.

Figure 1. Identification and Regulation of a P. entomophila Protease

(A) Survival analysis of wild-type (Or^R) larvae following feeding with 60-fold concentrated sterile filtered culture supernatants of wild-type P. entomophila and its gacA mutant. Survival analysis was repeated three times and performed on 50 larvae. Log-rank analysis demonstrated a statistically significant difference in survival of flies fed with supernatant from wild-type P. entomophila and flies fed with supernatant from the gacA mutant (p < 0.0001). (B) Survival analysis of wild-type (Or^R) adult flies (n = 60) injected with 69 nl of non-concentrated supernatants of wild-type P. entomophila and its gacA mutant. Log-rank analysis demonstrated a statistically significant difference in survival of flies injected with supernatant from wild-type P. entomophila and flies injected with supernatant from the gacA mutant (p < 0.0001). (C) SDS-PAGE analysis of culture supernatants from P. entomophila derivatives. Protein extracts from culture supernatants (OD₆₀₀ = 2) following growth at 29 °C for 24 h, were loaded and stained with Coomassie blue. The genotypes of the bacterial strains used are indicated on the top. The first lane represents the molecular weight marker (indicated in kDa on the left). CL25 and CU1 are two independent Tn5 insertions in the prtR locus [10].The last lane displays the aprA mutant complemented with a plasmid expressing the aprA locus. AprA corresponds to the major band at 51 kDa. (D) Purification of P. entomophila AprA. Lane 1, molecular weight marker. Lane 2, purified peak fraction of AprA. (E) Survival analysis of wild-type adult flies (n = 60) following microinjection of 9.2 nl of purified AprA protease (50 μg/ml) or PBS. Flies succumbed within 4 hr after microinjection of pure protease. Log-rank analysis demonstrated a statistically significant difference in survival of flies injected with AprA and flies injected with PBS (p < 0.0001). (F) Proteolytic activity of supernatant of P. entomophila derivatives as measured at 440 nm by the azocasein test.

Figure 2. The aprA Mutant Exhibits Attenuated Virulence

(A) Genetic organization of the P. entomophila apr locus and its associated Type 1 transporter. The locus contains the structural gene for the protease (aprA), followed by the genes encoding its putative inhibitor (aprI) and those coding for the associated Type 1 transporter (ABC Transporter, aprD; Membrane Fusion Protein, aprE; Outer Membrane Protein, aprF). The apr operon organization was deduced from [10]. (B) Survival analysis of Drosophila larvae (n = 60) after feeding with wild-type P. entomophila, gacA, prtR, and aprA mutants; the aprA mutant complemented with the wild-type apr operon (pUCP20-apr); and an aprA mutant complemented with the apr operon carrying a non-polar mutation in the aprA gene (pUCP20-aprΔaprA). This experiment was repeated twice and yielded similar results. Log-rank analysis demonstrated a statistically significant difference in survival of flies fed with wild-type P. entomophila and flies fed with the aprA mutant (p < 0.0001). (C) Diptericin expression measured by RT-qPCR in Drosophila larvae following natural infection with wild-type P. entomophila, aprA, and gacA mutants. Infection with wild-type P. entomophila and the aprA mutant induced sustained Diptericin expression unlike the gacA mutant. Larvae were collected at different time intervals after oral infection. Diptericin expression was normalized to rp49 mRNA. For each time point the values represented are the mean of three independent experiments (± standard deviation). 100% value corresponds to the level of Dpt mRNA obtained 24 h after infection with wild-type P. entomophila. rp49: ribosomal protein 49. (D) Ingestion of aprA or prtR mutant bacteria induces a food-uptake cessation in larvae in contrast to animals fed with the gacA mutant. Larvae fed with medium containing 0.5% (w/v) bromophenol blue displayed a clearly discernable blue coloration throughout the gut whereas larvae fed with both P. entomophila wild-type, aprA, or prtR mutants and bromophenol blue showed only a pale blue coloration. Images were taken 6 h after infection. This visual effect was not due to a change of gut pH (acidification) that would result in yellow rather than blue coloration since the overall level of bromophenol blue in dissected and homogenized intestines was confirmed by measuring the absorbance of blue dye with larval extracts in a HEPES Buffer at [pH 8] (unpublished data).

Figure 3. Bacterial Persistence in Wild-Type and Rel Flies

(A) Bacterial persistence was measured in live wild-type flies by plating appropriate dilutions of homogenates of five surface-sterilized adults on LB medium containing rifampicin. The flies had been previously orally infected with rif^R strains of wild-type P. entomophila and its aprA and gacA derivatives. AprA mutants persisted less than wild-type P. entomophila but better than the gacA mutant. (B) Persistence of wild-type P. entomophila aprA and gacA mutants in live Rel flies. AprA mutant bacteria persisted at a level similar to wild-type bacteria in Rel mutant flies whereas gacA bacterial levels decreased with time. The number of cfus per fly represented in each histogram corresponds to the average of six independent experiments (± standard deviation). cfu, colony-forming unit.

Figure 4. AprA Confers Protection against the Imd-Dependent Immune Response

(A) Over-expression of the Imd immune pathway protects against P. entomophila infection during gut infection. The genotypes of the flies are as indicated. Over-expression of the UAS-imd construct with an hs-GAL4 driver induces strong expression of the Diptericin gene in the absence of infection [39]. The expression was triggered once (1 h heat-shock at 37 °C) 6 h and 12 h prior to infection. The percentage of surviving flies (n = 60) after infection with wild-type P. entomophila is shown. This experiment was repeated three times and gave similar results. Log-rank analysis demonstrated a statistically significant difference in survival of wild-type flies and UAS-imd, hs-Gal4/+ (HS 12 h) flies fed with wild-type P. entomophila (p < 0.0001). HS, heat-shock. (B) Persistence of aprA, gacA, and wild-type P. entomophila in adults over-expressing imd 12 h prior to infection. AprA bacterial titers decrease faster with time than the ones of wild-type bacteria. The gacA mutant fails to survive in the intestine of flies over-expressing imd. The number of cfus per fly represented in each histogram corresponds to the average of six independent experiments (± standard deviation). cfu, colony-forming unit.

Figure 5. The Local Immune Response Plays a Critical Role against P. entomophila

(A) Time course analysis of Diptericin expression measured by RT-qPCR in Drosophila fat body and gut extracted from females following natural infection with wild-type P. entomophila, aprA, and gacA mutants. Infection with wild-type P. entomophila and the aprA mutant induced a rapid and sustained Diptericin expression unlike the gacA mutant. Fat bodies (carcasses) and digestive tracts were dissected from adults collected at different time intervals after oral infection. Diptericin expression was normalized to rp49 mRNA. 100% value corresponds to the level of Dpt mRNA obtained 4 h after infection with wild-type P. entomophila. rp49: ribosomal protein 49; Dpt, Diptericin (B) P. entomophila induces Diptericin expression in the cardia of adult Drosophila. Histochemical staining of β-galactosidase activity shows that Dpt-lacZ is expressed in the anterior midgut at the level of the proventriculus of infected flies carrying the Dpt-lacZ reporter gene (left panel). Similar results were obtained with a Dpt-GFP transgene (right panel). Adults were collected 24 h after infection. The control pictures display the cardia of uninfected flies. A constitutive expression was observed in the anterior part of the cardia. Dpt, Diptericin. (C) Survival of wild-type flies (n = 60) to P. entomophila after previous infection with Ecc15. Flies were first infected with Ecc15 either orally (OD₆₀₀ = 100) or by septic injury (OD₆₀₀ = 200). 20 h after this Ecc15 infection, flies were fed with P. entomophila. Survival curves demonstrate that Drosophila flies primed with Ecc15 were protected from a subsequent P. entomophila infection only when Ecc15 was orally administrated. Log-rank analysis demonstrated a statistically significant difference in survival to P. entomophila infection of flies primed with Ecc15 by septic injury (SI) or natural infection (NI) (p < 0.0001). (D) Survival of wild-type and Rel flies (n = 60) to P. entomophila after previous infection with Ecc15 evf-. Flies were first infected with Ecc15 evf- orally (OD₆₀₀ = 100). 20 h after this Ecc15 infection, flies were fed with P. entomophila. Survival curves demonstrate that wild-type, but not Rel Drosophila flies primed with Ecc15 evf-, were protected from a subsequent P. entomophila infection. This experiment was repeated three times and gave similar results. Log-rank analysis demonstrated a statistically significant difference in survival to P. entomophila infection of flies primed with Ecc15 evf- by natural infection (NI) and flies without previous priming (p < 0.0001). (E) Rel flies expressing a UAS-Rel transgene in the midgut under the control of the gut-specific cad-Gal4 driver survive better to P. entomophila infection than Rel mutant flies. Survival experiments were performed on 30 flies orally infected with P. entomophila (OD₆₀₀ = 50). This experiment was repeated three times and yielded similar results. Log-rank analysis demonstrated a statistically significant difference in survival of UAS-Rel/caudal-Gal4; Rel flies, and UAS-Rel/Rel; Rel flies to P. entomophila infection (p < 0.01).

Figure 6. AprA Confers Protection against Diptericin

(A) imd flies over-expressing Diptericin or Attacin A show increased resistance against P. entomophila infection. Survival rates were monitored on imd mutants, wild-type, and imd;UAS-AMP flies orally infected with P. entomophila (OD₆₀₀ = 25). The genotypes of the utilized flies are: imd: imd/imd; wild-type: imd/+; da-Gal4/+; imd; da>AMP: imd, UAS-AMP/imd; UAS-AMP/ da-GAL4. Log-rank analysis demonstrated a statistically significant difference in survival of imd;UAS-AMP flies and wild-type flies to P. entomophila oral infection (p < 0.01). (B) Persistence of wild-type P. entomophila and aprA mutants in imd flies over-expressing Diptericin under the control of the da-Gal4 driver. aprA mutants persist less than wild-type P. entomophila in lines over-expressing Diptericin in contrast to flies mutated in the imd locus. The number of cfu per fly represented in each histogram corresponds to the average of six independent experiments (± standard deviation). cfu, colony-forming unit.