Genetic evidence for a protective role of the peritrophic matrix against intestinal bacterial infection in Drosophila melanogaster

Abstract

The peritrophic matrix (PM) forms a layer composed of chitin and glycoproteins that lines the insect intestinal lumen. This physical barrier plays a role analogous to that of mucous secretions of the vertebrate digestive tract and is thought to protect the midgut epithelium from abrasive food particles and microbes. Almost nothing is known about PM functions in Drosophila, and its function as an immune barrier has never been addressed by a genetic approach. Here we show that the Drosocrystallin (Dcy) protein, a putative component of the eye lens of Drosophila, contributes to adult PM formation. A loss-of-function mutation in the dcy gene results in a reduction of PM width and an increase of its permeability. Upon bacterial ingestion a higher level of expression of antibacterial peptides was observed in dcy mutants, pointing to an influence of this matrix on bacteria sensing by the Imd immune pathway. Moreover, dcy-deficient flies show an increased susceptibility to oral infections with the entomopathogenic bacteria Pseudomonas entomophila and Serratia marcescens. Dcy mutant flies also succumb faster than wild type upon ingestion of a P. entomophila toxic extract. We show that this lethality is due in part to an increased deleterious action of Monalysin, a pore-forming toxin produced by P. entomophila. Collectively, our analysis of the dcy immune phenotype indicates that the PM plays an important role in Drosophila host defense against enteric pathogens, preventing the damaging action of pore-forming toxins on intestinal cells.

Fig. 1.

Dcy expression is induced in the midgut upon oral bacterial infection. (A) Dcy expression upon Ecc15 oral infection in wild-type and dcy mutant flies. Dcy mRNA was measured by real-time qPCR in whole flies at indicated time points, and results are shown as a relative Dpt/rpL32 ratio. (B) Real-time qPCR analysis of dcy mRNA expression from the indicated tissues of wild-type Drosophila. Note that adult carcasses do not include gut. Data are representative of three (A) or two (B) independent experiments (shown are error bars). (C) Transversal sections of wild-type or dcy adult anterior midgut were analyzed by immunostaining with an anti-Dcy serum. Arrows indicate the PM (Scale bars, 50 μm.)

Fig. 2.

The dcy mutation induces PM defects. (A) Left: Ultrathin sections of adult anterior midguts derived from wild-type or dcy¹ mutant flies were observed by transmission electron microscopy. A2 and A5 are magnified views of A1 and A4. A4 and A6 show another section at high magnification. Arrows indicate the PM. M, mucus; EC, enterocytes; L, lumen with ingested food. (Scale bars, 10 μm in A1 and A4, 1 μm in A2, A3, A5, and A6.) Right: Quantitative measurements of the thickness of the PM in wild-type or dcy¹ flies. Data show means and SEs from six and nine different midgut sections for the control and the mutant, respectively. (B) Dextran-feeding assay of wild-type or dcy¹ flies. Adult flies were fed with 70-kDa or 500-kDa FITC-labeled dextran beads. Guts were dissected and examined under a fluorescence microscope. The FITC signal is retained in the lumen if the dextran beads cannot pass through the PM. The FITC signal is observed in contact with epithelial cells (indicated as positive) if beads can cross the PM. Bar graph shows the number of “positive” guts for each genotype when 500-kDa molecules were fed. Means and SEs from four independent experiments are shown. *P < 0.05.

Fig. 3.

Dcy is required for protection against oral infection with entomopathogenic bacteria. (A) Survival analysis of wild-type, dcy^Rev, homozygous dcy¹, and dcy^1/Df(dcy) hemizygous flies upon oral infection of P. entomophila. Means and SEs of four independent experiments are shown (P < 0.0001, log–rank test). (B) Survival analysis of wild-type, homozygous dcy¹, and the dcy¹ mutants expressing the dcy gene in the midgut upon oral infection of P. entomophila. Means and SEs of three independent experiments are shown (P < 0.0002, log–rank test). (C) Survival analysis of wild-type, dcy^Rev, and homozygous dcy¹ flies upon oral infection with S. marcescens Db11. Graphs show the means of 60 flies, bars show the SE. This experiment was repeated three times and yielded similar results (P < 0.0001, log–rank test). (D) Bacterial persistence in wild-type and dcy¹ flies. Bacterial persistence in dcy¹ and dcy^Rev at 30 min and 18 h upon oral infection with P. entomophila (OD₆₀₀ = 100), as the number of cfu per fly. No difference was observed between the two strains. Each histogram corresponds to the average of three independent experiments.

Fig. 4.

Dpt expression upon oral bacterial infection is higher in dcy¹ flies compared with wild-type flies. (A) Dpt expression in the midgut (Left) or the fat body (Right) of wild-type and dcy¹ flies upon oral infection with P. entomophila was measured by real-time qPCR at the indicated time points. Data are the mean of four independent experiments, and error bars show the SE. **P < 0.01 vs. dcy^Rev. Results are shown as a percentage of the Dpt/rpL32 ratio normalized to the levels observed in wild-type flies collected 8 h after septic injury (SI) with P. entomophila. (B) β-Galactosidase staining reveals lacZ gene expression in the fat body of wild-type or dcy¹ flies carrying a Dpt-lacZ reporter. Flies were collected 4 h after oral infection with P. entomophila.

Fig. 5.

Dcy¹ flies succumbed to ingestion of a P. entomophila extract. (A) Survival analysis of dcy^Rev and dcy¹ flies upon feeding of the extract from P. entomophila (Pe extract) or buffer (PBS 1% Triton X-100). (B) Survival analysis of wild-type, dcy^Rev, and dcy¹ flies upon feeding with extracts derived from wild-type and gacA P. entomophila derivatives. (C) Survival analysis of wild-type, dcy^Rev, and dcy¹ flies upon feeding with extracts derived from wild-type and AprA P. entomophila derivatives. (D) Survival analysis of wild-type, dcy^Rev, and dcy¹ flies upon feeding with extracts derived from wild-type and monalysin (mnl) P. entomophila derivatives. In A–D, graphs show the means of 60 flies, and bars show the SE. These experiments were repeated three times and yielded similar results (*P < 0.0001, **P < 0.005, log–rank test. ns, not significant).