Pseudomonas aeruginosa RhlR is required to neutralize the cellular immune response in a Drosophila melanogaster oral infection model

Abstract

An in-depth mechanistic understanding of microbial infection necessitates a molecular dissection of host-pathogen relationships. Both Drosophila melanogaster and Pseudomonas aeruginosa have been intensively studied. Here, we analyze the infection of D. melanogaster by P. aeruginosa by using mutants in both host and pathogen. We show that orally ingested P. aeruginosa crosses the intestinal barrier and then proliferates in the hemolymph, thereby causing the infected flies to die of bacteremia. Host defenses against ingested P. aeruginosa included an immune deficiency (IMD) response in the intestinal epithelium, systemic Toll and IMD pathway responses, and a cellular immune response controlling bacteria in the hemocoel. Although the observed cellular and intestinal immune responses appeared to act throughout the course of the infection, there was a late onset of the systemic IMD and Toll responses. In this oral infection model, P. aeruginosa PA14 did not require its type III secretion system or other well-studied virulence factors such as the two-component response regulator GacA or the protease AprA for virulence. In contrast, the quorum-sensing transcription factor RhlR, but surprisingly not LasR, played a key role in counteracting the cellular immune response against PA14, possibly at an early stage when only a few bacteria are present in the hemocoel. These results illustrate the power of studying infection from the dual perspective of host and pathogen by revealing that RhlR plays a more complex role during pathogenesis than previously appreciated.

Fig. 1.

Systemic and cellular immune responses contribute to host defense against orally ingested P. aeruginosa PA14. (A and B) Survival following PA14 oral infection. IMD pathway mutants [imd (P = 0.0003, n = 8), key (P = 0.00005, n = 22)] and Toll pathway mutants [MyD88 (P = 0.0001, n = 22), spätzle (spz) (P = 0.01, n = 4)] succumbed faster to the infection than wild-type (wt) flies (A). Flies defective for phagocytosis [eater (P = 0.01, n = 3); latex bead-injected flies: wtΔphag (P = 8 × 10⁻⁷, n = 9)] also died faster than wild type (B). (C) Flies were either fed continuously or fed for the indicated period on the bacterial solution and then fed on a sterile sucrose solution that was changed daily; survival data are shown. At least 4 consecutive days of feeding were required to develop a lethal infection. (D) Bacterial titers measured in the hemolymph collected from batches of 10 flies in seven independent experiments are shown on a logarithmic scale. The values shown correspond to the bacterial titer per fly. Error bars are ±SD.

Fig. 2.

An early-activated local IMD response and a late systemic IMD response both contribute to host defense against orally ingested P. aeruginosa PA14. (A) qRT-PCR analysis of the induction of Diptericin, a classic IMD pathway readout, in infected flies. Results are expressed as a percentage of the induction measured 6 h after a septic injury challenge with E. coli. P values (*) refer to the comparison between infected and noninfected flies of the same genotype: *P < 0.05; **P < 0.01; ***P < 0.001; n = 7. Other P values (°) refer to the comparison between mutant and wild-type flies at the same day of infection: °P < 0.05; n = 7. (B–G) β-Galactosidase staining of Diptericin-LacZ flies. Diptericin is induced in the proventriculus (arrows) throughout the infection (B–D), whereas systemic Diptericin induction in the fat body (arrowheads) of the fly occurs in later stages of the infection (E–G). (H) Rescue of the imd PA14 susceptibility phenotype by overexpression of a UAS-imd⁺ transgene (>IMD) with a gut (NPG4G80), a hemocyte (hmlG4G80), or a fat body (ylkG4)-specific driver as documented by the average time it takes to kill 50% of the flies (LT50). Note that AMPs synthesized in hemocytes and the fat body are secreted into the hemocoel. In this series of experiments, wild-type flies succumbed somewhat earlier than usual. P values computed by comparison with imd mutant flies: *P < 0.05; **P < 0.01; ***P < 0.001; n = 5. Error bars are ±SD. ns: not significant. (Scale bars: 500 μm.)

Fig. 3.

Late Toll pathway activation contributes to systemic host defense against orally ingested P. aeruginosa PA14. (A) qRT-PCR analysis of the induction of Drosomycin, a classical readout of Toll pathway activation, in infected flies. Results are expressed as a percentage of the induction measured 24 h after a septic injury challenge with M. luteus. P values (*) refer to the comparison between infected and noninfected flies of the same genotype: *P < 0.05; **P < 0.01; ***P < 0.001; n = 6. No significant difference was observed between wild-type (wt) and key flies with respect to Drosomycin expression levels. (B and C) Drosomycin-GFP reporter induction in the fat body upon infection. (D) Overactivation of the Toll pathway and rescue of the MyD88 susceptibility phenotype by overexpression of a UAS-MyD88⁺ transgene (>MyD88) with a gut (NPG4G80) or a hemocyte (hmlG4 or hmlG4G80)-specific driver, as documented by the average time it takes to kill 50% of the flies (LT50). Rescue was observed by overactivation of the Toll pathway in hemocytes, but not in the gut. Note that AMPs synthesized in hemocytes are secreted into the hemocoel. The UAS-Toll^10B transgene (>Toll10B) expresses a gene encoding a constitutively active form of the Toll receptor. In this series of experiments, wild-type flies succumbed somewhat earlier than usual. P values are compared with MyD88 mutant flies: *P < 0.05; **P < 0.01; n = 5. Error bars are ±SD. ns: not significant. (Scale bar: 500 μm.)

Fig. 4.

RhlR, but not LasR, is required to counteract the cellular immune response against P. aeruginosa PA14. (A) Survival experiments in wild-type Drosophila to analyze virulence of P. aeruginosa mutants in known virulence factors. The average time that it takes to kill 50% of flies (LT50) is plotted. Two rhlR transposon insertion mutants [37943 (referred to as rhlR) and 34255] and a deletion (ΔrhlR) displayed the same attenuated virulence phenotype, whereas other mutants were not significantly less virulent than wild-type (wt) PA14. pscD is a deletion mutation that affects the secretion machinery and thus prevents the secretion of all T3SS effectors, including ExoT. exoT mutant bacteria were tested in independent experiments using flies of a different genetic background and also did not show a phenotype (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; n = 3 or 4 depending on the mutant tested. (B–F) In the Insets, the genotype of the host [wild-type (wt) or mutant flies] and the genotype of the pathogen (PA14 refers to wild-type PA14) are indicated. (B) Bacterial counts per fly measured in the hemolymph collected from PA14 and rhlR infected wild-type (wt) and latex bead-injected flies (wtΔphag) expressed on a logarithmic scale (n = 3). (C) Survival experiments using wild-type PA14 and rhlR mutant bacteria. rhlR mutants are less virulent (P values PA14 vs. rhlR in wild-type flies: P = 0.0017, n = 7; key flies: P = 0.0020, n = 6; MyD88 flies: P = 0.0001, n = 7). (D) rhlR mutant bacteria killed phagocytosis-deficient latex bead-injected flies as rapidly as wild-type bacteria (P > 0.05, n = 6). (E) Survival experiments using wild-type PA14 and lasR mutant bacteria. lasR mutants are as virulent as PA14 in wild-type flies and less virulent in key and MyD88 flies (P values PA14 vs. lasR in wild-type flies: P = 0.33, n = 5; key flies: P = 0.027, n = 2; MyD88 flies: P = 0.025, n = 3). (F) lasR mutant bacteria killed phagocytosis-deficient latex bead-injected flies somewhat less rapidly than wild-type bacteria (P = 0.013, n = 4). Error bars in A and B are ±SD.