Secreted Bacterial Effectors and Host-Produced Eiger/TNF Drive Death in aSalmonella-Infected Fruit Fly

Abstract

Death by infection is often as much due to the host's reaction as it is to the direct result of microbial action. Here we identify genes in both the host and microbe that are involved in the pathogenesis of infection and disease in Drosophila melanogaster challenged with Salmonella enterica serovartyphimurium (S. typhimurium). We demonstrate that wild-typeS. typhimurium causes a lethal systemic infection when injected into the hemocoel of D. melanogaster. Deletion of the gene encoding the secreted bacterial effect or Salmonella leucine-rich (PslrP)changes an acute and lethal infection to one that is persistent and less deadly. We propose a model in which Salmonella secreted effectors stimulate the fly and thus cause an immune response that is damaging both to the bacteria and, subsequently, to the host. In support of this model, we show that mutations in the fly gene eiger, a TNF homolog, delay the lethality of Salmonella infection. These results suggest that S. typhimurium-infected flies die from a condition that resembles TNF-induced metabolic collapse in vertebrates. This idea provides us with a new model to study shock-like biology in a genetically manipulable host. In addition, it allows us to study the difference in pathways followed by a microbe when producing an acute or persistent infection.

Figure 1. Growth of Salmonella in Drosophila melanogaster

(A) Survival of wild-type flies injected with S. typhimurium. Three sets of 60 flies were injected with approximately 10,000 cfu of SL1344 (Hoiseth and Stocker 1981) from an overnight 37 °C standing culture. Injected flies were incubated at 29 °C. Survival was monitored daily. Circles, S. typhimurium-injected; squares, LB-injected; triangles, uninjected. (B) Survival of immune pathway mutant flies injected with S. typhimurium. Three sets of 20 flies were injected with approximately 100,000 cfu SL1344 and incubated at 29 °C. Fly survival was monitored at 0, 12, and 24 h postinfection. Flies infected: Black, wild-type (Oregon R); gray, imd¹/imd¹; white,Dif¹/Dif¹. (C) Salmonella growth in infected immunocompromised flies. Flies were injected with approximately 100,000 cfu SL1344 and incubated at 29 °C for the indicated times. Flies were homogenized in LB with 1% Triton X-100 to release bacteria from cells, and the homogenates were plated on LB-streptomycin plates. Only living flies were homogenized. Flies infected: Black, wild-type; gray, imd¹/imd¹; white, Dif¹/Dif¹. (D) Effects of gentamicin on S. typhimurium growth in the fly. Wild-type flies were injected with 10,000 cfu of S. typhimurium and then incubated at 29 °C for 7 d. Flies were then injected with either a solution containing 50 nl of 1 mg/ml gentamicin (black) or water (gray). A second group of previously uninfected flies was preinjected with gentamicin or water 15 min before bacterial challenge to determine the effects of the drug on bacteria before they were phagocytosed. All flies were then incubated after gentamicin or water injection at 29 °C for 4 h. Flies were then homogenized and plated. All error bars show standard deviation. (E) Effects of S. typhimurium inoculation size on bacterial growth in the fly. Flies were injected with SL1344 over a 1,000-fold dilution range. Flies were incubated at 29 °C. Flies were homogenized immediately after injection or following 7 d of incubation before plating. Injected concentrations: Black, 0.1 optical density at 600 nm (OD₆₀₀); dark gray, 0.01 OD₆₀₀; light gray, 0.001 OD₆₀₀; white, 0.0001 OD₆₀₀.

Figure 2. Location of S. typhimurium in the Fly

(A–D) Induction of pmig-1 in Drosophila hemocytes. SL1344 carrying pmig-1 grown standing at 37 °C overnight were dried to a slide, fixed with formaldehyde and photographed using (A) differential intereference contrast (DIC) optics and (B) GFP optics. The bacteria are not highly fluorescent under these conditions. SL1344 carrying pmig-1 and grown as described above were injected into D. melanogaster larvae and the hemocytes were isolated and fixed after 24 h incubation at 29 °C. A hemocyte is shown in (C) DIC and (D) GFP optics using the same exposures as in A and B. Intensely fluorescent bacteria in the hemocytes are visible. Bar equals 5 um. (E–H) S. typhimurium growth in living flies. SL1344 carrying pmig-1 were injected into wild-type flies and incubated for 2 d at 29 °C. (E and F) Uninfected flies are compared to (G and H) infected flies with (E and G) DIC and (F and H) GFP optics. The arrowhead highlights GFP-expressing Salmonella associated with hemocytes on the dorsal side of the fly.

Figure 3. Effects of Salmonella Virulence Mutations on Disease in the Fly

(A) Mean time to death for infected flies. Identical quantities of S. typhimurium strains were injected into Oregon R flies and survival was monitored daily (i). Infections with slrP and rescuing construct were performed separately and are thus reported separately (ii). (B) Growth of mutant Salmonella in the fly. Approximately 10,000 cfu of each bacterial strain were injected into flies. Flies were then homogenized and plated at the time of injection (black bars) or following a 7-d incubation (white bars). All error bars show standard deviation. (C–H) Phagocytosis assays in living Drosophila. To assay the effects of Salmonella infections on phagocyte function, flies were injected with approximately 10,000 cfu of each strain of S. typhimurium. Following a 7-d incubation, the flies were first injected with FITC-labeled dead E. coli and incubated for 60 min to permit them to be phagocytosed. Trypan blue was then injected to quench the fluorescence of extracellular bacteria. The area boxed in (C) was photographed using FITC optics (D–H). The flies were injected with the following bacterial strains: (D) SL1344; (E) LB (control); (F) BJ66 (SPI1); (G) P3F4 (SPI2); (H) slrP (Table 1).

Figure 4. Effects of Eiger Mutations on S. typhimurium Infections in the Fly

(A) Survival of eiger-mutant flies infected with S. typhimurium. Three sets of 20 flies were infected with 10,000 cfu of SL1344 and incubated at 29 °C. Two eiger mutants (eiger¹ and eiger³) and the background strain (w¹¹⁸) were assayed. Survival was monitored daily. Circle, eiger¹/eiger¹; square, eiger³/eiger³; triangle, w¹¹⁸. Solid shapes indicate LB-injected flies; open shapes indicate SL1344-injected flies. Mantel-Cox analysis demonstrated p < 0.001 when comparing infected wild-type to eiger-mutant flies. (B) Growth of S. typhimurium in eiger-mutant flies. Flies were infected with 10,000 cfu of SL1344 and plated at the time of injection or following a 7-d incubation. Black, time = 0; white, time = 7 d. All error bars show standard deviation. (C) Effects of Salmonella infection on eiger RNA transcript levels. Total RNA was extracted from five flies per sample on days 0, 1, 3, 5, and 7 postinjection with (open circles) SL1344 or (closed squares) LB. Quantitative real-time RT-PCR was performed. Relative eiger transcript quantity is expressed as the fold-difference in comparison to the day 0 value. All error bars show the standard deviation of three RNA preparations.