We developed a model of percutaneous infection in D. melanogaster to mimic humans recovering from sepsis and followed immune and functional outcomes over a course of 7 days and lifespan up to 60 days. We had four experimental groups, which were unmanipulated, sham, infected without treatment, and infected with antibiotics groups.
Drosophila melanogaster strains and maintenance
The flies were raised at 23 °C, 60 % humidity, and 12-h light/dark cycle on standard cornmeal-yeast medium and changed every 3–5 days. We selected male flies 2–3 days after eclosion for experiments. We obtained cec κB-luc flies from Dr. Williams at the University of Pennsylvania . Wild-type (WT) Canton S and Drosomycin-green fluorescent protein (GFP) reporter (Dipt-lacZ, Drs-GFP, y/CyO) along with appropriate control flies were obtained from Bloomington stock.
We prepared Staphylococcus aureus suspension in Luria-Bertani (LB) broth from frozen glycerol stock. Following an overnight culture, we transferred bacteria to a fresh tube to achieve the exponential growth phase (4–5 h) and then washed in phosphate-buffered serum (PBS). The bacterial pellet was resuspended in PBS to an optical density (OD) of 1.0 at 600 nm. At OD of 1.0, there were 1.67 × 106 CFU of S. aureus. We anesthetized the flies with CO2 and pricked with a tungsten needle (0.01 mm at the tip and 0.25 mm across the needle body) into their thorax. The sham group was pricked with a sterile needle while the infected group had the needle dipped into the bacterial solution . We pricked the flies at room temperature (23 °C) and placed the flies back into the vials to recover. We pricked the sham flies first to coat the tungsten needle with hemolymph to achieve consistent bacterial coating with the infection group.
We prepared linezolid-containing (500 μg/ml) fly food (cornmeal-yeast medium) by adding 50 mg of linezolid into 100 ml of liquefied food. We transferred unmanipulated, sham, and infected with antibiotics flies to antibiotic-containing vials immediately after infection and kept in these vials for 18 h before transferring the flies back to antibiotic-free vials. The infected without treatment group was placed into antibiotic-free new vials immediately after infection.
Fly survival and lifespan
After infection, we assessed fly survival by visual inspection of living flies every 1 h (except night time) for the first 72 h. Infected without treatment flies started dying usually after 16 h of infection. We excluded flies that died within 6 h after inoculation from survival analysis. During lifespan observations over 60 days, we changed fly media and assessed survival every 2 days.
We determined the bacterial burden of flies by sampling three groups of 10 files immediately, 6, 18, 36, 48, and 72 h and 7 and 20 days after inoculation and homogenized in LB broth. Serial 10× dilutions of final fly extracts were cultured on LB agar and colony forming units (CFU) were counted after 24-h incubation in 37 °C.
NF-κB luciferase activity following infection
We measured luciferase activity in flies expressing cec NF-κB reporter. We prepared 96-well microwell plates for flies as described . Each well contained two layers of food medium with the top layer containing luciferin. We added 300 μl of 5 % sucrose and 2 % agar solution to each well and allowed to solidify. Then, we added a 50-μl top layer to each well containing 5 % sucrose, 1 % agar, and 2 mM luciferin to detect NF-κB activation in infected, treated, and control flies. Similar to our other experiments, we added linezolid (500 μg/ml) to the top layer with the same concentration of sucrose, agar, and luciferin. We allowed plates to dry thoroughly to prevent flies from adhering to the condensates. Because luciferin is light sensitive, we avoided exposing plates to light more than necessary. Then, we applied a clear adhesive film to a 96-well microwell plate and made two holes per well for air exchange with a 25-gauge needle. We initially placed flies into vials containing 5 % sucrose and 2 % agar 2 days before experiment to acclimatize them to the food. Before needle pricking, we loaded flies into microwell plate under constant ambient light for 3 h to adjust to the new environment and to observe that they consume the luciferin substrate. After flies acclimatized and consumed luciferin, we anesthetized them with CO2 in groups of 24 and performed infection assay as described above. We returned each fly to its original well and resealed the microplate. We measured luminescence emission with a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA) at 25 °C every 10 min and 1.5 s per well. We followed flies for up to 72 h in the microwell plates. We expressed the luminescence data as relative luciferase activity per group of 24 surviving flies.
Patterns of host response gene expression
We determined inflammatory gene expression, pattern recognition receptors, anti-microbial peptides, NF-kB, and insulin pathway members by collecting flies 6, 18, and 48 h and 1 week after needle pricking. We extracted RNA using RNeasy Mini kit (RNeasy Mini Kit/74104, QIAGEN, Germantown, MD). Gene expression of Toll, PGRP-SD, defensin, drosomycin, metchnikowin, cecropin A, JNK, dorsal, Relish, Dif, IRS, FOXO, dAkt1, dPTEN, InR, TORC1, Glut-1, and Glut-3 were determined with quantitative real-time polymerase chain reaction (qRT-PCR) protocol using actin-5C as the housekeeping gene; all data were normalized to unmanipulated group. We used 20 flies per group in triplicates. Primers that we used in qRT-PCR were from the TaqMan® Gene Expression Assay: toll, Dm02151201_g1; PGRP-SD, Dm01840723_s1; defensin, Dm01818074_s1; drosomycin, Dm01822006_s1; metchnikowin, Dm01821460_s1; cecropin A, Dm02151846_gH; JNK(bsk), Dm01803999_g1; dorsal, Dm01810803_g1; Relish, Dm02134843_g1; Dif (Dorsal related immunity factor), Dm01810797_g1; IRS (Chico), Dm01803991_g1; FOXO, Dm02140207_g1; dAkt1, Dm02149559_g1; dPTEN, Dm01844965_g1; TORC1(CTRC), Dm01806284_s1; InR, Dm02136224_g1; Glut-1, Dm01821914_g1; Glut3, Dm02152390_s1; and actin-5C, Dm02361909_s1.
Western immunoblotting for Drosomycin-GFP and Akt
We decapitated 10–12 flies and homogenized in 125 μl of RIPA buffer with phosphatase and proteinase inhibitors. Samples were then heated at 95 °C for 5 min, sonicated, and loaded into the wells of an 18 % SDS-PAGE gel. We used GFP antibody (Santa Cruz Biotechnology Inc., TX) at 1:2500, Phospho-Akt (Ser473) Antibody #9271, and Akt Antibody #9272 (Cell Signaling Technology, Danvers, MA) at 1:5000 dilutions. Anti-ATP- α (a5-c antibody, Developmental Studies Hybridoma Bank, University of Iowa, USA) was used as a loading control. ATP-α is a nuclear encoded plasma membrane protein (the catalytic subunit of the Na+/K+ ATPase). Secondary detection was performed using anti-mouse (1:4000) or anti-rabbit (1:5000) (Bio-Rad, Hercules, CA) HRP conjugated antibodies.
Rapid iterative negative geotaxis (RING)
We joined two empty polystyrene vials by tape vertically facing each other forming an 18.5-cm-long tube . We transferred groups of 20 flies into the vials and allowed to acclimatize to the new setting for 5 min before conducting the assay. Flies were gently tapped down to the bottom of the vial for 10 s with the same interval and strength by the same operator throughout the whole experiment. Pictures of the flies were taken with a digital camera at 5 s. We repeated each geotaxis experiment six times, allowing for 1-min rest periods between each trial and pictures were analyzed by counting the number of flies that climb above the 10-cm mark in 5 s after the tap. We calculated the average of the number of flies crossing the 10-cm threshold and expressed the results as percentage of the total number of flies in the tube (=% climbing index). Each geotaxis experiment was performed 1 h before the needle pricking (0 h baseline) and at 18, 48, 72, and 96 h after needle pricking. The data are presented as percent of the baseline at time 0 h. All of the control groups were kept in antibiotic-containing media.
Protein and metabolite measurements
We measured total protein (Bio-Rad DC), glucose (GAGO20-1KT, Sigma-Aldrich), glycogen (A1602, Sigma-Aldrich), lactate (MAK064-1KT, Sigma-Aldrich), pyruvate (MAK071-1KT, Sigma-Aldrich), and ATP (MAK190-1KT, Sigma-Aldrich) concentrations and lactate dehydrogenase activity (MAK066-1KT, Sigma-Aldrich) in flies at 18, 48, and 168 h (1 week) after infection in triplicate vials with 10–15 flies in each vial, according to the manufacturer’s recommendations. We decapitated flies to minimize red eye color effect on outcomes of the colorimetric assays.
Kaplan-Meyer survival analysis was performed using GraphPad Pad 6 (La Jolla, CA). Statistical analysis between different groups was accomplished with t test analysis and timed changes in geotaxis and NF-κB with ANOVA using GraphPad Pad 6.