Gaspak ez

Strains and plasmids used in this work are listed in Table S1 in the supplemental material. EHEC was routinely grown in LB medium and under conditions where expression of the T3SS was desirable. Dulbecco’s modified Eagle’s medium with 1 g/liter glucose (DMEM-low glucose) was used. E. faecalis strains were routinely cultured in brain heart infusion (BHI) medium. Anaerobic growth was performed using a GasPak EZ anaerobe container system (Becton, Dickinson). HeLa cells were routinely cultured in complete DMEM (cDMEM), defined as DMEM with 4.5 g/liter glucose, 10% fetal bovine serum (FBS), and penicillin/streptomycin/glutamine.

Bacteria were obtained from clinical isolates from children with tooth decay. S. mutans was identified using the methodology proposed by Salazar et al. [22 (link)]. The cultures were made in Petri plates with Columbia agar (Becton Dickinson and Co., NY, and USA) supplied with sucrose (1%) in an anaerobic container (GasPak EZ. Becton Dickinson and Co., NY, USA) and it was incubated at 37°C and 5% of CO₂, for 24 hours. The inoculum was adjusted using optical density comparison from 1.0 to 550 nm which corresponds to 2 × 10⁸ CFU·mL⁻¹ (stock suspension).

Tween 80 was provided from Fluka (Milan, Italy). Hydrocortisone and all chemicals and solvents (HPLC grade ≥ 98%) were purchased from Sigma-Aldrich (Milan, Italy). Buffer solution at pH 7.4 was composed of 7.4 mM Na₂HPO₄×12 H₂O, 1.1 mM KH₂PO₄, and 136 mM NaCl. The culture media and GasPak EZ were supplied by Becton Dickinson and Company (Sparks, MD, USA). l-cysteine hydrochloride monohydrate was purchased from Merck (Darmstadt, Germany).

Metronidazole and DMSO controls were added to the pre-spotted 1536-well Jump-stARter library plates using a Multidrop Combi reagent dispenser (Thermo Scientific), resulting in final assay concentrations of 25 μM and 0.5% (v/v) respectively. Cultured E. histolytica trophozoites were then seeded into the same plates at a density of 400 cells per well, also using a Multidrop Combi reagent dispenser, resulting in a final test compound concentration of 25 μM with the total volume of 2 μL. The plates were sealed into GasPak EZ (Becton-Dickinson) bags and incubated at 37°C for 48hr. 2 μL of CellTiter-Glo luminescence-based cell viability assay reagent was added using a Multidrop Combi reagent dispenser, and plates were incubated in the dark for 10 minutes. Luminescence was measured using an EnVision plate reader (Perkin Elmer). Percent inhibition values for each well were calculated and plotted using Microsoft Excel. Outlying values and plates resulting from observed technical malfunctions such as clogging or bubbling during liquid handling were excluded from the final analysis.

Following a previously-published approach [14 (link)] E. histolytica trophozoites maintained in the logarithmic phase of growth were seeded into 96-well plates (Greiner Bio-One) at 5,000 cells/well to a total volume of 100 μl/well. 8- or 16-point two-fold dilution series of the treatment compounds were prepared, beginning at a maximum final treatment concentration of 50 μM. 0.5 μl of each drug concentration was added to triplicate wells for each treatment group. 0.5 μl of DMSO was used as a negative control, and 0.5 μl of 10 mM metronidazole dissolved in DMSO was used as a positive control, giving a final concentration of 50 μM. Alternatively, wells with only media were used as a negative control. The plates were placed in GasPak EZ (Becton-Dickinson) bags and incubated at 37°C for 48hr. Plates were removed and 50 μl of CellTiter-Glo (Promega) was added to each well. Plates were shaken and incubated in darkness for 20 minutes and the luminescence value of each well was read by a luminometer (EnVision, PerkinElmer). Percent inhibition was calculated by subtracting the luminescence values of each experimental data point from the average minimum signal obtained from positive control values and dividing by the difference between the average maximum signal negative control and the positive control. The resulting decimal value was then multiplied by 100 to give a percentage.

A brain-heart infusion (BHI) broth supplemented with 5 g yeast extract/L and 5% v/v vitamin K+ hemin (BHI-YE; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used to grow the bacteria. Bacterial strains were grown at 37°C in an anaerobic environment, using gas-generating sachets (Gas-Pak EZ; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) to produce the required environment.

All bacterial strains and plasmids used in this study are listed in Table S1. EHEC and E. coli HS were routinely grown in Luria-Bertani (LB) medium. E. faecalis V583 was routinely grown in Brain Heart Infusion (BHI) medium. B. thetaiotaomicron was routinely grown in TYG medium (41 (link)). For coculture experiments, Dulbecco’s modified Eagle’s medium with 1 g/L glucose (low-glucose DMEM) was used, as these conditions are known to induce EHEC virulence gene expression (42 (link)). Anaerobic growth was performed using a GasPak EZ anaerobe container system (Becton, Dickinson). When adequate, media were supplemented with ampicillin (100 μg/mL), gentamicin (50 μg/mL), chloramphenicol (20 μg/mL), erythromycin (10 μg/mL), or streptomycin (100 μg/mL). Bacterial stocks were kept on LB supplemented with glycerol 30% (vol/vol) at −80°C.
HeLa (human cervical adenocarcinoma) cells were maintained in DMEM with 4.5 g/L glucose (high-glucose DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin-glutamine. The Lifeact::GFP-expressing HeLa cell line was obtained with the Flip-In system (Invitrogen) as previously described (43 (link)), and maintained in high-glucose DMEM supplemented with 10% FBS, penicillin-streptomycin-glutamine, and hygromycin (50 μg/mL). Hygromycin was not added when cells were split before infection. Cells were routinely grown at 37°C and 5% CO₂.