Irgasan

TCS (Irgasan, 5-chloro-2-(2, 4-dichlorophenoxy) phenol, ≥ 97.0 % purity (HPLC), CAS no. 3380-34-5) and dimethyl sulfoxide (DMSO, 99 % purity) were purchased from Sigma-Aldrich (St. Louis, MO).

The disinfectant soap used was liquid soap often used for domestic cleaning. It includes triclosan (Irgasan), 97% granular, as the active ingredient purchased from Sigma- Aldrich (St. Louis, MO, AC abstract 3380-34-5). It was once evaluated against larvae of Cx. quinquefasciatus mosquitoes and proved to have a lethal effect [21 (link)].

A Tn-Seq pool was constructed in P. aeruginosa strain PAO1 using the vector pBT20. First, E. coli SM10λpir + pBT20 was grown overnight at 37°C on LB agar containing 100 μg/ml ampicillin (Sigma-Aldrich). P. aeruginosa PAO1 was grown at 42°C overnight on LB agar with no antibiotic. The bacteria were scraped off the plates and mixed in equal volumes of each at a ratio of OD₆₀₀ 40:20 donor (E. coli): recipient (P. aeruginosa). Conjugation mixtures were spotted on to LB agar plates and incubated for 2 h at 37°C. All conjugation spots were then scraped into LB and plated on LB agar with 25 μg/ml irgasan (Sigma-Aldrich) and 25 μg/ml gentamicin (Sigma-Aldrich). The cells were grown overnight then counted and collected in LB, and stored in 20% glycerol at −80°C. The procedure was repeated until at least 200,000 mutants were collected. The collected mutants were thawed at 37°C, normalized to equalize the number of colonies per mL per conjugation, and then pooled and recovered at 37°C. They were recovered by adding 0.5 ml of pooled cells into 50 ml LB with 15 μg/ml gentamicin. 20% glycerol was added to the final pool and it was aliquoted into 1 ml volumes and flash frozen with liquid nitrogen before storing at −80°C.

PBMCs were incubated overnight with the following compounds to inhibit specific metabolic pathways: glycolysis, 5 nM 2-deoxy-D-glucose (2-DG, Sigma-Aldrich); glutaminolysis, 10 μM bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES, Sigma-Aldrich); mTOR, 5 nM rapamycin (Sigma-Aldrich); autophagy, 10 μM spautin-1 (a kind gift from Dr. Stephanie Graff-Dubois); FA oxidation, 100 μM etomoxir (Sigma-Aldrich); FA synthesis, 25 μM irgasan (Sigma-Aldrich); and cholesterol synthesis, 1 μM simvastatin (Sigma-Aldrich). Pre-treated cells were cultured under resting conditions or activated for 24 h with plate-bound αCD3, then surface stained as described above to measure the expression of activation markers by flow cytometry. The activation/inhibition ratio was measured for each T-cell subset using the following formula: 1 – (% HLA-DR⁺ on activated cells with inhibitors – % HLA-DR⁺ on resting cells)/(% HLA-DR⁺ on activated cells – % HLA-DR⁺ on resting cells). Spanning-tree progression analysis of density-normalized events (SPADE) was conducted using three activation markers (CD134, HLA-DR, and PD-1), and t-distributed stochastic neighbor embedding (t-SNE) was used to check the clustering generated via SPADE.