Kh2po4

E. timonensis SN18 was purchased from Leibniz Institute DSMZ. E. timonensis SN18 culturing was performed in Hungate or Balch tubes (Chemglass Life Sciences) at 37 °C and set up in an anaerobic chamber (Coy Laboratory Products) under an atmosphere of 2% to 4% H₂, 2% to 4% CO₂, and N₂ as the balance. Standard cultures were grown in peptone-yeast-glucose (PYG) medium, modified (medium recipe DSM 104, DSMZ Germany) that was sparged with N₂ after autoclaving. Basal medium lacking electron acceptors was prepared as described previously (57 (link)), containing 1 g/L tryptone (trypticase peptone; BD Biosciences), 1 g/L yeast extract (BD Biosciences), 0.4 mM l-cysteine, 2.5 g/L NaHCO₃, 1 g/L NaCl, 0.5 g/L MgCl₂•6H₂O, 0.2 g/L KH₂PO₄, 0.3 g/L NH₄Cl, 0.3 g/L KCl, 0.015 g/L CaCl₂•2H₂O, 0.25 mL/L of 0.1% resazurin, and 1% ATCC vitamins and trace mineral solutions (ATCC). NCE medium lacking carbon sources was prepared as described previously (59 (link)), containing 4 g/L KH₂PO₄, 5 g/L K₂HPO₄, 3.5 g/L NaNH₄PO₄ 40 mM sodium fumarate dibasic, 1 mM MgSO₄•7H₂O, 0.1% casamino acids (VWR Life Science), and 1% ATCC vitamin and trace mineral solutions (ATCC). All chemicals were purchased from Sigma-Aldrich unless otherwise indicated.

pfhsp70-1-mCherry was subcloned into a P. falciparum expression vector, pfYC103 as previously reported (Wagner et al., 2013 (link)). The plasmid was purified using a Qiagen Maxi kit and resuspended in CytoMix (25 mM HEPES, pH 7.6, 2 mM EGTA (Sigma), 5 mM MgCl₂ (Fisher Scientific), 8.66 mM K₂HPO₄ (Fisher Scientific), 1.34 mM KH₂PO₄ (VWR International), 120 mM KCl (Fisher Scientific), 0.15 mM CaCl₂ (Sigma)) (Rug and Maier, 2013 (link); Crabb et al., 2004 (link)). For transfection of P. falciparum 3D7, 100 μL ring-stage parasites (14–18 hpi) at 5% parasitemia was resuspended in 300 μL CytoMix containing 75 μg plasmid DNA in a 0.2 cm electroporation cuvette (Bio-Rad). Electroporation was performed at 0.31 kV and 950 μF with maximal capacitance using a Gene Pulser II system (Bio-Rad). The parasites were then cultured in complete medium at 1% hematocrit at 37°C. Selection of transfectants with 200 nM pyrimethamine (Sigma) started at 3 days post-transfection. PfHsp70-1-mCherry expression in transfected parasites was verified by fluorescence microscopy.

Most chemicals were provided by Sigma Aldrich (Oakville, ON, Canada). EDTA, KCl, K₂HPO₄, and KH₂PO₄ were obtained from VWR (Ville Mont-Royal, QC, Canada), cytochrome c from equine heart was provided by Alfa Aesar (Tewksbury, MA, United States), and the protein standard was from Bio-Rad (Mississauga, ON, Canada).

For the receptor solution preparation, PBS (free from Ca²⁺ and Mg²⁺, VWR), propan-1,2-diol (ACROS Organics^TM), and sodium azide (VWR) were used. Methanol HPLC grade (Fischer Scientific, Thermo Fischer Scientifics^TM, Waltham, MA, USA), KH₂PO₄ (VWR), and ortho-phosphoric acid (VWR) were used for the chromatographic analysis. Glass beads with 3–5 mm and syringe PTFE filters with a 0.45 µm pore diameter both from VWR. Biological membranes EPISJ13 U (EPISKIN^®, Lyon, France) were purchased along with maintenance medium MAIN3.

All reagents used were of analytical grade. All solutions were prepared using ultra-high pure (UHP) 18 Ω water. All laboratory ware was soaked for 24 h in an acid bath containing 10% (v/v) nitric acid and then rinsed with UHP water. Concentrated hydrochloric acid (12 mol L⁻¹), HNO₃ 69%, NaCl, NH₄Cl, anhydrous Na₂SO₄, CaCl₂.2H₂O, NaHCO₃, anhydrous D ( + )-glucose, sodium arsenate dibasic heptahydrate, cadmium acetate and lead acetate trihydrate were obtained from Fisher Scientific. d-glucuronic acid, Pancreatin (pig), pepsin (pig), Bovine serum albumin (BSA), KSCN, NaH₂PO₄, mucin (pig), D-glucosamine hydrochloride, lipase (pig), α-amylase (Bacillus species), urea and bile salts (bovine), modified eagle medium (DMEM) containing 4.5 g/L glucose, penicillin-streptomycin solution (10, 000 units penicillin and 10 mg streptomycin per mL), and Hank’s Balanced Salt Solution (HBSS) and glycine were obtained from Sigma–Aldrich (St. Louis, MO, USA). KH₂PO₄, MgCl₂·6H₂O, KCl, and uric acid were obtained from VWR. Caco-2 cells (HTB-37™) were purchased at passage 18 from the American Type Culture Collection (ATCC, Manassas, VA, USA). Fetal bovine serum and trypsin-EDTA were purchased from Invitrogen (Burlington, ON, Canada).

Sodium carbonate (Na₂CO₃), sodium hydrogen phosphate (Na₂HPO₄), and potassium dihydrogen phosphate (KH₂PO₄) were acquired from VWR (Saint-Prix, France). Nitric acid (HNO₃) was obtained from Panreac (Barcelona, Spain). Methanol, absolute ethanol (EtOH) and hexane were purchased from VWR Chemicals (Rosny-sous-Bois, France). CO₂ was obtained from Carburos Metálicos (Massalfassar, Valencia, Spain). EtOH 96° was obtained from Guinama (La Pobla de Vallbona, Valencia, España). Folin–Ciocalteu reagent, potassium persulfate (K₂S₂O₈), gallic acid, ABTS (2,2-Azino-Bis-3-eThylbenzothiazoline-6-Sulphonic Acid), fluorescein, 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH), and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma Aldrich (Steinheim, Baden-Württemberg, Germany).

Strains and plasmids used in this study are listed in Supplementary Tables 4 and 5. E. coli strains were grown on Luria–Bertani agar (VWR) or in Luria–Bertani broth (VWR) with aeration at 37 °C. S. aureus strains were grown on tryptic soy agar (VWR), in TSB (Difco) or in M9 minimal medium (KH₂PO₄ 3.4 g l⁻¹, VWR; K₂HPO₄ 2.9 g l⁻¹, VWR; di-ammonium citrate 0.7 g l⁻¹, Sigma-Aldrich; sodium acetate 0.26 g l⁻¹, Merck; glucose 1% (w/v), Merck; MgSO₄ 0.7 mg l⁻¹, Sigma-Aldrich; CaCl₂ 7 mg l⁻¹, Sigma-Aldrich; casamino acids 1% (w/v), Difco; minimum essentrial medium amino acids 1×, Thermo Fisher Scientific; and minimum essential medium vitamins 1×, Thermo Fisher Scientific) at 200 rpm with aeration at 37 °C, 30 °C or 25 °C. When necessary, culture media were supplemented with antibiotics (100 μg ml⁻¹ ampicillin, Sigma-Aldrich; 10 μg ml⁻¹ erythromycin, Apollo Scientific; and 10 μg ml⁻¹ chloramphenicol, Sigma-Aldrich). Unless otherwise specified, 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal, Apollo Scientific) was used at 100 μg ml⁻¹, IPTG (Apollo Scientific) was used at 0.5 mM and Atc (Sigma-Aldrich) was used at 2 ng ml⁻¹.