2 2 bipyridyl

Bc407 was grown in LB medium at 37 °C with agitation (200 rpm) until the end of the exponential growth phase (OD₆₀₀ = 2). Then, bacteria were diluted 1/100 in RPMI 1640 Medium GlutaMAX^TM (SKU 61870-010, Thermofisher), and exposed to diverse stresses for 15 min to 60 min at 37 °C without agitation.
NOC5 (146724-82-5, Calbiochem) was used as NO donor at a final concentration of 10 and 50 µM. Sodium nitrite (7632-00-0 Sigma-Aldrich) was used at 2.5 mM, Potassium nitrate (7757-79-1, Merck) at 20 mM and hydrogen peroxide solution (7722-84-1, Sigma-Aldrich) at 0.03% (V/V). The quantity of nitrate in solution was measured using the Griess Reagent System (G2930, Promega) following the manufacturer protocol.
Bacteria were starved from iron during incubation in iron-free RMPI medium for one hour at 37 °C with agitation. Then iron stresses (excess or starvation) were induced during 15 min with Iron III citrate (6100-05-6, Sigma-Aldrich) at 81 µM, and 2,2′-Bipyridyl (366-18-7, Sigma-Aldrich) at 4,4 μM as an iron chelator, respectively [25 (link),26 (link)].

To minimize the heterogeneity of expressed VSPs and HCMPs in the culture, we started off by creating a clonal trophozoite population from the original WB-C6 clone using serial dilution. Briefly, a trophozoite culture was diluted in TYDK and seeded as single cells into the wells of a 96-well plate. Upon reaching 70–80% confluence, a clonal culture was selected used to seed 10 ml tubes containing TYDK, TYDK without added ferric (i.e., ferric ammonium citrate), and TYDK without ferric and supplemented with 50 μM of the metal ion chelator 2,2′-Bipyridyl (Sigma-Aldrich, MO, United States). The concentration of 2,2′-Bipyridyl was the highest that did not affect growth of Giardia trophozoites in TYDK. Iron analyses at ALS (Umeå, Sweden) showed that the standard TYDK contained 5.5 mg/l iron, TYDK without added iron and with added chelator 1.6 mg/ml. It should be noted that the chelator reduces the available, free iron but the total level is not decreased. Upon reaching 80% confluence, trophozoites were detached on ice (10 min), collected by centrifugation (7 min, 750 × g, and 4°C), washed with ice-cold PBS, pelleted (7 min, 750 × g, and 4°C), lysed in 1.5 ml of Trizol and collected in Eppendorf tubes. All collected samples were frozen immediately in dry ice and kept at −80°C until RNA extraction. The experiment was repeated 3 times.

N-hexane (95%), THF (99.9%), and dichloromethane were purchased from Th. Geyer (Renningen, Germany); methanol and acetonitrile (HPLC-grade) were purchased from VWR (Darmstadt, Germany)); N,N-dimethylformamide (DMF, 99%) was purchased from Acros Organics (Geel, Belgium); anhydrous 1,2-dichlorobenzene (DCB, 99%), methanol (HPLC-grade), cobalt carbonyl (Co₂(CO)₈) containing 1%–5% n-hexane as stabilizer, hydrazine monohydrate (N₂H₂ 64%–65%), 2,2′-bipyridyl (99%), styrene (99%), α,α′-dichloro-p-xylene (98%), 18-crown-16 (99%), phthalimide potassium salt (99%), copper(I) chloride (97%), and alumina (activated, neutral, Brockmann Activity I) were purchased from Sigma Aldrich (Steinheim, Germany) and used as received without further purification. copper(I) chloride was purified by stirring in glacial acetic acid overnight, washed with ethanol and dried under vacuum. Co₂(CO)₈ was kept in the fridge of a glovebox, and n-hexane evaporated in the glovebox before use. styrene was passed through a short column of neutral alumina to remove inhibitors and deoxygenated by bubbling with N₂ before starting a polymerization.

2-Aminoethyl dihydrogen phosphate (AEP) (98%) was purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan); DAEMA (98%), copper (I) bromide, 2,2-bipyridyl (99%), carboxymethyl-dextran sodium salt (10-20 kDa), titanium(IV) oxide (anatase, <25 nm particle size, 99.7%), α-bromoisobutyryl bromide (98%), mPBA, methylthiazolyldiphenyl-tetrazolium bromide (MTT), DPBF, thiol tracker violet, and 2,7-dichlorofluorescin diacetate (DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and used without further purification. SCC7 and L929 cells were purchased from the American Type Culture Collection (Manassas, MD, USA). For cell culture, RPMI 1640 and fetal bovine serum (FBS) were purchased from Capricorn Scientific (Ebsdorfergrund, Germany). The antibiotic-antimycotic solution, trypsin-EDTA, and Dulbecco's phosphate-buffered saline (DPBS) were obtained from Welgene (Daegu, Korea). All experiments involving live animals were carried out in accordance with the relevant laws and institutional guidelines of Sungkyunkwan University (SKKUIACUC2019-08-22-1).

Polyclonal rabbit antibodies against W. chondrophila were produced in our laboratory as described previously [44 (link)]. Secondary antibody, Alexa Fluor^® 488 donkey anti-rabbit IgG, was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Clavulanic acid, deferoxamine, mecillinam, novobiocin, penicillin, phosphomycin, piperacillin, teicoplanin, and 2,2′-bipyridyl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vancomycin was obtained from AppliChem (Darmstadt, Germany). MP265 was purchased from American Custom Chemicals Corporation (San Diego, CA, USA). All drugs were diluted in deionized water with the exception of MP265 and 2,2′-bipyridyl, which were diluted in dimethyl sulfoxide (DMSO, AppliChem) and in ethanol, respectively, in order to obtain the specific concentrations described in Table 1. Finally, the solutions were filtered through a 0.22 μm pore filter and stored at −20 °C.