Lysine Hydrochloride

All chemicals were purchased from Fisher Scientific (Atlanta, GA), VWR International (Pittsburg, PA) or Sigma Aldrich (St. Louis, MO). Luria-Bertani (LB) liquid media, prepared from its components (5 g yeast extract, 10 g tryptone and 10 g sodium chloride in 1 L ultra-pure DI water), and Mueller-Hinton (MH) liquid media (21 g premixed MH in 1 L ultra-pure DI water) were used to grow E. coli and P. aeruginosa, respectively. LB agar media (40 g premixed LB agar in 1 L ultra-pure DI water) and MH agar media (38 g premixed MH agar in 1 L ultra-pure DI water) were used to enumerate the colony forming units (CFUs) of E. coli and P. aeruginosa strains, respectively (Keren et al., 2004a (link); Amato et al., 2013 (link); Orman and Brynildsen, 2015 (link)). Phosphate Buffered Saline (PBS) solution was used to wash the cells to remove the chemicals and antibiotics before plating them on agar media. For persister assays, 5 μg/ml ofloxacin and 200 μg/ml ampicillin were used (Keren et al., 2004a (link), b (link); De Groote et al., 2009 (link); Allison et al., 2011 (link)). For selection and retention of plasmids in bacteria, 50 μg/ml kanamycin was added in culture media (Orman and Brynildsen, 2015 (link)). To induce fluorescent protein expression, 1 mM IPTG was used (Orman and Brynildsen, 2015 (link)).
Primary drug screening was performed using Phenotype MicroArrays (PM11-20) in 96-well plate formats, containing various chemicals including FDA approved compounds (Biolog Inc., Hayward, CA). Eleven chemicals, identified as initial hits, were purchased separately for further investigation: amitriptyline hydrochloride (Fisher catalog# 50-144-4347), trifluoperazine hydrochloride (Fisher catalog# T28495G), thioridazine hydrochloride (Fisher catalog# 30-705-0), CPZ (Fisher catalog# C24815G), CCCP (Fisher catalog# 04-525-00), protamine sulfate (Fisher catalog# AAJ6292609), promethazine hydrochloride (Fisher catalog# P2029100G), dodecyltrimethyl ammonium bromide (Fisher catalog# D146825G), triclosan (Fisher catalog# 64-795-01GM), polymyxin B Sulfate (Fisher catalog# 52-915-GM) and poly-L-lysine hydrochloride (VWR catalog# IC15269080). All chemicals were dissolved in ultra-pure DI water followed by filter-sterilization, except for CCCP and triclosan which were dissolved in DMSO. All LB and MH media were sterilized by autoclaving. Overnight pre-cultures were prepared in 14-ml falcon tubes containing 2 ml LB broth inoculated from a 25% glycerol (−80°C) cells stock and grown for 24 h at 37°C with shaking (250 rpm). Overnight pre-cultures were diluted in fresh 2 ml media in 14-ml test tubes or 25 ml media in 250-ml baffled flasks for the subsequent assays as described below. Cells cultured in the presence of the solvent (DI water or DMSO) served as controls when the cultures were treated with chemical inhibitors.

For SILAC experiments, THP-1 or U937 cells were cultured in RPMI 1640 deficient in L-arginine and L-lysine, and supplemented with 10% dialyzed FBS. Two linages of cells were cultured in light medium (L-arginine (Arg 0) and L-lysine (Lys 0)) and heavy medium (L-arginine 13C6-15N4-HCl (Arg 10) (#89990, Thermo) and L-lysine 13C6-15N2-HCl (Lys 8)) (#88209, Thermo). Prior to infection, cells were treated with PMA (Phorbol 12-myristate-13-acetate) (#P1585, Sigma-Aldrich) for 24 h, then heavy-labeled cells were infected at 90% confluency with S. Typhimurium 14028S at MOI 100, while light-labeled cells were mock infected. After incubation for 1 h at 37°C in a 5% CO₂ atmosphere, extracellular bacteria were killed at 100 mg/ml gentamicin for 2 h, and then switched to medium containing 25 mg/ml of gentamicin for the remainder of the experiment. Cells were lysed at indicated time points in lysis buffer containing 8M urea, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM ethylene diamine tetra acetic acid (EDTA), 2 g/l Aprotinin, 10 g/l Leupeptin, 1×protease inhibitor cocktail (#CW2200, Cwbio), and 5 mM sodium butyrate. Protein concentration was determined using a BCA protein assay and 5 mg proteins in two states mixed at equal ratio. Samples were digested with trypsin, and peptides were enriched for lysine acetylation using the anti-KAc antibodies noncovalently coupled to protein A agarose beads (#13416, Cell Signaling Technology). Desalted peptides were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Q Exactive mass spectrometer. Heavy arginine (Arg10) and lysine (Lys8) were selected for SILAC quantification. The eXtracted Ion Current (XIC) of all isotopic clusters associated with the identified amino acids sequence in the light label cluster was summed up, and calculated the ratio between two heavy and light label partners. Then the ratio was normalized, the median of the total ratio population was shifted to 1 and data was analyzed using the MaxQuant software version 1.3.0.5. We used Significance A to assess the significance of outlier ratios. Only peptides with average 1.5-fold change of acetylation in two states and significance A values less than 0.05 were considered up or down regulated. The score at lysine acetylation of proteins greater than 20 is considered to be reliable.

A murine monocyte-macrophage cell line J774.2 (ATCC) was grown in DMEM supplemented with 10% fetal bovine serum. For metabolic labeling, the cells were transferred into arginine- and lysine-free DMEM (DMEM for SILAC, Thermo Fisher Scientific) supplemented with 10% dialyzed fetal bovine serum (Invitrogen) and heavy labeled amino acids L-arginine hydrochloride [¹³C₆ ¹⁵N₄] and L-lysine hydrochloride [¹³C₆ ¹⁵N₂] (Sigma-Aldrich) in the same concentrations as in the standard DMEM medium (“heavy” medium). After five cell divisions, the incorporation levels were checked by mass spectrometry (MS) and the labeled cells were stored as frozen stocks in DMEM with 10% DMSO at −150 °C. For the experiment, the labeled cell stock was cultivated in “heavy” medium supplemented with proline (300 mg/mL) to minimize the conversion of arginine to proline [30 (link)]. Volumes of 1 × 10⁷ of cells (heavy labeled and non-labeled) were washed in PBS, harvested in PBS completed with EDTA-free complete protease inhibitor mixture (Roche) and benzonase (Merck) and disrupted in French press by one passage at 2000 psi. Total protein concentration was determined as indicated above. The recombinant GapA-Twin-Strep tagged protein was added to freshly prepared “heavy” labeled cell lysate (6 µg of GapA to 1 mg of cell lysate), incubated for 40 min at 4 °C, and the GapA protein was purified together with its bound proteins using MagStrep “type3” XT beads, as described above. The purification was performed with the same number of lysates from non-labeled cells simultaneously (control of nonspecifically bound proteins onto the purification system). The obtained eluates were mixed and examined by LC-MS/MS analysis.

The construction of the N-terminal Halo-cnp1 strain is described in Vojnovic (2016) . An overnight Halo-cnp1 YES culture was harvested by centrifugation, resuspended in fresh YES, and fixed with 3.7% PFA at RT for 10 min. Afterwards, the sample was washed 3× and residual PFA was quenched using 1× PEM (100 mM Pipes [#P1851-100G; Sigma-Aldrich], pH 6.9, 1 mM EGTA [#EDS-100G; Sigma-Aldrich], and 1 mM MgSO₄ [#M2643-500G; Sigma-Aldrich]) containing 50 mg/ml NH₄Cl (#P726.1; Carl Roth). Next, the sample was permeabilized using 1.25 mg/ml Zymolyase (#320921; MP Biomedicals) in 1× PEM at 37°C for 10 min and subsequently washed 3× with 1× PEM for 10 min. The sample was incubated using a few drops of Image-iT FX signal enhancer (#I36933; Invitrogen) at RT for 1 h to prevent non-specific staining and afterwards stained with 50 nM Halo-CF647 in PEMBAL buffer (1× PEM containing 3% BSA [#A8549-10MG; Sigma-Aldrich], 0.1% NaN₃ [#4221.1; Carl Roth], and 100 mM lysine hydrochloride [#L5626; Sigma-Aldrich]) at RT for 3 h. The stained cells were then washed four times for 10 min with alternating 1× PEM and 1× PBS and incubated at RT for 15 min on a previously washed and with poly-L-lysine–coated Ibidi 8-well glass bottom slide. Finally, the attached cells were washed twice with 1× PEM for 10 min.