Disarming the virulence arsenal of Pseudomonas aeruginosa by blocking two-component system signaling† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc02496k

Pseudomonas aeruginosa infections have reached a “critical” threat status making novel therapeutic approaches required.

For purification, each pellet from 1 L of culture was resuspended in a total volume of ~50 mL lysis buffer (25 mM Tris-HCl, pH 8, 500 mM NaCl, 10% glycerol, and 2 mM DTT) containing 20 units Deoxyribonuclease I (Sigma) and four Complete Mini EDTA-free protease inhibitor tablets (Roche). Resuspended cells were lysed by a Branson Sonifier 250 with 1/8-inch tapered microtip (power setting 3.5, duty cycle 30%) for 1 h 20 min on ice. Lysate was centrifuged at 14,000 xg for 40 min at 4 °C. The supernatant was collected and filtered (0.22 µm). Using an ÄKTApurifier (GE Healthcare) at 4 °C, HK853 was purified from lysate by nickel affinity on a nickel-nitriloacetic acid column (Ni-NTA; Qiagen). Ni-NTA buffer was 25 mM Tris-HCl, pH 8, 500 mM NaCl, 10% glycerol, and 2 mM DTT. An elution gradient of 5 mM imidazole (buffer A) to 1 M imidazole (buffer B) was used to elute His-tagged protein. Eluted HK853 was concentrated for size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg column (GE Healthcare) using 10 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, 0.5 M NaCl, 12% glycerol, and 2 mM DTT. This buffer was also used for storage of protein at -80 °C, in which protein was flash frozen on dry ice/isopropanol. Protein concentration was determined using the DC Protein Assay (Bio-Rad).

Inhibition of HK853 Activity
Commercially available probes (BODIPY-ATPγS) and inhibitors (Rilu-1-12) were obtained from Sigma-Aldrich, Thermo Fisher Scientific, Alfa Aesar and TCI America at >97% purity. Unless otherwise indicated, all commercially available chemicals were used without further purification.
BODIPY-ATPγS competition screening was performed at inhibitor concentrations that did not cause aggregation. Triton X-100 was premixed with reaction buffer to yield 0.1% (v/v) in final 25-µL reactions. In reaction buffer, 1 µM HK853 was preincubated with test compounds (final concentration, 0.01−1250 µM) in 24 µL for 30 min. 1 µL BODIPY-ATPγS was added to bring the final 25-µL reactions to 0.96 µM HK853 and 2 µM BODIPY-ATPγS in the presence of competitors and 5% DMSO. Samples were mixed and incubated in the dark at RT for 1 h before quenching with 8.6 µL 4× SDS-PAGE sample loading buffer and loading 15 µL on a 10% stacking gel. After SDS-PAGE, in-gel fluorescence detection elucidated HK853 activity, and coomassie staining of the gels ensured even protein loading. Integrated density values of the fluorescent gel bands were normalized as "% Activity" with respect to a control that contained no inhibitor. Data were plotted in GraphPad Prism with relation to the log of molar inhibitor to determine IC 50 values (Equation 2).

Data analysis.
Integrated density measurements of in-gel fluorescence and phosphorescence were performed in ImageJ. 2 Data were prepared and analyzed in GraphPad Prism (version 7.0 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com). For all DRCs (control FP competition and activity assays), data were fit to a four-parameter logistic equation, where y is the response, Bottom and Top are plateaus in the units of the y-axis, x is the log of the molar concentration of inhibitor, HillSlope is the slope of the curve, and IC 50 is the concentration of compound required for 50% inhibition (a response half way between Bottom and Top). Some compounds exhibited incomplete DRCs because going to higher concentrations would increase the required DMSO or cause protein aggregation. Visually, this meant there was no curve plateau for the "Bottom" value. However, IC 50 values were desirable for purposes of comparison to other compounds. As a result, IC 50 values were estimated by constraining the bottom of the curve to "0." Figure S1: (a-i) Inhibition of HK853 activity with various Rilu-inhibitors.  Figure S2. Structures of non-leads from the HTS that contain the benzothiazole scaffold. These compounds were found to be protein aggregators and were not pursued further (100 µM-1250 µM). 3

HK853 Aggregation Analysis
To analyze the propensity for compounds to cause aggregation, each was mixed at six concentrations (0−1250 µM) with purified 0.5 µM HK853 in 25 µL of 20 mM HEPES buffer (5% (v/v) DMSO final). After incubating at RT for 30 min, 8.6 µL native-PAGE sample loading buffer was added, and 15 µL was loaded onto a 7.5% polyacrylamide gel. Proteins were resolved by native-PAGE and silver staining. Compound-induced aggregation was detected by the disappearance of the dimeric HK853 band. NH125 (Tocris Bioscience) was used as a positive aggregation control.

Figure S3
: Native-PAGE of HK853 in presence of Rilu-4, 12 and known HK aggregator, NH125 (for comparison). Protein aggregation with NH-125 causes disappearance of the protein dimer band at ≥ 10 µM.

Growth of PA14 cultures with inhibitors
P. aeruginosa PA14 strain was grown overnight (~16 h) in LB media at 37 °C and 200 RPM. The overnight culture was diluted 1:100 in fresh LB media. 4 mL of this culture was then put into a 15 mL borosilicate glass tube, to which Rilu-inhibitors in DMSO were added to a final concentration of 200 µM and 500 µM (for the control experiments, the same amount of DMSO was added). These cultures were grown for 24 h at 37 °C and 200 RPM, and their OD 620 values were measured using a spectrophotometer at various time points. These experiments were done in triplicate.

Heat-shock protein (HSP90) inhibition assay with inhibitors
Commercially available BPS Bioscience kit was used for HSP90α inhibition analysis at different concentrations of inhibitors (0.00001 -1.25 mM) and in triplicates (n=3). For the positive control, geldanamycin, a known HSP90 inhibitor was used (IC 50 ~50 nM).  These cultures were grown for 9 h (late exponential phase) or 24 h (stationary phase) at 37 °C and 200 RPM. The OD 620 values were measured using a spectrophotometer. 1 mL of each culture was placed in a 1.7 mL Eppendorf tube, centrifuged at 5,000 g for 5 min and the supernatant was removed from the cell pellets. The supernatants were directly analyzed on an ESI-LC-MS/MS instrument for metabolites and the cell pellets (from 9 h cultures) were further used for RNA extraction in q-PCR experiments.
ESI-LC-MS/MS analysis was performed on an Agilent 1290 Infinity/MS Agilent 6540 instrument. Injections of 2 µL were separated on Zorbax Eclipse plus C18 column (2.1 mm x 50 mm) and with mobile phases A (5% MeOH:95% water with 0.1% formic acid) and B (95% MeOH:5% water with 0.1% formic acid). The metabolites were separated using an isocratic elution of 100% for 0-3 min, then a 3-17 min linear gradient to 100% B, and 17-20 min of 100% B at a constant flow rate of 0.4 ml/min. Samples were measured in positive ion mode and data was collected in triplicate. Data were analyzed using the Agilent MassHunter Quantitative Analysis suite. From the extracted ion chromatograms (EICs) of the respective metabolite signals, the peak areas were normalized to the OD 620 growth values of the respective cultures. All of the peak areas of the treated samples were plotted by considering the DMSO-control sample to be 100% abundance. The following ions were analyzed:

Rapid attachment assays 5
P. aeruginosa PA14 strain was grown overnight (~16 h) in LB media at 37 °C and 200 RPM. The overnight cultures were washed and diluted in LB medium to an OD 620 value of 1.0. 100 µL of this suspension was used to inoculate each well of a microtiter plate in presence of Riluinhibitors in DMSO at 200 µM or 500 µM (for the control experiments, same amount of DMSO was added). Eight wells per plate were used for each sample. Cells were allowed to adhere for 60 min at 37 °C and 200 RPM. After the planktonic cells were removed, staining with crystal violet was carried out, as described in the biofilm assay procedure. Assays were repeated in triplicate.

Swarming assays and quantification of rhamnolipids in swarm-plates
We followed the revised protocol developed by Trembley et al. for P. aeruginosa swarm assays. 6 Plates consisted of modified M9 medium [20 mM NH 4 Cl; 12 mM Na 2 HPO 4 ; 22 mM KH 2 PO 4 ; 8.6 mM NaCl; 1 mM MgSO 4 ; 1 mM CaCl 2• 2H 2 O; 11 mM dextrose; 0.5% casamino acids (Difco)] solidified with Bacto-agar (Difco). The M9 media without MgSO 4 and CaCl 2 •2H 2 O was autoclaved, which were added after cooling the media ~60 °C. The pH was adjusted after autoclaving with HCl and NaOH (pH = 7.5). Then, a 1-in-100 dilution of filter-sterile stock containing 1 mg/mL Nile red (Sigma Aldrich) dissolved in 85% ethylene glycol and added (prepared the day of use to limit photoinactivation). 7 Twenty mL of this media was poured into Petri dishes, along with the Rilu-inhibitors in DMSO at final concentrations, 125 µM or 200 µM (for the control experiments, same amount of DMSO was added). Plates were allowed to dry for 60 min, with the first 20 min under UV light to ensure plates were sterile following pH adjustment. P. aeruginosa PA14 strain was grown overnight (~16 h) in LB media at 37 °C and 200 RPM. Five µl of bacteria were spotted at the center of each plate, which were incubated at 30 °C in the dark and ~20% humidity. The plates were intermittently taken out for fluorescence scanning [scan settings: GE Typhoon Variable Mode Imager 9500, using 532-nm laser with DY-520XL (λex: 520 nm, λem: 664 nm)]. The scans were visualized and integrated density measurements of whole-plate fluorescence was measured using ImageJ software. Images of these plates were also taken with a Nikon D5200 DSLR camera, which were processed in Adobe Photoshop Lightroom 5 (Mac version) for phase-contrast.     Error bars represent the standard error of three independent experiments (n = 3). Statistical analysis performed with non-parametric one-way ANOVA (α= 0.05; ****p < 0.0001; ***p < 0.001; **p < 0.02, *p < 0.05). Asterisks closest to the bars denote the significance of the difference between inhibitor-treated samples and the DMSO-treated sample, while the asterisks outside the bracket denote the significance of the difference between the two inhibitor concentrations.