Short-chain reactive probes as tools to unravel the <i>Pseudomonas aeruginosa</i> quorum sensing regulon
In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed "quorum sensing" (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-L-homoserine lactone (C 4 -HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a 'click' tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.
Introduction<!>General methods<!>Materials<!>Strain and cell culture conditions<!>Growth and luminescent activity assays<!>Pyocyanin production assays<!>His 6 -TEV-PhzB1 expression<!>Purication of His 6 -TEV-PhzB1 protein using immobilized metal affinity chromatography (IMAC)<!>The specicity of labeling towards His 6 -TEV-PhzB1<!>StageTips preparation and desalting<!>Probe design and structure-activity relationship studies<!>Proteome analysis and protein identications<!>PhzB2 transposon mutant evaluation assays<!>Conclusion<!>Conflicts of interest
<p>Most Gram-positive and Gram-negative bacteria use a type of cell to cell communication called QS in which bacteria produce, secrete and respond to small signaling molecules called autoinducers. [1][2][3] These compounds help bacteria sense the bacterial population density during the growth of the culture. With the increase in the bacterial population, the concentration of the autoinducers increases, and at a certain population threshold (quorum), the bacteria start to act collectively and begin to express QS-regulated genes. The ensuing phenotypic changes help the bacteria to adapt to environmental challenges. 4,5 QS is involved in the regulation of virulence factors, development of genetic competence, transfer of conjugative plasmids, sporulation, antimicrobial peptides, secondary metabolite synthesis and symbiosis with animals, bacteria and plants, 6 both in Gram-positive and Gram-negative bacteria. 5 Among the QSregulated factors one can nd bioluminescence in Vibrio scheri, pyocyanin and biolm production in Pseudomonas aeruginosa, [7][8][9] pneumolysin and polysaccharide capsule production in Streptococcus pneumonia, 10 etc. In P. aeruginosa, a Gram-negative pathogen, the QS systems are regulated by two LuxI-LuxR homolog systems, 11 the long-chain AHL-based, LasI-LasR system, and the short-chain AHL-based RhlI-RhlR system. In addition to the AHL-based systems, P. aeruginosa uses the PQS system, and in recent years, additional signals have been proposed to contribute to the complexity of the large cellular network of QS systems in P. aeruginosa. 12 The Las and the Rhl systems regulate P. aeruginosa gene expression and are organized hierarchically. In the Las system, the LasR receptor protein responds to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) autoinducer which is synthesized by the LasI synthase protein. The Rhl system responds to N-butyryl-Lhomoserine lactone (C 4 -HSL) autoinducer, which is synthesized by the RhlI synthase protein. C 4 -HSL is thought to bind to the RhlR receptor protein. The LasR-3-oxo-C 12 -HSL complex regulates the expression of lasI and induces a positive feedback loop through the up-regulation of 3-oxo-C 12 -HSL synthesis by the Las system. In addition, the expression of rhlI and rhlR is also regulated by the LasR-3-oxo-C 12 -HSL complex, placing the Las system higher up the hierarchy compared with the Rhl system. 13 However, under certain conditions, the Las system is secondary to the Rhl system. 14 The PQS (2-heptyl-3-hydroxy-4-quinolone) signal system is dependent on, and regulated by the balanced production of 3-oxo-C 12 -HSL and C 4 -HSL, and is thus linked with the Las and Rhl QS systems. 15 In addition, PQS itself plays a role in the regulation and expression of virulence genes. From a biochemical perspective, the Rhl QS sub-system is the least well-understood of the three main QS signaling pathways and here we show another possible path for this system (Fig. 1).</p><p>To begin to access the C 4 -HSL interactome, we used a powerful method termed Activity-Based Protein Proling (ABPP), to help gain a deeper understanding of how C 4 -HSL impacts on cell physiology. This method has become a major tool for the identication and functional characterization of individual proteins in complex proteomes. 16,17 We have synthesized a series of probes based on the scaffold of C 4 -HSL and introduced to the molecular structure a diazirine photoreactive moiety and ketone or alkyne analytical handles for oxime ligation or copper-mediated azide-alkyne coupling (CuAAC) reactions, respectively. We evaluated the biomimetic activity of these analogues with reporter strains, and with the use of the most suitable probe, we proceeded to label intact bacteria. Aer cell lysis and reaction with rhodamine azide, we evaluated the labeled proteome by SDS-PAGE. Finally, using a biotinylated tag, we captured the C 4 -HSL interactome by affinity chromatography and analyzed the labeled proteins by mass spectrometry. The resulting positive hits were evaluated through assays with dened transposon mutants and recombinant proteins, revealing a previously unknown connection between several proteins and the P. aeruginosa virulence regulon 18 (Fig. 2).</p><!><p>For ash chromatography, Sigma-Aldrich silica gel, high-purity grade, pore size 60 Å, 230-400 mesh particle size, 40-63 mm was used. TLC was performed using Merck TLC silica gel 60 F254 glass plates (20 Â 20 cm), detected by UV (l ¼ 254 nm), or stained using potassium permanganate and heated at 150 C. All compounds MS analyses were performed with LCQ Fleet mass spectrometer (Thermo Fisher Scientic) with an ESI source. Spectra were collected in positive ion mode and analyzed by Xcalibur soware (Thermo Fisher Scientic). For LC-MS (ESI) analyses, a Surveyor Plus HPLC System (Thermo Fisher Scientic) was used, equipped with a Luna 5 mm C18(2), 100 Å, (150 Â 4.6 mm) column at a ow rate of 0.5 mL min À1 , using a mobile phase gradient of H 2 O containing 0.1% FA (solvent A) and acetonitrile containing 0.1% FA (solvent B). 1 H and 13 C NMR spectra were recorded on a Bruker DPX-400 MHz spectrometer. Chemical shis are reported in d ppm relative to TMS or residual solvent peak. Multiplicities were reported as singlet (s), broad singlet (br s), doublet (d), doublet of doublet (dd), doublet of doublet of doublet (ddd), doublet of triplet of doublet (dtd), triplet (t), triplet of doublet (td), quintet (quint), sextet (sext), multiplet (m) and broad multiplet (br m). NMR data were analyzed using MestReNova 6.0.2. Optical density and luminescence measurements were performed on a Varioskan Flash Spectral Scanning Multimode Plate Reader (Thermo Fisher Scientic) and SpectraMax M2 Microplate reader (Molecular Devices). EC 50 values were calculated using Graph-Pad prism 9.0.0 and conrmed as reported. 19 Protein concentrations were determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientic). UV irradiation was performed on TFX-20 LM 365/312 nm UV table (100% intensity). Cell lysates were prepared by sonication with a Sonics VCX-750 Vibra-Cell Ultra Sonic processor with a 3 mm probe. SDS-PAGE was separated with BIO-RAD Mini-PROTEAN Tetra system and PowerPac Basic power supply. Fluorescence readouts were obtained using an ImageQuant Fuji LAS4000 scanner. Quantitative ABPP analysis was performed on LTQ Orbitrap XL ETD (Thermo Fisher Scientic)/NanoLC-2D (Eksigent) and analyzed with MaxQuant 1.6.0.16.</p><!><p>All chemicals were obtained from Sigma Aldrich, Acros organics, Alfa Aesar, Bio-Lab, J.T. Baker, and Fluka unless otherwise noted, and were used without additional purication. Solvents were used as received from DAEJUNG, Bio-Lab, and J.T. Baker.</p><!><p>All bacterial strains were cultured from cryo stocks (maintained in 50% glycerol at À80 C) and grown in LB (BACTO™) medium or LB agar (BACTO™). Wild-type P. aeruginosa strain PAO1 was grown in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) for liquid bacterial cultures, and on LB agar medium (LB with 1.5% agar) for solid bacterial cultures. To monitor Rhl signaling activity, P. aeruginosa double mutant, PAO-JP2 (DlasI/rhlI) carrying pKD-rhlA (encoding the rhlA promoter fused to the luxCDABE luminescence reporter gene 20 ) was grown in LB medium and on LB agar supplemented with 300 mg mL À1 of trimethoprim (Sigma). P. aeruginosa transposon mutants, PW4328 and PW4329, were obtained from the University of Washington PAO1 transposon mutant library 21,22 and grown in LB medium and on LB agar supplemented with 5 mg mL À1 of tetracycline (Fluka). Verication of transposon insertion was done via PCR as described (http://www.gs.washington.edu/labs/ manoil/libraryindex.htm). Escherichia coli BL21 (DE3) carrying pRARE2 and phzB1 cloned into the expression vector, pET-19m, was grown in LB medium supplemented with 25 mg mL À1 of</p><!><p>The P. aeruginosa Rhl reporter strain (PAO-JP2 pKD-rhlA), was inoculated into 20 mL of fresh LB medium in a 50 mL PP sterile conical centrifuge tube (Greiner Bio-One) and incubated aerobically at 37 C with 180 rpm shaking overnight. The cultures were grown to late stationary phase and were diluted into fresh LB to OD 600 of 0.05. Flat-bottomed, white polystyrene, 96-well, microtiter plates (Greiner Bio-One) were prepared with the desired range of probe concentrations (0-1 mM) from 10 mM DMSO stock solutions and the diluted culture was added to obtain a bacterial OD 600 of 0.025. The plates were then incubated in a Varioskan Plate Reader for a period of 15 h at 37 C with good aeration (vigorous agitation). During this time, luminescence and OD 600 measurements were made at 20 min intervals. The luminescence and OD 600 data were plotted against probe concentration, and EC 50 values were calculated. For agonist control experiments, a nal concentration of 10 mM C 4 -HSL was used.</p><!><p>Triplicate samples of wild-type P. aeruginosa strain PAO1, and the otherwise isogenic phzB2 transposon mutants, PW4328 and PW4329, were incubated separately with good aeration in 50 mL PP sterile conical centrifuge tubes, each containing 15 mL of LB medium. The bacteria were grown overnight at 37 C with shaking at 180 rpm to late stationary phase. Following this, 10 mL of the overnight-grown cultures (OD 600 z 1.0) were inoculated in triplicate into 25 mL LB in 250 mL autoclaved Erlenmeyer asks. Various concentrations of C 4 -HSL, (0-100 mM, in DMSO) were added to each ask. The highest concentration (1% v/v) of DMSO (with no C 4 -HSL) employed in the doseresponse experiment, was added to the control asks. The bacteria were incubated aerobically for 24 h at 37 C with shaking at 180 rpm. OD 600 measurements were taken and pyocyanin was extracted directly from bacterial cultures as follows: ve mL of bacterial culture and 3 mL of chloroform were added to a 15 mL PP sterile conical centrifuge tube (Greiner Bio-One). The mixture was agitated to extract the pyocyanin and claried by centrifugation at 4 C, 3220 Â g for 10 min. The organic and aqueous phases were separated, and 2 mL of the greenish-blue chloroform layer was transferred to a new 5 mL glass test tube. This was mixed with 0.3 mL HCl (0.2 M) which gave the pyocyanin-containing aqueous layer a pinkish color. An aliquot (150 mL) of the aqueous layer from each sample was transferred to a TC Microwell 96F Nunclon D SI plate and the absorption was measured at 520 nm. HCl (0.2 M) was used as blank and subtracted from the absorption of the extracted pyocyanin samples.</p><!><p>A single bacterial colony of E. coli BL21 (DE3) carrying pRARE2 and phzB1::pET-19m strain was inoculated into 10 mL of fresh LB medium supplemented with 25 mg mL À1 chloramphenicol (Sigma Aldrich) and 50 mg mL À1 carbenicillin disodium salt (CHEM-IMPEX INT'L INC) in a 50 mL PP sterile conical centrifuge tube. The culture was incubated with good aeration at 37 C overnight. Five mL of this starter culture was used to inoculate 800 mL of fresh LB medium supplemented with 25 mg mL À1 chloramphenicol and 50 mg mL À1 carbenicillin disodium salt in a 2 L sterile Erlenmeyer ask, and the culture was once again grown at 37 C with good aeration on a rotary shaker (180 rpm) until OD 600 reached 0.5-1. Protein expression was induced by addition of IPTG (ACROS ORGANICS) to a nal concentration of 1 mM. Following induction, the temperature was lowered to 16 C and growth (with vigorous aeration, shaking at 180 rpm) was continued overnight. The bacterial pellet expressing recombinant protein was collected by sedimentation (3220 Â g, 15 min, 4 C, or room temperature). The supernatant was discarded and the cell pellet was washed with ice-cold 1Â phosphate-buffered saline solution (PBS). The washed cell pellet was resuspended in ice-cold 5 mL of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 2 mL mL À1 protease inhibitor cocktail (Merck), pH 8.0) and transferred to 15 mL PP sterile conical centrifuge tube. The bacterial suspension was sonicated for 7.5 min in een 30 s pulses with 60 s rest between runs at 40% amplitude setting while maintaining the sample on ice. The cell debris was removed by sedimentation (20 000 Â g, 15 min, 4 C, or room temperature), and the lysate was ltered through a 0.22 mm membrane lter (Millex®).</p><!><p>An aliquot (1.6-2 mL) of Ni-NTA agarose slurry (QIAGEN) was added to a 1 Â 10 cm glass column (Glass Econo-Column®) and the resin was allowed to settle for a few minutes. The column was equilibrated with 10 resin-bed volumes of lysis buffer. The cleared lysate was applied to the equilibrated column and the ow rate was adjusted to 0.5-1 mL min À1 . The column was rinsed with wash buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) until the eluate A 280 had returned to baseline. The bound protein was eluted in elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, glycerol (5% v/v), 5 mM b-mercaptoethanol, pH 8.0) and 1 mL fractions were collected. The A 280 of the fractions was measured. Fractions containing protein were pooled and analyzed by SDS-PAGE (4% stacking and 12% resolving gel). The puried protein samples were dialyzed in Snakeskin Dialysis tubing 7000 MWCO (Thermo scientic) against 2 L of stirred PBS at 4 C. Aer 8 h, the reservoir buffer was changed (again, 2 L of PBS) and dialysis continued overnight. The dialyzed protein sample was divided into aliquots in clear PP tubes (BIOLOGIX®) and frozen in liquid nitrogen. The frozen samples were stored at À80 C.</p><p>In situ proteome oxime ligation PAO-JP2 (pKD-rhlA) was inoculated into 200 mL of LB media in a sterile Erlenmeyer ask. The culture was grown overnight at 37 C with good aeration to late stationary phase and harvested at 3220 Â g at 4 C. The pellet was washed and resuspended twice with fresh LB, then sedimented once more. The pellet was resuspended in 10 mL of fresh LB media. Aliquots of 1 mL were transferred to clear 15 mL PP sterile conical centrifuge tubes, and the bacteria were incubated in the presence of probe 13 (1, 0.5, 0.1, 0 mM) for 4 h. The cells were then pelleted for 10 min at 3220 Â g (4 C) and resuspended in 1 mL of PBS. The cell suspension was irradiated at 365 nm for 3.5 min. The samples were then mixed with 3 mL of EDTA-free protease inhibitor cocktail (Merck) and sonicated for a total of 2 min in twelve 10 second pulses with 45 s rest between runs at 40% amplitude setting. Protein concentrations were determined as was mentioned above. For the subsequent ligation reactions, the reaction mixtures (100 mL nal volume) contained 0.5 mg mL À1 protein, 10 mM aniline, 100 mM sodium acetate buffer (pH 5.1), and 700 nM aminooxy bodipy. 23 Aer 12 h at room temperature with vigorous mixing (shaking at 1000 rpm), the reactions were quenched and precipitated by the addition of methanol/ chloroform/water mixture (2 reaction volumes of methanol, 1/ 2 reaction volume of chloroform and 1 reaction volume of H 2 O). The precipitate was washed once with ice-cold methanol to remove unreacted aminooxy bodipy and other reagents. The precipitate was pelleted and the supernatant was removed. The proteins were resuspended in the same reaction volume (100 mL) of 1Â SDS sample buffer and heated for 10 min at 95 C. The redissolved proteins were resolved by SDS-PAGE (4% stacking and 10% separating gels) alongside a protein sizing ladder (3 mL SMOBIO PM2500 Excel Band 3-color Regular Range Protein Marker). Fluorescent bands in the gels were revealed using a atbed scanner and Cy3 lter. Following this, the gels were stained with Coomassie Brilliant Blue R-250, destained, and scanned in non-uorescent mode.</p><p>In situ Cu 1+ -mediated azide-alkyne coupling (CuAAC) reactions PAO-JP2 (pKD-rhlA) was inoculated into 200 mL of LB media in a sterile Erlenmeyer ask. The culture was grown overnight at 37 C with good aeration to late stationary phase and harvested at 3220 Â g (4 C). The pellet was washed and resuspended twice with fresh LB. The pellet was then resuspended in 10 mL of fresh LB media. Aliquots of 1 mL were transferred to clear 15 mL PP sterile conical centrifuge tubes, and the bacteria were incubated in the presence of probe 12 (1, 0.5, 0.1, 0 mM) for 4 h. The cells were then pelleted for 10 min at 3220 Â g (4 C) and resuspended in 1 mL of PBS. The cell suspension was irradiated at 365 nm for 3.5 min. The samples were then mixed with 3 mL of EDTA-free protease inhibitor cocktail (Merck) and sonicated for a total of 2 min in twelve 10 s pulses with 45 s rest between runs, and 40% amplitude setting. Protein concentrations were determined as mentioned above. CuAAC reactions (200 mL total reaction volume (100 mL proteome + 100 mL click reagents)) contained nal reagent concentrations of 0.5 mg mL À1 protein, 100 mM of rhodamine azide, 24,25 100 mM TBTA (Sigma-Aldrich), 1 mM CuSO 4 (Sigma-Aldrich) and 1 mM TCEP (Sigma-Aldrich) were performed. Aer 1 h at room temperature with vigorous mixing (shaking at 1000 rpm), the reactions were quenched and precipitated by the addition of 2 reaction volumes of methanol, 1/2 reaction volume of chloroform and 1 reaction volume of H 2 O. The resulting protein pellet was washed once with ice-cold methanol and pelleted. The pelleted proteins were resuspended in the same reaction volume (100 mL) of 1Â SDS sample buffer, heated for 10 min at 95 C, and analyzed as described previously.</p><p>Concentration-dependent His 6 -TEV-PhzB1 labeling Puried His 6 -TEV-PhzB1 protein samples were thawed, and the protein concentration was measured. Labeling reactions were carried out in 1.5 mL transparent PP tubes (Greiner Bio-One) in a nal volume of 100 mL. Samples adjusted to contain 0.5 mg mL À1 of His 6 -TEV-PhzB1 in PBS, were incubated in the presence of probe 12 (dissolved in DMSO) at the indicated concentrations of 0-50 mM for 45 min at room temperature, with vigorous mixing (shaking at 600 rpm). During this time, the samples were wrapped in aluminium foil to shield them from light. DMSO was used as control and never exceeded 2% nal concentration. The reaction mixtures were UV irradiated at 365 nm for 30 s. Aer irradiation was completed, the reactions were quenched by the addition of 500 mL of acetone (À20 C). The reaction mixtures were placed in the dark at À20 C for 20 min and the precipitated protein was pelleted (20 000 Â g, 10 min, 25 C). The supernatant was removed and the pellets were washed with 500 mL of cold acetone (À20 C). The washed pellets were air-dried and redissolved in 2.5% w/v aqueous SDS. The samples were heated at 95 C and vigorously vortexed to ensure complete dissolution for 5 min. CuAAC reactions (100 mL total reaction volume) contained a nal concentration of 50 mM rhodamine azide, 50 mM TBTA (Sigma-Aldrich), 0.5 mM CuSO 4 (Sigma-Aldrich), and 0.5 mM TCEP-HCl (Combi Blocks) were performed. Aer 1 h of vigorous mixing (shaking at 1000 rpm) at room temperature, the proteins were precipitated with 500 mL of cold acetone (À20 C), pelleted, and the pellets were washed once with cold acetone. The resulting pellets were air-dried and resuspended in 100 mL SDS sample buffer and analyzed as described previously.</p><!><p>His 6 -TEV-PhzB1, bovine serum albumin (BSA, Chem Cruz) and lysozyme (Sigma-Aldrich) were each prepared in PBS and subjected to the labeling reaction as described above for puried His 6 -TEV-PhzB1. All nal protein concentrations in the reaction mixtures were adjusted to 0.5 mg mL À1 and probe 12 concentration xed at 20 mM.</p><p>Labeling of His 6 -TEV-PhzB1 in the presence of competing C 4 -HSL His 6 -TEV-PhzB1 protein samples were labeled as described above except that the reactions contained 0-1000 mM C 4 -HSL (dissolved in DMSO) and a xed concentration (20 mM) of probe 12. The probe was added last and the labeling reaction was allowed to proceed for 5 min, aer which, 8 s UV irradiation (365 nm) was performed and the proteins were analyzed by SDS-PAGE as described previously.</p><p>In vitro quantitative ABPP Wild-type P. aeruginosa strain PAO1 was inoculated into 200 mL of LB media in a sterile Erlenmeyer ask. The culture was grown with good aeration (shaking at 180 rpm) at 37 C overnight to late stationary phase. The cells were pelleted at 3220 Â g (4 C). The pellet was resuspended and washed three times with PBS. The nal pellet, aer washing, was frozen in liquid nitrogen. When required, the pellet was resuspended in 10 mL of PBS supplemented with EDTA-free protease inhibitor cocktail (Merck) (3 mL per mL of resuspended cells) and sonicated for 2 min in twelve 10 s pulses with 45 s rest between runs, at 40% amplitude setting. The protein concentration was determined. For the ABPP, two sets of triplicate reaction mixtures were set up in 1.5 mL transparent PP tubes. The mixtures contained 500 mL of bacterial protein extract, adjusted to 1 mg mL À1 . One group was incubated in the presence of probe 12 (dissolved in DMSO) at a nal concentration of 200 mM and the other group in the presence of C 4 -HSL (dissolved in DMSO) at a nal concentration of 10 mM. Aer 1 h of incubation at room temperature, the samples were irradiated at 365 nm for 1.5 min and CuAAC reagents were added to make a nal volume of 1 mL. The nal concentration of CuAAC reagents in the reaction mixtures was 50 mM biotin azide, 50 mM TBTA (Sigma-Aldrich), 0.5 mM CuSO 4 (Sigma-Aldrich) and 0.5 mM TCEP (Sigma-Aldrich). Aer 1 h at room temperature with vigorous agitation (shaking at 1000 rpm), the reaction mixtures were transferred to 15 mL transparent PP sterile conical centrifuge tube and the proteins were precipitated with methanol/chloroform/H 2 O (2 reaction volumes of methanol 1/2 reaction volume of chloroform and 1 reaction volume of H 2 O). The precipitated proteins were pelleted and washed once with cold methanol. The protein pellet was resuspended in 500 mL of 6 M urea (Sigma-Aldrich) and TCEP was introduced to a nal concentration of 10 mM. The mixture was incubated for 0.5 h at 37 C with shaking at 180 rpm, followed by the addition of IAA (Sigma-Aldrich) to a nal concentration of 55 mM. The mixtures were le shielded from light at room temperature for 0.5 h with gentle agitation on a at-bed shaker. Finally, 5 mL of PBS containing 0.2% of SDS was added to each of the tubes, thereby diluting the urea but retaining the labeled proteins in soluble form. In parallel, 100 mL aliquots of streptavidin-agarose bead suspension (Merck Novagen) were introduced to 1.5 mL transparent PP tubes. The beads were washed three times with PBS before use, and the overburden of supernatant was removed. For affinity enrichment, the washed beads were introduced to the SDS-solubilized reaction mixtures and the resulting suspensions were incubated for 1.5 h at room temperature with gentle agitation on a at-bed shaker. The beads were then gently pelleted (1400 Â g) for 3 min, and the overburden of liquid was decanted off. The beads were then washed with 5 mL of 0.2% SDS and re-pelleted. This was repeated three times. Following this, the beads were washed (three times) with 5 mL PBS, followed by three washes in pure water. The beads from each reaction were then transferred to new low-binding 1.5 mL transparent PP tubes (Eppendorf) and pelleted for 2 min at 1400 Â g. The supernatant was removed and replaced with 200 mL of 2 M urea (Sigma-Aldrich) in PBS containing 1 mM CaCl 2 (J.T. Baker) and 10 ng mL À1 trypsin (Promega). The mixture was incubated overnight at 37 C with shaking at 180 rpm. Following this, the beads were pelleted at 1400 Â g for 2 min and the supernatant (containing the released tryptic peptides) from each reaction mixture was transferred, separately, to a new low-binding 1.5 mL transparent PP tubes. The pelleted beads were washed once with 100 mL PBS and the supernatant was pooled to the corresponding reaction mixture containing the tryptic peptides. Each tryptic peptide mixture was supplemented with (5% nal) HPLC grade formic acid (FA, Bio-Lab) and the samples were puried and desalted using StageTips (see below). Finally, the mixtures were dried in a speed-vac and stored at À20 C until LC-MS/MS analysis. Data processing was performed with Max-Quant and peptides were identied with the Uniprot PAO1 proteome. Carbamidomethylation of cysteine residues was considered as a xed modication and methionine oxidation (M) and acetyl (protein N-terminal) were treated as variable modications. Otherwise, the default settings in the soware were applied. For probe 12 and C 4 -HSL (control) triplicates, the experimental data were analyzed with Microso Excel and proteins represented by >1 peptide were ltered and quantied for all replicates.</p><!><p>StageTips were prepared according to a modied protocol 26 with C 18 solid-phase extraction disc (3M Empore). Between 4-6, 1 mm cut discs were loaded into 200 mL pipette tips. The discs were packed tightly with the help of a truncated needle, and washed once with MeOH, twice with 80% ACN containing 0.5% FA (Bio-Lab) and four times with H 2 O containing 0.5% FA (50 mL each buffer). The tips were spun at low speed (40 Â g for 2 min) between the washes, allowing the C 18 column to stay wet with a thin layer of liquid aer each spin. The tryptic peptides from each sample were loaded in two consecutive batches onto each StageTip, and the tips were desalted once with 50 mL of H 2 O containing 0.5% FA. Peptides were eluted from the StageTips into 1.5 mL low-binding transparent PP tubes by the addition of four successive 50 mL aliquots of 80% ACN containing 0.5% FA. For subsequent MS analysis, the samples were resuspended (with sonication in a water bath) in 10 mL of H 2 O containing 0.5% FA.</p><!><p>In this study, we aimed to determine which proteins in the P. aeruginosa proteome interact with the C 4 -HSL autoinducer. This way, we hoped to reveal potentially novel enzymes/proteins that might be regulated by C 4 -HSL. We synthesized molecular probes based on the C 4 -HSL structure, harboring handles that allow for bioorthogonal reactions. In all luminescent bioassays, we employed PAO-JP2 (pKD-rhlA). PAO-JP2 contains deletions in the lasI and rhlI genes, and therefore cannot synthesize endogenous 3-oxo-C 12 -HSL and C 4 -HSL (respectively). However, the chromosomally-encoded rhlR gene is intact in this mutant, enabling it to sense exogenously-added C 4 -HSL. In addition, the pKD-rhlA plasmid borne in this strain, is a reporter for RhlR activity. Here, the RhlR-regulated rhlA promoter is fused upstream of a cluster of lux genes, enabling facile monitoring of C 4 -HSL and C 4 -HSL agonist/antagonist activities through bioluminescence. We introduced the different structural probes to identical cultures of PAO-JP2 (pKD-rhlA) and compared their ability to stimulate lux gene expression with the natural autoinducer C 4 -HSL (Table 1). This allowed us to assess which probes best mimic the activity of the natural autoinducer. In terms of probe design, we adhered to three main principles of ABPP: (a) the affinity-based structural element should be minimally modied compared with the natural ligand, (b) the reactive group that allows for covalent attachment of the probe to the binding site on the target protein should be small and not overly reactive (here, we chose to employ the diazirine moiety, which, upon irradiation at 365 nm, forms a reactive carbene capable of binding covalently to the nearest carbon or heteroatom), and (c) the analytical "handle" assisting in visualization, separation, and analysis of the bound proteins should be minimally-reactive with any of the functional moieties naturally present in proteins. The "handles" we chose to employ in the current study were the alkyne group (which has been successfully used in copper-assisted azide-alkyne coupling (CuAAC)) and a ketone group for aniline-catalyzed oxime ligation reactions. 27 The analysis of the probe activities is shown in Fig. S29-S32. † As outlined in Table 1, we evaluated differences in aliphatic chain length, diazirine position, ketone position, and ring size. In the rst analogue that we synthesized, 2, we substituted the four-carbon chain of C 4 -HSL with a ve-carbon chain. This change slightly increased the bioactivity of the compound. Based on this result, we decided to append the alkyne "handle" to the end of the aliphatic chain of C 4 -HSL. The resulting compound (3) showed increased bioactivity as well.</p><p>We next tested whether replacement of the lactone with a ketone moiety was feasible. This was done in order to prevent possible lactonolysis of the probes. 28 However, for both ve and six-membered ring analogues this led to a large decrease in bioactivity (compounds 4 and 5). Next, we examined how the introduction of a diazirine moiety affected probe bioactivity. The diazirine moiety had strong impact on bioactivity compared to the alkyne containing probes (compounds 6 and 7). Interestingly, we observed that the reduced precursors of 6, 7, 13, and 14 (containing hydroxyl moieties in the ring, 8-11) also exhibited bioactivity compare to the corresponding ketones, indicating that small changes in the polar head group can be tolerated (notably, compounds 8 and 9 exhibited low activation levels of the system but still display activation of Rhl with low EC 50 values). These combined observations led us to focus on three nal probe designs; in compound 12, we introduced the diazirine moiety into the six-carbon ring and the alkyne moiety into the side chain, and in compounds 13 and 14, shorter-chain analogues of 7 and 6, respectively, we introduced the diazirine moiety into the side chain, and the ketone moiety into the ring. As all three ABPP probes, 12, 13 and 14 could potentially be used to label the P. aeruginosa proteome, we carried out a deeper quantitative analysis of their ability to activate the Rhl QS system (Fig. 3) and compare them to C 4 -HSL bioactivity as control. 29 Interestingly 13 and 14 showed more robust activation of the Rhl system than the natural autoinducer C 4 -HSL. Compound 12 displayed lower maximal activation than 13 and 14, and lower potency (EC 50 of 57.7 mM vs. 11.3 and 10.7 mM, respectively). However, based on these data we concluded that all three probes (12, 13 and 14) are capable of activating the Rhl QS system (albeit, to differing extents) and are able to mimic the natural autoinducer C 4 -HSL, at least in terms of RhlR-dependent activation of rhlA transcription. Based on these efficacy data, and taking into account the anticipated selectivity of the ligation reactions (CuAAC and oxime ligation), we chose to further examine compounds 12 and 13 in order to identify novel C 4 -HSL-binding proteins. Compound 12 was selected as the presence of an alkyne analytical handle enables a CuAAC click reaction with an azide-containing uorophore or affinity tag, and 13 was selected due to its ability to activate rhl strongly, and the presence of a ketone moiety which can be used in oxime ligation reactions.</p><p>Rhodamine azide and bodipy aminooxy uorescent dye labeling of P. aeruginosa PAO-JP2 (pKD-rhlA)</p><p>Our goal was to identify and characterize proteins that might play a hitherto unrecognized role in the P. aeruginosa QS response.</p><p>Based on the fact that C 4 -HSL is membrane permeable, 30 we used a two-step ABPP approach 31 in which the C 4 -HSL based probes 12 and 13, which contain a diazirine reactive group and an alkyne (12) or ketone (13) analytical handle, were incubated at varying concentrations with cultures of P. aeruginosa PAO-JP2 (pKD-rhlA). The cells were washed, resuspended in PBS, irradiated, homogenized and then CuAAC click or oxime ligation reactions were performed. Fig. 4A, B and S33 † show the results of the photo labeling experiments with probe 12 and probe 13. The uorescent scans of the proteins resolved aer SDS-PAGE show a clear difference in the selectivity of protein labeling between the two reactions, CuAAC with rhodamine azide and the oxime ligation with bodipy aminooxy and the two probes; probe 13 (or the uorescent bodipy aminooxy tag) labels the P. aeruginosa proteome much less selectively due to the poor selectivity of the oxime ligation reaction, whereas probe 12 appears less reactive, but labeling is ultimately more specic due to the more selective CuAAC reaction. In the latter case, two weakly-labeled bands were observed with masses of z50 kDa and z42 kDa, and a more strongly-labeled band of mass z29 kDa.</p><!><p>Aer these initial labeling and optimization experiments, we repeated the labeling experiments with probe 12 but instead of conjugation with rhodamine azide, we now used biotin azide in order to isolate the labeled proteins. Wild-type P. aeruginosa strain PAO1 was grown to late stationary phase and harvested. The cell lysate was prepared and incubated separately with probe 12 and with C 4 -HSL (as negative control). The samples were then irradiated with UV light at 365 nm and subjected to CuAAC reactions with biotin azide. The biotinylated proteins were captured with streptavidin agarose beads and subjected to tryptic digestion, as described in the Experimental section. The tryptic digests were analyzed by LC-MS/MS and the proteins  were quantied and identied using MaxQuant. Proteins that were identied in the negative controls were subtracted from the positive probe 12 experimental results. Although many proteins were identied in these experiments, only a few were seen in all seven replicates (Table 2), and among these are the virulence-related phenazine biosynthetic proteins, PhzB1 and PhzB2. These enzymes, which are almost identical at a sequence level, are expressed of different operons and comprise part of the pyocyanin biosynthesis pathway. 32,33 Pyocyanin is a major virulence factor in P. aeruginosa, and is used by the pathogen to establish successful colonization in new niches. Another virulence-related protein that was identied is the putative transcriptional regulatory protein AlgP. AlgP, together with algR and algQ, is a key factor in the transcription of the biosynthesis gene algD. This protein is a key component of the production machinery for exopolysaccharide alginate, a critical virulence factor in P. aeruginosa. The alginate is associated with respiratory infections in cystic brosis patients and it is essential for adhesion and anti-phagocytosis and contributes to the persistence of the bacteria. 34,35 Another labeled protein that was identied is a putative ClpA/B protease ATP-binding subunit. The ClpA and ClpB proteins belong to the Clp/Hsp100 ATPase family and function as chaperone proteins. Both ClpA and ClpB are involved in protein folding and stabilization; ClpA is a regulatory subunit in the ClpAP protease, which participates in the degradation of protein aggregates. ClpB is known to be required for colonization and in-host survival by several bacterial pathogens. 36,37 We also identied an uncharacterized protein (Q9HU11/PA5178), which, aer BLAST analysis, was found to contain a domain belonging to the LysM family of peptidoglycan-binding proteins. LysM homologs are present in a large number of proteins in both prokaryotes and eukaryotes, although the majority of LysM-homologs are bacterial peptidoglycan hydrolases. Several LysM domain-containing proteins from bacterial pathogens such as Staphylococcus aureus, Listeria monocytogenes, Streptococcus agalactiae are known virulence factors, involved in cell separation/autolysis and adherence to human cells, etc. 38</p><!><p>Phenazines are redox-active, heterocyclic compounds with distinctive coloration. They serve as both soluble electron mediators, and as virulence factors. They are secreted by a variety of different bacterial species, although the position of various substituents on the phenazine ring system determines the toxicity of the phenazine toward prokaryotic and eukaryotic organisms. In P. aeruginosa the biosynthesis of phenazines is carried out by enzymes expressed from two almost identical, but differentially regulated operons (phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2). The enzymes encoded by PhzA-G convert chorismic acid to phenazine-1-carboxylic acid, and the latter is then converted to phenazine-1-carboxamide, 1-hydroxyphenazine and 5-methylphenazine-1-carboxylic acid betaine through the action of enzymes encoded by the unlinked genes phzH, phzS, and phzM, respectively. In a downstream reaction, 5methylphenazine-1-carboxylic acid betaine is transformed by PhzS into pyocyanin. Pyocyanin is a strongly-colored blue phenazine and a potent P. aeruginosa virulence factor. 32,39 Pyocyanin catalyzes the formation of reactive oxygen species, which can inhibit microbial growth 40 and induce rapid apoptosis of human neutrophils. 41 In people with cystic brosis, pyocyanin inhibits the ciliary function of respiratory epithelial cells and alters the host immune and inammatory response. 42 It can also stimulate the release of prostaglandin (prostacyclin), disrupt calcium homeostasis and inactivate the antioxidant enzyme, catalase. 43 The synthesis of pyocyanin is ultimately regulated by several different QS systems in P. aeruginosa (among them, the PQS, and indirectly the Las and Rhl systems). In the Rhl system, the N-butyryl-L-homoserine lactone autoinducer binds to RhlR, which activates the transcription of pyocyanin biosynthesis genes. 7 In addition, through a coordinated production of 3-oxo-C 12 -HSL and C 4 -HSL, the production of the PQS (2-heptyl-3-hydroxy-4-quinolone) signal is regulated, and this, in turn, affects the regulation and expression of virulence genes of the Rhl system, and thus, the production of pyocyanin. 44 The PhzB2 protein has been suggested to be a major contributor to the synthesis of phenazine-1-carboxylic acid (PCA), the precursor of pyocyanin. 45 To examine whether C 4 -HSL might inuence pyocyanin production, we compared the effect of adding exogenous C 4 -HSL to cultures of wild-type P. aeruginosa strain PAO1 and cultures of two transposon mutants (PW4328 and PW4329) which both lack functional PhzB2 (Fig. 5). While we can observe a C 4 -HSL concentrationdependent inhibition of pyocyanin production in wild-type P. aeruginosa strain PAO1, this inhibitory effect is less pronounced in strain PW4328 and is almost absent in strain PW432s9 (Fig. 5A1, A2 and A3 resp. and Fig. S34 †). This C 4 -HSL concentration-dependent inhibition of pyocyanin production indicates that in high concentrations of C 4 -HSL there appears to be a "switch off" effect on pyocyanin production, a phenomenon that could be linked to energy and resource-saving mechanisms in the bacteria in order to enable non-virulent survival in the host environment. Importantly, while the relatively high C 4 -HSL concentrations, at which we observe a signicant decrease in pyocyanin production, is generally not reached in planktonic cultures, in biolms, the local concentrations of autoinducers can be much higher (>600 mM). 46 To our knowledge, this is the rst indication that direct interaction between C 4 -HSL and factors of the pyocyanin biosynthetic machinery may enable P. aeruginosa to down-regulate the synthesis of its major virulence factor in a population-density dependent manner. Most likely P. aeruginosa has additional tools in its arsenal of factors that enable the correct and bene-cial assessment of its environment under changing conditions, such as the other C 4 -HSL binding proteins that we identied (LysM, AlgP and ClpA/B), and this is the subject of ongoing investigations.</p><p>Cu 1+ -Mediated azide-alkyne coupling (CuAAC) reactions; bioorthogonal labeling of PhzB1</p><p>As mentioned above, P. aeruginosa has two almost identical operons, the phzA1-G1 (phz1) and phzA2-G2 (phz2), with $98% similarity at the nucleotide level. 47,48 In P. aeruginosa, PCA is modied to yield bioactive phenazine derivatives by the enzymes PhzH, PhzS, and PhzM. 49 PhzB1 is one of the enzymes that are responsible for the conversion of chorismic acid, an intermediate product of the shikimate pathway, 50,51 to PCA and PDC via various phenazine intermediates with the help of the other mainly involved enzymes PhzA/B, PhzD, PhzE, PhzF, and PhzG, the crystal structures of which are known. 52 The general role of PhzB in the phenazine biosynthesis is to catalyze the condensation of two precursor molecules of 6-amino-5oxocyclohex-2-ene-1-carboxylic acid (AOCHC, see Fig. S39 †) to form the tricyclic phenazine (HHPDC). This tricyclic phenazine intermediate can undergo enzymatic/nonenzymatic transformations to yield DHPCA and DHPDC, the precursors of more than 150 structural derivatives of strain-specic phenazines. 53,54 To directly investigate the labeling of PhzB1 by the C 4 -HSL mimic, probe 12, we expressed recombinant His 6 -TEV-PhzB1 in E. coli Rosetta strain BL21 (DE3). The puried His 6 -TEV-PhzB1 protein migrated as a single band with a molecular mass of z20 kDa (Fig. S35B and C †) which corresponds to the predicted molecular mass of the protein based on the DNA sequence. The puried His 6 -TEV-PhzB1 protein was used to assess compound 12 and C 4 -HSL binding in vitro. First, we established how much probe 12 was required to label His 6 -TEV-PhzB1 in vitro (Fig. S36A and B †). Based on these data, we chose to work with 20 mM of probe 12 in subsequent experiments. This was a tradeoff between the concentration required to give an intense uorescent signal, and ensuring that the probe concentration would be comparable with the physiological range of C 4 -HSL concentration in vivo. Having selected the optimal concentration of probe 12 to use, we proceeded to assess the specicity of labeling towards His 6 -TEV-PhzB1. Probe 12 was incubated with His 6 -TEV-PhzB1 in the presence of BSA and lysozyme as control proteins (Fig. S37A and B †). Probe 12 efficiently labeled the z20 kDa His 6 -TEV-PhzB1 protein (lanes 2, 4 and 6) with much lower labeling of the BSA control (corresponding to the band with a molecular mass z60-62 kDa). No labeling of the lysozyme control (z14 kDa) was observed. We conclude that probe 12 preferentially interacts with the His 6 -TEV-PhzB1 protein. We next examined whether C 4 -HSL would compete with probe 12 and block the labeling of His 6 -TEV-PhzB1. This was indeed the case (Fig. 6 and S38 †); increasing concentrations of C 4 -HSL led to markedly lower labeling of the protein by probe 12 (Fig. 6A lanes 2-5 and S38A, lanes 1-4 †). Quantitation of the uorescent band intensity (Fig. 6C) conrmed this observation. From these experiments we conclude that C 4 -HSL binds to PhzB1, and currently we examine in more detail the mechanisms underlying the consequent reduction in pyocyanin biosynthesis (see phenazine biosynthetic pathway, Fig. S39 †). 52</p><!><p>Using a C 4 -HSL-based ABBP strategy, we report here on the identication and validation of PhzB1/2 as C 4 -HSL binding proteins, and we propose that binding of the autoinducer to PhzB1/2 may result in a decrease of pyocyanin production. As such, this binding event may serve an important role in providing information to the bacteria regarding their population density. Both PhzB1 and PhzB2 are engaged in C 4 -HSLinduced phenazine biosynthesis, and upon an increase of population density, when P. aeruginosa reaches its quorum, a strong increase in production of pyocyanin can be observed. At higher population densities, however, this production is reduced, a phenomenon that can be mimicked by the addition of increasing concentrations of C 4 -HSL to growing P. aeruginosa cultures. Our studies show that C 4 -HSL binds PhzB1/2 and inhibits pyocyanin production, and while at lower concentrations this autoinducer activates the Rhl QS system, resulting in the production of virulence factors, the interaction with PhzB1/ 2 at higher concentrations could provide a negative feedback loop to this system. We surmise that this mechanism enables the bacteria to correctly sense and adjust their population density-dependent behaviour, such that the optimal amount of energy is spent on virulence factor production when needed, and these efforts can be reduced when the population has reached an optimal density.</p><!><p>There are no conicts to declare.</p>