Dissecting the antibacterial activity of oxadiazolone-core derivatives against Mycobacterium abscessus

Mycobacterium abscessus (M. abscessus), a rapidly growing mycobacterium, is an emergent opportunistic pathogen responsible for chronic bronchopulmonary infections in individuals with respiratory diseases such as cystic fibrosis. Most treatments of M. abscessus pulmonary infections are poorly effective due to the intrinsic resistance of this bacteria against a broad range of antibiotics including anti-tuberculosis agents. Consequently, the number of drugs that are efficient against M. abscessus remains limited. In this context, 19 oxadiazolone (OX) derivatives have been investigated for their antibacterial activity against both the rough (R) and smooth (S) variants of M. abscessus. Several OXs impair extracellular M. abscessus growth with moderated minimal inhibitory concentrations (MIC), or act intracellularly by inhibiting M. abscessus growth inside infected macrophages with MIC values similar to those of imipenem. Such promising results prompted us to identify the potential target enzymes of the sole extra and intracellular inhibitor of M. abscessus growth, i.e., compound iBpPPOX, via activity-based protein profiling combined with mass spectrometry. This approach led to the identification of 21 potential protein candidates being mostly involved in M. abscessus lipid metabolism and/or in cell wall biosynthesis. Among them, the Ag85C protein has been confirmed as a vulnerable target of iBpPPOX. This study clearly emphasizes the potential of the OX derivatives to inhibit the extracellular and/or intracellular growth of M. abscessus by targeting various enzymes potentially involved in many physiological processes of this most drug-resistant mycobacterial species.


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controls consisting of amikacin (50 µg/mL) and 1% DMSO (i.e., infected macrophages only); as well as positive controls containing amikacin (50 µg/mL) plus 80 μg/mL (i.e., 267 µM) IMP were also included. Plates were incubated for 24 h at 37 °C, 5% CO2. In each case, the viability of infected macrophages was checked by addition of trypan blue [6] before cell lysis. Cells were washed three times with PBS and lysed by adding 200 μL of 0.1% Triton X-100. Serial dilutions of each culture were then plated at least in triplicate on 7H9 agar medium supplemented with 0.2% glucose. Colonies were counted after 4 to 5 days of incubation at 37 °C to check intracellular bacterial viability following treatment with each compound concentration. DMSO-treated infected macrophages corresponded as control representing 100% of bacterial viability. Intracellular MICRaw values were estimated by fitting the CFU% sigmoidal dose-response curves in Kaleidagraph 4.2 software (Synergy Software). The lowest compound concentration inhibiting 50% of intracellular bacterial growth was defined as the MIC50Raw. Experiments were done three times independently.

Activity-Based Protein Profiling (ABPP) for iBpPPOX target enzymes identification. From 300
mL of culture at the logarithmically growth stage (OD600 ~1.5), M. abscessus R cells were harvested by centrifugation at 4,000 g for 15 min, and adjusted to a final concentration of 6  10 9 cells/mL (i.e., OD600 of 40) in 7H9-S (7H9 broth + 0.2% glycerol + 0.05% Tween 80 + 0.2% glucose). One mL of this suspension was incubated with iBpPPOX (400 µM final concentration), or DMSO (control) at 37 °C for 2-3 h. under gentle shaking at 75 rpm. Bacteria were then washed 3 times with PBS containing 0.05% Tween 80, and resuspended in PBS buffer at a 1:1 (w/v) ratio. The bacterial cells (500 µL) were mixed with 350 µL of 0.1 mm diameter glass beads (BioSpec) in a 2-mL Eppendorf tube and disrupted during 2 × 4 min of violent shaking, with ice cooling between each run, using mini-Beadbeater-96 (BioSpec). The lysate was cooled down in ice for 5 min and then centrifuged at 4°C and at 13,500 g for 15 min to remove the cell debris and unbroken cells. Supernatants were adjusted to a concentration of 1 mg/mL of total proteins, snap frozen in liquid nitrogen and stored at -80°C until further use.
Both iBpPPOX-treated M. abscessus R and DMSO-control lysate samples (750 µL -0.75 mg total proteins) were incubated with 2 µM ActivX™ Desthiobiotin-FP probe for 90 min at room temperature. The reaction was next stopped by adding 0.45 g of urea (10 M final concentration) to complete denaturation of proteins. Unreacted probes were removed using Zeba Spin desalting column (7K MWCO, ThermoFisher Scientific) and labelled proteins were further captured by 200 µg Nanolink streptavidin magnetic beads 0.8 µm (Solulink), according to the manufacturer's instructions. First, 20 µL of a 10 mg/mL NanoLink streptavidin magnetic beads was transferred into a 1.5 mL Eppendorf tube. The Wash Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) was then added to bring the final volume to 250 µL and the resulting mixture was mixed gently S4 to resuspend and wash the beads. The tube was placed on a magnetic stand for 2 min. and the supernatant was discarded. The tube was removed from the magnetic stand and the beads were washed two more times with the Wash Buffer (250 µL). Each M. abscessus treated-lysate sample was enriched for labelled proteins by transfer to the previously washed beads (around 200 µg). The lysate/beads suspensions were incubated for 1 h. at room temperature with mild shaking. The tubes were then placed on the magnetic stand for 2 min to collect the beads, and the supernatant was removed. The beads containing bound, biotinylated proteins were washed three times carefully with the Wash Buffer, as described above, and resuspended in 30 µL PBS buffer pH 7.4 containing 50 mM free biotin. The resulting solution was mixed with 4X Laemmli reducing sample buffer, and heated at 95°C for 5 min. This step allowed the recovery of the captured labelled proteins by exchanging the initially captured desthiobiotin/streptavidin complex to the greater affinity biotin/streptavidin complex. Each sample was snap frozen in liquid nitrogen and stored at -80°C before mass spectrometry experiments.
To check for unspecific binding, a DMSO-treated lysate sample was also incubated only with the streptavidin-magnetic beads in absence of Desthiobiotin-FP probe treatment, and processed as described above. Mass spectrometry analysis. Protein extract were loaded on NuPAGE 4-12% Bis-Tris acrylamide gels (Life Technologies) to stack proteins in a single band that was stained with Imperial Blue (Pierce, Rockford, IL) and cut from the gel. Gels pieces were submitted to an in-gel trypsin digestion [7] with slight modifications. Briefly, gel pieces were washed and destained using 100 mM NH4HCO3.

Capture of M. abscessus potential target enzymes from iBpPPOX-treated total lysate via
Destained gel pieces were shrunk with 100 mM ammonium bicarbonate in 50% acetonitrile and dried at room temperature. Protein spots were then rehydrated using 100 mM ammonium bicarbonate pH S5 8.0 buffer containing 10 mM DTT for 45 min at 56 C. This solution was replaced by 100 mM ammonium bicarbonate pH 8.0 buffer containing 55 mM iodoacetamide and the gel pieces were incubated for 30 min at room temperature in the dark. They were then washed twice in 100 mM ammonium bicarbonate buffer and finally shrunk by incubation for 5 min with 100 mM ammonium bicarbonate buffer in 50% acetonitrile. The resulting alkylated gel pieces were dried at room temperature. The dried gel pieces were re-swollen by incubation in same buffer supplemented with trypsin (12.5 ng/µL; Promega) for 1 h at 4 °C and then incubated overnight at 37 °C. Peptides were harvested by collecting the initial digestion solution and carrying out two extractions; first in 5% formic acid and then in 5% formic acid in 60% acetonitrile. Pooled extracts were dried down in a

Protein identification and quantification. Relative intensity-based label-free quantification (LFQ)
was processed using the MaxLFQ algorithm [8] from the freely available MaxQuant computational proteomics platform, version 1.5.3.8 [9]. The acquired raw LC Orbitrap MS data were first processed using the integrated Andromeda search engine [10]. Spectra were searched against a UniProt M. using a minimum ratio count of 1 (unique+razor) and the second peptide option to allow identification of two co-fragmented co-eluting peptides with similar masses. The false discovery rate (FDR) at the peptide and protein levels were set to 1% and determined by searching a reverse database to limit the list of identified proteins. All proteins that cannot be distinguished based on their identified peptides were assembled into a single protein group according to the MaxQuant rules. The statistical analysis was done with Perseus program (version 1.5.6.0) from the MaxQuant environment S7 (www.maxquant.org). The LFQ normalised intensities were uploaded from the proteinGroups.txt file.
First, proteins marked as contaminant, reverse hits, and "only identified by site" were discarded.
Quantifiable proteins were defined as those detected in at least 100% of samples in at least one condition. Protein LFQ normalized intensities were base 2 logarithmized to obtain a normal distribution. Missing values were replaced using data imputation by randomly selecting from a normal distribution centred on the lower edge of the intensity values that simulates signals of low abundant proteins using default parameters (a downshift of 1.8 standard deviation and a width of 0.3 of the original distribution). In this way, imputation of missing values in the controls allows statistical comparison of protein abundances that are present only in the inhibitor's samples. To determine whether a given detected protein was specifically differential a two-sample t-test was done using permutation-based FDR-controlled at 0.01 and 0.05, and employing 250 permutations. The p value was adjusted using a scaling factor s0 with a value of 1 [11].
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (www.proteomexchange.org) [12] via the PRIDE partner repository with the dataset identifier PXD015680.
Mass spectrometry analysis of Ag85CMabs-iBpPPOX complex. Purified Ag85CMabs recombinant protein (14 μM -100 µg) was further incubated for 1 h in its native form with iBpPPOX, using an enzyme/inhibitor molar ratio E/I= 1:100 to ensure total inhibition. Samples of the resulting Ag85CMabs-iBpPPOX complex were analysed on a MALDI-TOF-TOF Bruker Ultraflex III spectrometer (Bruker Daltonics, Wissembourg, France) controlled by the Flexcontrol 3.0 package (Build 51), as described previously [13]. This instrument was used at a maximum accelerating potential of 25 kV and was operated in linear mode using the m/z range from 20,000 to 100,000 (LP_66 kDa Method). Five external standards (Protein Calibration Standard II, Bruker Daltonics) were used to calibrate each spectrum to a mass accuracy within 200 ppm. Peak picking was performed with Flexanalysis 3.0 software (Bruker) with an adapted analysis method. To eliminate salts from the samples, 10 µL of each preparation was submitted to a desalting step on a C4 Zip-Tip µcolumn (Millipore). 1 µL of desalted sample was mixed with 1 µL α-cyano-4-hydoxycinnamic acid matrix in a 50% acetonitrile/0.3% TFA mixture (1:1, v/v). 1 µL was spotted on the target, dried and analysed with the LP_66 kDa method. Peak picking was performed with Flexanalysis 3.0 software (Bruker) with an adapted analysis method. Parameters used were as follows: SNAP peak detection algorithm, S/N threshold fixed to 6 and a quality factor threshold of 30. pMyc Multicopy E. coli -mycobacterium shuttle vector, hygromycin cassette, acetamide inducible promoter [14] pUX1 Plasmid produced by ligating the colE1 origin and kanamycin cassettes-containing XmnI fragment of pMV261-AflII to the blunted SpeI fragment from pTEC27 containing the tdTomato and hygromycin cassettes. [15] pVV16 Multicopy E. coli -mycobacterium shuttle vector, kanamycin cassette, hsp60 constitutive promoter [14]