Culture conditions and REMA assay of various bacteria. Mycobacteria (Mycobacterium bovis BCG, M. marinum strain M, M. smegmatis MC2155) were grown in 7H9 broth (Difco) supplemented with Middlebrook albumin-dextrose-catalase (ADC) enrichment, 0.2% glycerol, 0.05% Tween 80. Bacillus subtilis, Candida albicans, Corynebacterium glutamicum ATCC13032, Micrococcus luteus, Pseudomonas putida, Salmonella typhimurium and Staphylococcus aureus were grown in Luria broth base (Sigma). Corynebacterium diphtheriae, Enterococcus faecalis, Listeria monocytogenes and Pseudomonas aeruginosa were grown in brain heart infusion broth (Difco). Two-fold serial dilutions of each test compound were prepared in 96-well plates containing bacteria in a total volume of 100 μl and then incubated at 37°C or 30°C (depend on the strain) before addition of 10 μl of 0.025%
resazurin. After incubation, fluorescence of the resazurin metabolite resorufin was determined (excitation at 560 nm and emission at 590 nm, Gain 80) by using a TECAN Infinite M200 microplate reader.
Dimethyl Labeling and SAX fractionation and digestion of culture filtrate proteins. For the secretome analysis 10 μg of protein was reconstituted in 200 μl of 4 M Urea, 10% acetonitrile and buffered with Tris-HCl pH 8.5 to a final concentration of 30 mM. Proteins were reduced in 10 mM dithioerythritol (DTE) at 37°C for 60 min.
and then alkylated in 40 mM iodoacetamide at 37°C for 45 min. Reactions were quenched by addition of DTE to a final concentration of 10 mM. First, protein digestion was performed using Lys-C (1:50 enzyme: protein) for 2 hours at 37°C. The lysates were then diluted 5-fold and a second digestion was performed overnight at 37°C using mass spectrometry grade trypsin gold (1:50 enzyme: protein) and 10 mM CaCl2. Reactions were stopped by addition of 8 μl of pure formic acid and peptides were concentrated by vacuum centrifugation to a final volume of 70 μl. After digestions, samples were dimethyl-labeled as described previously (Boersema et al., 2009). In brief, culture filtrates from bacteria treated with DMSO were labeled with light dimethyl reactants (CH2O + NaBH3CN) and Culture filtrates from bacteria treated with BBH7 were labeled with medium reactants (CD2O + NaBH3CN). In the
"reverse" experiment, the labelling of the culture filtrates from bacteria treated with DMSO and BBH7 samples were reversed. As a final step of labeling the procedure, samples were mixed in a 1: 1 (Light:Medium) and lyophilized.
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SAX fractionation was performed as previously described with minor modifications (Wisniewski et al., 2009). Stage Tips were prepared by placing six layers of a 3M Empore™ anion exchange disk (3M) into a P200 pipette tips. SAX buffers were freshly prepared and titrated (pH 2, 4, 5, 6, 8, 11) with NaOH. Tips were first conditioned successively with 100% Methanol, 1M NaOH and Phosphoric acid buffer (pH 11). Samples were reconstituted in SAX buffer (pH 11) and loaded into the conditioned tips. The loading flow-through as well as the pH step elutions (in decreasing order of pH) were on-line captured on Empore™ C18 stage tips. Each collected fraction was washed with 0.1% TFA and eluted with acidified high organic content solvent. Eluted fractions were finally dried by vacuum centrifugation and used for LC-MS/MS analysis.
Mass Spectrometry and Data Analysis. Each SAX fraction was resuspended in 2% acetonitrile, 0.1% FA and loaded on a capillary pre-column (Magic AQ C18; 3μm by 200Å; 2 cm x 100 μm ID). Separations were performed on a C18 tip-capillary column (Nikkyo Technos Co; Magic AQ C18; 3μm by 100Å; 15 cm x 75 μm) using a Dionex Ultimate 3000 RSLC nano UPLC system. Data were acquired in data-dependent mode (over a 4 hr acetonitrile 2–42% gradient) on an Orbitrap Elite Mass spectrometer. Acquired RAW files were processed using MaxQuant version 1.3.0.5 (Cox et al., 2009) and its internal search engine Andromeda (Cox et al., 2011). The Mtb strain H37Rv R26 database (http://tuberculist.epfl.ch/) (Lew et al., 2011) was used for the search and MaxQuant default identification settings were applied in combination with specific dimethyl labeling parameters. Search results were filtered with a false-discovery rate of 0.01. Known contaminants and reverse hits were removed before statistical analysis. Relative quantification within different conditions was obtained calculating the significance B values for each of the identified proteins using Perseus (Cox et al., 2009).
Genome annotation and RNA-seq data analysis. All analyses in this study were carried out using the M. tuberculosis H37Rv annotation from the TubercuList database (http://tuberculist.epfl.ch/) (Lew et al., 2011). There are 4019 protein coding sequences (CDS) currently annotated in the genome, 73 genes encoding for stable RNAs, small RNAs and tRNAs. In order to quantify protein occupancy and transcription across the entire genome, 3080 intergenic regions (regions flanked by two non-overlapping CDS) were included, resulting in a total of 7172 features.
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The single-ended sequence reads generated from RNA-seq experiments were aligned to the M. tuberculosis H37Rv genome (NCBI accession NC_000962.2) using Bowtie2 with default parameters (Langmead and Salzberg, 2012). Read counts for all annotated features were obtained with the htseq-count program (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html). Regions where genes overlapped were excluded from counting. Reads spanning more than one feature were counted for each feature. Since the RNA library was strand-specific, the orientation of sequence reads had to correspond to the orientation of annotated features to be counted. Analysis of differential gene expression was carried out using the DESeq package (Anders and Huber, 2010).
Cloning and purification of His6-tagged MprB. Cloning of the mprB PCR-product into pQE80L (Qiagen) was performed using the In-Fusion PCR Cloning kit (Clontech). Two litres of mid-log phase E. coli BL21 (DE3) culture were induced with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) and incubated for 12 hours at 16°C.
cells were lysed in lysis buffer (50 mM Tris pH 8, 500 mM NaCl, 5 mM imidazole, 10% glycerol, 1% Tween 20) using a French press. After clearance by centrifugation, the lysates were incubated with 1 g of PrepEase resin (USB, Cleveland, USA) for 1 hour at 4°C followed by separation on a PolyPrep chromatography column (Biorad).
The resin was washed with two column volumes of buffer containing 10 mM imidazole and eluted with 250 mM imidazole. After dialysis against 25 mM Tris pH7.5 and 200 mM NaCl the protein was further purified by gel filtration on a HiLoad 16/60 Superdex 200 column (Amersham Biosciences).
Data processing for intracellular quantification of bacteria using confocal microscopy. For quantification of intracellular bacteria the DAPI-channel was filtered using a median filter of 2 pixels (radius), and a Gaussian blur with a sigma of 2 pixels. Afterwards, an automatic threshold using Huang's fuzzy thresholding method (Fiji, “Huang” auto threshold) was applied on this modified image of the DAPI-channel and an automatic threshold using Tsai's thresholding method (Fiji,
"Moments" auto threshold) was applied on the bacteria-channel. Finally, the area of each segmented image was measured. Areas or their ratio can be plotted and are indicative of the bacterial load within macrophage.
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