Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance

In recent years, the treatment of tuberculosis is once again facing a severe situation because the existing antituberculosis drugs have become weaker and weaker with the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The studies of cell division and cell cycle-related factors in Mtb are particularly important for the development of new drugs with broad-spectrum effects. Mycobacterium smegmatis (Msm) has been used as a model organism to study the molecular, physiological, and drug-resistant mechanisms of Mtb. Bioinformatics analysis has predicted that MSMEG_6171 is a MinD-like protein of the septum site-determining protein family associated with cell division in Mycobacterium smegmatis. In our study, we use ultrastructural analysis, proteomics, metabolomics, and molecular biology techniques to comprehensively investigate the function of MSMEG_6171. Overexpression of MSMEG_6171 in Msm resulted in elongated cells, suggesting an important role of MSMEG_6171 in regulating cell wall morphology. The MSMEG_6171 overexpression could enhance the bacterial resistance to vancomycin, ethionamide, meropenem, and cefamandole. The MSMEG_6171 overexpression could alter the lipid metabolism of Msm to cause the changes on cellular biofilm property and function, which enhances bacterial resistance to antibiotics targeting cell wall synthesis. MSMEG_6171 could also induce the glyceride and phospholipid alteration in vivo to exhibit the pleiotropic phenotypes and various cellular responses. The results showed that amino acid R249 in MSMEG_6171 was a key site that can affect the level of bacterial drug resistance, suggesting that ATPase activity is required for function.

electroporated into the msmeg_6171-deleted strains, select were performed as previously described [3].
BODIPY-FL conjugate of vancomycin (Van-FL, Molecular Probes, Invitrogen) and unlabelled vancomycin (sigma) each at 1 mg/ml (This was added to a final concentration of vancomycin was 2 mg/ml) were added directly to 1~2 ml Logarithmic phase culture and incubated under standard growth conditions for 90 min.
Cells were collected and washed twice in 0.5 ml PBS, and finally resuspended in 50 ml PBS. Cell aliquots (10 μL) were spread on poly-L-lysine coated slides (BDH) follow on adding a drop of Prolong Antifade reagent with 100 ng/ml of DAPI (Sigma).
Samples were mounted under a coverslip and examined using a Zeiss LSM880 inverted widefield micrcope, cells measured using Zen Blue 2.3 software.

Flag-based pulldown assay
The in vivo interactions for MSMEG_6171 were analyzed by Flag-based pulldown assay according to previously published procedures with some modifications. Full length msmeg_6171 gene was cloned into the vector pMV261 plasmid with an N-terminus 3X-Flag tag. Vector construction, screening, and confirmation were performed in E. coli. Completed vectors were confirmed via colony PCR and direct sequencing. Vectors were transformed into M. smegmatis, as described above. Samples were centrifuged at 12,000 × g for 5 min and the supernate was transferred to a clean tube. 50 μl of Ni 2+ coated magnetic beads (Promega, Madison, WI) were added to each sample. Samples were incubated on a rotating mixer at 4°C for 2 h.
After incubation, samples were washed three times in denaturing buffer. Each sample was split into two aliquots, one for exposure to the prey lysate and the other for exposure to the control lysate, M. smegmatis with an unmodified pMV261 plasmid.
Lysate from M. smegmatis carrying the pMV261-MSMEG_6171-3X Flag prey construct was prepared in a urea-based denaturing buffer, as described above. 1.5 ml of prey lysate was added to one aliquot of each sample containing immobilized bait protein. Proteins were allowed to refold together through sequential dilution of the denaturing buffer with HBSS. Samples were diluted by the addition of 1 ml aliquots of HBSS (pH 7.4, 10 mM imidazole), followed by 5 min incubations until the concentration of urea reached 350 mM (sample volume 30 ml). Proteins immobilized on magnetic beads were purified with a magnetic rack (Promega) and washed three times with HBSS. The beads were resuspended in laemmli buffer (+5% βbME) and proteins were separated by SDS-PAGE. Proteins were visualized with silver staining.
The unique band was isolated and processed by in-gel trypsin digest before mass spectrometry analysis.

Drug susceptibility testing (MIC)
The MICs of the drugs were determined as described by Luciano Mengatto et al. [7].
To prepare the inoculum, the bacterial suspension adjusted to equal the density of a 1 McFarland standard was diluted 1:25 with 7H9 broth and 100 ml was used as an inoculum. 200 μl sterile water was added to all outer wells of sterile 96-well plates. 100 μl 7H9 broth was added in each well and serial two-fold dilutions of each drug were prepared directly on the plate by adding 100 ml of the drug working solutions.
The final drug concentration ranges were 0.125 to 128 μg/ml. 100 μl of the inoculum were added to the wells. Six growth controls containing no antibiotic were included for each isolate. The plates were sealed, placed in plastic bags and incubated at 37°C at room tempreture. On day-3, 50 ml of the tetrazolium-Tween 80 mixture {1.5 ml of tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide] [ICN Biomedicals, Aurora, Oh, US] at a dilution of 1 mg/ml in absolute ethanol and 1.5 ml of 10% Tween 80 was added to one growth control and the plate was then incubated at 37°C for 12 h. If this well turned purple, the tetrazolium-Tween 80 mixture was added to all wells and the color was recorded at 12 h. Otherwise, if the growth control well remained yellow the plate was then incubated at 37°C for 12 h, after which tetrazolium-Tween 80 mixture was added to another control before the plate was incubated for another 12 h. If this well remained yellow, incubation was continued and tetrazolium-Tween 80 solutions was added to each of the remaining four controls on days 5, 6, and 7.

Protein preparation and iTRAQ Labeling
The proteins were extracted using a mechanical crushing method [8].

Strong cation exchange (SCX) chromatography
The iTRAQ-labeled peptide mixtures were dissolved in buffer A (NH3 in H2O, pH=10) and loaded onto a C18 column (ACQUITY UPLC CSH, 130Å, 1.7 µm, 2.1 mm X 150 mm, 1/pkg, Waters, USA). The peptides were eluted at a flow rate of 200 μL/min with a gradient of 5-10% buffer B (80%ACN NH3 in H2O, pH=10) for 10 min, 10-38% buffer B for 60 min and 38-95% buffer B for 10 min. A total of twelve SCX fractions were collected for one run. The system was then maintained at 5% buffer B for 30 min before the next injection. The eluted peptides vacuum dried.
The fractions above were dissolved in an aqueous solution containing 0.1% FA.
Five Microliter supernatant was loaded on an EASY nLC1000 (Thermo Fisher The final proteins that were deemed to be differentially expressed were filtered as a P value <0.05 and 1.5-fold changes (>1.50 or <0.667) relative to the control group.

Metabolite extraction for LC-MS
The collected Msm were washed 3 times with PBS and then quenched immediately by liquid nitrogen for 10 min stored at -80°C. For intracellular metabolites, samples were reconstituted by dissolving in 1 mL solvent mixture containing water/methanol/acetonitrile (1∶ 2∶ 2) and disrupted in a Fastprep-24 (MP Biomedicals).
The resulting whole extract was subjected to ultracentrifugation at 12,000 × g for 15 min using an SW 41 Ti rotor (Sigma). Supernatants were filtered twice with a 0.22 μm pore Millex filter unit (Millipore) and transferred to LC vial for LC-MS analysis.
For lipids extraction, Chloroform/methanol solution was added to each sample. The mixture was mixed by a vortex mixer for 5 min and carried out by Ultrasonic cleaner for 10 min. Then the mixture was centrifuged at 12000 rpm, 4℃ for 10 min. The supernatant was taken in the clean test tube and the precipitates were extracted by 2 ml chloroform/methanol solution twice. All supernatant was dried by N2 and then dissolved by chloroform/methanol (2:1, v:v). 200 μl supernatant was transferred to sampler vials for detected. As for culture supernatant, the secondary subcultures were centrifuged at 3000 rpm for 30 min to obtain the supernatant which was filtered twice Representative MS/MS spectra were exported in abf format for MS-DIAL, and compound identification was performed against MS/MS libraries including MassBank [9]and MONA [10].
Lipids metabolites -Raw data were converted the common (mz.data) format by Agilent Masshunter Qualitative Analysis B.08.00 software (Agilent Technologies, USA). In the R software platform, the XCMS program was used in peak identification, retention time correction, automatic integration pretreatment. Then the data were subjected to internal standard normalization and weight normalization. Visualization matrices containing sample name, m/z-RT pair and peak area was obtained. 1801 features were got in positive mode and 636 features in negative mode. After editing, the data matrices were imported into SIMCA-P 13.0 [11](Umetrics, Umea, Sweden), mean-centered and scaled to Pareto variance. Then, a multivariate analysis was conducted.