Overexpressing OmpA strains construction

The Mtb reference strain H37Rv genomic DNA was used as a template, and the ompA gene was amplified by PCR using OmpA981-F and OmpA981-R primers (Supplementary Table S1). The shuttle-plasmid pMV261 and ompA were digested with EcoRI and SalI restriction endonuclease enzymes (Thermo Fisher, USA). The products were ligated and transformed into E.coli DH5α, and the positive clones were verified by PCR and DNA sequencing. The recombinant pMV261-ompA plasmid was electroporated into Mycobacterium smegmatis (mc2155) and M. bovis(C68004) [11, 24, 25], and the kanamycin-resistant colonies were confirmed by PCR. After obtaining recombinant strains, OmpA protein expression was detected by Western blotting. The experiments related to Mb and Mb-OmpA virulent strains were conducted at the Biosafety Level-3 (BSL-3) Laboratory at China Agricultural University.

Western blot

Ms-OmpA and Mb-OmpA were cultured to OD600 = 0.8 ∼ 1.0, centrifuged at 8000 rpm at 4℃ for 10 min to collect bacteria, and washed twice with PBS. Use a bacterial crusher to break the bacterial cells, 15 cycles and 30 s each time. The lysates were centrifuged at 12,000 rpm at 4℃ and for 30 min. The supernatant and precipitates were separated, added the loading buffer individually, and boiled at 100 ℃ for 10 min. The sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature and then incubated with the stocked OmpA polyclonal antibody for 2 h at room temperature. After washing three times with TBST, the secondary antibodies were added and incubated for 1 h at room temperature.

Growth curve

The WT Ms, Ms-pMV-261, and Ms-OmpA strains at the log phase were inoculated into Middle brook 7H9 medium (BD, USA) at the dilution of 1:100 and cultured in a shaker (37 °C, 180 rpm). The strains’ growth was monitored by measuring OD600 by Eppendorf BioSpectrometer [11, 25]. For checking the strains’ growth under the antibiotic pressure, streptomycin was added to a final concentration of 0.5 µg/ml. The cultures were incubated and the absorbance at 600 nm was measured every 4 h.

MICs determination

The MICs of the strains in this study were tested by the microplate-based Alamar Blue assay (MABA) method [26,27,28]. The strains were incubated in Middle brook 7H9 medium (BD, USA) to the logarithmic growth stage, and the turbidity of the bacterial solution was adjusted to 2 × 107 CFU/ml, and the MIC was determined by 2-fold dilution method: 50 µl 7H9 medium was added to each well of the 96-well plate, 48 µl 7H9 medium was added to the first column which prefilled with 2 µl antibiotics solution. For example, rifampicin (200 µg/ml), streptomycin (16 µg/ml), amikacin (4 µg/ml), gentamicin (4 µg/ml), rifabutin (4.8 µg/ml), rifapentine (48 µg/ml) were diluted twice at one time, and the last column was listed as a blank (without drugs); 50 µl bacterial suspension aliquots were transferred into each well. After incubating at 37℃ for 24 h, 32.5 µl chromogenic reagent [AlamarBlue 20 µl (Biorad, USA), 20% Tween-80 12.5 µl (Amresco, USA)]was added. The 96-well plate was placed in an incubator at 37 ℃, and the color change was observed after 6 h. The MIC was defined as the drug concentration at the corresponding well when the blue color no longer changed to pink. Antibiotics used in this study: Aminoglycosides: streptomycin (STR, Topscience, T0060), amikacin (AMI, Topscience, T1013), gentamicin (GEN, Topscience, T1326); Rifamycins: rifampicin (RIF, Sigma, R3501), rifabutin (RIFB, Topscience, T1501), rifapentine (RIFP, Topscience, T1629); isoniazid(INH, Sigma V900688);ethambutol (EMB, Topscience, T0810); Fluoroquinolones: ofloxacin (OFX, Topscience, T0902).

The plate assay

The strains were grown to logarithmic phase, collected after centrifuging at 4000 rpm for 5 min, and washed three times in PBS. The 25-gauge sterile syringe was used to prepare single-cell suspensions, and the turbidity of each bacterial solution was adjusted to OD600 at 0.1. The bacterial solution was continuously diluted 10-fold, and 10 µl of each dilution was spotted onto LB agar plates containing different concentrations of antibiotics including streptomycin (0.5 µg/ml and 0.75 µg/ml), amikacin (0.5 µg/ml), rifampicin (1.5 µg/ml, 5 µg/ml, 7.5 µg/ml and 10 µg/ml), rifabutin (0.15 µg/ml), isoniazid (2 µg/ml), ethambutol (0.5 µg/ml), and ofloxacin (0.25 µg/ml) individually. LB agar plates without antibiotics served as controls. The percentage of bacterial survival rate (%) was calculated by dividing the number of bacterial clones on plates with antibiotics to clones counts on the plates without antibiotics [11, 12].

The macrophage infection assay

The J774a.1 macrophage cell line was obtained from Cell Resource Center, Peking Union Medical College. The cells were seeded with 2.5 × 105 cells per well and cultured overnight in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Solarbio, China) in a 24-well plate. After that, the cells were washed with blank DMEM and infected with Ms using a MOI of 10:1 for 4 h. After cells were washed three times with blank DMEM to remove extracellular bacteria, the cells were incubated with streptomycin (0.5 µg/ml) for 6 and 12 h, respectively. Then the cells were lysed by 0.1% Triton- 100 (Solarbio, China) for 10 min, and the lysates were spread onto LB plates. After 3 days, CFU counts were performed [24, 25].

Spermidine experiment

Bacteria were grown to the log phase, then harvested by centrifugation at 4000 rpm for 5 min, and the cell pellet was washed with PBS. Single-cell suspensions were prepared using a 25-gauge sterile syringe, and the OD600 of the bacterial solution was adjusted to 1.0. Bacteria were seeded in 24-well plates with an initial OD600 of 0.01 in each well. The growth curves of the experimental groups were monitored after adding the streptomycin and spermidine (Solarbio, China) at the final concentration of 0.1 µg/ml and 0.5 mM/ml individually [29,30,31]. The control group was only incubated with streptomycin.

Ethidium bromide/nile red uptake experiments

Ethidium bromide and Nile red were used as substitutes for hydrophilic and hydrophobic substrates respectively to verify the changes in the permeability of hydrophilic substances and lipid permeability of the cell membrane [32, 33]. The bacteria were cultured until the OD600 = 0.8 ∼ 1.0, and washed with PBS three times, then the OD600 of the bacteria solution was adjusted to 0.5, and glucose with the final concentration of 25 mM was added. After standing at room temperature for 5 min, 200 µl bacterial solution was added to the 96-well plate, and the final concentration of Ethidium bromide (2 µg/ml) (Topscience, China) and Nile Red (2 µM) (Topscience, China) were added, respectively. The fluorescence intensity was measured under the Tecan Sunrise.

Real-time PCR

Total RNA was extracted from bacterial cells using the Trizol method [34, 35]. The bacterial suspension was harvested by centrifugation at 4℃, 12,000 rpm for 5 min. The cells were then re-suspended in a freshly prepared mixture of methanol and chloroform (V: V = 1:3) and shaken vigorously for 1 min. After the addition of 1 ml Trizol solution from TRIzolTM Plus RNA Purification Kit (Invitrogen, Cat No: 12183555CN) and vortex for 15 s, 200 µl of chloroform was immediately mixed. After incubating for 30 min at room temperature, the mixture was centrifuged at 12,000 rpm for 15 min at 4℃. The resulting supernatant was then transferred to an RNase-free centrifuge tube. An equal volume of isopropanol was added and mixed well, and the mixture was placed at -20℃ for 2 h. The supernatant was discarded by centrifugation at 12,000 rpm for 15 min at 4℃. The RNA precipitate was then washed twice with 1 ml of 75% ethanol, gently inverted, and mixed 4–6 times. After resuspension, genome DNA was removed and the cDNA was synthesized by HiScript Ш 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme, Cat No: R312). Real-time quantitative PCR was performed with an AceQ qPCR SYBR Green Master Mix kit (Vazyme, Cat No: Q121). Reactions were performed in a volume of 20 µl, and the reaction mixtures consisted of 0.4 µM forward and reverse primers (Supplementary Table S1), 10 µl of 2 × master mix, and 2 µl of cDNA. The control with no RT was included in each run. An additional sample was included to measure 16 S rRNA. Amplification was performed in Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher, USA). After the Ct values were obtained by qPCR, 16sRNA was used as the internal reference gene, and the 2−ΔΔCt method was used to calculate the gene expression level.

Transmission electron microscope (TEM)

The strains were grown to the log phase and collected. The bacteria were fixed in 2.5% glutaraldehyde (SPI Supplies, USA) for 2 h, washed in PBS, and then fixed in 1% osmium tetroxide (Thermo Fisher, USA) for 1 h. Dehydrate bacterial samples in 30%, 50%, 70%, and 90% ethanol for 15 min, respectively, then 100% acetone for 15 min. Embed, solidify, section, and stain the samples. Forty-four fields were randomly selected for observation and photographed for preservation by Hitachi HT7800 RuliTEM Electron Microscopy [36, 37]. Each bacterial cell membrane thickness was then measured using ImageJ software and analyzed statistically. Ensure experiment repetition and credibility by randomly analyzing individual experiments.

Statistical analysis

Data are expressed as mean ± SD. Data were compared by two-tailed unpaired Student’s t-tests using the computer program Prism version 8.0 (GraphPad Software, USA). Differences were considered statistically significant when p-values < 0.05. n represents the number of experiments.