Bacterial strains

A total of 225 Mycobacterium tuberculosis strains, including 110 MDR-TB and 115 pre-XDR strains, used in this study were collected from Beijing Chest Hospital, Capital Medical University, between January 2016 and December 2018. Each isolate was obtained from an individual patient and identified at the species level through multilocus sequence analysis, as previously described [9].

Minimal inhibitory concentration determinations

The MICs of MTB isolates against SQ109 were determined using the 2-fold broth dilution method as previously reported [10]. Briefly, the turbidity of 4-week MTB cultures on L-J medium was adjusted 1.0 using a McFarland standard. Followed by 1:20 dilution with sterile Middlebrook 7H9 broth containing 10% oleic acid, albumin, dextrose, and catalase (OADC), a total volume of 100 µL of this inoculum was added into the wells of a 96-well plate containing serial two-fold dilutions of SQ109 (16–0.016 mg/L) in 100 µl of 7H9 Middlebrook medium. After incubation at 37 °C for 7 days, 70 µL of alamarBlue solution was added and assessment of color development was performed whereby a change from blue to pink indicated bacterial growth. The MIC of each strain against SQ109 was defined as the minimal drug concentration at which no growth was observed. Additionally, MIC90, MIC95 and MIC99 were designated as the minimum concentration of the drugs that inhibited growth by 90%, 95% or 99% of the isolates tested, respectively. For quality control, a control MTB strain H37Rv (ATCC 27249) was included with every MIC experiment. Each isolate was tested in triplicate to assess reproducibility. The synthesis and purification of pure SQ109 powder were carried out by HanXiang Biotech Co., Ltd (Shanghai, China). The ECOFFs were determined on the basis of the distribution profile of MIC values. For unimodal MIC distributions, ECOFFs were defined as concentrations representing ≥ 99.9% of the bacterial population; For bimodal MIC distributions, ECOFFs were set between the two populations [9]. For the drug sensitivity test involving inhibitor treatment, three efflux pump inhibitors, namely verapamil (VP), carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine (RP), were selected. Each inhibitor was tested at three different concentrations: VP at 40 mg/L, 80 mg/L, and 160 mg/L, CCCP at 0.5 mg/L, 2 mg/L, and 8 mg/L, and RP at 6 mg/L, 12 mg/L, and 24 mg/L. In vitro MIC tests were conducted on six pre-XDR isolates with no mutations in the mmpL3 gene. The drug sensitivity test of Mycobacterium smegmatis, which carries the pMV261-MmpL5 and pMV261-MmpL7 plasmids, was conducted for SQ109 as previously reported [11]. All the experiments were performed in triplicate.

DNA amplification and sequencing

The crude genomic DNA was extracted from freshly culture mycobacteria as previously reported [12]. The entire fragment of mmpL3 gene conferring SQ109 resistance was analyzed by Sanger sequencing. The DNA fragments were amplified with primers listed in Table S1. The 50 µL PCR mixture was prepared as follows: 25 µL 2×PCR Mixture (CWBio, Beijing, China), 5 µL of DNA template, and 0.2 µM of each primer pair. PCR cycling conditions consisted of preincubation at 94 °C for 5 min, and then 35 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min followed by a final cycle of 72 °C for 10 min. PCR product was purified using a Mag-Bind PCR Purification Kit (CWBio, Beijing, China) and amplicons were sent to Qingke Company (Beijing, China) for DNA sequencing. DNA sequences were aligned with the homologous sequences of the reference MTB strain H37Rv (ATCC 27249) using BioEdit Sequence Alignment Editor Version 7.1.3 (www.mbio.ncsu.edu/bioedit.bioedit.html).

Quantitative real-time PCR assay

The mRNA levels of the efflux pump gene in the isolates were measured using a qPCR assay. The isolates in the logarithmic phase were first placed at -80 °C for 10 min, followed by thawing in boiling water and subjecting to 240 W ultrasound treatment for 3 cycles, each lasting 3 min with intervals of 15 s, and subsequently underwent RNA extraction. The total RNA was extracted using the Trizol method following the instructions of the manufacturers [13]. After DNaseI treatment (TaKaRa, Dalian, China), cDNAs were synthesized in reverse transcription from 5 µg of total RNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qPCR) was performed in triplicates for each sample using TB Green® Premix Ex Taq™ II (TaKaRa, Dalian, China) on the Real-Time PCR System (Bio-Rad). The Real-Time PCR System was programmed as follows: 94 °C for 2 min to denature template; 94 °C for 45 s for denaturation; at 60 °C for 45 s for annealing; and 68 °C for 1 min for extension. Primers for transcriptional level analysis were listed in Table S1 and 16 S rRNA was used as the internal control of respective qPCR assay. The fold change values in the qPCR experiments were calculated using the 2(−Delta Delta C(T)) method [14] and were further used to create a heatmap with log2 fold changes (log2 FC), with Table S2 reporting the values from the heatmap.

Construction of Msm/pMV261-MmpL5 and Msm/pMV261-MmpL7

The DNA of H37Rv and the empty plasmid pMV261 were sent to Beijing TSINGKE biotechnology co., Ltb for plasmid construction and attachment of a 3*Flag sequence at the C terminal. In brief, the mmpL5 and mmpL7 genes were separately cloned into the empty plasmid pMV261 through homologous recombination and then transformed into E. coli DH5α competent cells. Confirmation was done by sequencing. The constructed plasmid was introduced into Mycobacterium smegmatis mc2 155 through electroporation [11], and the presence of the vector was confirmed by PCR and western blot. The empty plasmid pMV261 was also electroporated into Mycobacterium smegmatis mc2 155, but without the Flag sequence.

PCR and western blot analysis

The bacterial cultures containing Mycobacterium smegmatis with the pMV261-MmpL5 and pMV261-MmpL7 plasmids and empty plasmid pMV261 were centrifuged at 12,000 rpm for 2 min. The resulting pellet was then resuspended in 200 µL of sterile water and heated in a 100℃ water bath for 10 min. After centrifugation, the supernatant was collected as the template for PCR amplification. For the PCR system, A solution of 25 µL PCR reaction mixture was prepared containing 12.5 µL 2×Phanta Flash Master Mix, 5 µL of DNA template, 1 µL of each primer set and 5.5 µL ddH2O. Amplification was conducted with an initial denaturation at 98 °C for 30 s, followed by 35 cycles of amplification (denaturation at 98 °C for 10 s, annealing at 70 °C for 5 s and extension at 72 °C for 15 s, with a final extension at 72 °C for 1 min). The PCR products were subjected to gel electrophoresis.

For western blot analysis, the protein expression of MmpL5 and MmpL7 in Msm/pMV261-MmpL5 and Msm/pMV261-MmpL7 was verified by western blotting as previously reported [11]. Msm/pMV261-MmpL5 and Msm/pMV261-MmpL7 were centrifugated at 12,000 rpm for 2 min and the pellet was resuspended in 500 µl 1×PBS buffer and 5 µl MCE protease inhibitor “Cocktail”. After blending, ultrasound was performed for 2 min with low frequency, and then the cells lysates were centrifuged at 12,000 rpm for 2 min to collect the supernatant. Add 1× protein loading buffer and boil at 95 °C for 10 min. Proteins were separated by 8% SDS-PAGE at 80 V for 2 h and transferred to a PVDF membrane (Millipore). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 2 h at room temperature and then incubated for 2 h at room temperature with anti-DDDDK-tag mAb-HRP-DirecT antibody (#M185-7, MBL, 1:5000) in TBS with Tween-20 (TBST) and 5% (w/v) bovine serum albumin. Following three washes of 10 min each with TBST, the blots were developed with Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) and detected using an enhanced chemiluminescence detection system (iBright CL1500 imaging system; Thermo Fisher Scientific, Inc.).