Study on the interaction between Mycobacterium tuberculosis membrane protein MmpS5/MmpL5 and bedaquiline

doi:10.19982/j.issn.1000-6621.20240584

Abstract

Abstract:

Objective: To express and purify the Mycobacterium tuberculosis membrane proteins MmpS5 and MmpL5, investigate their interaction with bedaquiline (Bdq), and elucidate the specific transport mechanism of the MmpS5/MmpL5 efflux pump system in conferring Bdq resistance. Methods: Expression plasmids for MmpS5 and MmpL5 were constructed. MmpS5 and MmpL5 proteins were expressed and purified in HEK293F mammalian cells and Mycobacterium smegmatis, respectively. The interactions between Bdq and MmpS5/MmpL5 were assessed using differential scanning fluorimetry (DSF) and competitive enzyme-linked immunosorbent assay (ELISA). Results: The expression plasmids for MmpS5 and MmpL5 were successfully constructed. MmpS5 was purified in HEK293F cells, and MmpL5 was purified in Mycobacterium smegmatis. DSF results showed that as the concentration of Bdq increased, the melting temperature (Tm) of MmpS5 slightly decreased. Whereas the Tm of MmpL5 significantly increased, by up to 5.98 ℃, suggesting a binding interaction between MmpL5 and Bdq. Competitive ELISA further demonstrated that the absorbance of MmpL5 decreased significantly with increasing concentrations of unlabeled Bdq, indicating that MmpL5 interacts with biotin-labeled Bdq, and this interaction can be competitively inhibited by unlabeled Bdq. Conclusion: In the MmpS5/MmpL5 efflux pump system, MmpL5 directly interacts with Bdq, while MmpS5 shows no significant interaction. These findings suggest that MmpL5 may directly mediate the efflux of Bdq, whereas MmpS5 likely plays an auxiliary role without direct involvement in Bdq transport. This study provides a critical foundation for understanding the mechanistic role of the MmpS5/MmpL5 efflux pump in Bdq resistance.

Key words: Mycobacterium tuberculosis, Membrane proteins, Molecular mechanisms of pharmacological action, Bedaquiline

CLC Number:

Zheng Zhuangbin, Bi Lijun, Zhang Liqun. Study on the interaction between Mycobacterium tuberculosis membrane protein MmpS5/MmpL5 and bedaquiline[J]. Chinese Journal of Antituberculosis, 2025, 47(7): 884-892. doi: 10.19982/j.issn.1000-6621.20240584

Figures/Tables 8

References 39

[1]	World Health Organization. Global tuberculosis report 2024. Geneva: World Health Organization, 2024.
[2]	Cox E, Laessig K. FDA approval of bedaquiline-the benefit-risk balance for drug-resistant tuberculosis. N Engl J Med, 2014, 371(8):689-691. doi:10.1056/NEJMp1314385.
[3]	Borisov SE, Dheda K, Enwerem M, et al. Effectiveness and safety of bedaquiline-containing regimens in the treatment of MDR- and XDR-TB: a multicentre study. Eur Respir J, 2017, 49(5): 1700387. doi:10.1183/13993003.00387-2017.
[4]	Ahmad N, Ahuja SD, Akkerman OW, et al. Treatment correlates of successful outcomes in pulmonary multidrug-resistant tuberculosis: an individual patient data meta-analysis. Lancet, 2018, 392(10150): 821-834. doi:10.1016/S0140-6736(18)31644-1. pmid: 30215381
[5]	Olayanju O, Limberis J, Esmail A, et al. Long-term bedaquiline-related treatment outcomes in patients with extensively drug-resistant tuberculosis from South Africa. Eur Respir J, 2018, 51(5): 1800544. doi:10.1183/13993003.00544-2018.
[6]	梁晨, 唐神结, 林明贵. 结核病综合治疗研究进展. 结核与肺部疾病杂志, 2024, 5(1): 70-80. doi:10.19983/j.issn.2096-8493.20230112.
[7]	Guglielmetti L. Bedaquiline for the treatment of multidrug-resistant tuberculosis: another missed opportunity?. Eur Respir J, 2017, 49(5): 1700738. doi:10.1183/13993003.00738-2017.
[8]	Mallick JS, Nair P, Abbew ET, et al. Acquired bedaquiline resistance during the treatment of drug-resistant tuberculosis: a systematic review. JAC Antimicrob Resist, 2022, 4(2): dlac029. doi:10.1093/jacamr/dlac029.
[9]	孙慧娟, 苏伟, 陈伟. 利福平耐药结核病患者不良治疗结局及其影响因素研究进展. 结核与肺部疾病杂志, 2024, 5(6): 573-582. doi:10.19983/j.issn.2096-8493.2024111.
[10]	Veziris N, Bernard C, Guglielmetti L, et al. Rapid emergence of Mycobacterium tuberculosis bedaquiline resistance: lessons to avoid repeating past errors. Eur Respir J, 2017, 49(3): 1601719. doi:10.1183/13993003.01719-2016.
[11]	Huitric E, Verhasselt P, Koul A, et al. Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor. Antimicrob Agents Chemother, 2010, 54(3): 1022-1028. doi:10.128/AAC.01611-09. pmid: 20038615
[12]	Almeida D, Ioerger T, Tyagi S, et al. Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis. Antimicrob Agents Chemother, 2016, 60(8): 4590-4599. doi:10.1128/AAC.00753-16. pmid: 27185800
[13]	Villellas C, Coeck N, Meehan CJ, et al. Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or beda-quiline. J Antimicrob Chemother, 2017, 72(3): 684-690. doi:10.1093/jac/dkw502. pmid: 28031270
[14]	Salfinger M, Somoskövi A. Multidrug-resistant tuberculosis and bedaquiline. N Engl J Med, 2014, 371(25): 2435-2436. doi:10.1056/NEJMc1412235.
[15]	Omar Shaheed V, Ismail F, Ndjeka N, et al. Bedaquiline-Resistant Tuberculosis Associated with Rv0678 Mutations. N Engl J Med, 2022, 386(1): 93-94. doi:10.1056/NEJMc2103049.
[16]	Liu Y, Gao J, Du J, et al. Acquisition of clofazimine resis-tance following bedaquiline treatment for multidrug-resistant tuberculosis. Int J Infect Dis, 2021, 102: 392-396. doi:10.1016/j.ijid.2020.10.081.
[17]	Snobre J, Villellas MC, Coeck N, et al. Bedaquiline- and clofazimine-selected Mycobacterium tuberculosis mutants: further insights on resistance driven largely by Rv0678. Sci Rep, 2023, 13(1): 10444. doi:10.1038/s41598-023-36955-y.
[18]	Dheda K, Mirzayev F, Cirillo DM, et al. Multidrug-resistant tuberculosis. Nat Rev Dis Primers, 2024, 10(1): 22. doi:10.1038/s41572-024-00504-2. pmid: 38523140
[19]	Cuthbert BJ, Mendoza J, de Miranda R, et al. The structure of Mycobacterium thermoresistibile MmpS 5 reveals a conserved disulfide bond across mycobacteria. Metallomics, 2024, 16(3):mfae011. doi:10.1093/mtomcs/mfae011.
[20]	史静华, 李东硕, 岑山, 等. 结核分枝杆菌MmpL5蛋白与贝达喹啉及氯法齐明相互作用的关键结合区域研究. 中国抗生素杂志, 2024, 49(8): 890-897. doi:10.13461/j.cnki.cja.007743.
[21]	Bailo R, Bhatt A, Aínsa JA. Lipid transport in Mycobacterium tuberculosis and its implications in virulence and drug development. Biochem Pharmacol, 2015, 96(3): 159-167. doi:10.1016/j.bcp.2015.05.001.
[22]	Briffotaux J, Huang W, Wang X, et al. MmpS5/MmpL5 as an efflux pump in Mycobacterium species. Tuberculosis (Edinb), 2017, 107: 13-19. doi:10.1016/j.tube.2017.08.001.
[23]	Sandhu P, Akhter Y. The internal gene duplication and interrupted coding sequences in the MmpL genes of Mycobacterium tuberculosis: Towards understanding the multidrug transport in an evolutionary perspective. Int J Med Microbiol, 2015, 305(3): 413-423. doi:10.1016/j.ijmm.2015.03.005.
[24]	Farnia P, Besharati S, Farina P, et al. The Role of Efflux Pumps transporter in Multi-drug Resistant Tuberculosis: Mycobacterial memberane protein (MmpL5). Int J Mycobacteriol, 2024, 13(1): 7-14. doi:0.4103/ijmy.ijmy_37_24.
[25]	Andries K, Villellas C, Coeck N, et al. Acquired resistance of Mycobacterium tuberculosis to bedaquiline. PLoS One, 2014, 9(7): e102135. doi:10.1371/journal.pone.0102135.
[26]	Milano A, Pasca MR, Provvedi R, et al. Azole resistance in Mycobacterium tuberculosis is mediated by the MmpS5-MmpL 5 efflux system. Tuberculosis (Edinb), 2009, 89(1): 84-90. doi:10.1016/j.tube.2008.08.003.
[27]	Xu J, Li D, Shi J, et al. Bedquiline Resistance Mutations: Correlations with Drug Exposures and Impact on the Proteome in M.tuberculosis. Antimicrob Agents Chemother (Bethesda), 2023, 67(7): e0153222. doi:10.1128/aac.01532-22.
[28]	Yamamoto K, Nakata N, Mukai T, et al. Coexpression of MmpS5 and MmpL 5 Contributes to Both Efflux Transporter MmpL5 Trimerization and Drug Resistance in Mycobacterium tuberculosis. mSphere, 2021, 6(1): e00518-20. doi:10.1128/mSphere.00518-20.
[29]	Jing W, Zhang F, Shang Y, et al. Deciphering the possible role of MmpL 7 efflux pump in SQ109 resistance in Mycobacterium tuberculosis. Ann Clin Microbiol Antimicrob, 2024, 23(1): 87. doi:10.1186/s12941-024-00746-8.
[30]	李东硕, 王彬, 陆宇, 等. 结核分枝杆菌膜蛋白MmpS5-MmpL5的表达及功能研究. 中国防痨杂志, 2022, 44(3): 227-233. doi:10.19982/j.issn.1000-6621.20210587.
[31]	Matagne A, Joris B, Frère JM. Anomalous behaviour of a protein during SDS/PAGE corrected by chemical modification of carboxylic groups. Biochem J, 1991, 280 (Pt 2): 553-556. doi:10.1042/bj2800553.
[32]	Hu CC, Ghabrial SA. The conserved, hydrophilic and arginine-rich N-terminal domain of cucumovirus coat proteins contributes to their anomalous electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels. J Virol Methods, 1995, 55(3): 367-379. doi:10.1016/0166-934(95)00085-1. pmid: 8609202
[33]	Papageorgiou FT, Soteriadou KP. Expression of a novel Leishmania gene encoding a histone H1-like protein in Leishmania major modulates parasite infectivity in vitro. Infect Immun, 2002, 70(12): 6976-6986. doi:10.1128/IAI.70.12.6976-86.2002. pmid: 12438377
[34]	Rath A, Glibowicka M, Nadeau VG, et al. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A, 2009, 106(6): 1760-1765. doi:10.073/pnas.0813167106.
[35]	Tiwari P, Kaila P, Guptasarma P. Understanding anomalous mobility of proteins on SDS-PAGE with special reference to the highly acidic extracellular domains of human E- and N-cadherins. Electrophoresis, 2019, 40(9): 1273-1281. doi:10.002/elps.201800219. pmid: 30702765
[36]	Gooran N, Kopra K. Fluorescence-Based Protein Stability Monitoring-A Review. Int J Mol Sci, 2024, 25(3): 1764. doi:10.3390/ijms25031764.
[37]	Gan SD, Patel KR. Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. J Invest Dermatol, 2013, 133(9): e12. doi:0.1038/jid.2013.287.
[38]	Sandhu P, Akhter Y. Siderophore transport by MmpL5-MmpS5 protein complex in Mycobacterium tuberculosis. J Inorg Biochem, 2017, 170: 75-84. doi:10.1016/j.jinorgbio.2017.02.013.
[39]	Vargas R Jr, Freschi L, Spitaleri A, et al. Role of Epistasis in Amikacin, Kanamycin, Bedaquiline, and Clofazimine Resis-tance in Mycobacterium tuberculosis Complex. Antimicrob Agents Chemother, 2021, 65(11): e0116421. doi:10.1128/AAC.01164-21.