Abstract
EmbR, a substrate of pknH in Mycobacterium tuberculosis (Mtb), is related to the ethambutol (EMB) resistance. This study aimed to investigate the relationship between acetylation of pknH and the resistance of EMB mono-resistant Mtb. The EMB mono-resistant Mtb strain was constructed based on the MYCOTB and the Löwenstein–Jensen (LJ) proportion method. The growth kinetics was used to evaluate the bacterial growth. Escherichia coli, as the host of Mtb, was used for cloning and protein purification. Moreover, the immunoprecipitation was performed along with western blot to evaluate the EmbR phosphorylation and pknH acetylation. Each independent experiment was conducted in triplicate. EMB mono-resistant Mtb strain was successfully constructed according to the results of MIC values of 14 anti-Mtb drugs. The EMB resistant (ER) Mtb strain showed faster growth than the wild-type (WT) Mtb strain, and the difference was statistically significant. Moreover, pknH robustly phosphorylates EmbR, and pknH and acetylated pknH protein levels were downregulated in ER strain. The acetylation of pknH may reduce the phosphorylation of EmbR to inhibit the growth of Mtb strain. Enhancing the acetylation of pknH may be a promising method to inhibit the EMB resistance against Mtb.
Similar content being viewed by others
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Alam MS, Guan P, Zhu Y, Zeng S, Fang X, Wang S, Yusuf B, Zhang J, Tian X, Fang C et al (2022) Comparative genome analysis reveals high-level drug resistance markers in a clinical isolate of Mycobacterium fortuitum subsp. fortuitum MF GZ001. Front Cell Infect Microbiol 12:1056007. https://doi.org/10.3389/fcimb.2022.1056007
Andres S, Gröschel MI, Hillemann D, Merker M, Niemann S, Kranzer K (2019) A diagnostic algorithm to investigate pyrazinamide and ethambutol resistance in rifampin-resistant Mycobacterium tuberculosis isolates in a low-incidence setting. Antimicrob Agents Chemother 63(2):10–1128. https://doi.org/10.1128/AAC.01798-18
Baeza J, Smallegan MJ, Denu JM (2016) mechanisms and dynamics of protein acetylation in mitochondria. Trends Biochem Sci (amsterdam. Regular Ed.) 41(3):231–244. https://doi.org/10.1016/j.tibs.2015.12.006
Boni FG, Hamdi I, Moukendza KL, Dai Y, Shrestra K, Abokadoum MA, Ekomi MU, Suleiman IM, Xie J (2023) The gene and regulatory network involved in ethambutol resistance in Mycobacterium tuberculosis. Microb Drug Resist 29(5):175–189. https://doi.org/10.1089/mdr.2021.0239
Brossier F, Sougakoff W, Bernard C, Petrou M, Adeyema K, Pham A, Amy DLBD, Vallet M, Jarlier V, Sola C et al (2015) Molecular analysis of the embCAB locus and embR gene involved in ethambutol resistance in clinical isolates of Mycobacterium tuberculosis in france. Antimicrob Agents Chemother 59(8):4800–4808. https://doi.org/10.1128/AAC.00150-15
Diallo I, Seve M, Cunin V, Minassian F, Poisson JF, Michelland S, Bourgoin-Voillard S (2019) Current trends in protein acetylation analysis. Expert Rev Proteomics 16(2):139–159. https://doi.org/10.1080/14789450.2019.1559061
Garcia MJ, Hans F, von Zweydorf F, Feederle R, Elsasser SJ, Skodras AA, Gloeckner CJ, Buratti E, Neumann M, Kahle PJ (2022) Sirtuin-1 sensitive lysine-136 acetylation drives phase separation and pathological aggregation of TDP-43. Nat Commun 13(1):1223. https://doi.org/10.1038/s41467-022-28822-7
Kumar S, Khan MZ, Khandelwal N, Chongtham C, Singha B, Dabla A, Behera D, Singh A, Gopal B, Arimbasseri GA et al (2022) Mycobacterium tuberculosis transcription factor embr regulates the expression of key virulence factors that aid in Ex Vivo and In Vivo survival. Mbio 13(3):e383621. https://doi.org/10.1128/mbio.03836-21
Lee J, Armstrong DT, Chien G, Cho S, Via LE, Barry CE, Ellner JJ, Alland D, Dorman SE, Joloba ML et al (2014) Sensititre MYCOTB MIC plate for testing Mycobacterium tuberculosis susceptibility to first- and second-line drugs. Antimicrob Agents Chemother 58(1):11–18. https://doi.org/10.1128/AAC.01209-13
Marinescu GC, Popescu RG, Stoian G, Dinischiotu A (2018) beta-nicotinamide mononucleotide (NMN) production in Escherichia coli. Sci Rep 8(1):12278. https://doi.org/10.1038/s41598-018-30792-0
Miotto P, Zhang Y, Cirillo DM, Yam WC (2018) Drug resistance mechanisms and drug susceptibility testing for tuberculosis. Respirology 23(12):1098–1113. https://doi.org/10.1111/resp.13393
Na I, Dai H, Li H, Gupta A, Kreda D, Zhang P, Chen X, Zhang L, Alterovitz G (2021) Computational prediction and validation of specific EmbR binding site on PknH. Synth Syst Biotechnol 6(4):429–436. https://doi.org/10.1016/j.synbio.2021.11.006
Oliveira M, Silva Y, Azevedo LA, Linhares LA, Montenegro L, Alves SJ, Amorim R (2021) Antimycobacterial compound of chitosan and ethambutol: ultrastructural biological evaluation in vitro against Mycobacterium tuberculosis. Appl Microbiol Biotechnol 105(24):9167–9179. https://doi.org/10.1007/s00253-021-11690-4
Olmo ED, Molina-Salinas GM, Bini EI, Gonzalez-Hernandez S, Bustos LA, Escarcena R, Marquina-Castillo B, Mata-Espinosa D, Barrios-Payan J, Gonzalez-Ramirez D et al (2016) Efficacious In Vitro and In Vivo effects of dihydrosphingosine-ethambutol analogues against susceptible and multi-drug-resistant Mycobacterium tuberculosis. Arch Med Res 47(4):262–270. https://doi.org/10.1016/j.arcmed.2016.07.004
Papavinasasundaram KG, Chan B, Chung JH, Colston MJ, Davis EO, Av-Gay Y (2005) Deletion of the Mycobacterium tuberculosis pknH gene confers a higher bacillary load during the chronic phase of infection in BALB/c mice. J Bacteriol 187(16):5751–5760. https://doi.org/10.1128/JB.187.16.5751-5760.2005
Plinke C, Cox HS, Zarkua N, Karimovich HA, Braker K, Diel R, Rusch-Gerdes S, Feuerriegel S, Niemann S (2010) embCAB sequence variation among ethambutol-resistant Mycobacterium tuberculosis isolates without embB306 mutation. J Antimicrob Chemother 65(7):1359–1367. https://doi.org/10.1093/jac/dkq120
Sharma K, Gupta M, Krupa A, Srinivasan N, Singh Y (2006) EmbR, a regulatory protein with ATPase activity, is a substrate of multiple serine/threonine kinases and phosphatase in Mycobacterium tuberculosis. Febs J 273(12):2711–2721. https://doi.org/10.1111/j.1742-4658.2006.05289.x
Sun Q, Xiao TY, Liu HC, Zhao XQ, Liu ZG, Li YN, Zeng H, Zhao LL, Wan KL (2018) Mutations within embCAB are associated with variable level of ethambutol resistance in Mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother 62(1):10–128. https://doi.org/10.1128/AAC.01279-17
Sun L, Zhang L, Wang T, Jiao W, Li Q, Yin Q, Li J, Qi H, Xu F, Shen C et al (2019) Mutations of Mycobacterium tuberculosis induced by anti-tuberculosis treatment result in metabolism changes and elevation of ethambutol resistance. Infect Genet Evol 72:151–158. https://doi.org/10.1016/j.meegid.2018.09.027
Velayati AA, Farnia P, Hoffner S (2018) Drug-resistant Mycobacterium tuberculosis: epidemiology and role of morphological alterations. J Glob Antimicrob Resist 12:192–196. https://doi.org/10.1016/j.jgar.2017.10.006
Walker TM, Miotto P, Koser CU, Fowler PW, Knaggs J, Iqbal Z, Hunt M, Chindelevitch L, Farhat M, Cirillo DM et al (2022) The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis. Lancet Microbe 3(4):e265–e273. https://doi.org/10.1016/S2666-5247(21)00301-3
Wang S, Osgood AO, Chatterjee A (2022) Uncovering post-translational modification-associated protein-protein interactions. Curr Opin Struct Biol 74:102352. https://doi.org/10.1016/j.sbi.2022.102352
Wu X, Tan G, Yang J, Guo Y, Huang C, Sha W, Yu F (2022a) Prediction of Mycobacterium tuberculosis drug resistance by nucleotide MALDI-TOF-MS. Int J Infect Dis 121:47–54. https://doi.org/10.1016/j.ijid.2022.04.061
Wu Z, Tan Q, Zhang C, Zhao Y, Liao Q, Yu M, Xu L, Wang J, Liang H, Li H et al (2022b) mbtD and celA1 association with ethambutol resistance in Mycobacterium tuberculosis: a multiomics analysis. Front Cell Infect Microbiol 12:959911. https://doi.org/10.3389/fcimb.2022.959911
Zhao LL, Sun Q, Liu HC, Wu XC, Xiao TY, Zhao XQ, Li GL, Jiang Y, Zeng CY, Wan KL (2015) Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother 59(4):2045–2050. https://doi.org/10.1128/AAC.04933-14
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by YZ, QF, ZG, MS, ML and ZC. The first draft of the manuscript was written by QS and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
Ethical approval
Not applicable.
Consent to participate
Not applicable.
Consent to publish
Not applicable.
Additional information
Communicated by Yusuf Akhter.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Sun, Q., Zou, Y., Feng, Q. et al. The acetylation of pknH is linked to the ethambutol resistance of Mycobacterium tuberculosis. Arch Microbiol 205, 337 (2023). https://doi.org/10.1007/s00203-023-03676-9
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00203-023-03676-9