Toxin-antitoxin Genes Expression in Multidrug-resistant Mycobacterium tuberculosis Isolates under Drug Exposure

Article ID: e240523217289 Pages: 7

  • * (Excluding Mailing and Handling)

Abstract

Introduction: Toxin–antitoxin systems (TAs) are highly conserved in Mycobacterium tuberculosis (Mtb). The TAs role in maintaining and disseminating drug resistance in bacterial populations has been indicated. So, we aimed to analyze the expression level of mazEF-related genes in drugsusceptible and multidrug-resistant (MDR) Mtb isolates under isoniazid (INH) and rifampin (RIF) stress.

Methods: We obtained 23 Mtb isolates, including 18 MDR and 5 susceptible isolates, from the Ahvaz Regional TB Laboratory collection. The expression levels of mazF3, mazF6, and mazF9 toxin genes, and mazE3, mazE6, and mazE9 antitoxin genes in MDR and susceptible isolates were evaluated by quantitative real-time PCR (qRT-PCR) after exposure to RIF and INH.

Results: The mazF3, F6, and F9 toxin genes were overexpressed in at least two MDR isolates in the presence of RIF and INH, in contrast to mazE antitoxin genes. More MDR isolates were induced to overexpress mazF genes by RIF than INH (72.2% vs. 50%). Compared to the H37Rv strain and susceptible isolates, the expression levels of mazF3,6 by RIF and mazF3,6,9 by INH were significantly upregulated in MDR isolates (p<0.05), but no remarkable difference was detected in the expression level of mazF9 genes by INH between these groups. In susceptible isolates, the expression levels of mazE3,6 by RIF and mazE3,6,9 by INH were induced and enhanced significantly compared to MDR isolates, but there was no difference between MDR and H37Rv strain.

Conclusion: Based on the results, we propose that mazF expression under RIF/INH stress may be associated with drug resistance in Mtb in addition to mutations, and the mazE antitoxins may be related to enhanced susceptibility of Mtb to INH and RIF. Further experiments are needed to investigate the exact mechanism underlying the TA system's role in drug resistance.

Graphical Abstract

[1]
Kanabalan RD, Lee LJ, Lee TY, et al. Human tuberculosis and Mycobacterium tuberculosis complex: A review on genetic diversity, pathogenesis and omics approaches in host biomarkers discovery. Microbiol Res 2021; 246: 126674.
[http://dx.doi.org/10.1016/j.micres.2020.126674] [PMID: 33549960]
[2]
Chakaya J, Khan M, Ntoumi F, et al. Global Tuberculosis Report 2020 – Reflections on the Global TB burden, treatment and prevention efforts. Int J Infect Dis 2021; 113 (Suppl. 1): S7-S12.
[http://dx.doi.org/10.1016/j.ijid.2021.02.107] [PMID: 33716195]
[3]
B Cadmus SI. Falodun OI, Fagade OE, Murphy R, Taiwo B, Van Soolingen D. The problem of resistance in Mycobacterium tuberculosis may be underestimated in Africa. Int J Mycobacteriol 2018; 7(2): 148-51.
[http://dx.doi.org/10.4103/ijmy.ijmy_29_18] [PMID: 29900891]
[4]
Global tuberculosis report WHO 2020, Geneva, Switzerland. 2020. Available From: https://www.who.int/publications/i/item/ 9789240013131
[5]
Torfs E, Piller T, Cos P, Cappoen D. Opportunities for overcoming Mycobacterium tuberculosis drug resistance: Emerging mycobacterial targets and host-directed therapy. Int J Mol Sci 2019; 20(12): 2868.
[http://dx.doi.org/10.3390/ijms20122868] [PMID: 31212777]
[6]
Yang QE, Walsh TR. Toxin–antitoxin systems and their role in disseminating and maintaining antimicrobial resistance. FEMS Microbiol Rev 2017; 41(3): 343-53.
[http://dx.doi.org/10.1093/femsre/fux006] [PMID: 28449040]
[7]
Równicki M, Lasek R, Trylska J, Bartosik D. Targeting type II toxin-antitoxin systems as antibacterial strategies. Toxins 2020; 12(9): 568.
[http://dx.doi.org/10.3390/toxins12090568] [PMID: 32899634]
[8]
Beck IN, Usher B, Hampton HG, Fineran PC, Blower TR. Antitoxin autoregulation of M. tuberculosis toxin-antitoxin expression through negative cooperativity arising from multiple inverted repeat sequences. Biochem J 2020; 477(12): 2401-19.
[http://dx.doi.org/10.1042/BCJ20200368] [PMID: 32519742]
[9]
Boldrin F, Provvedi R, Cioetto Mazzabò L, Segafreddo G, Manganelli R. Tolerance and persistence to drugs: A main challenge in the fight against Mycobacterium tuberculosis. Front Microbiol 2020; 11: 1924.
[http://dx.doi.org/10.3389/fmicb.2020.01924] [PMID: 32983003]
[10]
Keren I, Minami S, Rubin E, Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2011; 2(3): e00100-11.
[http://dx.doi.org/10.1128/mBio.00100-11] [PMID: 21673191]
[11]
Eroshenko DV, Polyudova TV, Pyankova AA. VapBC and mazEF toxin/antitoxin systems in the regulation of biofilm formation and antibiotic tolerance in nontuberculous mycobacteria. Int J Mycobacteriol 2020; 9(2): 156-66.
[http://dx.doi.org/10.4103/ijmy.ijmy_61_20] [PMID: 32474537]
[12]
Slayden RA, Dawson CC, Cummings JE. Toxin–antitoxin systems and regulatory mechanisms in Mycobacterium tuberculosis. Pathog Dis 2018; 76(4): fty039.
[http://dx.doi.org/10.1093/femspd/fty039] [PMID: 29788125]
[13]
Zhao J-L, Liu W, Xie W-Y, Cao X-D, Yuan L. Viability, biofilm formation, and mazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China. Infect Drug Resist 2018; 11: 345-58.
[http://dx.doi.org/10.2147/IDR.S148648]
[14]
Chen R, Zhou J, Sun R, Du C, Xie W. Conserved conformational changes in the regulation of Mycobacterium tuberculosis mazEF-mt1. ACS Infect Dis 2020; 6(7): 1783-95.
[http://dx.doi.org/10.1021/acsinfecdis.0c00048] [PMID: 32485099]
[15]
Tiwari P, Arora G, Singh M, Kidwai S, Narayan OP, Singh R. mazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs. Nat Commun 2015; 6(1): 6059.
[http://dx.doi.org/10.1038/ncomms7059] [PMID: 25608501]
[16]
Coskun USS, Cicek AC, Kilinc C, et al. Effect of mazEF, higBA and relBE toxin-antitoxin systems on antibiotic resistance in Pseudomonas aeruginosa and Staphylococcus isolates. Malawi Med J 2018; 30(2): 67-72.
[http://dx.doi.org/10.4314/mmj.v30i2.3] [PMID: 30627331]
[17]
Shahi F, Khosravi AD, Tabandeh MR, Salmanzadeh S. Investigation of the Rv3065, Rv2942, Rv1258c, Rv1410c, and Rv2459 efflux pump genes expression among multidrug-resistant Mycobacterium tuberculosis clinical isolates. Heliyon 2021; 7(7): e07566.
[http://dx.doi.org/10.1016/j.heliyon.2021.e07566] [PMID: 34337183]
[18]
Singh R, Singh M, Arora G, Kumar S, Tiwari P, Kidwai S. Polyphosphate deficiency in Mycobacterium tuberculosis is associated with enhanced drug susceptibility and impaired growth in guinea pigs. J Bacteriol 2013; 195(12): 2839-51.
[http://dx.doi.org/10.1128/JB.00038-13] [PMID: 23585537]
[19]
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. J Bacteriol Methods 25(4): 402-8.
[http://dx.doi.org/10.1006/meth.2001.1262]
[20]
Li G, Zhang J, Guo Q, et al. Study of efflux pump gene expression in rifampicin-monoresistant Mycobacterium tuberculosis clinical isolates. J Antibiot 2015; 68(7): 431-5.
[http://dx.doi.org/10.1038/ja.2015.9] [PMID: 25690361]
[21]
Li G, Zhang J, Guo Q, et al. Efflux pump gene expression in multidrug-resistant Mycobacterium tuberculosis clinical isolates. PLoS One 2015; 10(2): e0119013.
[http://dx.doi.org/10.1371/journal.pone.0119013] [PMID: 25695504]
[22]
Chattopadhyay G, Bhasin M, Ahmed S, et al. Functional and biochemical characterization of the mazEF6 toxin-antitoxin system of Mycobacterium tuberculosis. J Bacteriol 2022; 204(4): e00058-22.
[http://dx.doi.org/10.1128/jb.00058-22] [PMID: 35357163]
[23]
Kazemian H, Heidari H, Kardan-Yamchi J, et al. Comparison of toxin-antitoxin expression among drug-susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis. Adv Respir Med 2021; 89(2): 110-4.
[http://dx.doi.org/10.5603/ARM.a2021.0033] [PMID: 33966258]
[24]
Fu Z, Tamber S, Memmi G, Donegan NP, Cheung AL. Overexpression of mazFsa in Staphylococcus aureus induces bacteriostasis by selectively targeting mRNAs for cleavage. J Bacteriol 2009; 191(7): 2051-9.
[http://dx.doi.org/10.1128/JB.00907-08] [PMID: 19168622]
[25]
Pedersen K, Christensen SK, Gerdes K. Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol Microbiol 2002; 45(2): 501-10.
[http://dx.doi.org/10.1046/j.1365-2958.2002.03027.x] [PMID: 12123459]
[26]
Erental A, Sharon I, Engelberg-Kulka H. Two programmed cell death systems in Escherichia coli: An apoptotic-like death is inhibited by the mazEF-mediated death pathway. PLoS Biol 2012; 10(3): e1001281.
[http://dx.doi.org/10.1371/journal.pbio.1001281] [PMID: 22412352]