Mutation EthAW21R confers co-resistance to protionamide and ethionamide in both Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv

Abstract:

Ethionamide (ETA) and prothionamide (PRO) are interchangeably used in tuberculosis (TB) chemotherapy regimens. Subtle discrepancies between biochemical and genetic information on the modes of sensitivity and resistance of isoniazid (INH) and ETA warrants further studies. We report a new mutation – EthA_W21R – in Mycobacterium bovis Bacillus Calmette-Guérin that corresponds with co-resistance to both PRO and ETA, which to the best of our knowledge has not been reported before. Our findings suggest that mutation EthA_W21R could be used as a marker site for testing PRO and ETA cross-resistance.

Keywords::

• mutation
• EthA_W21R
• isoniazid
• co-resistance
• thioamides
• molecular marker

The thioamide, ethionamide (ETA), and its propyl-analog prothionamide (PRO) are interchangeably used in tuberculosis (TB) chemotherapy regimens to treat multidrug-resistant TB (MDR-TB),^1–3 drug-susceptible TB meningitis (TBM), and miliary TB in some settings, due to their good cerebrospinal fluid (CSF) penetration ability.⁴

PRO is associated with better tolerance compared with ETA in the treatment of MDR-TB, and both are structurally similar to isoniazid (INH).^2,5,6 The only notable distinction in their mechanism(s) of action is the lack of cross-resistance to INH.^7,8

Both ETA and PRO are prodrugs whose enzymatic activation by Mycobacterium tuberculosis’ EthA inhibits InhA, which subsequently inhibits the M. tuberculosis’ mycolic acid synthesis (Figure 1).^2,9 Mutations in the ethA gene often underlie ETA and PRO monoresistance.² Hanoulle et al¹⁰ postulated that both are further transformed by EthA enzyme to a metabolite that accumulates intracellularly and acts as the final toxic compound. As illustrated in Figure 1,¹¹ activated ETA and PRO form adducts with nicotinamide adenine dinucleotide (NAD), which is the inhibitor of the InhA enzyme in M. tuberculosis.^1,12,13 Thee et al² suggested that the correlation between mutations conferring ETA resistance and the MIC warrants further studies because of the subtle discrepancies between biochemical and genetic information on the modes of sensitivity and resistance in the cases of INH and ETA.

Figure 1 The mechanism of action for ETA.

Notes: ETA is activated by monooxygenase EthA to form a reactive species that binds to NAD+. The resulting ETH–NAD adduct inhibits the enoyl-ACP reductase InhA of the FASII system, resulting in mycolic acid biosynthesis inhibition. ©2014 American Society for Microbiology. Used with permission. No further reproduction or distribution is permitted without the prior written permission of American Society for Microbiology.11
Abbreviations: ETA, ethionamide; NAD, nicotinamide adenine dinucleotide.

Here, we report a new mutation – EthA_W21R – in Mycobacterium bovis Bacillus Calmette-Guérin (BCG) that corresponds with co-resistance to PRO and ETA, which to the best of our knowledge has not been reported before.

We screened wild-type M. bovis BCG Tice on high PRO concentrations and obtained one drug-resistant colony at 30 µg/mL PRO-containing 7H11 plate. To confirm the phenotypic resistance of the single colony, we similarly re-tested it on 30 and 40 µg/mL PRO-containing 7H11 plate. We sequenced the six reported genes (ethR, ethA, inhA katG, ndh, and ahpC; Table 1; BGI, Shenzhen, China) associated with ETA and PRO resistance and found a single-nucleotide mutation in ethA gene leading to W21R mutation while the other five genes had no mutation(s).

Table 1 PCR and sequencing primers used to delineate target-based spontaneous genotypic resistance mechanisms of M. bovis BCG Tice

Resistance to	Primer pairs	Nucleotide sequences (5'-3')	Upstream extension (base)	Downstream extension (base)	Product length (bp)
PRO	EthRf5/EthRr5	TTTTCCAGGATGGCGTAGC/CCGACCGGATCGTCAACA	185	263	1099
	EthAf/EthAr	CCTGGCAGCTTACTACGTGTC/CGGCATCATCGTCGTCTG	75	54	1599
	inhAf/inhAr	TCACGGCGGTAGAAGAGCA/CCACGCAGATGTCGCAAAGA	548	326	1684
	KatGf/KatGr	TGCGAAAGATCCAACCCTC/AGACCAACCGTGTAGGCAAAT	276	317	2816
	Ndhf/Ndhr	ACTTGGCTCCGCACGGCTAT/ATCCGGCGACGGCATTCA	217	109	1718
	ahpCf/ahpCr	CGACTGGCTCATATCGAGAAT/AATACCTGCGGATTTCGTGT	216	180	984
EthAf2	GGAATTCCATATGACCGAGCACCTCGACGTT
EthAr2	CCCAAGCTTCTAAACCCCCACCGGGGCA
hyg-f	GTGACACAAGAATCCCTG
hyg-r	TCAGGCGCCGGGGGCGGTG

Note: Primers for each gene amplification were extended with ~150 bp upstream and downstream of start and stop codons.

Abbreviations: M. bovis, Mycobacterium bovis; PRO, prothionamide.

We then overexpressed this mutated 1.4kb ethA^W21R and the M. bovis ethA^wt genes by cloning them at the NdeI and HindIII sites of extrachromosomal p60LuxN plasmid bearing the M. tuberculosis hsp60 promoter (Figure 2).¹⁴ Recombinant plasmids p60ethA^W21Rand p60ethA^wt constructs were verified by enzyme digestion and sequencing (BGI). Wild-type M. bovis BCG Tice and M. tuberculosis H37Rv strains were transformed with the plasmids p60ethA^W21Rand p60ethA^wt through electroporation as described previously with some modifications.¹⁵ Positive selection was confirmed by PCR amplification of the hygromycin resistance marker gene (hyg) in p60ethA^W21Randp60ethA^wt using primers hyg-r and hyg-f (Table 1).

Figure 2 E. coli–mycobacteria shuttle plasmids p60ethA^Mt/Wt.

Notes: OriE, origin of replication region in E. coli; OriM, origin of replication in mycobacteria; hyg, hygromycin-resistant gene; ethA, ethA^wt or ethA^W21R.
Abbreviation: E. coli, Escherichia coli.

We then evaluated the MICs of PRO and ETA against the recombinant and parental strains (control) using the classical agar plate method.¹⁶ We show that after overexpressing the mutated ethA^W21R in wild-type BCG and M. tuberculosis H37Rv, both PRO and ETA MIC rose by 256- and 128-fold, respectively (Table 2). Additionally, no observable differences were noted in the MICs of the overexpressed ethA^wt recombinants and the parent strains (MIC =0.25 and 0.5 µg/mL; Table 2). Our findings suggest that the mutation ethA^W21R could be used as a marker site for testing PRO and ETA cross-resistance.

Table 2 MICs of PRO and ETA for wild-type and recombinant strains

Serial number	Strain	Mutations	MICs (μg/mL)
			PRO	ETA
1	M. tuberculosis H37Rv:p60ethAMt	W2IR	32	32
2	M. tuberculosis H37Rv:p60ethAWt	-	0.25	0.25
3	M. tuberculosis H37Rv Wt	-	0.5	0.5
4	M. bovis BCG Tice BCG:p60ethAMt	W2IR	32	32
5	M. bovis BCG Tice BCG:p60ethAWt	-	0.5	0.5
6	M. bovis BCG Tice Wt	-	0.25	0.25

Abbreviations: ETA, ethionamide; M. bovis, Mycobacterium bovis; M. tuberculosis, Mycobacterium tuberculosis; PRO, prothionamide.

Acknowledgments

This work was supported by the National Mega project of China for Innovative Drugs (2018ZX09721001-003-003) and for Main Infectious Diseases (2017ZX10302301-003-002), the National Natural Science Foundation of China (81572037), the Chinese Academy of Sciences Grants (154144KYSB20150045, KFZD-SW-207, and YJKYYQ20170036) and Guangzhou Municipal Industry and Research Collaborative Innovation Program (201508020248 and 201604020019). It was also partially supported by the Public Research and Capacity Building Project of Guangdong Province (2017A020212004), the Guangzhou Municipal Clinical Medical Centre Program (155700012) and the Key Project Grant (SKLRD2016ZJ003) from the State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Diseases, First Affiliated Hospital of Guangzhou Medical University. TZ received support from Science and Technology Innovation Leader of Guangdong Province (2016TX03R095). JM is a recipient of the University of Chinese Academy of Sciences PhD Fellowship Program while GM and GC are recipients of the CAS-TWAS President’s PhD Fellowship Program for International Students. The work was undertaken at the following institutes, which are mandated to undertake tuberculosis studies in Southern China: 1) State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China, and 2) State Key Laboratory of Respiratory Disease, Department of Clinical Laboratory, Guangzhou Chest Hospital, Guangzhou, China. The facilities are compliant with biosafety level 2+ and 3 requirements for handling infectious materials.

Disclosure

The authors report no conflicts of interest in this work.