Mutations in Efflux Pump Rv1258c (Tap) Cause Resistance to Pyrazinamide, Isoniazid, and Streptomycin in M. tuberculosis

Abstract

Although drug resistance in Mycobacterium tuberculosis is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in the possible role of efflux in causing drug resistance. Previously, efflux genes have been shown to be upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance in M. tuberculosis. We constructed point mutations V219A and S292L in Rv1258c in the chromosome of M. tuberculosis and the point mutations were confirmed by DNA sequencing. The susceptibility of the constructed M. tuberculosis Rv1258c mutants to different tuberculosis drugs was assessed using conventional drug susceptibility testing in 7H11 agar in the presence and absence of efflux pump inhibitor piperine. A C14-labeled PZA uptake experiment was performed to demonstrate higher efflux activity in the M. tuberculosis Rv1258c mutants. Interestingly, the V219A and S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs in M. tuberculosis. While V219A point mutation conferred low-level drug resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. The S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance, including PZA resistance, and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance in M. tuberculosis and address their role in affecting treatment outcome in vivo .

Recent updates on drug resistance in Mycobacterium tuberculosis

“…; Liu et al . ). SMR transporter, Mmr was identified in Mtb and found to confer in vitro resistance to acriflavine, erythromycin and ethidium bromide (Nguyen ; Nasiruddin et al .…”

“…; Liu et al . ). Although MATE transporters are common in Escherichia coli and Vibrio sp., they were not reported in Mtb (Wang et al .…”

“…Upon exposure to isoniazid and rifampicin, overexpression of Rv1258c ( Tap ) was found in clinical MDR‐TB isolates, which might be responsible for clinical drug resistance (Liu et al . ). Furthermore, point mutations (V219A and S292L) were identified in efflux pump Rv1258c ( Tap ) and reported to play an important role in conferring drug resistance in clinical isolates to multiple drugs including isoniazid, pyrazinamide and streptomycin (Liu et al .…”

“…; Liu et al . ) and since these proteins exhibited characteristics specific to Mycobacteria , they were called mycobacterial membrane proteins large ( mmpL ) (Melly and Purdy ). The putative RND transporter, mmpL17 conferred in vitro resistance to isoniazid (Wang et al .…”

Evaluation of a membrane hybridization array for detection of Mycobacterium tuberculosis complex and resistance to isoniazid and rifampin in sputum specimens, mycobacterial liquid cultures, and clinical isolates

“…Furthermore, there are several novel mutation sites and mechanism which may cause RIF and INH resistance.Twenty-two different types of mutations in the rpoB gene were identified from the 58 RIF-resistant strains from a current research article and the other recent study revealed that mutations in efflux pump Rv1258c may cause resistance to pyrazinamide, isoniazid, and streptomycin in MTBC. The updated information may be useful for further supplement of probes to minimize false-negative result 21,22. (b) The genotypic changes detected by the array were not always correlated with phenotypic changes determined by drug susceptibility testing.…”