A NiCoT family metal transporter of Mycobacterium tuberculosis (Rv2856/NicT) behaves as a drug efflux pump that facilitates cross-resistance to antibiotics

Abstract

Metals often act as a facilitator in the proliferation and persistence of antibiotic resistance. Efflux pumps play key roles in the co-selection of metal and antibiotic resistance. Here, we report the ability of a putative nickel/cobalt transporter (NiCoT family), Rv2856 or NicT of Mycobacterium tuberculosis (Mtb), to transport metal and antibiotics and identified some key amino acid residues that are important for its function. Ectopic expression of NicT in Escherichia coli CS109 resulted in the increase of intracellular nickel uptake. Additionally, enhanced tolerance towards several antibiotics (norfloxacin, sparfloxacin, ofloxacin, gentamicin, nalidixic acid and isoniazid) was observed with NicT overexpression in E. coli and Mycobacterium smegmatis . A comparatively lower intracellular accumulation of norfloxacin upon NicT overexpression than that of the cells without NicT indicated the involvement of NicT in an active efflux process. Although expression of NicT did not alter the sensitivity towards kanamycin, doxycycline, tetracycline, apramycin, neomycin and ethambutol, the presence of a sub-inhibitory dose of Ni2+ resulted in the manifestation of low-level tolerance towards these drugs. Further, substitution of four residues (H77I, D82I, H83L and D227I) in the conserved regions of NicT by isoleucine and leucine resulted in reduced to nearly complete loss of the transport function for both metals and antimicrobials. Therefore, the study suggests that nickel transporter Rv2856/NicT may actively export different drugs and the presence of nickel might drive the cross-resistance to some of the antibiotics.

P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity

“…Interestingly, Adhikary et al . [ 8 ] emphasized that a nickel/cobalt efflux pump, NicT, might use the electrochemical gradient created by Ni 2+ uptake to transport drugs. Similarly, we have observed that in the presence of varying concentrations of Zn 2+ , Rv3270 could enhance the efflux of levofloxacin, which otherwise had no effect in the absence of the metal ion in the concerned host.…”

“…MICs of various structurally unrelated classes of antibiotics, such as fluoroquinolones, beta-lactams, tetracycline, anti-tubercular drugs, aminoglycosides, metal salts of magnesium, cobalt, and zinc, and natural dyes [such as ethidium bromide (EtBr), acriflavine and rhodamine B] were determined. The micro-broth dilution assay was performed in 96-well micro-titre plates [ 8 ]. The antibiotics, metal salts and dyes were two-fold serially diluted, and the total volume was made up to 300 µl with MH broth containing 0.2 % arabinose for E. coli whereas for M. smegmatis cells (10 5 per well), the volume was made up to 200 µl with 7H9 media and 20 ng µl –1 tetracycline was used for induction of the gene.…”

“…The cells were further centrifuged and the fluorescence of the supernatant was measured by using a spectrofluorimeter (FluoroMax 4; HORIBA Scientific Instruments) for norfloxacin (at an excitation wavelength 281 nm and emission wavelength 447 nm), levofloxacin (excitation wavelength of 285 nm and emission wavelength of 496 nm) and for bocillin FL (excitation and emission wavelength of 488 and 530 nm, respectively). Relative efflux (RE) was calculated concerning the control strain as follows: RE=1+(N control – N test )/N control , where N control represents antibiotic accumulated by the control strain harbouring only the vector control and N test represents antibiotic uptake by the cells harbouring Rv3270 [ 8 ]. The relative percentage of efflux was calculated by: (N control − N test )/ N control × 100.…”

“…There are multiple reports describing the dual-activity efflux pumps as a cause of imparting resistance to antibiotics and eliminating metal toxicity. One such finding revealed the role of an established NiCoT family metal transporter, NicT (rv2856) of M. tuberculosis , in influencing antibiotic resistance [ 8 ]. It is interesting to note that the Czcs–CzcR two-component system in controlling expression of the CzcCBA efflux pump system is reportedly involved in heavy metal resistance such as with zinc, cadmium and cobalt along with imparting resistance towards carbapenems such as imipenem [ 9 ].…”

The role of transcriptional regulators in metal ion homeostasis of Mycobacterium tuberculosis

“…Overexpression of NicT in M. smegmatis enhances tolerance to several antibiotics, including norfloxacin, sparfloxacin, ofloxacin, gentamicin, nalidixic acid, and isoniazid. The relatively low accumulation of norfloxacin in cells overexpressing NicT proteins suggests its involvement in the process of active exocytosis ( Adhikary et al., 2022 ). NicT may serve as a promising target for new drugs or vaccines, offering potential for the development of novel therapies against drug-resistant TB.…”