Characterization of the Mycobacterial MSMEG-3762/63 Efflux Pump in Mycobacterium smegmatis Drug Efflux

Siena, Barbara De; Campolattano, Nicoletta; D’Abrosca, Gianluca; Russo, Luca; Cantillon, Daire; Marasco, Rosangela; Muscariello, Lidia; Waddell, Simon J.; Sacco, Margherita

doi:10.3389/fmicb.2020.575828

Cited by 9 publications

(9 citation statements)

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“…Such other efflux pumps could be, for example, the homologs of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c ( efpA ), and Rv3065 ( mmr and emrE ) [ 4 ]. In the study by De Siena et al [ 33 ], two other ortholog operons, Rv1687/86/85c in M. tuberculosis and MSMEG_3762/63/65 in M. smegmatis , both designated as ABC efflux pump systems, were identified, sharing a high percentage of identity. It was shown that the protein complex MSMEG-3762/63, annotated as an efflux pump, is involved in the resistance toward first- and second-line anti-TB drugs, such as rifampicin and ciprofloxacin, as well as in the formation of mycobacterial biofilms.…”

Section: Discussionmentioning

confidence: 99%

“…There has been much research performed on genes involved in the intrinsic and acquired antimicrobial drug resistance of mycobacteria, including MDR/XDR M. tuberculosis. Besides the role of efflux pumps in antibiotic resistance, they might also be involved in bacterial behaviors associated with virulence, biofilm development, and quorum sensing-dependent expression of virulence factors [ 33 , 37 , 38 ]. Based on the findings of this study, the interplay of 4 and 6 in P. ostruthium fractions seems to be crucial for efflux pump inhibitory-, antibacterial, and resistance-modulatory effects of this plant.…”

Section: Discussionmentioning

confidence: 99%

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Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins

et al. 2021

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Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread and, due to antibiotic resistance, also hard to treat and a major cause of mortality. Natural compounds have the potential to increase antibiotic effectiveness due to their resistance modulatory and antimicrobial effects. In this study, Peucedanum ostruthium extracts, fractions, and isolated compounds were investigated regarding their antimicrobial and resistance-modulatory effects as well as efflux pump inhibition in Mycobacterium smegmatis. P. ostruthium extracts were found to have anti-mycobacterial potential and resistance modulating effects on ethidium bromide activity. The major antibacterial effect was attributed to ostruthin, and we found that the more lipophilic the substrate, the greater the antimicrobial effect. Imperatorin caused potent modulatory effects by interfering with the action of the major LfrA efflux pump in M. smegmatis. The plant P. ostruthuim has a complex effect on M. smegmatis, including antibacterial, efflux pump inhibition, resistance modulation, and membrane permeabilization, and its major constituents, ostruthin and imperatorin, have a distinct role in these effects. This makes P. ostruthium and its coumarins promising therapeutics to consider in the fight against drug-resistant mycobacteria.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins

et al. 2021

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“…coli strains and 200 µl per well for M. smegmatis strains [27]. Two-fold serial dilutions of different antibiotics were made in cation-adjusted MH broth and 7H9 media and each well was inoculated with 10 5 cells.…”

Section: Methodsmentioning

confidence: 99%

“…MICs of different compounds were determined using micro-broth dilution method and assessed according to Clinical and Laboratory Standards Institute (CLSI) guidelines [26]. The assays were performed in 96-well micro-titre plates with an assay volume of 300 µl per well for E. coli strains and 200 µl per well for M. smegmatis strains [27]. Two-fold serial dilutions of different antibiotics were made in cation-adjusted MH broth and 7H9 media and each well was inoculated with 10 5 cells.…”

Section: Determination Of Susceptibility Towards Antibiotics and Meta...mentioning

confidence: 99%

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

et al. 2022

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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.

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“…We have previously characterized the ABC-type MSMEG-3762/63 e ux pump in Mycobacterium smegmatis, a mycobacterium widely used as a model system for M. tuberculosis physiology [17,18]. This e ux pump shares a high percentage identity with the M. tuberculosis Rv1687/86c pump [19,20]. In our previous studies, we demonstrated that the TetR-like MSMEG-3765 repressor was able to negatively regulate the expression of the MSMEG_3762/63/65 operon and of the homologous Rv1687/86/85c operon, encoding the e ux pump and its regulator Rv1685c in M. tuberculosis [19].…”

Section: Introductionmentioning

confidence: 99%

Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis

Campolattano,

D'Abrosca,

Russo

et al. 2023

Preprint

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Drug resistance is one of the most difficult challenges facing tuberculosis (TB) control. Drug efflux is among the mechanisms leading to drug resistance. In our previous studies, we partially characterized the ABC-type MSMEG-3762/63 efflux pump in Mycobacterium smegmatis, which shares high percentage identity with the Mycobacterium tuberculosis Rv1687/86c pump. MSMEG-3762/63 was shown to have extrusion activity for rifampicin and ciprofloxacin, used in first and second-line anti-TB treatments. Moreover, we described the functional role of the TetR-like MSMEG-3765 protein as a repressor of the MSMEG_3762/63/65 operon and orthologous Rv1687/86/85c in M. tuberculosis. Here we show that the operon is upregulated in the macrophage environment, supporting a previous observation of induction triggered by acid-nitrosative stress. Expression of the efflux pump was also induced by sub-inhibitory concentrations of rifampicin or ciprofloxacin. Both these drugs also prevented the binding of the MSMEG-3765 TetR repressor protein to its operator in the MSMEG_3762/63/65 operon. The hypothesis that these two drugs might be responsible for the induction of the efflux pump operon was assessed by bioinformatics analyses. Docking studies using a structural model of the regulator MSMEG-3765 showed that both antibiotics abolished the ability of this transcriptional repressor to recognize the efflux pump operon by interacting with the homodimer at different binding sites within the same binding pocket. Reduced binding of the repressor leads to induction of the efflux pump in M. smegmatis, and reduced efficacy of these two anti-mycobacterial drugs.

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Characterization of the Mycobacterial MSMEG-3762/63 Efflux Pump in Mycobacterium smegmatis Drug Efflux

Cited by 9 publications

References 54 publications

Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins

Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins

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

Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis

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