Efflux Pumps of Mycobacterium tuberculosis Play a Significant Role in Antituberculosis Activity of Potential Drug Candidates

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cThe inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed.

Section: Energy Metabolism and Drug Effluxmentioning

confidence: 99%

Energy Metabolism and Drug Efflux in Mycobacterium tuberculosis

Black

Warren

Louw

et al. 2014

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“…We have constructed knockout (KO) mutants lacking the different classes of efflux pumps of M. tuberculosis as follows. Rv1258c (KO1) and Rv0849 (KO6) are major facilitator superfamily (MFS) efflux pumps, Rv1218c (KO5) belongs to the ATP-binding cassette (ABC) superfamily, and Rv3065 (K07) belongs to the small multidrug resistance family (6,7). We investigated the contributions of these efflux pumps of M. tuberculosis towards intrinsic resistance to various PSI by comparing the in vitro activities of selected drugs on wild-type (WT) M. tuberculosis and the efflux pump KO mutants.…”

mentioning

confidence: 99%

Involvement of Efflux Pumps in the Resistance to Peptidoglycan Synthesis Inhibitors in Mycobacterium tuberculosis

Dinesh

Sharma

Balganesh

2013

Self Cite

We evaluated the contributions of Mycobacterium tuberculosis efflux pumps towards intrinsic resistance to different classes of peptidoglycan synthesis inhibitors (PSI). Our study indicates that the efflux pump knockout strains are more susceptible to PSI than the wild type. Vancomycin and ceftriaxone exhibited up to 3 log increased kill on efflux pump mutants compared to the wild-type strain, strongly suggesting an important role for efflux pumps in the intrinsic resistance of M. tuberculosis to PSI. Intrinsic resistance of Mycobacterium tuberculosis to peptidoglycan synthesis inhibitors (PSI) is generally attributed to the poor permeability of the mycobacterial cell wall and to the presence of the ␤-lactamase encoded by blaC (1, 2, 3). It is of concern that although ␤-lactam antibiotics are the most successful and widely used antibacterial agents; they have little or no use in the treatment of mycobacterial infections, including tuberculosis (4).The intrinsic resistance of mycobacteria to PSI could also be attributed to the presence of multiple drug efflux pumps (5). We have constructed knockout (KO) mutants lacking the different classes of efflux pumps of M. tuberculosis as follows. Rv1258c (KO1) and Rv0849 (KO6) are major facilitator superfamily (MFS) efflux pumps, Rv1218c (KO5) belongs to the ATP-binding cassette (ABC) superfamily, and Rv3065 (K07) belongs to the small multidrug resistance family (6, 7). We investigated the contributions of these efflux pumps of M. tuberculosis towards intrinsic resistance to various PSI by comparing the in vitro activities of selected drugs on wild-type (WT) M. tuberculosis and the efflux pump KO mutants. MICs were determined by the resazurin-based method as previously described (8). Bactericidal activities were evaluated by the CFU-based method as previously described (6).The ␤-lactams exhibited increased potency against all four of the efflux pump KOs tested compared to the WT (Table 1). The MIC of penicillin was 64 g/ml for WT M. tuberculosis. KO5 and KO7 were the most sensitive to penicillin, as measured by the MICs, which dropped 4-fold (16 g/ml), while KO1 and KO6 exhibited a 2-fold drop (32 g/ml) in the MIC. WT M. tuberculosis had an ampicillin MIC of 16 g/ml; KO1, KO5, KO6, and KO7 were equally susceptible to this ␤-lactam, displaying 2-to 4-fold drops in the MICs (8 g/ml). The MICs of meropenem for KO1 and KO5 dropped 2-to 4-fold, KO7 showed a 0-to 2-fold drop, and the MIC for KO6 remained the same as that for the WT, which was 2 to 4 g/ml. Ceftriaxone had an MIC of 2 to 4 g/ml for WT M. tuberculosis. The drop in the MICs for all four of the KOs was uniformly 4-fold (0.5 g/ml). Cefotaxime had an MIC of 2 to 4 g/ml for WT M. tuberculosis. The MICs for KO1, KO5, and KO7 dropped 2-to 4-fold, and that for KO6 dropped 0-to 2-fold. Vancomycin had an MIC of 2 g/ml for WT M. tuberculosis. The MICs for KO1 and KO7 dropped 4-fold, while a MIC decrease of only 2-fold for the KO5 and KO6 strains was observed. Interestingly, the decrease in the bacitracin MIC was noticeable onl...

“…A variety of compounds that have demonstrated activity against either resistance-nodulation-division (RND) pumps or ABC transporter-type systems were tested. Specifically, phenylalanine-arginine ␤-naphthylamide (PA␤N) (11), 1-(1-naphthylmethyl)-piperazine (NMP) (12), reserpine (13), probenecid (14), verapamil (15), and gemfibrozil (16) were all tested for their abilities to potentiate MB-1 activity in the adaptation assay. Using concentrations that were previously shown to affect the activities of various efflux pumps, improvements in MB-1 activity were not observed when it was combined with PA␤N, NMP, probenecid, verapamil, or gemfibrozil.…”

mentioning

confidence: 99%

Potentiation of Antibacterial Activity of the MB-1 Siderophore-Monobactam Conjugate Using an Efflux Pump Inhibitor

Tomaras

Crandon

McPherson

et al. 2015

Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability.T he extent and severity of antibiotic resistance in Gram-negative pathogens require the development of innovative therapeutic interventions that not only circumvent existing clinically relevant resistance mechanisms but will also result in limited spontaneous resistance emergence. Although standard in vitro methodologies exist for predicting resistance development, translational deficiencies that can stem from their lack of in vivo physiological relevance are becoming increasingly apparent. We previously described the siderophore-monobactam conjugate MB-1 ( Fig. 1 [1]), which has broad-spectrum in vitro activities against multidrug-resistant (MDR) Gram-negative pathogens, including Pseudomonas aeruginosa, when tested using standard methods established by the Clinical and Laboratory Standards Institute (CLSI) (1). Unfortunately, we have also demonstrated that despite these encouraging in vitro activities, the efficacy of MB-1 in a neutropenic murine infection model was highly variable and not directly predictable from traditional in vitro activity assays (2). Instead, the development of a modified in vitro resistance frequency assay, which relies on the utilization of more in vivo-relevant iron conditions, has proven to be more predictive of the efficacy of MB-1 in vivo. Additionally, this assay has demonstrated a role for endogenous siderophores, such as pyoverdine, in mediating the adaptive cellular response that enables P. aeruginosa to transiently resist MB-1 activity (2). While these results suggest that the use of MB-1 as monotherapy is not a viable option, we have speculated about alternative strategies that may mitigate this adaptive response and resurrect MB-1 as a legitimate antibacterial agent. Among these potentiation-type approaches, we considered the idea that inhibiting endogenous siderophore functionality should circumvent the adaptation phenotype mediated by the competition for iron by native siderophore systems. Siderophore secretion into the extracellular milieu has been shown to be dependent on functional efflux pumps in Gram-negative bacteria (3-5). In the case of P. aeruginosa, the secretion of newly synthesized pyoverdine is accomplished by the ABC transporter-type efflux system encoded by pvdR, pvdT, and opmQ (6). Additionally, this system is responsible for pyoverdine recycling after successive rounds of iron acquisition and delivery (7).To prove that inhibiting the activity of this pump can potentiate the activity of MB-1, we generated a pvdRT-op...