scholarly journals Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Jichan Jang ◽  
Ryangyeo Kim ◽  
Minjeong Woo ◽  
Jinsun Jeong ◽  
Da Eun Park ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Ex Vivo ◽  
Drug Candidate ◽  
Content Type ◽  
Tb Treatment ◽  
Pump Activity ◽  
Efflux Pump Inhibitors ◽  
Combinatorial Strategy

ABSTRACT New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo, indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis.

scholarly journals Developing Synergistic Drug Combinations To Restore Antibiotic Sensitivity in Drug-Resistant Mycobacterium tuberculosis

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Charles Omollo ◽  
Vinayak Singh ◽  
Elizabeth Kigondu ◽  
Antonina Wasuna ◽  
Pooja Agarwal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Ex Vivo ◽  
Antibiotic Sensitivity ◽  
Infectious Agent ◽  
Drug Combinations ◽  
Major Facilitator Superfamily ◽  
Drug Resistant ◽  
Content Type

ABSTRACT Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis. We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S rRNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis. Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant. These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yumeng Zhang ◽  
Jia Zhang ◽  
Peng Cui ◽  
Ying Zhang ◽  
Wenhong Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Binding Studies ◽  
Content Type ◽  
Clinical Strains ◽  
Efflux Pump Inhibitors ◽  
Level Resistance ◽  
Pyrazinoic Acid ◽  
Increased Susceptibility ◽  
Efflux Proteins

ABSTRACT Pyrazinamide (PZA) is a critical drug used for the treatment of tuberculosis (TB). PZA is a prodrug that requires conversion to the active component pyrazinoic acid (POA) by pyrazinamidase (PZase) encoded by the pncA gene. Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1 mutations, clinical strains without these mutations are known to exist. While efflux of POA was demonstrated in Mycobacterium tuberculosis previously, the efflux proteins involved have not been identified. Here we performed POA binding studies with an M. tuberculosis proteome microarray and identified four efflux proteins (Rv0191, Rv3756c, Rv3008, and Rv1667c) that bind POA. Overexpression of the four efflux pump genes in M. tuberculosis caused low-level resistance to PZA and POA but not to other drugs. Furthermore, addition of efflux pump inhibitors such as reserpine, piperine, and verapamil caused increased susceptibility to PZA in M. tuberculosis strains overexpressing the efflux proteins Rv0191, Rv3756c, Rv3008, and Rv1667c. Our studies indicate that these four efflux proteins may be responsible for PZA/POA efflux and cause PZA resistance in M. tuberculosis. Future studies are needed to assess their roles in PZA resistance in clinical strains.

scholarly journals Correlates between Models of Virulence for Mycobacterium tuberculosis among Isolates of the Central Asian Lineage: a Case for Lysozyme Resistance Testing?

2015 ◽  
Vol 83 (6) ◽  
pp. 2213-2223 ◽  
Author(s):  
Claire Pardieu ◽  
Nicola Casali ◽  
Simon O. Clark ◽  
Richard Hooper ◽  
Ann Williams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Resistance Testing ◽  
Snp Analysis ◽  
Single Nucleotide ◽  
Content Type ◽  
Differential Outcomes ◽  
Central Asian ◽  
Strain H37rv

Virulence factors (VFs) contribute to the emergence of new humanMycobacterium tuberculosisstrains, are lineage dependent, and are relevant to the development ofM. tuberculosisdrugs/vaccines. VFs were sought withinM. tuberculosislineage 3, which has the Central Asian (CAS) spoligotype. Three isolates were selected from clusters previously identified as dominant in London, United Kingdom. Strain-associated virulence was studied in guinea pig, monocyte-derived macrophage, and lysozyme resistance assays. Whole-genome sequencing, single nucleotide polymorphism (SNP) analysis, and a literature review contributed to the identification of SNPs of interest. The animal model revealed borderline differences in strain-associated pathogenicity.Ex vivo, isolate C72 exhibited statistically significant differences in intracellular growth relative to C6 and C14. SNP candidates inducing lower fitness levels included 123 unique nonsynonymous SNPs, including three located in genes (lysX,caeA, andponA2) previously identified as VFs in the laboratory-adapted reference strain H37Rv and shown to confer lysozyme resistance. C72 growth was most affected by lysozymein vitro. A BLAST search revealed that all three SNPs of interest (C35F, P76Q, and P780R) also occurred in Tiruvallur, India, and in Uganda. Unlike C72, however, no single isolate identified through BLAST carried all three SNPs simultaneously. CAS isolates representative of three medium-sized human clusters demonstrated differential outcomes in models commonly used to estimate strain-associated virulence, supporting the idea that virulence varies within, not just across,M. tuberculosislineages. Three VF SNPs of interest were identified in two additional locations worldwide, which suggested independent selection and supported a role for these SNPs in virulence. The relevance of lysozyme resistance to strain virulence remains to be established.

scholarly journals Transcriptional Profiling of Mycobacterium tuberculosis Exposed toIn VitroLysosomal Stress

2016 ◽  
Vol 84 (9) ◽  
pp. 2505-2523 ◽  
Author(s):  
Wenwei Lin ◽  
Paola Florez de Sessions ◽  
Garrett Hor Keong Teoh ◽  
Ahmad Naim Nazri Mohamed ◽  
Yuan O. Zhu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Metabolic Reprogramming ◽  
Human Macrophage ◽  
Host Immune System ◽  
Activated Macrophages ◽  
Intrinsic Resistance ◽  
Macrophage Infection ◽  
Content Type

Increasing experimental evidence supports the idea thatMycobacterium tuberculosishas evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge ofM. tuberculosisresponse to the hostile lysosomal environment, we profiled the global transcriptional activity ofM. tuberculosiswhen exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication,M. tuberculosisexpressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified theglgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection andin vitrostress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding geneRv1258cwas selected for validation. AnM. tuberculosis ΔRv1258cmutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1.

scholarly journals In VitroandIn VivoActivities of Three Oxazolidinones against Nonreplicating Mycobacterium tuberculosis

2014 ◽  
Vol 58 (6) ◽  
pp. 3217-3223 ◽  
Author(s):  
Ming Zhang ◽  
Claudia Sala ◽  
Neeraj Dhar ◽  
Anthony Vocat ◽  
Vasan K Sambandamurthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis ◽  
Drug Susceptibility ◽  
New Combination ◽  
Drug Candidate ◽  
Bacteriostatic Effect ◽  
Content Type ◽  
New Class ◽  
Modest Activity

ABSTRACTOxazolidinones represent a new class of antituberculosis drugs that exert their function by inhibiting protein synthesis. Here, we compared the activities of three oxazolidinones, linezolid, PNU-100480, and AZD5847, against latent tuberculosis using a simple model employing the streptomycin-starvedMycobacterium tuberculosisstrain 18b. Thein vitrodrug susceptibility results showed that the three oxazolidinones had a bacteriostatic effect against actively growing bacilli but potent bactericidal activity against nonreplicating cells. In the murine model of latent infection withM. tuberculosis18b, the efficacy of the three compounds varied greatly. Indeed, AZD5847 or its prodrug exhibited no activity or only modest activity, respectively, after 2 months of treatment, whereas both linezolid and PNU-100480 were effective against latent bacilli in mice and showed promising outcomes in combination therapy with rifampin. Moreover, the potency of PNU-100480 was significantly greater than that of linezolid, making it an attractive drug candidate in the development of new combination therapies for latent tuberculosis.

scholarly journals MmpL3 Is the Cellular Target of the Antitubercular Pyrrole Derivative BM212

2011 ◽  
Vol 56 (1) ◽  
pp. 324-331 ◽  
Author(s):  
Valentina La Rosa ◽  
Giovanna Poce ◽  
Julio Ortiz Canseco ◽  
Silvia Buroni ◽  
Maria Rosalia Pasca ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Efflux Pump ◽  
Protein A ◽  
Multidrug Resistant ◽  
Whole Genome ◽  
Content Type ◽  
Genomic Libraries ◽  
Cellular Target ◽  
Efflux Pump Inhibitors

ABSTRACTThe 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates andMycobacterium tuberculosisresiding within macrophages as well as againstMycobacterium aviumand other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. SpontaneousMycobacterium smegmatis,Mycobacterium bovisBCG, andM. tuberculosisH37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in themmpL3gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterialmembraneprotein,large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanidem-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [14C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.

scholarly journals In VitroActivity of AZD5847 against Geographically Diverse Clinical Isolates of Mycobacterium tuberculosis

2014 ◽  
Vol 58 (7) ◽  
pp. 4222-4223 ◽  
Author(s):  
Jim Werngren ◽  
Maria Wijkander ◽  
Nasrin Perskvist ◽  
V. Balasubramanian ◽  
Vasan K. Sambandamurthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lavage Fluid ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Drug Candidate ◽  
Content Type ◽  
Geographical Regions ◽  
Lung Biopsies

ABSTRACTThe MIC of the novel antituberculosis (anti-TB) drug AZD5847 was determined against 146 clinical isolates from diverse geographical regions, including eastern Europe, North America, Africa, and Asia, using the automated Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system. These isolates originated from specimen sources such as sputum, bronchial alveolar lavage fluid, pleural fluid, abscess material, lung biopsies, and feces. The overall MIC90was 1.0 mg/liter (range, 0.125 to 4 mg/liter). The MICs of AZD5847 for isolates ofMycobacterium tuberculosiswere similar among drug-sensitive strains, multidrug-resistant (MDR) strains, and extensively drug resistant (XDR) strains. The goodin vitroactivity of AZD5847 againstM. tuberculosisand the lack of cross-resistance make this agent a promising anti-TB drug candidate.

scholarly journals Phenotypically Adapted Mycobacterium tuberculosis Populations from Sputum Are Tolerant to First-Line Drugs

2016 ◽  
Vol 60 (4) ◽  
pp. 2476-2483 ◽  
Author(s):  
Obolbek Turapov ◽  
Benjamin D. O'Connor ◽  
Asel A. Sarybaeva ◽  
Caroline Williams ◽  
Hemu Patel ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liquid Medium ◽  
Culture Supernatant ◽  
Ex Vivo ◽  
First Line ◽  
Content Type ◽  
Host Environment ◽  
Solid Media ◽  
Bactericidal Effects

ABSTRACTTuberculous sputum contains multipleMycobacterium tuberculosispopulations with different requirements for isolationin vitro. These include cells that form colonies on solid media (plateableM. tuberculosis), cells requiring standard liquid medium for growth (nonplateableM. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependentM. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of theseM. tuberculosispopulations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10reductions), and SN-dependentM. tuberculosiswas more tolerant to streptomycin and isoniazid than the plateable and nonplateableM. tuberculosisstrains. Susceptibility of plateableM. tuberculosisto bactericidal drugs was significantly increased after passagein vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simpleex vivosystem for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.

scholarly journals Vitamin C Potentiates the Killing of Mycobacterium tuberculosis by the First-Line Tuberculosis Drugs Isoniazid and Rifampin in Mice

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Catherine Vilchèze ◽  
John Kim ◽  
William R. Jacobs
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Vitamin C ◽  
Mouse Serum ◽  
First Line ◽  
Content Type ◽  
Tb Treatment ◽  
High Concentrations

ABSTRACT The treatment of drug-susceptible tuberculosis (TB) is long and cumbersome. Mismanagement of TB treatment can lead to the emergence of drug resistance in patients, so shortening the treatment duration could significantly improve TB chemotherapy and prevent the development of drug resistance. We previously discovered that high concentrations of vitamin C sterilize cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis . Here, we tested subinhibitory concentration of vitamin C in combination with TB drugs against M. tuberculosis in vitro and in a mouse model of M. tuberculosis infection. In vivo , we showed that the vitamin C level in mouse serum can be increased by intraperitoneal injection of vitamin C to reach vitamin C levels close to the concentrations required for activity in vitro . Although vitamin C had no activity by itself in M. tuberculosis -infected mice, the combination of vitamin C with the first-line TB drugs isoniazid and rifampin reduced the bacterial burden in the lungs of M. tuberculosis -infected mice faster than isoniazid and rifampin combined in two independent experiments. These experiments suggest that the addition of vitamin C to first-line TB drugs could shorten TB treatment. Vitamin C, an inexpensive and nontoxic compound, could easily be added to the TB pharmacopeia to substantially improve chemotherapy outcome, which would have a significant impact on the worldwide TB community.

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