Binding Pocket Alterations in Dihydrofolate Synthase Confer Resistance topara-Aminosalicylic Acid in Clinical Isolates of Mycobacterium tuberculosis

ABSTRACTThe mechanistic basis for the resistance ofMycobacterium tuberculosistopara-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursorpara-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance inM. tuberculosisstrain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates ofM. tuberculosisandMycobacterium bovis. From a panel of 85 multidrug-resistantM. tuberculosisclinical isolates, 5 were found to harbor mutations in thefolCgene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy offolCfully restored PAS susceptibility infolCmutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics forM. tuberculosisclinical isolates and for further defining the mode of action of this important tuberculosis drug.

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Sulfamethoxazole Susceptibility of Mycobacterium tuberculosis Isolates from HIV-Infected Ugandan Adults with Tuberculosis Taking Trimethoprim-Sulfamethoxazole Prophylaxis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01101-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5844-5846 ◽

Cited By ~ 3

Author(s):

Sam Ogwang ◽

Caryn E. Good ◽

Brenda Okware ◽

Mary Nsereko ◽

Michael R. Jacobs ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Sub Saharan Africa ◽

Content Type ◽

Pulmonary Tb ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Sub Saharan

ABSTRACTAdditional drugs are needed for the treatment of multidrug-resistant tuberculosis (TB). Sulfamethoxazole has been shown to havein vitroactivity againstMycobacterium tuberculosis; however, there is concern about resistance given the widespread use of trimethoprim-sulfamethoxazole prophylaxis among HIV-infected patients in sub-Saharan Africa. Thirty-eight of 40Mycobacterium tuberculosisisolates (95%) from pretreatment sputum samples from Ugandan adults with pulmonary TB, including HIV-infected patients taking trimethoprim-sulfamethoxazole prophylaxis, were susceptible with MICs of ≤38.4 μg/ml.

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Bazedoxifene Suppresses Intracellular Mycobacterium tuberculosis Growth by Enhancing Autophagy

mSphere ◽

10.1128/msphere.00124-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 3

Author(s):

Qi Ouyang ◽

Kehong Zhang ◽

Dachuan Lin ◽

Carl G. Feng ◽

Yi Cai ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Antimycobacterial Activity ◽

Multidrug Resistant ◽

Drug Resistant ◽

New Host ◽

Drug Resistant Tuberculosis ◽

Content Type ◽

Receptor Modulator ◽

Resistant Tuberculosis ◽

Negative Side Effects

ABSTRACT Tuberculosis (TB) is still the leading killer caused by Mycobacterium tuberculosis infection. There is a clear need for new treatment strategy against TB. It has been reported that tamoxifen, known as a selective estrogen receptor modulator (SERM), exhibits antimycobacterial activity and inhibits M. tuberculosis growth in macrophages. However, it remains unknown whether such antimicrobial activity is a general property of all SERMs and how it works. In this study, we identified that bazedoxifene (BZA), a newer SERM, inhibits intracellular M. tuberculosis growth in macrophages. BZA treatment increases autophagosome formation and LC3B-II protein expression in M. tuberculosis-infected macrophages. We further demonstrated that the enhancement of autophagy by BZA is dependent on increased reactive oxygen species (ROS) production and associated with phosphorylation of Akt/mTOR signaling. In summary, our data reveal a previously unappreciated antimicrobial function of BZA and suggest that future investigation focusing on the mechanism of action of SERMs in macrophages may lead to new host-directed therapies against TB. IMPORTANCE Since current strategies for the treatment of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have low efficacy and highly negative side effects, research on new treatments including novel drugs is essential for curing drug-resistant tuberculosis. Host-directed therapy (HDT) has become a promising idea to modulate host cell responses to enhance protective immunity against pathogens. Bazedoxifene (BZA), which belongs to a new generation of SERMs, shows the ability to inhibit the growth of M. tuberculosis in macrophages and is associated with autophagy. Our findings reveal a previously unrecognized antibacterial function of BZA. We propose that the mechanism of SERMs action in macrophages may provide a new potential measure for host-directed therapies against TB.

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Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolated in Nepal

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06418-11 ◽

2012 ◽

Vol 56 (6) ◽

pp. 2831-2836 ◽

Cited By ~ 38

Author(s):

Ajay Poudel ◽

Chie Nakajima ◽

Yukari Fukushima ◽

Haruka Suzuki ◽

Basu Dev Pandey ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Promoter Region ◽

Multidrug Resistant ◽

Content Type ◽

Molecular Tests ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Mdr Tb ◽

Substitution Mutations ◽

Drug Resistant Strains

ABSTRACTDespite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance inMycobacterium tuberculosisis required. In the present study, we investigated the prevalence of mutations inrpoBandkatGgenes and theinhApromoter region in 158M. tuberculosisisolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) ofrpoBwere identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in thekatGgene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in theinhApromoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance inM. tuberculosisin Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.

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Unique Regulation of the DosR Regulon in the Beijing Lineage of Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.00696-16 ◽

2016 ◽

Vol 199 (2) ◽

Cited By ~ 17

Author(s):

Pilar Domenech ◽

Jason Zou ◽

Alexandra Averback ◽

Nishath Syed ◽

Daniele Curtis ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Treatment Programs ◽

Nucleotide Polymorphisms ◽

Content Type ◽

Fitness Advantage ◽

Mechanistic Basis ◽

Dramatic Shift ◽

Dosr Regulon ◽

The Impact

ABSTRACT The DosR regulon, a set of 48 genes normally expressed in Mycobacterium tuberculosis under conditions that inhibit aerobic respiration, is controlled via the DosR-DosS/DosT two-component system. While the regulon requires induction in most M. tuberculosis isolates, for members of the Beijing lineage, its expression is uncoupled from the need for signaling. In our attempts to understand the mechanistic basis for this uncoupling in the Beijing background, we previously reported the identification of two synonymous single-nucleotide polymorphisms (SNPs) within the adjacent Rv3134c gene. In the present study, we have interrogated the impact of these SNPs on dosR expression in wild-type strains, as well as a range of dosR-dosS-dosT mutants, for both Beijing and non-Beijing M. tuberculosis backgrounds. In this manner, we have unequivocally determined that the C601T dosR promoter SNP is the sole requirement for the dramatic shift in the pattern of DosR regulon expression seen in this globally important lineage. Interestingly, we also show that DosT is completely nonfunctional within these strains. Thus, a complex series of evolutionary steps has led to the present-day Beijing DosR phenotype that, in turn, potentially confers a fitness advantage in the face of some form of host-associated selective pressure. IMPORTANCE Mycobacterium tuberculosis strains of the Beijing lineage have been described as being of enhanced virulence compared to other lineages, and in certain regions, they are associated with the dramatic spread of multidrug-resistant tuberculosis (TB). In terms of trying to understand the functional basis for these broad epidemiological phenomena, it is interesting that, in contrast to the other major lineages, the Beijing strains all constitutively overexpress members of the DosR regulon. Here, we identify the mutational events that led to the evolution of this unique phenotype. In addition, our work highlights the fact that important phenotypic differences exist between distinct M. tuberculosis lineages, with the potential to impact the efficacy of diagnosis, vaccination, and treatment programs.

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Mode of Action of Clofazimine and Combination Therapy with Benzothiazinones against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00395-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4457-4463 ◽

Cited By ~ 57

Author(s):

Benoit Lechartier ◽

Stewart T. Cole

Keyword(s):

Mycobacterium Tuberculosis ◽

Bacterial Load ◽

Multidrug Resistant ◽

Content Type ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Drug Resistant Strains ◽

Transfer Chain

ABSTRACTClofazimine (CZM) is an antileprosy drug that was recently repurposed for treatment of multidrug-resistant tuberculosis. InMycobacterium tuberculosis, CZM appears to act as a prodrug, which is reduced by NADH dehydrogenase (NDH-2), to release reactive oxygen species upon reoxidation by O2. CZM presumably competes with menaquinone (MK-4), a key cofactor in the mycobacterial electron transfer chain, for its reduction by NDH-2. We studied the effect of MK-4 supplementation on the activity of CZM againstM. tuberculosisand found direct competition between CZM and MK-4 for the cidal effect of CZM, against nonreplicating and actively growing bacteria, as MK-4 supplementation blocked the drug's activity against nonreplicating bacteria. We demonstrated that CZM, like bedaquiline, is synergisticin vitrowith benzothiazinones such as 2-piperazino-benzothiazinone 169 (PBTZ169), and this synergy also occurs against nonreplicating bacteria. The synergy between CZM and PBTZ169 was lost in an MK-4-rich medium, indicating that MK-4 is the probable link between their activities. The efficacy of the dual combination of CZM and PBTZ169 was testedin vivo, where a great reduction in bacterial load was obtained in a murine model of chronic tuberculosis. Taken together, these data confirm the potential of CZM in association with PBTZ169 as the basis for a new regimen against drug-resistant strains ofM. tuberculosis.

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Disparities in Capreomycin Resistance Levels Associated with therrsA1401G Mutation in Clinical Isolates of Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04438-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 444-449 ◽

Cited By ~ 19

Author(s):

Analise Z. Reeves ◽

Patricia J. Campbell ◽

Melisa J. Willby ◽

James E. Posey

Keyword(s):

Mycobacterium Tuberculosis ◽

Strong Predictor ◽

Critical Concentration ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Cross Resistance ◽

Second Line ◽

Content Type

ABSTRACTAs the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. TherrsA1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between therrsA1401G mutation and CAP resistance, with up to 40% ofrrsA1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring therrsA1401G mutation and found that the CAP MICs ranged from 8 μg/ml to 40 μg/ml. These results were drastically different from engineered A1401G mutants generated in isogenicMycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 μg/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 μg/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02718-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 4222-4223 ◽

Cited By ~ 7

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.

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In VitroandIn VivoActivities of the Nitroimidazole TBA-354 against Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03823-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 136-144 ◽

Cited By ~ 48

Author(s):

A. M. Upton ◽

S. Cho ◽

T. J. Yang ◽

Y. Kim ◽

Y. Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Pharmacokinetic Profile ◽

Multidrug Resistant ◽

Phase Iii ◽

Drug Resistant ◽

Next Generation ◽

Content Type ◽

Resistant Tuberculosis

ABSTRACTNitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 againstMycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidalin vitroagainst replicating and nonreplicatingMycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity againstMycobacterium tuberculosisH37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10−7.In vitrostudies andin vivostudies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life.In vitrostudies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependentin vivobactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole.

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Structure–activity relationship of natural and synthetic coumarin derivatives against Mycobacterium tuberculosis

Future Medicinal Chemistry ◽

10.4155/fmc-2018-0281 ◽

2020 ◽

Vol 12 (17) ◽

pp. 1533-1546 ◽

Cited By ~ 2

Author(s):

Claudia TA Pires ◽

Regiane BL Scodro ◽

Diógenes AG Cortez ◽

Mislaine A Brenzan ◽

Vera LD Siqueira ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Coumarin Derivatives ◽

Resistant Tuberculosis ◽

Diphenyltetrazolium Bromide ◽

Relationship Of ◽

Microtiter Assay ◽

Tuberculosis Activity ◽

Natural And Synthetic

Aim: Eight coumarin derivatives (1a–h) obtained from natural (-)-mammea A/BB (1) and 13 synthetic coumarins (2–14) had their cytotoxicity and biological activity evaluated against Mycobacterium tuberculosis H37Rv reference strain and multidrug-resistant clinical isolates. Materials & methods: Anti- M. tuberculosis activity was evaluated by resazurin microtiter assay plate, and the cytotoxicity of natural and synthetic products using J774A.1 macrophages by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Results: Compounds 1g, 5, 6, 12 and 14 were more active against M. tuberculosis H37Rv and multidrug-resistant clinical isolates with MIC values ranging from 15.6 to 62.5 μg/ml. Conclusion: These results demonstrate that the coumarin derivatives were active against multidrug-resistant clinical isolates, becoming potential candidates to be used in the treatment of resistant tuberculosis.

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Some Synonymous and Nonsynonymous gyrA Mutations in Mycobacterium tuberculosis Lead to Systematic False-Positive Fluoroquinolone Resistance Results with the Hain GenoType MTBDRsl Assays

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02169-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 12

Author(s):

Adebisi Ajileye ◽

Nataly Alvarez ◽

Matthias Merker ◽

Timothy M. Walker ◽

Suriya Akter ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Specific Resistance ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Resistance Mutations ◽

Content Type ◽

Synonymous Mutations ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis

ABSTRACT In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

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