Novel mutations conferring resistance to kanamycin in Mycobacterium tuberculosis clinical isolates from Northern India

Kaur, Swayamjot; Rana, Vibhuti; Singh, Pooja; Trivedi, Garima; Anand, Shashi; Kaur, Amanpreet; Gupta, Pawan; Jain, Amita; Sharma, Charu

doi:10.1016/j.tube.2015.10.012

Cited by 12 publications

(14 citation statements)

References 29 publications

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“…WhiB7/WblC confers antibiotic resistance in actinomycetes such as antibiotic‐producing Streptomyces species and saprophytic or pathogenic species of Mycobacterium (Fowler‐Goldsworthy et al., 2011; Hurst‐Hess et al., 2017; Morris et al., 2005; Pryjma et al., 2017; Ramon‐Garcia et al., 2013). Several mutations in the whiB7 gene were also identified in clinical isolates of drug‐resistant M. tuberculosis (Chakravorty et al., 2015; Kaur et al., 2016; Koser et al., 2013), suggesting that its contribution to antibiotic resistance is physiologically relevant. In agreement with its requirement for antibiotic resistance, transcription of the whiB7/wblC gene is strongly induced up to several 1,000‐fold by various classes of translation‐inhibitory antibiotics (Burian et al., 2012) including second‐line antituberculosis drugs (World Health Organization, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Treatment of translation‐inhibitory antibiotics decreases uORF : whiB7 mRNA ratio by approximately threefold, suggesting that there is more transcriptional readthrough in the presence of antibiotics (Burian & Thompson, 2018). Third, even though uORF exhibits little sequence homology among species (Burian & Thompson, 2018), mutations carrying base insertion/deletions in the uORF or substitutions in the predicted stem region of Rho‐independent terminator (RIT) increased whiB7 transcription (Burian & Thompson, 2018; Chakravorty et al., 2015; Kaur et al., 2016; Reeves et al., 2013), suggesting that disturbing translation of uORF or the formation of the predicted RIT is required for whiB7 induction. However, curiously enough, whether the predicted RIT can cause premature transcription termination has not been formally demonstrated.…”

Section: Introductionmentioning

confidence: 99%

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uORF‐mediated riboregulation controls transcription of whiB7/wblC antibiotic resistance gene

Lee

Roe

2021

Molecular Microbiology

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WhiB7/WblC is a transcriptional factor of actinomycetes conferring intrinsic resistance to multiple translation‐inhibitory antibiotics. It positively autoregulates its own transcription in response to the same antibiotics. The presence of a uORF and a potential Rho‐independent transcription terminator in the 5′ leader region has suggested a possibility that the whiB7/wblC gene is regulated via a uORF‐mediated transcription attenuation. However, experimental evidence for the molecular mechanism to explain how antibiotic stress suppresses the attenuator, if any, and induces transcription of the whiB7/wblC gene has been lacking. Here we report that the 5′ leader sequences of the whiB7/wblC genes in sub‐clades of actinomycetes include conserved antiterminator RNA structures. We confirmed that the putative antiterminator in the whiB7/wblC leader sequences of both Streptomyces and Mycobacterium indeed suppresses Rho‐independent transcription terminator and facilitates transcription readthrough, which is required for WhiB7/WblC‐mediated antibiotic resistance. The antibiotic‐mediated suppression of the attenuator can be recapitulated by amino acid starvation, indicating that translational inhibition of uORF by multiple signals is a key to induce whiB7/wblC expression. Our findings of a mechanism leading to intrinsic antibiotic resistance could provide an alternative to treat drug‐resistant mycobacteria.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

uORF‐mediated riboregulation controls transcription of whiB7/wblC antibiotic resistance gene

Lee

Roe

2021

Molecular Microbiology

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“…Out of these 68 isolates, the mutational analysis with respect to KAN resistance has been reported earlier for 58 isolates from Lucknow. 37 Remaining 10 new SLD resistant isolates were obtained in March 2016 from Lucknow and evaluated in this study. Therefore, a total of 68 isolates from Lucknow have been compared with 58 isolates of Maharashtra for analyzing the target specific mutational prevalence and lineage identification.…”

Section: Methodsmentioning

confidence: 99%

“…The genomic DNA was isolated from all resistant and sensitive isolates as described previously. 37 , 42 Briefly, loopful of freshly subcultured colonies from each of the LJ medium bottles were resuspended in 200 µL of double distilled water in 1.5 mL microcentrifuge tubes (MCTs). The bacterial suspension was vortexed thrice for 10sec at 30sec intervals to allow proper dissolution of colonies and lysed by boiling at 95°C for 20 min in water bath.…”

Section: Methodsmentioning

confidence: 99%

Molecular Epidemiology and Polymorphism Analysis in Drug-Resistant Genes in M. tuberculosis Clinical Isolates from Western and Northern India

Rana¹,

Singh²,

Nikam³

et al. 2022

IDR

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Introduction The mechanistic details of first line drug (FLD) resistance have been thoroughly explored but the genetic resistance mechanisms of second line injectables, which form the backbone of the combinatorial drug resistant tuberculosis therapy, are partially identified. This study aims to highlight the genetic and spoligotypic differences in the second line drug (SLD) resistant and sensitive Mycobacterium tuberculosis ( Mtb) clinical isolates from Mumbai (Western India) and Lucknow (Northern India). Methods The rrs, eis, whiB7, tlyA, gyrA and gyrB target loci were screened in 126 isolates and spoligotyped. Results The novel mutations were observed in whiB7 loci (A43T, C44A, C47A, G48T, G59A and T152G in 5’-UTR; A42C, C253T and T270G in gene), tlyA (+CG200, G165A, C415G, and +G543) and gyrB (+G1359 and +A1429). Altogether, the rrs, eis , and whiB7 loci harbored mutations in ~86% and ~47% kanamycin resistant isolates from Mumbai and Lucknow, respectively. Mumbai strains displayed higher prevalence of mutations in gyrA (~85%) and gyrB loci (~13%) as compared to those from Lucknow (~69% and ~3.0%, respectively). Further, spoligotyping revealed that Beijing lineage is distributed equally amongst the drug resistant strains of Mumbai and Lucknow, but EAI-5 is existed at a higher level only in Mumbai. The lineages Manu2, CAS1-Delhi and T1 are more prevalent in Lucknow. Conclusion Besides identifying novel mutations in whiB7, tlyA and gyrB target loci, our analyses unveiled a potential polymorphic and phylogeographical demarcation among two distinct regions.

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“…Since partial loss of ettA function appears to confer acquired drug resistance by constitutive activation of whiB7, we next mined our clinical strain genome database to identify putative gain-of-function whiB7 mutations that may be associated with acquired drug resistance (Reeves et al, 2013). We identified numerous putative gain-of-function mutations in the whiB7 promoter, 5'UTR, and upstream ORF (uORF), most of which have not been previously recognized as potential determinants of acquired drug resistance (Figure 6A; Supplemental Data 7) (Chakravorty et al, 2015;Kaur et al, 2016;Reeves et al, 2013). Unexpectedly, however, the most common whiB7 variant in our database was a putative loss-of-function allele.…”

Section: Tuberculosis Hypersusceptible To Macrolidesmentioning

confidence: 99%

A chemical-genetic map of the pathways controlling drug potency in Mycobacterium tuberculosis

Poulton

Chang

et al. 2021

Preprint

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Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtb’s intrinsic drug resistance, as well as its ability to evolve acquired resistance to all antituberculars in clinical use. A deeper understanding of the bacterial pathways that govern drug efficacy could facilitate the development of more effective therapies to overcome resistance, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. To define these pathways, we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. Mining this dataset, we discovered diverse and novel mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical-genetics with comparative genomics of Mtb clinical isolates, we further identified numerous new potential mechanisms of acquired drug resistance, one of which is associated with the emergence of a multidrug-resistant tuberculosis (TB) outbreak in South America. Lastly, we make the unexpected discovery of an “acquired drug sensitivity.” We found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat TB. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future TB drug development and treatment.

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Novel mutations conferring resistance to kanamycin in Mycobacterium tuberculosis clinical isolates from Northern India

Cited by 12 publications

References 29 publications

uORF‐mediated riboregulation controls transcription of whiB7/wblC antibiotic resistance gene

uORF‐mediated riboregulation controls transcription of whiB7/wblC antibiotic resistance gene

Molecular Epidemiology and Polymorphism Analysis in Drug-Resistant Genes in M. tuberculosis Clinical Isolates from Western and Northern India

A chemical-genetic map of the pathways controlling drug potency in Mycobacterium tuberculosis

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