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Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.

ABSTRACT: Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics-even below this breakpoint-is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.

SUBMITTER: Hicks ND

PROVIDER: S-EPMC7738170 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.

Hicks Nathan D ND Giffen Samantha R SR Culviner Peter H PH Chao Michael C MC Dulberger Charles L CL Liu Qingyun Q Stanley Sydney S Brown Jessica J Sixsmith Jaimie J Wolf Ian D ID Fortune Sarah M SM

PLoS pathogens 20201130 11

Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics-even below this breakpoint-is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug su ...[more]

PMID: 33253310

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Project description:RationaleMinority drug-resistant Mycobacterium tuberculosis subpopulations can be associated with phenotypic resistance but are poorly detected by Sanger sequencing or commercial molecular diagnostic assays.ObjectivesTo determine the role of targeted next-generation sequencing in resolving these minor variant subpopulations.MethodsWe used single molecule overlapping reads (SMOR), a targeted next-generation sequencing approach that dramatically reduces sequencing error, to analyze primary cultured isolates phenotypically resistant to rifampin, fluoroquinolones, or aminoglycosides, but for which Sanger sequencing found no resistance-associated variants (RAVs) within respective resistance-determining regions (study group). Isolates also underwent single-colony selection on antibiotic-containing agar, blinded to sequencing results. As a positive control, isolates with multiple colocalizing chromatogram peaks were also analyzed (control group).Measurements and main resultsAmong 61 primary culture isolates (25 study group and 36 control group), SMOR described 66 (49%) and 45 (33%) of 135 total heteroresistant RAVs at frequencies less than 5% and less than 1% of the total mycobacterial population, respectively. In the study group, SMOR detected minor resistant variant subpopulations in 80% (n = 20/25) of isolates with no Sanger-identified RAVs (median subpopulation size, 1.0%; interquartile range, 0.2-3.9%). Single-colony selection on drug-containing media corroborated SMOR results for 90% (n = 18/20) of RAV-containing specimens, and the absence of RAVs in 60% (n = 3/5) of isolates. Overall, Sanger sequencing was concordant with SMOR for 77% (n = 53/69) of macroheteroresistant (5-95% total population), but only 5% of microheteroresistant (<5%) subpopulations (n = 3/66) across both groups.ConclusionsCryptic minor variant mycobacterial subpopulations exist below the resolving capability of current drug susceptibility testing methodologies, and may explain an important proportion of false-negative resistance determinations.

Project description:Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. In this study, we performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand the genomic diversity. A retrospective collection of 498 M. tuberculosis isolates submitted to the National Institute for Research in Tuberculosis for phenotypic susceptibility testing between 2014 to 2016 were sequenced. Genotypic resistance prediction was performed using known resistance-conferring determinants. Genotypic and phenotypic results for 12 antituberculosis drugs were compared, and sequence data were explored to characterize lineages and their association with drug resistance. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction. IMPORTANCE Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. We performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand genomic diversity. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction.

Dataset Information

Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.

Publications

Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.

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