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.

An evolutionary functional genomics approach identifies novel candidate regions involved in isoniazid resistance in Mycobacterium tuberculosis

“…The resistance mechanisms involved seem to be multiple and diverse. For instance, two of the genes with the most mutations in resistant strains are dnaA and mutT4 , which are involved in DNA replication and may affect the acquisition of resistance by increasing mutation rate or indirect mechanisms such as decreasing expression levels of katG 24 . Other interesting genes in the candidate set are those encoding epoxide hydrolases ( ephD and ephF ), which are involved in detoxification following oxidative damage to lipids.…”

Adaptation and compensation in a bacterial gene regulatory network evolving under antibiotic selection

“…Mutations in DnaA that compromise its ATPase activity would help achieve this. Second, DnaA mutations in clinical and laboratory strains of M. tuberculosis have been associated with isoniazid resistance due to altered gene expression profiles ( Hicks et al, 2020 ), and a similar mechanism may be operative in the case of E. coli and trimethoprim. The appearance of mutations at these loci only at high antibiotic pressure is likely to be a result of the higher pressure for resistance.…”

Adaptation and Compensation in a Bacterial Gene Regulatory Network Evolving Under Antibiotic Selection

“…Mutations in DnaA that compromise its ATPase activity would help achieve this. Secondly, DnaA mutations in clinical and laboratory strains of Mycobacterium tuberculosis have been associated with isoniazid resistance due to altered gene expression profiles [66], and a similar mechanism may be operative in the case of E. coli and trimethoprim. The appearance of mutations at these loci only at high antibiotic pressure are likely to be a result of the higher pressure for resistance.…”