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

Author summary Tuberculosis disease is treated with a combination of antibiotics targeting the bacterial pathogen Mycobacterium tuberculosis (Mtb). In response to widespread use of antibiotics, Mtb has evolved resistance mutations that increase the amount of antibiotic required to inhibit its growth and undermine effective treatment. The bacterial mutations that cause high-level drug resistance have largely been identified allowing for the development of rapid diagnostics. Recent studies have shown that intermediate levels of resistance can also affect patient outcomes, however, we do not yet know the range of mutations that can cause intermediate resistance. Here we utilize a genome-wide association study approach and identify that mutations in the bacterial DNA replication initiation factor dnaA are associated with drug resistance in clinical isolates. By generating precision dnaA mutant strains we identify that these mutations confer intermediate levels of resistance to the first-line drug isoniazid. We also find that mutations at a second site in the genome physically bound by dnaA can confer the same effect on isoniazid, likely acting through the same pathway. This study provides insight into previously unidentified clinically prevalent variants that may help explain patient outcome and guide therapy to reduce treatment failure and the subsequent evolution of high-level resistance. 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.