Discovery and functional characterization of katG mutations mediating Isoniazid resistance in Mycobacterium tuberculosis.

OBJECTIVE

To identify isoniazid (INH)-resistance-associated mutations in the katG of Mycobacterium tuberculosis (MTB) using whole genome sequencing (WGS) and validate mutations through functional studies.

METHODS

Clinical MTB isolates from tuberculosis patients at Shijiazhuang Fifth Hospital (June 1, 2020-June 30, 2022) were stratified into INH-resistant and INH-susceptible groups based on minimum inhibitory concentration (MIC). 67 isolates from each group underwent WGS. Based on the WHO drug resistance database, select katG mutation sites that are not yet confirmed as resistance-conferring in the WHO 2024 catalog, and predict their interactions with INH through molecular docking and molecular dynamics simulations. Functional validation was performed by overexpressing mutant katG alleles in the model organism Mycobacterium smegmatis mc²155 and assessing MIC shifts.

RESULTS

WGS revealed 15 katG mutation sites, including 9 variants not catalogued in WHO databases. Structural modeling demonstrated reduced binding stability between INH and the G124R mutant compared to wild-type katG. Functional assays confirmed that mc²155 overexpressing katG-G124R exhibited a two-fold MIC increase, while V151I and G124D mutants showed no significant resistance changes.

CONCLUSIONS

9 katG mutations were identified in INH-resistant MTB, with experimental validation confirming that the G124R substitution enhances INH resistance through impaired drug-target interaction.