Characterization of a novel mutation in the overlap of tlyA and ppnK involved in capreomycin resistance in Mycobacterium.

Capreomycin (CAP) is an important second-line drug for multidrug-resistant tuberculosis. To further define the drug resistance mechanism of CAP, a Mycobacterium smegmatis transposon mutant library was constructed using Tn5 transposon for screening isolates with enhanced CAP resistance. A mutant (named C4) with fourfold increased CAP resistance was isolated and characterized. Tn5 was found to be inserted into MSMEG_0841, an annotated pseudogene. However, knockout demonstrated that MSMEG_0841 was not responsible for CAP resistance. We further sequenced the whole genome of C4 and found an A to G substitution in the overlap region between tlyA and ppnK, which leads a stop codon mutation in upstream tlyA and a T2A mutation in downstream ppnK. Mutation in the overlap might confer the dysfuction of both genes. tlyA is a known gene involved in CAP action. Overexpression of ppnK in both Escherichia coli and M. smegmatis confer subtle susceptible to CAP. Taken together, our study found that a novel mutation involved in CAP resistance.