A Glutamine Insertion at Codon 432 of RpoB Confers Rifampicin Resistance in Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious respiratory disease caused by Mycobacterium tuberculosis and one of the top 10 causes of death worldwide. Treating TB is challenging; successful treatment requires a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant M. tuberculosis makes treatment even more difficult. To determine the mechanism of RIF resistance in these strains, we searched for novel mutations by sequencing. Four isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and unique mutations encoding RpoB G¹⁵⁸R, RpoB V¹⁶⁸A, RpoB S¹⁸⁸P, and RpoB Q⁴³²insQ, respectively. To evaluate their correlation with RIF resistance, plasmids carrying rpoB genes encoding these mutant proteins were transfected into the H₃₇Rv reference strain. The plasmid complementation of RpoB indicated that G¹⁵⁸R, V¹⁶⁸A, and S¹⁸⁸P did not affect the MIC of RIF. However, the MIC of RIF was increased in H₃₇Rv carrying RpoB Q⁴³²insQ. To confirm the correlation between RIF resistance and Q⁴³²insQ, we cloned an rpoB fragment carrying the insertion (encoding RpoB Q⁴³²insQ) into H₃₇Rv by homologous recombination using a suicide vector. All replacement mutants expressing RpoB Q⁴³²insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q⁴³²insQ causes RIF resistance in M. tuberculosis.