Variation in multiple classes of simple sequence repeats can alter drug susceptibility in Mycobacterium tuberculosis.

Insertions and deletions (INDELs) in simple sequence repeats (SSRs) generate relatively high-frequency reversible genetic changes that facilitate bacterial adaptation to changing environments. Analyses of global Mycobacterium tuberculosis (Mtb) isolates indicate that many SSRs are under diversifying selection, and several of the resulting INDELs in homopolymer tracts (HTs) can increase the pathogen's fitness during exposure to host and antibiotic stresses. However, the functional impact of most variable SSRs, particularly those within more complex repeat sequences than HT, remains unclear.  Here, we combine phylogenomic analysis of clinical Mtb strains from Vietnam and Peru with in vitro experimental validation of engineered strains to identify SSR INDELs that alter antibiotic susceptibility. Our findings demonstrate that INDELs across multiple SSRs of differing repeat composition are highly variable and correlate with clinical antibiotic resistance. These variants included frameshifting HT INDELs in ppe13, glpK, Rv2081c, and ppsA, and in-frame trinucleotide (triplet) SSR INDELS in ponA1, ppe53, and ppe59 that produce much more subtle changes in protein structure. Reconstruction of these INDELs in an isogenic background identified four variants that directly reduce drug potency, including a triplet SSR deletion in ppe53 that conferred intermediate resistance to isoniazid, rifampicin, and streptomycin. The clinically prevalent ppe53 CGCdel mutation shortens a polyalanine stretch adjacent to the conserved WxG domain and impairs the processing and secretion of the full-length protein. Overall, our work provides additional evidence of selective pressure across Mtb SSRs and demonstrates the significance of in-frame INDELs within triplet SSRs, highlighting their contribution to the evolution of antibiotic resistance.