Insertions and deletions (INDELs) within simple sequence repeats (SSRs) create relatively frequent, reversible genetic changes that can enable bacterial adaptation to environmental stresses. In Mycobacterium tuberculosis (Mtb), prior analyses have indicated many SSRs are under diversifying selection. Some homopolymer tract (HT) INDELs have been associated with increased fitness during host or antibiotic exposure, but the functional significance of most variable SSRs—particularly in more complex repeat classes such as trinucleotide (triplet) SSRs—remains unclear. This study combines phylogenomic analysis of clinical Mtb isolates with experimental reconstruction to identify SSR INDELs that alter antibiotic susceptibility.
The authors examined clinical Mtb strain collections from Vietnam and Peru to assess SSR diversity and its relationship to clinical antibiotic resistance. Across these datasets, multiple SSR loci exhibited variability consistent with diversifying selection. The phylogenomic approach enabled identification of SSR variants that correlated with resistance phenotypes observed in the clinical isolates, guiding selection of candidate INDELs for experimental testing.
Variable SSRs included both frameshifting HT INDELs and in-frame triplet SSR INDELs. Specific loci highlighted in the analysis were:
The presence of both types of SSR variation across diverse repeat compositions indicates multiple SSR classes are mutable and potentially relevant to Mtb adaptation.
To test whether specific SSR INDELs directly affect drug susceptibility, the authors reconstructed selected variants in an isogenic Mtb background and measured antibiotic potency. Four engineered variants were identified that directly reduced drug susceptibility in vitro. Among these, a triplet SSR deletion in ppe53 produced intermediate resistance to multiple first-line drugs. The experimental approach established causality by moving from correlative phylogenomic signals in clinical strains to controlled validation in a common genetic background.
The clinically prevalent ppe53 CGCdel mutation is a triplet deletion that shortens a polyalanine stretch adjacent to the conserved WxG domain of the protein. Functional assays showed this change impaired processing and secretion of the full-length Ppe53 protein. Phenotypically, reconstruction of the CGCdel mutation in an isogenic strain conferred intermediate resistance to isoniazid, rifampicin, and streptomycin, indicating that an in-frame triplet SSR change can have measurable effects on multiple antibiotic classes. The findings emphasize that in-frame SSR INDELs need not be silent: subtle alterations in repeat-encoded amino acid tracts can affect protein maturation and export and thereby influence drug susceptibility.
This work expands the understanding of SSR variation in Mtb by showing that multiple SSR classes—including HTs and triplet SSRs—are variable, under selection, and can alter antibiotic susceptibility. Key implications are:
Overall, the combination of phylogenomic association and experimental reconstruction in this study provides direct evidence that SSR INDELs across multiple repeat classes contribute to clinically relevant changes in Mtb drug susceptibility. The source article reports detailed data, methods, and metrics supporting these conclusions; readers seeking exact frequencies, statistical analyses, or experimental protocols should consult the full preprint for those specifics.