Phase variation in Mycobacterium tuberculosis glpK produces transiently heritable drug tolerance.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity of Mycobacterium tuberculosis to develop drug resistance, are major barriers to global TB control efforts. M. tuberculosis is known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a mechanism of genetically encoded but rapidly reversible drug tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of 7 cytosines (7C) in the glpK gene. Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multidrug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the same glpK HT region. These reversible frameshift mutations in the 7C HT of M. tuberculosis glpK occur in clinical isolates, accumulate in M. tuberculosis-infected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction of dosR and sigH and repression of kstR regulons, similar to that observed in other in vitro models of M. tuberculosis tolerance. These results suggest that GlpK phase variation may contribute to drug tolerance, treatment failure, and relapse in human TB. Drugs effective against phase-variant M. tuberculosis may hasten TB treatment and improve cure rates.

Rapidly Correcting Frameshift Mutations in the Mycobacterium tuberculosis orn Gene Produce Reversible Ethambutol Resistance and Small-Colony-Variant Morphology.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosisorn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 µg/ml) produces an SCV with an EMB MIC of 32 µg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 µg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

Geographic Differences in the Contribution of ubiA Mutations to High-Level Ethambutol Resistance in Mycobacterium tuberculosis.

Ethambutol (EMB) resistance can evolve through a multistep process, and mutations in the ubiA (Rv3806c) gene appear to be responsible for high-level EMB resistance in Mycobacterium tuberculosis We evaluated the prevalence of ubiA and embB (Rv3795) mutations in EMB-resistant strains originating from Africa and South Korea. No differences in embB mutation frequencies were observed between strains from both origins. However, ubiA mutations were present in 45.5% ± 6.5% of the African EMB-resistant isolates but in only 9.5% ± 1.5% of the South Korean EMB-resistant isolates. The ubiA mutations associated with EMB resistance were localized to regions encoding the transmembrane domains of the protein, whereas the embB mutations were localized to regions encoding the extramembrane domains. Larger studies are needed to investigate the causes of increased ubiA mutations as a pathway to high-level EMB resistance in African countries, such as extended EMB usage during tuberculosis treatment.

Lack of association of novel mutation Asp397Gly in aftB gene with ethambutol resistance in clinical isolates of Mycobacterium tuberculosis.

To discover additional genotypic indicators for ethambutol (EMB) resistant M. tuberculosis, we studied polymorphisms in arabinofuranosyl transferase encoding genes aftA (Rv3792), aftB (Rv3805) and aftC (Rv2673) in 38 EMB resistant and 34 EMB susceptible isolates from India and a repository established by the World Health Organization (WHO) Special Programme for Research and Training in Tropical Disease (TDR) by DNA sequencing. The results were correlated with the minimum inhibitory concentration (MIC) of EMB and mutations in embB (Rv3795). The most common non-synonymous polymorphism identified in aftB was Asp397Gly in 12/38 (31.6%) EMB resistant and 3/34 (8.8%) EMB susceptible isolates. Interestingly, 10/12 (83.3%) EMB resistant isolates with aftB Asp397Gly mutation also carried embB306, embB402 or embB497 mutations. Association of Asp397Gly polymorphism with EMB resistance was statistically significant (p 0.0216). However, overexpression of the mutant aftB in M. tuberculosis H37Rv did not exhibit any change in the MIC. Whole genome sequencing of a panel of Indian isolates and SNP cluster grouping (SCG) of TDR strains revealed an association between aftB mutation Asp397Gly and Beijing genotype or SCG2, a cluster group representing the Beijing genotype. To conclude, though aftBAsp397Gly mutation is not associated with EMB resistance, this mutation may be a phylogenetic marker for the Beijing clade.

Polymorphisms in Rv3806c (ubiA) and the upstream region of embA in relation to ethambutol resistance in clinical isolates of Mycobacterium tuberculosis from North India.

Mutations at embB306 are the most prevalent polymorphisms associated with ethambutol (EMB) resistance, responsible for 40-60% of EMB resistant clinical cases of tuberculosis (TB). The present study analyzed additional mutations associated with EMB resistance in the embB, embC, embA and Rv3806c (ubiA) genes in 29 EMB resistant and 29 EMB susceptible clinical isolates of M. tuberculosis selected from 360 patients with TB. The entire ubiA gene, mutational hotspot regions of embB, embC, and upstream region of embA were screened for polymorphisms by DNA sequencing and the results correlated with minimum inhibitory concentrations (MIC) of EMB. The most common polymorphism identified in ubiA was at codon 149 (GAA to GAC), occurring in 5/29 (17.2%) resistant isolates and 7/29 (24%) susceptible isolates. Mutations in embB were most common at codon 306 (ATG to ATC/GTG), occurring only in EMB resistant isolates (20/29; 69%). Mutations in the upstream region of embA at -8, -11, -12 and -60 codons also occurred in EMB resistant strains (8/29; 27.5%) of which 6/8 (75%) were observed in isolates with EMB MIC ≥16 µg/ml. Though no polymorphisms associated with EMB resistance were identified in ubiA, polymorphisms upstream to embA may contribute to high level EMB resistance.

Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-ß-D-arabinose biosynthetic and utilization pathway genes.

To study the evolution of drug resistance, we genetically and biochemically characterized Mycobacterium tuberculosis strains selected in vitro for ethambutol resistance. Mutations in decaprenylphosphoryl-ß-D-arabinose (DPA) biosynthetic and utilization pathway genes Rv3806c, Rv3792, embB and embC accumulated to produce a wide range of ethambutol minimal inhibitory concentrations (MICs) that depended on mutation type and number. Rv3806c mutations increased DPA synthesis, causing MICs to double from 2 to 4 µg/ml in a wild-type background and to increase from 16 to 32 µg/ml in an embB codon 306 mutant background. Synonymous mutations in Rv3792 increased the expression of downstream embC, an ethambutol target, resulting in MICs of 8 µg/ml. Multistep selection was required for high-level resistance. Mutations in embC or very high embC expression were observed at the highest resistance level. In clinical isolates, Rv3806c mutations were associated with high-level resistance and had multiplicative effects with embB mutations on MICs. Ethambutol resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance.