Common Variants in the Glycerol Kinase Gene Reduce Tuberculosis Drug Efficacy

Abstract

Despite the administration of multiple drugs that are highly effective in vitro, tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify Mycobacterium tuberculosis genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the glpK gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of M. tuberculosis to antibiotics in vitro, and glpK-deficient bacteria persisted during antibiotic treatment in vivo, particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the glpK gene were found to be a specific marker of multidrug resistance in clinical M. tuberculosis isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the glpK coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance. IMPORTANCE TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for M. tuberculosis mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the glpK gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance.

Distinct Bacterial Pathways Influence the Efficacy of Antibiotics against Mycobacterium tuberculosis

“…The genes identified are involved in a range of functions and include pathways known to alter antibiotic efficacy. For example, pncA and glpK mutants were found to be less sensitive to PZA treatment, consistent with previous studies (36). Conversely, we identified mutants in ppe50/51, which had previously been shown to increase the efficacy of a multidrug regimen (36).…”

“…For example, pncA and glpK mutants were found to be less sensitive to PZA treatment, consistent with previous studies (36). Conversely, we identified mutants in ppe50/51, which had previously been shown to increase the efficacy of a multidrug regimen (36). We also identified multiple mutants lacking putative antibiotic efflux pumps, including ABC transporters Rv1747 and Rv1273, which were more susceptible to INH and RIF, respectively.…”

Tolerance and Persistence to Drugs: A Main Challenge in the Fight Against Mycobacterium tuberculosis

“…GlpK is an enzyme required for glycerol catabolism via the lower glycolytic pathway and is essential for growth in media with glycerol as the sole carbon source ( Bellerose et al, 2019 ). A M. tuberculosis mutant missing this gene was recently found to be tolerant to several drugs when bacteria were grown on glycerol ( Bellerose et al, 2019 ).…”

“…GlpK is an enzyme required for glycerol catabolism via the lower glycolytic pathway and is essential for growth in media with glycerol as the sole carbon source ( Bellerose et al, 2019 ). A M. tuberculosis mutant missing this gene was recently found to be tolerant to several drugs when bacteria were grown on glycerol ( Bellerose et al, 2019 ). Interestingly, two independent groups recently showed that glpK is subjected to phase variation due to frequent and reversible frameshift mutations in a homopolymeric 7G sequence present at the 5' half of its open reading frame.…”

“…As a consequence of this phase variation mechanism, some clinical strains produce subpopulations forming small colonies with a smooth phenotype, with heritable and rapidly revertible tolerance to several drugs. Interestingly, these variants accumulate during drug treatment, suggesting their relevance in treatment failure and relapse ( Bellerose et al, 2019 ; Safi et al, 2019 ).…”