Phase variation in Mycobacterium tuberculosis glpK produces transiently heritable drug tolerance

Abstract

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 novel mechanism of genetically encoded but rapidly reversible drug-tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of seven cytosines (7C) in the glpK gene. Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multi-drug 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.\n\nSIGNIFICANCEThe ability of M. tuberculosis to survive during prolonged treatment has been attributed to either transient stress responses or fixed heritable drug-resistance producing mutations. We show that phase-variation in the M. tuberculosis glpK gene represents a third type of resistance mechanism. The ability of these glpK mutants to grow slowly and then rapidly revert suggests that these transiently-heritable changes may also explain how a hidden population of drug-tolerant bacteria develops during TB treatment. As a genetically trackable cause of drug-tolerance, M. tuberculosis glpK mutants provides a unique opportunity to study these phenomena at a cellular and mechanistic level. These mutants could also be used for developing drugs that target tolerant populations, leading to more rapid and effective TB treatments.