Biology subjects

Dick, T.

Publications and source records attributed to Dick, T..

3 recordsLinked to original sources

bioRxiv2019

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 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.

microbiology↗

bioRxiv2019

Pyrazinamide triggers degradation of its target aspartate decarboxylase

The introduction of pyrazinamide (PZA) in the tuberculosis drug regimen shortened treatment from 12 to 6 months 1. PZA is a prodrug that is activated by a Mycobacterium tuberculosis (Mtb) amidase to release its bioactive component pyrazinoic acid (POA) 2. Aspartate decarboxylase PanD, a proenzyme activated by autocatalytic cleavage (Supplementary Fig. 1A, 3) and required for Coenzyme A (CoA) biosynthesis, emerged as a target of POA 4-7. In vitro and in vivo screening to isolate spontaneous POA-resistant Mtb mutants identified missense mutations in either panD or the unfoldase clpC1, encoding a component of the caseinolytic protease ClpC1-ClpP 4,6-9. Overexpression and binding studies of PanD or ClpC1 pointed to PanD as the direct target of POA whereas clpC1 mutations appeared to indirectly cause resistance 4,5,7,9,10. Indeed, supplementing growth media with CoA precursors downstream of the PanD catalyzed step conferred POA resistance 4,7,11. Metabolomic analyses and biophysical studies using recombinant proteins confirmed targeting of PanD by POA 5. However, the exact molecular mechanism of PanD inhibition by POA remained unknown. While most drugs act by inhibiting protein function upon target binding, we show here that POA is not a bona fide enzyme inhibitor. Rather, POA binding to PanD triggers degradation of the protein by ClpC1-ClpP. Thus, the old tuberculosis drug PZA promotes degradation of its target. While novel for an antibacterial, drug-induced target degradation has recently emerged as a strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA.

microbiology↗

bioRxiv2019

Identification of new MmpL3 inhibitors by untargeted and targeted mutant screens defines MmpL3 domains with differential resistance

The Mycobacterium tuberculosis (Mtb) mycolic acid flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole genome sequencing of isolated resistant mutants. Here we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel Mtb growth inhibitors for differential growth inhibition of wild type Mtb as compared to a pool of twenty-four unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular Mtb. Mtb cells treated with these compounds were shown to accumulate trehalose monomycolate and have reduced levels of trehalose dimycolate, supporting MmpL3 as the target. The inhibitors were mycobacteria specific with several also showing activity against the non-tuberculosis mycobacterial species M. abscessus. Cluster analysis of cross resistance profiles generated by dose response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance substitutions to the MmpL3 crystal structure revealed clade specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small molecule inhibitors.

microbiology↗