Loss of glycerol catabolism confers carbon-source-dependent artemisinin resistance in Mycobacterium tuberculosis.

In view of the urgent need for new antibiotics to treat human infections caused by multidrug-resistant pathogens, drug repurposing is gaining strength due to the relatively low research costs and shorter clinical trials. Such is the case of artemisinin, an antimalarial drug that has recently been shown to display activity against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To gain insight into how Mtb is affected by artemisinin, we used RNAseq to assess the impact of artemisinin on gene expression profiles, revealing the induction of several efflux pumps and the KstR2 regulon. To anticipate the artemisinin resistance-conferring mutations that could arise in clinical Mtb strains, we performed an in vitro evolution experiment in the presence of lethal concentrations of artemisinin. We obtained artemisinin-resistant isolates displaying different growth kinetics and drug phenotypes, suggesting that resistance evolved through different pathways. Whole-genome sequencing of nine isolates revealed alterations in the glpK and glpQ1 genes, both involved in glycerol metabolism, in seven and one strains, respectively. We then constructed a glpK mutant and found that loss of glpK increases artemisinin resistance only when glycerol is present as a major carbon source. Our results suggest that mutations in glycerol catabolism genes could be selected during the evolution of resistance to artemisinin when glycerol is available as a carbon source. These results add to recent findings of mutations and phase variants that reduce drug efficacy in carbon-source-dependent ways.

Evolution and function analysis of glycerol kinase GlpK in Pseudomonasaeruginosa.

Pseudomonas aeruginosa is a Gram-negative bacterium capable of widespread niches, which is also one of the main bacteria that cause patient infection. The metabolic diversity of Pseudomonas aeruginosa is an essential factor in adapting to a variety of environments. Based on the previous studies, adaptive genetic variation in the glycerol kinase GlpK, the glycerol 3-phosphotransferase, contributes to the fitness of bacteria in human bodies, such as Mycobacterium tuberculosis and Escherichia coli. Thus, this study aimed to explore the molecular evolution and function of glpK in P. aeruginosa. Using extensive population genomic data, we have identified the prevalence of two glpK copies in P. aeruginosa that clustered into distinct branches, which were later known as Clade 1 and 2. The evolution analysis revealed that glpK in Clade 1 derived from an ancestral P. aeruginosa species and the other from an ancient horizontal gene transfer event. In addition, we confirmed that the GlpK in Clade 2 still retained glycerol kinase activity but was much weaker than that of GlpK in Clade 1. We demonstrated the importance of the critical amino acid Q70 in GlpK glycerol kinase activity by point mutation. Furthermore, Co-expression network analysis implied that the two glpK copies of P. aeruginosa regulate separate networks and may be a strategy to improve fitness in P. aeruginosa.

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.

Development of 3-hydroxypropionic-acid-tolerant strain of Escherichia coli W and role of minor global regulator yieP.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, but its toxic effect at high concentrations (> 200 mM) is a serious challenge for commercial production. In this study, a highly 3-HP-tolerant strain of Escherichia coli W (tolerance concentration: 400 mM in M9 minimal medium and 800 mM when yeast extract was added) was developed by adaptive laboratory evolution (ALE) with glycerol as the carbon source. Genome analysis of the adapted strain (designated as E. coli WA) indicated the presence of mutations in 13 genes, including glpK (glycerol kinase) and yieP (a less-studied global regulator). The mutant GlpK (K67T) exhibited a higher activity than the wild-type enzyme, but it was not beneficial for 3-HP production due to its causing carbon overflow metabolism. Interestingly, among the other 12 genes, the mutation in yieP alone was almost fully responsible for the improved tolerance to 3-HP. When the mutant yieP was substituted with the wild-type counterpart, the adapted E. coli WA strain completely lost its tolerance to 3-HP, showing a tolerance similar to the wild-type W strain. Deletion of yieP conferred 3-HP tolerance to several other E. coli strains including K-12 W3110, K-12 MG1655, and B except BL21 (DE3). The E. coli WA with wild-type glpK showed, as compared with its parental strain, better 3-HP production, indicating that improved tolerance is beneficial for 3-HP production.

Elevated production of 3-hydroxypropionic acid by metabolic engineering of the glycerol metabolism in Escherichia coli.

3-Hydroxypropionic acid (3-HP) is a renewable-based platform chemical which may be used to produce a wide range of chemicals including acrylic acid, 1,3-propanediol, and acrylamide. Commercialization of microbial 3-HP production from glycerol, which is produced inexpensively as a by-product of biodiesel production, could be expedited when global biodiesel production increases significantly. For enhancing 3-HP production, this study aimed to investigate metabolic engineering strategies towards eliminating by-products of 3-HP as well as optimizing the glycerol metabolism. The removal of genes involved in the generation of major by-products of 3-HP including acetate and 1,3-propanediol increased both 3-HP production level (28.1g/L) and its average yield (0.217g/g). Optimization of l-arabinose inducible expression of glycerol kinase GlpK, which catalyzes the conversion of glycerol to glycerol-3-phosphate, was also made to increase the metabolic flow from glycerol to 3-HP. To activate the whole glycerol metabolism towards 3-HP, the regulatory factor repressing the utilization of glycerol in Escherichia coli, encoded by glpR was eliminated by knocking-out in its chromosomal DNA. The resulting strain showed a significant improvement in the glycerol utilization rate as well as 3-HP titer (40.5g/L). The transcriptional analysis of glpR deletion mutant revealed the poor expression of glycerol facilitator GlpF, which is involved in glycerol transport in the cell. Additional expression of glpF in the glpR deletion mutant successfully led to an increase in 3-HP production (42.1g/L) and an average yield (0.268g/g).

Diversity of glpK Gene and Its Effect on Drug Sensitivity in Mycobacterium bovis.

Background: Glycerol kinase (glpK) is essential for the first step of glycerol catabolism in Mycobacterium tuberculosis. However, Mycobacterium bovis has been known to grow poorly in glycerol media because of a base insertion in the glpK gene. Methods: We analyzed the glpK gene sequences of 60 clinical M. bovis isolates, and determined the minimum inhibitory concentration of 14 drugs by microdilution method to evaluate the effect of frameshift mutations on drug sensitivity. The effect of M. bovis growth rate on its drug sensitivity was investigated using bacteria grown on glycerol or pyruvate. Results: A total of 44 (73.33%) clinical M. bovis isolates have frameshift mutations in a homopolymeric tract of 7 cytosines in the glpK gene. 15.00% M. bovis isolates showed phenotypic drug resistance. Glycerol metabolism-deficient M. bovis showed reduced susceptibility to 9 out of 14 tested drugs. Mutations in the glpK gene can lead to impaired growth in glycerol-based media, while the minimal inhibitory concentration values of slow-growing M. bovis were higher. Conclusion: Mutations in the glpK gene can lead to slowed growth and reduced susceptibility to drugs in M. bovis, which may contribute to the emergence of drug-resistant M. bovis and pose a threat to human health owing to the zoonotic capacity of M. bovis.

Whole Genome Sequencing Identifies Novel Mutations Associated With Bedaquiline Resistance in Mycobacterium tuberculosis.

Bedaquiline (BDQ), a new antitubercular agent, has been used to treat drug-resistant tuberculosis (TB). Although mutations in atpE, rv0678, and pepQ confer major resistance to BDQ, the mechanisms of resistance to BDQ in vitro and in clinical settings have not been fully elucidated. We selected BDQ-resistant mutants from 7H10 agar plates containing 0.5 mg/L BDQ (the critical concentration) and identified mutations associated with BDQ resistance through whole genome sequencing and Sanger sequencing. A total of 1,025 mutants were resistant to BDQ. We randomly selected 168 mutants for further analysis and discovered that 157/168 BDQ-resistant mutants harbored mutations in rv0678, which encodes a transcriptional regulator that represses the expression of the efflux pump, MmpS5-MmpL5. Moreover, we found two mutations with high frequency in rv0678 at nucleotide positions 286-287 (CG286-287 insertion; accounting for 26.8% [45/168]) and 198-199 (G198, G199 insertion, and G198 deletion; accounting for 14.3% [24/168]). The other mutations were dispersed covering the entire rv0678 gene. Moreover, we found that one new gene, glpK, harbors a G572 insertion; this mutation has a high prevalence (85.7%; 144/168) in the isolated mutants, and the minimum inhibitory concentration (MIC) assay demonstrated that it is closely associated with BDQ resistance. In summary, we characterized 168/1,025 mutants resistant to BDQ and found that mutations in rv0678 confer the primary mechanism of BDQ resistance. Moreover, we identified a new gene (glpK) involved in BDQ resistance. Our study offers new insights and valuable information that will contribute to rapid identification of BDQ-resistant isolates in clinical settings.