An official website of the United States government
Antibiotic resistance is a growing health concern. Efforts to control resistance would benefit from an improved ability to forecast when and how it will evolve. Epistatic interactions between mutations can promote divergent evolutionary trajectories, which complicates our ability to predict evolution. We recently showed that differences between genetic backgrounds can lead to idiosyncratic responses in the evolvability of phenotypic resistance, even among closely relatedEscherichia colistrains. In this study, we examined whether a strain's genetic background also influences the genotypic evolution of resistance. Do lineages founded by different genotypes take parallel or divergent mutational paths to achieve their evolved resistance states? We addressed this question by sequencing the complete genomes of antibiotic-resistant clones that evolved from several different genetic starting points during our earlier experiments. We first validated our statistical approach by quantifying the specificity of genomic evolution with respect to antibiotic treatment. As expected, mutations in particular genes were strongly associated with each drug. Then, we determined that replicate lines evolved from the same founding genotypes had more parallel mutations at the gene level than lines evolved from different founding genotypes, although these effects were more subtle than those showing antibiotic specificity. Taken together with our previous work, we conclude that historical contingency can alter both genotypic and phenotypic pathways to antibiotic resistance.
more »
« less
Protein Flexibility and Dissociation Pathway Differentiation Can Explain Onset of Resistance Mutations in Kinases**
Abstract Understanding how mutations render a drug ineffective is a problem of immense relevance. Often the mechanism through which mutations cause drug resistance can be explained purely through thermodynamics. However, the more perplexing situation is when two proteins have the same drug binding affinities but different residence times. In this work, we demonstrate how all‐atom molecular dynamics simulations using recent developments grounded in statistical mechanics can provide a detailed mechanistic rationale for such variances. We discover dissociation mechanisms for the anti‐cancer drug Imatinib (Gleevec) against wild‐type and the N368S mutant of Abl kinase. We show how this point mutation triggers far‐reaching changes in the protein's flexibility and leads to a different, much faster, drug dissociation pathway. We believe that this work marks an efficient and scalable approach to obtain mechanistic insight into resistance mutations in biomolecular receptors that are hard to explain using a structural perspective.
more »
« less
- Award ID(s):
- 2044165
- PAR ID:
- 10368546
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 61
- Issue:
- 28
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Microbes inhabit natural environments that are remarkably dynamic. Therefore, microbes harbor regulated genetic mechanisms to sense shifts in conditions and induce the appropriate responses. Recent studies suggest that the initial evolution of microbes occupying new niches favors mutations in regulatory pathways. However, it is not clear how this evolution is affected by how quickly conditions change (i.e. dynamics), or which mechanisms are commonly used to implement new regulation. Here, we perform experimental evolution on continuous cultures of Escherichia coli carrying the tetracycline resistance tet operon to identify specific mutations that adapt drug responses to different dynamic regimens of drug administration. We find that cultures evolved under gradually increasing tetracycline concentrations show no mutations in the tet operon, but instead a predominance of fine-tuning mutations increasing the affinity of an alternative efflux pump AcrB to tetracycline. When cultures are instead periodically exposed to large drug doses, all populations evolved transposon insertions in repressor TetR, resulting in loss of regulation and constitutive expression of efflux pump TetA. We use a mathematical model of the dynamics of antibiotic responses to show that sudden exposure to large drug concentrations overwhelm regulated responses, which cannot induce resistance fast enough, resulting in selection for constitutive expression of resistance. These results help explain the frequent loss of regulation of antibiotic resistance by pathogens evolved in clinical environments. Our experiment supports the notion that initial evolution in new ecological niches proceeds largely through regulatory mutations and suggests that transposon insertions are the main mechanism driving this process.more » « less
-
The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer’s oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC50 experiments and found to result in 8- and 72-fold increases in nirmatrelvir IC50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC50, confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral developmentmore » « less
-
Antibiotic resistance is a growing concern that has prompted a renewed focus on drug discovery, stewardship, and evolutionary studies of the patterns and processes that underlie this phenomenon. A resistant strain’s competitive fitness relative to its sensitive counterparts in the absence of drug can impact its spread and persistence in both clinical and community settings. In a prior study, we examined the fitness of tetracycline-resistant clones that evolved from five different Escherichia coli genotypes, which had diverged during a long-term evolution experiment. In this study, we build on that work to examine whether ampicillin-resistant mutants are also less fit in the absence of the drug than their sensitive parents, and whether the cost of resistance is constant or variable among independently derived lines. Like the tetracycline-resistant lines, the ampicillin-resistant mutants were often less fit than their sensitive parents, with significant variation in the fitness costs among the mutants. This variation was not associated with the level of resistance conferred by the mutations, nor did it vary across the different parental backgrounds. In our earlier study, some of the variation in fitness costs associated with tetracycline resistance was explained by the effects of different mutations affecting the same cellular pathway and even the same gene. In contrast, the variance among the ampicillin-resistant mutants was associated with different sets of target genes. About half of the resistant clones suffered large fitness deficits, and their mutations impacted major outer-membrane proteins or subunits of RNA polymerases. The other mutants experienced little or no fitness costs and with, one exception, they had mutations affecting other genes and functions. Our findings underscore the importance of comparative studies on the evolution of antibiotic resistance, and they highlight the nuanced processes that shape these phenotypes.more » « less
-
Miller, Samuel I. (Ed.)ABSTRACT The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii , the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis. IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii , which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.more » « less
