Gergely Fekete

Bacterial evolution of antibiotic hypersensitivity

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large‐scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole‐genome sequencing of laboratory‐evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton‐motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF‐dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade‐offs under antibiotic selection.

Genome-wide analysis captures the determinants of the antibiotic cross-resistance interaction network

Understanding how evolution of antimicrobial resistance increases resistance to other drugs is a challenge of profound importance. By combining experimental evolution and genome sequencing of 63 laboratory-evolved lines, we charted a map of cross-resistance interactions between antibiotics in Escherichia coli, and explored the driving evolutionary principles. Here, we show that (1) convergent molecular evolution is prevalent across antibiotic treatments, (2) resistance conferring mutations simultaneously enhance sensitivity to many other drugs and (3) 27% of the accumulated mutations generate proteins with compromised activities, suggesting that antibiotic adaptation can partly be achieved without gain of novel function. By using knowledge on antibiotic properties, we examined the determinants of cross-resistance and identified chemogenomic profile similarity between antibiotics as the strongest predictor. In contrast, cross-resistance between two antibiotics is independent of whether they show synergistic effects in combination. These results have important implications on the development of novel antimicrobial strategies.

The Genomic Landscape of Compensatory Evolution

The Genomic Landscape of Compensatory Evolution Laboratory selection experiment explains how organisms compensate for the loss of genes during evolution, and reveals the deleterious side-effects of this process when adapting to novel environments.

Antagonism between Bacteriostatic and Bactericidal Antibiotics Is Prevalent

ABSTRACT Combination therapy is rarely used to counter the evolution of resistance in bacterial infections. Expansion of the use of combination therapy requires knowledge of how drugs interact at inhibitory concentrations. More than 50 years ago, it was noted that, if bactericidal drugs are most potent with actively dividing cells, then the inhibition of growth induced by a bacteriostatic drug should result in an overall reduction of efficacy when the drug is used in combination with a bactericidal drug. Our goal here was to investigate this hypothesis systematically. We first constructed time-kill curves using five different antibiotics at clinically relevant concentrations, and we observed antagonism between bactericidal and bacteriostatic drugs. We extended our investigation by performing a screen of pairwise combinations of 21 different antibiotics at subinhibitory concentrations, and we found that strong antagonistic interactions were enriched significantly among combinations of bacteriostatic and bactericidal drugs. Finally, since our hypothesis relies on phenotypic effects produced by different drug classes, we recreated these experiments in a microfluidic device and performed time-lapse microscopy to directly observe and quantify the growth and division of individual cells with controlled antibiotic concentrations. While our single-cell observations supported the antagonism between bacteriostatic and bactericidal drugs, they revealed an unexpected variety of cellular responses to antagonistic drug combinations, suggesting that multiple mechanisms underlie the interactions.

Indispensability of horizontally transferred genes and its impact on bacterial genome streamlining

Why are certain bacterial genomes so small and compact? The adaptive genome streamlining hypothesis posits that selection acts to reduce genome size because of the metabolic burden of replicating DNA. To reveal the impact of genome streamlining on cellular traits, we reduced the Escherichia coli genome by up to 20% by deleting regions which have been repeatedly subjects of horizontal transfer in nature. Unexpectedly, horizontally transferred genes not only confer utilization of specific nutrients and elevate tolerance to stresses, but also allow efficient usage of resources to build new cells, and hence influence fitness in routine and stressful environments alike. Genome reduction affected fitness not only by gene loss, but also by induction of a general stress response. Finally, we failed to find evidence that the advantage of smaller genomes would be due to a reduced metabolic burden of replicating DNA or a link with smaller cell size. We conclude that as the potential energetic benefit gained by deletion of short genomic segments is vanishingly small compared with the deleterious side effects of these deletions, selection for reduced DNA synthesis costs is unlikely to shape the evolution of small genomes.

Phenotypic heterogeneity promotes adaptive evolution

Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress.

Adaptive evolution of complex innovations through stepwise metabolic niche expansion

A central challenge in evolutionary biology concerns the mechanisms by which complex metabolic innovations requiring multiple mutations arise. Here, we propose that metabolic innovations accessible through the addition of a single reaction serve as stepping stones towards the later establishment of complex metabolic features in another environment. We demonstrate the feasibility of this hypothesis through three complementary analyses. First, using genome-scale metabolic modelling, we show that complex metabolic innovations in Escherichia coli can arise via changing nutrient conditions. Second, using phylogenetic approaches, we demonstrate that the acquisition patterns of complex metabolic pathways during the evolutionary history of bacterial genomes support the hypothesis. Third, we show how adaptation of laboratory populations of E. coli to one carbon source facilitates the later adaptation to another carbon source. Our work demonstrates how complex innovations can evolve through series of adaptive steps without the need to invoke non-adaptive processes.

Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides

Antimicrobial peptides are promising alternative antimicrobial agents. However, little is known about whether resistance to small-molecule antibiotics leads to cross-resistance (decreased sensitivity) or collateral sensitivity (increased sensitivity) to antimicrobial peptides. We systematically addressed this question by studying the susceptibilities of a comprehensive set of 60 antibiotic-resistant Escherichia coli strains towards 24 antimicrobial peptides. Strikingly, antibiotic-resistant bacteria show a high frequency of collateral sensitivity to antimicrobial peptides, whereas cross-resistance is relatively rare. We identify clinically relevant multidrug-resistance mutations that increase bacterial sensitivity to antimicrobial peptides. Collateral sensitivity in multidrug-resistant bacteria arises partly through regulatory changes shaping the lipopolysaccharide composition of the bacterial outer membrane. These advances allow the identification of antimicrobial peptide–antibiotic combinations that enhance antibiotic activity against multidrug-resistant bacteria and slow down de novo evolution of resistance. In particular, when co-administered as an adjuvant, the antimicrobial peptide glycine-leucine-amide caused up to 30-fold decrease in the antibiotic resistance level of resistant bacteria. Our work provides guidelines for the development of efficient peptide-based therapies of antibiotic-resistant infections.Multidrug-resistant Escherichia coli have a high frequency of collateral sensitivity to antimicrobial peptides, which may arise from changes in lipopolysaccharide regulation.

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.In vivo, in vitro and in silico experiments demonstrate that interface residues of homomeric proteins are enriched toward protein C termini to avoid premature assembly and aggregation.

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs) that are ancient components of immune defence. Despite important medical relevance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here we show that AMP- and antibiotic-resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics revealed that AMP resistance genes originating from phylogenetically distant bacteria only have a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Third, the phenotypic impact of acquired AMP resistance genes heavily depends on the genetic background of the recipient bacteria. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota with implications for disease risks.