Clara Torres-Barceló

Evaluating evolutionary models of stress-induced mutagenesis in bacteria

Increased mutation rates under stress allow bacterial populations to adapt rapidly to stressors, including antibiotics. Here we evaluate existing models for the evolution of stress-induced mutagenesis and present a new model arguing that it evolves as a result of a complex interplay between direct selection for increased stress tolerance, second-order selection for increased evolvability and genetic drift. Further progress in our understanding of the evolutionary biology of stress and mutagenesis will require a more detailed understanding both of the patterns of stress encountered by bacteria in nature and of the mutations that are produced under stress.

From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro

RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.

Experimental evolution of plant RNA viruses

Undoubtedly, viruses represent a major threat faced by human and veterinary medicines and by agronomy. The rapid evolution of viruses enables them to escape from natural immunities and from state-of-the-art antiviral treatments, with new viruses periodically emerging with deadly consequences. Viruses have also become powerful and are increasingly used tools in the field of experimental evolution. A growing body of evidence points that the evolution of viruses is mainly determined by key features such as their compacted genomes, enormous population sizes, and short generation times. In addition, RNA viruses also present large selection coefficients, antagonistic epistasis, and high mutation rates. Most of this knowledge comes from studies that have used either bacteriophages or animal viruses in cell cultures as experimental systems. However, plant viruses provide almost identical advantages for evolutionary studies and, in addition, offer an invaluable tool for studying the interplay between viruses and pluricellular hosts. Without seeking to be exhaustive, here we summarize some peculiarities of plant viruses and review recent experiments that have explored important questions on evolution, such as the role of deleterious mutation and neutrality, the effect of different transmission modes in the evolution of virulence, and the heterogeneous selective constraints imposed by multiple hosts.

Evolutionary Rationale for Phages as Complements of Antibiotics

Antibiotic-resistant bacterial infections are a major concern to public health. Phage therapy has been proposed as a promising alternative to antibiotics, but an increasing number of studies suggest that both of these antimicrobial agents in combination are more effective in controlling pathogenic bacteria than either alone. We advocate the use of phages in combination with antibiotics and present the evolutionary basis for our claim. In addition, we identify compelling challenges for the realistic application of phage-antibiotic combined therapy.

A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining antibiotics and phages

The evolution of antibiotic resistance in bacteria is a global concern and the use of bacteriophages alone or in combined therapies is attracting increasing attention as an alternative. Evolutionary theory predicts that the probability of bacterial resistance to both phages and antibiotics will be lower than to either separately, due for example to fitness costs or to trade-offs between phage resistance mechanisms and bacterial growth. In this study, we assess the population impacts of either individual or combined treatments of a bacteriophage and streptomycin on the nosocomial pathogen Pseudomonas aeruginosa. We show that combining phage and antibiotics substantially increases bacterial control compared to either separately, and that there is a specific time delay in antibiotic introduction independent of antibiotic dose, that minimizes both bacterial density and resistance to either antibiotics or phage. These results have implications for optimal combined therapeutic approaches.

Compensatory Molecular Evolution of HC-Pro, an RNA-Silencing Suppressor from a Plant RNA Virus

RNA silencing is a eukaryotic mechanism involved in several cellular processes, one example being a sequence-specific antiviral defense. Many plant viruses have developed counterdefensive proteins that in many instances are multifunctional, such as helper component protease (HC-Pro) of Tobacco etch virus (TEV). In a previous work, a collection of mutants with amino acid replacements in TEV HC-Pro was generated, and their effects in the capacity of suppressing RNA silencing were quantified in a transient expression assay. In this study, three mutations that caused a reduction in suppression activity and three that increased it were used to create replicate experimental lineages that were evolved through serial passages. We have evaluated the number of genotypic changes that occurred during evolution in HC-Pro and their phenotypic effects on virus viability, virulence, and suppression of RNA silencing. In no instance did the original mutation revert to the wildtype (WT) sequence. In several cases, fixed mutations were canonical compensatory changes, returning the suppressor activity to the WT HC-Pro value, pointing to the existence of stabilizing selection pressures and pleiotropic effects of the introduced original mutations. However, in other instances, the fixed mutations were overcompensatory, driving the activity of the mutant beyond the optimal value. Negative epistatic effects among beneficial mutations as well as decompensatory epistasis also play an important role during compensatory evolution of RNA-silencing suppression.

Long‐term effects of single and combined introductions of antibiotics and bacteriophages on populations of Pseudomonas aeruginosa

With escalating resistance to antibiotics, there is an urgent need to develop alternative therapies against bacterial pathogens and pests. One of the most promising is the employment of bacteriophages (phages), which may be highly specific and evolve to counter antiphage resistance. Despite an increased understanding of how phages interact with bacteria, we know very little about how their interactions may be modified in antibiotic environments and, reciprocally, how phage may affect the evolution of antibiotic resistance. We experimentally evaluated the impacts of single and combined applications of antibiotics (different doses and different types) and phages on in vitro evolving populations of the opportunistic pathogen Pseudomonas aeruginosa PAO1. We also assessed the effects of past treatments on bacterial virulence in vivo, employing larvae of Galleria mellonella to survey the treatment consequences for the pathogen. We find a strong synergistic effect of combining antibiotics and phages on bacterial population density and in limiting their recovery rate. Our long‐term study establishes that antibiotic dose is important, but that effects are relatively insensitive to antibiotic type. From an applied perspective, our results indicate that phages can contribute to managing antibiotic resistance levels, with limited consequences for the evolution of bacterial virulence.

HC-Pro hypo- and hypersuppressor mutants: differences in viral siRNA accumulation in vivo and siRNA binding activity in vitro

Viruses have evolved mechanisms to suppress the RNA silencing defense of their hosts, allowing replication and systemic colonization. In a recent study, we found that the effect of mutations in the RNA silencing suppressor of tobacco etch virus (TEV) was variable, ranging from complete abolition of suppressor activity to significantly stronger suppression. Whereas hyposuppressor mutants were less virulent and accumulated fewer viral particles than the wild type, hypersuppressors induced symptoms similar to those of the wild type and accumulated particles to similar levels. Here, we further characterize a set of these mutants in terms of their ability to bind in vitro and induce accumulation in vivo of virus-derived siRNAs. Hyposuppressor alleles are less efficient at binding siRNAs than hypersuppressors, whereas the latter are not different from the wild type. As a consequence of lower viral accumulation, plants infected with virus bearing a hyposuppressor allele also accumulate less virus-derived siRNA.

Antibiotic stress selects against cooperation in the pathogenic bacterium Pseudomonas aeruginosa

Significance The evolution of cooperation is a central issue in biology and the social sciences. Study of model systems of social microbes has focused on how “cooperators” and “cheats” interact but rarely accounts for the surrounding environment. We demonstrate how environmental stress in the form of antibiotics alters the evolution of public goods cooperation in a bacterium. Antibiotics accentuate the costs to cooperators, resulting in their rapid demise relative to cheats. In a more applied vein, antibiotic resistance was maximal in the presence of both producers and cheats, suggesting that knowledge about social strategies can be used to improve therapies. Our work emphasizes ecoevolutionary feedback in social evolution and demonstrates that social interactions may be considerably modified in natural, stressful environments. Cheats are a pervasive threat to public goods production in natural and human communities, as they benefit from the commons without contributing to it. Although ecological antagonisms such as predation, parasitism, competition, and abiotic environmental stress play key roles in shaping population biology, it is unknown how such stresses generally affect the ability of cheats to undermine cooperation. We used theory and experiments to address this question in the pathogenic bacterium, Pseudomonas aeruginosa. Although public goods producers were selected against in all populations, our competition experiments showed that antibiotics significantly increased the advantage of nonproducers. Moreover, the dominance of nonproducers in mixed cultures was associated with higher resistance to antibiotics than in either monoculture. Mathematical modeling indicates that accentuated costs to producer phenotypes underlie the observed patterns. Mathematical analysis further shows how these patterns should generalize to other taxa with public goods behaviors. Our findings suggest that explaining the maintenance of cooperative public goods behaviors in certain natural systems will be more challenging than previously thought. Our results also have specific implications for the control of pathogenic bacteria using antibiotics and for understanding natural bacterial ecosystems, where subinhibitory concentrations of antimicrobials frequently occur.

Phage selection for bacterial cheats leads to population decline.

While predators and parasites are known for their effects on bacterial population biology, their impact on the dynamics of bacterial social evolution remains largely unclear. Siderophores are iron-chelating molecules that are key to the survival of certain bacterial species in iron-limited environments, but their production can be subject to cheating by non-producing genotypes. In a selection experiment conducted over approximately 20 bacterial generations and involving 140 populations of the pathogenic bacterium Pseudomonas aeruginosa PAO1, we assessed the impact of a lytic phage on competition between siderophore producers and non-producers. We show that the presence of lytic phages favours the non-producing genotype in competition, regardless of whether iron use relies on siderophores. Interestingly, phage pressure resulted in higher siderophore production, which constitutes a cost to the producers and may explain why they were outcompeted by non-producers. By the end of the experiment, however, cheating load reduced the fitness of mixed populations relative to producer monocultures, and only monocultures of producers managed to grow in the presence of phage in situations where siderophores were necessary to access iron. These results suggest that public goods production may be modulated in the presence of natural enemies with consequences for the evolution of social strategies.