ABSTRACT
The emergence of antibiotic resistance has raised serious concerns within scientific and medical communities, and has underlined the importance of developing new antimicrobial agents to combat such infections. Bacteriophages, naturally occurring bacterial viruses, have long been characterized as promising antibiotic alternatives. Although bacteriophages hold great promise as medical tools, clinical applications have been limited by certain characteristics of phage biology, with structural fragility under the high temperatures and acidic environments of therapeutic applications significantly limiting therapeutic effectiveness. This study presents and evaluates the efficacy of a new accelerated evolution platform, chemically accelerated viral evolution (CAVE), which provides an effective and robust method for the rapid enhancement of desired bacteriophage characteristics. Here, our initial use of this methodology demonstrates its ability to confer significant improvements in phage thermal stability. Analysis of the mutation patterns that arise through CAVE iterations elucidates the manner in which specific genetic modifications bring forth desired changes in functionality, thereby providing a roadmap for bacteriophage engineering.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriophages/genetics , Drug Resistance, Microbial/genetics , Genetic Engineering/methods , Animals , Evolution, Molecular , Gene Expression Regulation, Viral , Humans , Hydrogen-Ion Concentration , Phage Therapy , TemperatureABSTRACT
Protein evolution occurs via restricted evolutionary paths that are influenced by both previous and subsequent mutations. This effect, termed epistasis, is critical in population genetics, drug resistance, and immune escape; however, the effect of epistasis on the level of protein fitness is less well characterized. We generated and characterized a 6615-member library of all two-amino acid combinations in a highly mutable loop of a virus-like particle. This particle is a model of protein self-assembly and a promising vehicle for drug delivery and imaging. In addition to characterizing the effect of all double mutants on assembly, thermostability, and acid stability, we observed many instances of epistasis, in which combinations of mutations are either more deleterious or more beneficial than expected. These results were used to generate rules governing the effects of multiple mutations on the self-assembly of the virus-like particle.
Subject(s)
Epistasis, Genetic/genetics , Proteins/metabolism , Proteins/physiology , Amino Acids/metabolism , Biological Evolution , Epistasis, Genetic/physiology , Evolution, Molecular , Genetic Fitness/genetics , Genotype , Models, Genetic , Mutation , Peptide Library , Protein Conformation , Proteins/geneticsABSTRACT
Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.
