Optimizing bacteriophage engineering through an accelerated evolution platform

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.

After finding the values of n, k re f , and E a using a non-linear least squares optimization (see methods), these values can be used to calculate the half life of a phage variant for a given temperature as a function of initial concentration, using the following formula: Noting the dependence of degradation rate upon concentration at the initial time of thermal treatment, we may take note of the difference in the fraction of remaining wild-type phage after one hour at 60 • C between figures (1a) and (1c) , by considering the difference in the initial concentrations used in these two experiments (1a initial titer: 10 10 PFU/mL, 1c initial titer: 10 8 PFU/mL). While the theoretical prediction of half life values for reaction orders of n > 1 increases as initial concentration decreases, our experiments found that survival rates and associated half lives were higher in experiments with higher initial concentration. We hypothesize that this is due to the protein-rich environments of high titer solutions having more molecular resources to distribute the input heat throughout, leading to a diminished ability to reach the activation energy for molecular denaturation among each individual protein affected.

Degradation at different temperature ranges
Our assays for quantifying degradation rates monitored both short term degradation at high temperatures and long term degradation at low temperatures. Acknowledging the multiplicity of modes for energy input to be taken by bacteriophage molecular structures, and the innumerous denaturation pathways that can occur, a single energetic mechanism as described by our theoretical Arrhenius model is unlikely to adequately describe all classes of thermal denaturation. Likewise, the parametric fitting determined at low temperatures and high temperatures were inconsistent in value, suggesting that different mechanisms are responsible for the degradation process under vastly different thermal conditions.

Changes to pH sensitivity
Despite not applying a low-pH tolerance selection criteria in our protocol, the mutations that arose to confer heat resistance appeared to also improve survivability in acidic conditions. This effect can be rationalized as being related to changes to general structural stability, rather than specific changes in temperature sensitivity. In consideration of the limitations of bacteriophage therapy, which was a primary motivator for the development of this technique, these observations suggest that application of the CAVE protocol with high-temperature selection criteria can produce variant phages with tolerance to acidic conditions. The functional effects of such changes suggest that this method holds great promise for improving the efficacy of phages as orally administered therapeutic agents by promoting improvements in survivability under the acidic conditions of animal digestive systems, and thereby allow more active phages to reach their target pathogens.

Changes in plaque size
Previous analyses of the phenotypic traits that contribute to variation in plaque morphology have described that larger amorphous plaques are associated with lower binding efficiency of phages to their hosts [7][8][9]. This rationale provides a logical explanation in light of the physical principles that govern binding and diffusivity of interacting components within a system; if phages bind less efficiently to their hosts, then they will have more free diffusion through an aqueous medium, ultimately leading to larger regions of access and less uniform geometric distributions within their infections of the regions of bacterial occupancy. Analysis of mutation enrichment led us to note that the change in plaque size coincided with the occurrence of a mutation (33152[C>T]) in the tail fiber protein (T3p48), supporting the hypothesis that this morphological change is related to host adhesion. While an increase in adhesion propensity leads to noticeably decreased spread of phages when dried upon the surface of an agarose plate, we expect that this would not limit phage diffusion significantly in the context of liquid media (such as the internal environmental targets relevant to phage therapy), and would therefore not present limitations in phages' ability to reach the target hosts within their general vicinity. This is supported by our assays of infection dynamics in liquid media (Supplemental figure 2), where no difference in the rate at which infection spread between wild type and mutant phages. Phage degradation at room temperature.

Supplemental Results
Wild-type and mutant bacteriophages were kept at room temperature (25 • C) for 38 days in Dulbecco's phosphate-buffered saline, with a starting concentration of 1 × 10 8 PFU/mL. In this time, the mutant viruses which had been selected for improved thermal stability maintained higher levels of activity, indicating an improved shelf life relative to the wild-type. Relative concentrations were calculated using plaque-counting assays and linear regressions. Theoretical curves were fit to experimental data using a fractional-order kinetic model and the Arrhenius Law. Host ranges maintain consistency between wild type phages and mutants having undergone 10, 20, and 30 rounds of CAVE protocol. Columns represent wild type compared to mutant phages from specified number of mutagenic protocol iterations. Rows represent the host strain that lytic activity was assayed for, through coincubation. Enrichent of specific mutations within the mutant pool over the course of 30 rounds of CAVE application (initial T3 mutagenesis series). Primary conserved mutations appear within the first 10 rounds of mutagenesis, and reach enrichment saturation within the entire mutant pool shortly after. Survival fraction of T3 phages after 1 hour incubation at 60 • C as a function of normalized mutation frequency (R 2 = 0.968). Normalized mutation frequency calculated as the sum all mutation frequencies across a genome at a given round of CAVE applications, divided by the total net mutation frequency achieved at the final round of evolution. A linear correlation indicates the change in thermal tolerance being induced as a function of mutation enrichment.

Characteristics of Substituted Amino Acids
Physical characteristic groupings of amino acids at mutated residues; comparison between wild type and mutant genome data. Amino acid data compiled from both first and second T3 mutagenesis series, as well as the T7 mutagenesis series. Amino acids were grouped based on tabulated values of volume [10] and hydrophobicity [11]. Comparison of the percent survival following 1-hour incubation at 25 • C and 60 • C for: wild type T3 bacteriophage, T3 phage subjected to 20 rounds of directed evolution in the absence of chemical mutagen, and T3 phage subjected to 20 rounds of directed evolution in the absence of thermal selection.