Computationally Aided Discovery of LysEFm5 Variants with Improved Catalytic Activity and Stability

Abstract

Bacteriophage-derived lysin proteins are potentially effective antimicrobials that would benefit from engineered improvements to their bioavailability and specific activity. Here, the catalytic domain of LysEFm5, a lysin with activity against vancomycin-resistant Enterococcus faecium (VRE), was subjected to site-saturation mutagenesis at positions whose selection was guided by sequence and structural information from homologous proteins. A second-order Potts model with parameters inferred from large sets of homologous sequence information was used to predict the average change in the statistical fitness for mutant libraries with diversity at pairs of sites within the secondary catalytic shell. Guided by the statistical fitness, nine double mutant saturation libraries were created and plated on agar containing autoclaved VRE to quickly identify and segregate catalytically active (halo-forming) and inactive (non-halo-forming) variants. High-throughput DNA sequencing of 873 unique variants showed that the statistical fitness was predictive of the retention or loss of catalytic activity (area under the curve [AUC], 0.840 to 0.894), with the inclusion of more diverse sequences in the starting multiple-sequence alignment improving the classification accuracy when pairwise amino acid couplings (epistasis) were considered. Of eight random halo-forming variants selected for more sensitive testing, one showed a 1.8 (±0.4)-fold improvement in specific activity and an 11.5 ± 0.8°C increase in melting temperature compared to those of the wild type. Our results demonstrate that a computationally informed approach employing homologous protein information coupled with a mid-throughput screening assay allows for the expedited discovery of lysin variants with improved properties. IMPORTANCE Broad-spectrum antibiotics can indiscriminately kill most bacteria, including commensal species that are a part of the normal human flora. This can potentially lead to the proliferation of drug-resistant bacteria upon elimination of competing species and to unwanted autoimmune effects in patients. Bacteriophage-derived lysin proteins are an alternative to conventional antibiotics that have coevolved alongside specific bacterial hosts. Lysins are capable of targeting conserved substrates in the bacterial cell wall essential for its viability. To engineer these proteins to exhibit improved therapeutically relevant properties, homology-guided statistical approaches can be used to identify compelling sites for mutation and to quantify the functional constraints acting on these sites to direct mutagenic library creation. The platform described herein couples this informed approach with a visual plate assay that can be used to simultaneously screen hundreds of mutants for catalytic activity, allowing for the streamlined identification of improved lysin variants.

Deep Antimicrobial Activity and Stability Analysis Inform Lysin Sequence–Function Mapping

“…Finally, we compared τ D1 (Figure 2G) to previously reported lysin catalytic activities 20 to assess if the depletion assay correlated with activity. Two modes of performance were observed: very long τ D1 were observed for two minimally active variants (V4 and V14, Table S1 20 ), whereas a range of moderate times correlated with catalytic activity measurements among the other five variants (ρ = 0.66).…”

“…Finally, we compared τ D1 (Figure 2G) to previously reported lysin catalytic activities 20 to assess if the depletion assay correlated with activity. Two modes of performance were observed: very long τ D1 were observed for two minimally active variants (V4 and V14, Table S1 20 ), whereas a range of moderate times correlated with catalytic activity measurements among the other five variants (ρ = 0.66). We suspect the variable outcomes are caused by multiple factors, most significantly that growth inhibition in this assay is related to a combination of a molecule's catalytic activity, producibility, and stability.…”

“…Naturally occurring antimicrobial peptides and proteins offer compelling starting points for developing therapies, but they often require engineering to improve activity, stability, solubility, or other molecular properties to ultimately be translatable . While the field has developed HT assays for screening large (10 6 –10 9 variants) protein libraries for stability, , developability, and other functions of interest, − screening of antimicrobial activity has remained mostly limited given the complex and diverse modes of antimicrobial activity. − The complex nature of antimicrobial activity and the limited quantity of activity data have further limited the development of statistical models to predict activity and inform the design of improved variants.…”

A platform for deep sequence-activity mapping and engineering antimicrobial peptides

“…Multiple methods have advanced AMP engineering and discovery. Multiwell plate assays afford limited throughput 14,15 , which is only partially aided by recombinant AMP expression [16][17][18][19] . Innovative genotype-phenotype linkages, including surface-localized antimicrobial display (SLAY) 20 and host-target co-encapsulation 21 , expanded throughput to 10 6 variants, yet their current implementations provide a binary metric, active or inactive.…”

A Platform for Deep Sequence–Activity Mapping and Engineering Antimicrobial Peptides

“…As a result, traditional platforms for AMP characterization are constrained in throughput with typical evaluation of tens or hundreds of variants, and recent advancements to increase throughput have mechanistic constraints. Multiwell plate assays afford limited throughput, , which is only partially aided by recombinant AMP expression. − Innovative genotype-phenotype linkages, including surface-localized antimicrobial display (SLAY) and host–target coencapsulation, expanded throughput to 10 6 variants, yet their current implementations provide a binary metric, active or inactive. Moreover, SLAY tethers the AMP to the cell surface and coencapsulation utilizes cell permeability dye, which limits mechanisms of action.…”