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.2021 Mar 11;10(3):293.
doi: 10.3390/antibiotics10030293.

Rapid and High-Throughput Evaluation of Diverse Configurations of Engineered Lysins Using the VersaTile Technique

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Rapid and High-Throughput Evaluation of Diverse Configurations of Engineered Lysins Using the VersaTile Technique

Lisa Duyvejonck et al. Antibiotics (Basel)..

Abstract

Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targetingKlebsiella pneumoniae. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth ofKlebsiella pneumoniae could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP-linker-CBD-EAD) and the inverse configuration (CBD-EAD-linker-OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions.

Keywords: Klebsiella pneumoniae; VersaTile; bacteriophage; lysin; protein engineering.

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Conflict of interest statement

H.G., D.G., R.L., and Y.B. are inventors on a patent application related to this work filed by Ghent University and the University of Leuven (no. WO/2018/114980, filed on 19 February 2017, published on 28 June 2018). The authors declare that they have no other competing interests.

Figures

Figure 1
Figure 1
The different configurations tested in this study. Configuration 1 is the configuration analyzed in previous work [5]. OMP: outer membrane permeabilizing peptide, CBD: cell wall binding domain, EAD: enzymatically active domain. The total number of available tiles for configurations 1 through 5 are mentioned under each specific position. The resulting possible variants are indicated within the brackets.
Figure 2
Figure 2
The distribution of the growth inhibitory (GI) activities, expressed as percentages, for each configuration. (a) The replicates tested in the absence of 0.5 mM EDTA. (b) The replicates tested in the presence of 0.5 mM EDTA. The red dashed line represents the GI threshold set in this study to be considered a hit (80%). The first configuration (purple) shows the most active variants. Configurations 1 and 4 have a top cluster with strong active variants, whereas configuration 2 and 5 show a long tail of active variants with rising activity. EDTA does not significantly alter the specific distribution of the variants, but slightly increases the activity for configurations 1, 3, 4, and 5.
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