Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against Staphylococcus aureus Strains

Abstract

Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.

Rapid Antibacterial Activity Assessment of Chimeric Lysins

“…To compare the differences in gene expression, we prepared a chimeric lysin gene (ALS2-dA-L31), which we generated by deleting the amidase domain and seven amino acids of the linker of ALS2 (Figure 1F). The gene templates of ALS2-dA-L31, which were prepared using both the present (P_ALS2-L31+T term) and previous (V_ALS2-L31+TAG) methods, were expressed, and the antibacterial activities were compared using a turbidity reduction test [31]. In contrast to V_ALS2-dA-L31+TAG and the negative control, the P_ALS2-dA-L31+T term showed significantly lower OD 600 values, reflecting higher antibacterial activity against the different genotypes of human MRSA strains: CCARM3806 (RST10-2) and CCARM3840 (RST4-1) (Figure 1C) [31].…”

“…The gene templates of ALS2-dA-L31, which were prepared using both the present (P_ALS2-L31+T term) and previous (V_ALS2-L31+TAG) methods, were expressed, and the antibacterial activities were compared using a turbidity reduction test [31]. In contrast to V_ALS2-dA-L31+TAG and the negative control, the P_ALS2-dA-L31+T term showed significantly lower OD 600 values, reflecting higher antibacterial activity against the different genotypes of human MRSA strains: CCARM3806 (RST10-2) and CCARM3840 (RST4-1) (Figure 1C) [31]. Although we did not test the individual effects of the T7 terminator and the modified nucleotide sequences, they may increase the expression of ALS2-dA-L31 via the optimization of the transcription efficiency of T7 polymerase.…”

“…Sci. 2024, 25, x FOR PEER REVIEW 3 of 20 previous (V_ALS2-L31+TAG) methods, were expressed, and the antibacterial activities were compared using a turbidity reduction test [31]. In contrast to V_ALS2-dA-L31+TAG and the negative control, the P_ALS2-dA-L31+T term showed significantly lower OD600 values, reflecting higher antibacterial activity against the different genotypes of human MRSA strains: CCARM3806 (RST10-2) and CCARM3840 (RST4-1) (Figure 1C) [31].…”

“…Therefore, intensive efforts to find better chimeric lysins have been conducted via random domain shuffling and antibacterial activity tests using Escherichia coli (E. coli)-expressed proteins or via induced lysis-based screening [25][26][27][28][29]. Gene cloning into plasmid vectors and E. coli expression are laborious processes, and simpler and more rapid in vitro screening and activity-comparing protocols have been developed [30,31]. Previously, we developed a chimeric lysin (ALS2), which is composed of the CHAP domain of an autolysin (SsaA) and the amidase and SH3 domains of ϕK lysin (LysK), after screening multiple candidates using a simple and rapid cell-free expression system [31].…”

“…Gene cloning into plasmid vectors and E. coli expression are laborious processes, and simpler and more rapid in vitro screening and activity-comparing protocols have been developed [30,31]. Previously, we developed a chimeric lysin (ALS2), which is composed of the CHAP domain of an autolysin (SsaA) and the amidase and SH3 domains of ϕK lysin (LysK), after screening multiple candidates using a simple and rapid cell-free expression system [31]. However, the cell-free expression system needs to be improved for higher-expression-level proteins.…”