The lytic enzymes that target Gram-positive bacteria nearly universally have a two-domain structure, with the N-terminal catalytic domain bound through a short linker to a cell wall binding domain. We have exploited this modularity in designing new enzyme systems.
We have exploited the extraordinary specificity of lytic enzyme cell wall binding domains (CBD) when coupled with silver nanoparticles to kill target bacteria (Figure 1) [Kim et al. ACS Appl. Mater. Interfaces 10, 13,317-13,324 (2018)]. As a relevant example, CBDBA (binding domain from Bacillus anthracis) selectively bound to B. anthracis in a mixture with B. subtilis, as well as in a mixture with Staphylococcus aureus. As a result, the nonselective antimicrobial silver was converted into a highly selective antimicrobial targeting organisms that are bound selectively by a specific CBD. This new biologically-assisted hybrid strategy, therefore, has the potential to provide selective decontamination of pathogenic bacteria with minimal impact on normal microflora.
Figure 1. Schematic representation of cell wall binding domain-fluorescent protein-silver binding peptide complexation with AgNPs for selective recognition and specific killing of target pathogenic bacteria; cell wall binding domain CBDSA against S. aureus and CBDBA against B. anthracis. Antimicrobial Activity of various CBD-AgNP hybrids. (a) Inhibitory activity of EGFP-BP-AgNPs and CBDBA-mRUBY-BP-AgNPs on B. anthracis cell growth on BHI agar plate; (b) CFU assay showing dose-dependent growth inhibition of B. anthracis; (c) Effects of EGFP-BP-AgNPs and CBDBA-mRUBY-BP-AgNPs inhibition of S. aureus cell growth on BHI agar plate; (d) CFU assay showing dose-dependent growth inhibition of S. aureus [Kim et al. ACS Appl. Mater. Interfaces 10, 13,317-13,324 (2018)].
The modular nature of cell lytic enzymes can be exploited through unique assemblies (Figure 2). Cell lytic enzymes consist of catalytic and cell wall binding domains, which can be swapped among those of other lytic enzymes to produce unnatural chimeric enzymes. We have shown that microbially-generated biotinylated catalytic domains (CD) and cell wall binding domains (CBD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1) can be assembled with streptavidin to form chimeric lytic enzymes [Kim et al. Biomacromolecules 20, 4035-4043, 2019].
Figure 2. (A) Vector construction for enhanced green fluorescent protein (EGFP)-cell wall binding domain (CBDS) from SA1 and cell wall binding domain (CBDL) from lysostaphin fusions with Avi Tag at the N-terminus. (B) Vector construction for the CDS from SA1 and CDL from lysostaphin with Avi Tag at the C‐terminus. (C) Schematic representation of the modular assembly of recombinant catalytic domains (CDs) and cell wall binding domains (CBDs) with a biotinylated on streptavidin. Blue and red colors indicate catalytic and cell wall binding domains of lysostaphin, respectively, and magenta and brown colors indicate catalytic and cell wall binding domains of SA1, respectively. Gray color indicates linker regions. Cyan color indicates streptavidin.
