PlyC: A multimeric bacteriophage lysin

Abstract

Lysins are murein hydrolases produced by bacteriophage that act on the bacterial host cell wall to release progeny phage. When added extrinsically in their purified form, these enzymes produce total lysis of susceptible Gram-positive bacteria within seconds, suggesting a unique antimicrobial strategy. All known Grampositive lysins are produced as a single polypeptide containing a catalytic activity domain, which cleaves one of the four major peptidoglycan bonds, and a cell-wall-binding domain, which may bind a species-specific carbohydrate epitope in the cell wall. Here, we have cloned and expressed a unique lysin from the streptococcal bacteriophage C1, termed PlyC. Molecular characterization of the plyC operon reveals that PlyC is, surprisingly, composed of two separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA. Inhibitor studies predicted the presence of an active-site cysteine, and bioinformatic analysis revealed a cysteine, histidine-dependent amidohydrolase/peptidase domain within PlyCA. Point mutagenesis confirmed that PlyCA is responsible for the observed catalytic activity, and Cys-333 and His-420 are the active-site residues. PlyCB was found to self-assemble into an octamer, and this complex alone was able to direct streptococcal cell-wall-specific binding. Similar to no other proteins in sequence databases, PlyC defines a previously uncharacterized structural family of cell-wall hydrolases.multimeric protein ͉ cell-wall hydrolase ͉ Streptococcus

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…42 The two proteins are transcribed from two genes located in a single operon and separated by an intron-like sequence. 38,42 Furthermore, introns are rare in endolysin genes, but other exceptions do exist. 25 Engineering novel endolysin constructs The modular structure of endolysins provides an opportunity to engineer enzymes with altered bacteriolytic activity.…”

“…36,37 Endolysins with an intact CBD can also have a broad host range, such as with the streptococcal phage endolysin PlyC, which lyses several streptococcal species. 38 Even more promiscuous is the enterococcus phage f1 endolysin PlyV12, which lyses E. faecalis and several streptococcal and staphylococcal species. 39 Uniquely, the Bacillus phage endolysin PlyG lyses both vegetative cells and germinating spores of B. anthracis.…”

X-ray crystal structure of the streptococcal specific phage lysin PlyC

“…Previous research spanning over 50 y has shown that PlyC can rapidly lyse cultures of groups A, C, and E streptococci in addition to Streptococcus uberis and Streptococcus equi and has been shown to protect mice from streptococcal challenge (12,13). PlyC is furthermore unique among the Gram-positive lysins in that it consists of two separate proteins-a single 50-kDa PlyCA subunit that is suggested to form a complex with at least eight 8-kDa PlyCB subunits (14). The two proteins are transcribed from two genes located in a single operon (14).…”

“…PlyC is furthermore unique among the Gram-positive lysins in that it consists of two separate proteins-a single 50-kDa PlyCA subunit that is suggested to form a complex with at least eight 8-kDa PlyCB subunits (14). The two proteins are transcribed from two genes located in a single operon (14). PlyCA is known to contain an active cysteine, histidine-dependent amidohydrolases/peptidase (CHAP) domain, a fold distantly related to the papain-like cysteineprotease family, with Cys 333 and His 420 shown to be essential for amidase catalytic activity (14).…”

Insights into the regulation of bacteriophage endolysin: multiple means to the same end

“…A. Pohane and V. Jain Interestingly, however, most of the bacterial autolysins do not display peptidoglycan-binding specificity (Steen et al, 2003;Nelson et al, 2006;Fischetti, 2008). The G + phage endolysins have a very narrow range of substrate specificity, i.e.…”