Characterization of the catalytic activity of the γ-phage lysin, PlyG, specific forBacillus anthracis

Abstract

Bacillus anthracis causes anthrax, a lethal disease affecting humans that has attracted attention due to its bioterrorism potential. PlyG is a lysin of gamma-phage, which specifically infects B. anthracis and lyses its cell wall. PlyG contains a T7 lysozyme-like amidase domain, which appears to be the catalytic domain, in the N-terminal region and has a high degree of sequence similarity with PlyL, which is an N-acetylmuramoyl-l-alanine amidase encoded by the B. anthracis genome. Here, we demonstrated that two amino acid residues of PlyG, H29 and E90, are necessary for its catalytic activity in B. anthracis. These residues are structurally analogous to residues whose mutation in T7 lysozyme abolished its catalytic activity. A C-terminal deletion mutant of PlyG lacking the core sequence for binding to B. anthracis showed completely abolished binding activity, unlike PlyL, despite high sequence similarity with PlyL in the N-terminal region. This suggests that the C-terminal binding domain, as well as the N-terminal catalytic domain, is essential for the catalytic activity of PlyG. Our observations provide new insights into the mechanism of specific catalysis of PlyG in B. anthracis and may contribute to the establishment of new methods for anthrax therapy.

Endolysins as Antimicrobials

“…When analyzed on live bacteria, it was made clear that the B30 endolysin CHAP domain was the primary source of lytic activity from this dual domain endolysin when lysing 'from without' (Donovan et al, 2006b). Site-directed mutagenesis and deletion analysis of the Bacillus anthracis phage lysin PlyG were essential in defining the binding domain and active site residues (Kikkawa et al, 2007;Kikkawa et al, 2008), as for PlyC that was also examined in this way . Similarly, site-directed mutations altering histidine codons in the staphylococcal glycyl-glycine PG hydrolase ALE-1 have been used to define essential amino acids in the M23 endopeptidase domain (Fujiwara et al, 2005).…”

Bacillus anthracis physiology and genetics

“…The γ phage is the most well-studied B. anthracis phage due to its diagnostic use and more recently, the proposed use of the lysin enzyme PlyG for decontamination (Fischetti, 2006; Kikkawa et al, 2008; Schuch et al, 2002). γ is morphologically similar to the Siphoviridae family, possessing a long contractile tail and an icosohedral head.…”

Characteristics of a broad lytic spectrum endolysin from phage BtCS33 of Bacillus thuringiensis

“…Usually, one cell wall binding domain determines the endolysin strain specificity [11,12]; however, there are sometimes more than one [7,13,14] or even no cell wall binding domains [15,16]. The endolysin C-terminus nevertheless sometimes appears to be essential for catalytic activity, as several reports showed that the enzymatic activity is abolished after removal of the C-terminus [17,18]. …”

“…Because of the multidrug resistance of B. anthracis [21,22], several of its phage or prophage endolysins have been expressed, purified, and characterized. There have also been some attempts to use these endolysins to cure the disease caused by B. anthracis [8,9,11,17,18,23]. Practical applications of endolysins were enabled by studies on functional domain composition, optimal reaction conditions, and species- or strain-specificity.…”