Sensitive Detection of Bacillus anthracis Using a Binding Protein Originating from γ‐Phage

Abstract

Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Therefore, there is a pressing need to develop novel methods for rapid, simple, and precise detection of B. anthracis. Here, we report that the C‐terminal region of γ‐phage lysin protein (PlyG) binds specifically to the cell wall of B. anthracis and the recombinant protein corresponding to this region (positions, 156–233), PlyGB, is available as a bioprobe for detection of B. anthracis. Our detection method, based on a membrane direct blot assay using recombinant PlyGB, was more rapid and sensitive than the γ‐phage test and was simpler and more inexpensive than genetic methods such as PCR, or immunological methods using specific antibodies. Furthermore, its specificity was comparable to the γ‐phage test. PlyGB is applicable in conventional methods instead of antibodies and could be a potent tool for detection of B. anthracis.

Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells

“…The transmission electron microscopy assays and fluorescence assays of the sonicated spores indicated that the binding sites of the newly identified SBD are most probably on the exosporium of the spores. This result differs from that in a previous report, which suggested that PlyG could recognize only the germinating form, but not spores, of B. anthracis (9). This contradiction may be because only a shorter C-terminal fragment of PlyG (residues 156 to 233) was studied as the binding domain in the previous research, and the newly identified spore binding domain is located mainly within the catalytic domain previously considered.…”

“…PlyG, produced by gamma phage, has been reported to be an effective and specific agent for killing B. anthracis vegetative cells (40). The C terminus of PlyG (the site from residue 156 to residue 233) was identified as the cell wall binding domain (CBD), which recognizes vegetative cells, and the N terminus of PlyG (the site from residue 1 to residue 155) was previously considered to be the catalytic domain for destroying the target cells (9). Further research verified that the CBD of PlyG can recognize only the germinating form, but not spores, of B. anthracis (9).…”

“…The C terminus of PlyG (the site from residue 156 to residue 233) was identified as the cell wall binding domain (CBD), which recognizes vegetative cells, and the N terminus of PlyG (the site from residue 1 to residue 155) was previously considered to be the catalytic domain for destroying the target cells (9). Further research verified that the CBD of PlyG can recognize only the germinating form, but not spores, of B. anthracis (9). In vitro truncation analysis indicated that the residues from 190 to 199 of PlyG were necessary and sufficient for vegetative cell binding (21,39).…”

Pathology, Diagnosis, and Treatment of Anthrax in Humans

“…3 Assays detecting B. anthracis proteins: Several proteins specifi c for B. anthracis , such as galactose/ N -acetylglucosamaine cell wall -associated polysaccharide, capsule antigen, and EA1 can be detected using antibodies or the C -terminal region of gamma -phage lysin protein (Fujinami et al, 2007 ;Love et al, 2008 ). Detection can be performed using either direct fl uorescence or enzymatic labels.…”

Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

“…and 2% artificially contaminated milk, respectively (Tolba et al, 2012). Other methods based on endolysins have been developed to detect B. anthracis (Fujinami et al, 2007, Sainathrao et al, 2009. Using the C-terminal region of γ-phage lysin protein (PlyG), Fujinamii et al, (2007) developed a bioprobe to detect B. anthracis with a membrane direct blot assay which turned out to be simpler and less expensive than other genetic tools such as PCR, or immunological methods using specific antibodies.…”