SAR Endolysin Regulation in dsDNA Phage Lysis of Gram-Negative Hosts
SAR endolysins are a recently discovered class of muralytic enzymes that are regulated by dynamic membrane topology. They are synthesized as enzymatically inactive integral membrane proteins during the phage infection cycle and then are activated by conformational remodeling upon release from the membrane. This topological duality depends on N-terminal SAR (Signal-Anchor-Release) domains, which are enriched in weakly hydrophobic residues and require the proton motive force to be maintained in the bilayer. The first SAR endolysin to be characterized was P1 Lyz, of phage P1. Its activation requires a disulfide bond isomerization involving its catalytic Cys initiated by a free cysteine thiol from the newly-liberated SAR domain. A second mode of disulfide bond regulation, as typified by Lyz103 of the Erwinia Amylovora phage ERA103, has been demonstrated. In its membrane bound form, Lyz103 is inactivated by a disulfide that is formed between cysteine residues flanking a catalytic glutamate.
A second class of SAR endolysins, typified by R21, the lysozyme of the lambdoid phage 21, does not require disulfide bond isomerization for activation. Rather, these proteins are dependent on the release of the SAR domain for proper folding of the catalytic cleft. Bioinformatic analysis indicates that the regulatory theme of R21 is common in the SAR endolysins of dsDNA phages. Furthermore, bioinformatic study of endolysins of dsDNA phage of Gram-negative hosts revealed two new classes of SAR endolysins that are not homologous to T4 gpe, as all SAR endolysins were once thought to be. SAR endolysins were found in nearly 25% of sequenced dsDNA phages of Gram- negative hosts including 933W, which is involved in the release of Shiga toxin from EHEC strain EDL933. An inhibitor study against the SAR endolysin of 933W, R933W, was performed using a custom compound library in a high through-put, in vivo lysis assay. Of nearly 8,000 compounds screened, one compound, designated 67-J8, inhibited lysis but not growth. In vivo and in vitro experiments show that the compound has no effect on R933W activity, accumulation, or secretion. In vivo experiments suggest that 67-J8 increases the proton motive force, thereby presumably retaining the SAR domain in the membrane.