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.

There is an increasing incidence of antimicrobial-resistant organisms isolated from food and food products. Coupled with that rising incidence is increased media scrutiny and coverage of outbreaks of foodborne illnesses. Consequently, consumers increasingly demand safer food, and that the antimicrobial measures used be other than antimicrobial drugs. A possible solution is to use bacteriophages, or the purified holin and endolysin proteins that make them lethal and lytic, as antimicrobial food treatments or additives. The bacteriophage Felix O1 is a promising candidate for development as an anti-Salmonella food treatment. This dissertation describes the work done to determine if these proteins could be of value as bioremedial agents. Endolysin treatments of Gram negative bacteria require two agents: the lytic endolysin, and a second agent to permeabilize the outer membrane of the bacterium. The holin protein was proposed as an outer membrane permeabilization agent. Methods used to locate the holin gene included BLAST analysis, analysis of putative Felix O1 proteins for transmembrane domains, and examination of the lysin sequence for an N-terminal signal sequence. Analyses did not reveal a promising candidate. Cloning of rIIA as a potential holin was attempted without success. Results of various analyses are discussed, as are chemical alternatives to the use of purified holin as a permeabilization agent. The endolysin, LysO1, was successfully cloned and characterized. PHYRE analysis predicted that the enzyme structure is composed of α helices arranged into two lobes, with the active site in a cleft between them. The enzyme lysed all tested strains of Salmonella and a tested strain of the foodborne pathogen Escherichia coli. Campylobacter jejuni susceptibility remains ambiguous, and the enzyme had no effect on Listeria monocytogenes or Micrococcus luteus. LysO1 was most active at alkaline pH and low ionic strength; optimal activity was observed in 25 mM buffer at pH 10. If removed from frozen storage, the enzyme was most thermostable at 30 °C. Lytic activity was adversely affected by the presence of the divalent cations calcium, magnesium, and zinc, and by high ionic strength. Considerable time was devoted to development of the activity assay used to further characterize the enzyme, and details of those experiments are provided. Logical extensions of the research project, such as further characterization and testing needed to obtain government approval for widespread use of the treatment, and possible pursuit of treatment based on an enzyme derivative such as an antimicrobial peptide, are discussed. / Ph. D.

The N-terminal hydrophobic domain of the phage P1 endolysin Lyz was found to facilitate the export of Lyz in a sec-dependent fashion, explaining the ability of Lyz to cause lysis of E.coli in the absence of the P1 holin. The N-terminal domain of Lyz is demonstrated to be both necessary and sufficient not only for export to the membrane but also for release into the periplasm of this endolysin. We propose that this unusual N-terminal domain functions as a "signal arrest- release" (SAR) sequence, which first directs the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. To understand why release from the membrane is required for the physiological expression of the lytic activity of Lyz, we examined the role of its seven cysteine residues in the biogenesis of the active endolysin. The inactive, membrane-tethered and the active, soluble forms of Lyz differ in their pattern of intramolecular disulfide bonding. We conclude that the release of Lyz from the membrane leads to an intramolecular thiol-disulfide bond isomerization causing a dramatic conformational change in the Lyz protein. As a result, an active site cleft that is missing in nascent Lyz is generated in the mature form of the endolysin. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an SAR sequence is not unique to Lyz. Studies on holin and antiholin indicated that P1 encodes two holins, LydA and LydC. The antiholin LydB inhibits LydA by binding to it directly on the membrane. All above results demonstrate a new paradigm for control of phage lysis, which is, upon depolarization of the membrane by holin function at a programmed time, endolysin is released from the bilayer leading to the immediate lysis of the host.