Characterization of the Bacteriophage Felix O1 Endolysin and Potential Application for Salmonella Bioremediation

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.