Characterization of Tail Sheath Protein of N4-Like Phage phiAxp-3

Achromobacter phage phiAxp-3, an N4-like bacteriophage, specifically recognize Achromobacter xylosoxidans lipopolysaccharide (LPS) as its receptor. PhiAxp-3 tail sheath protein (TSP, ORF69) shares 54% amino acid sequence identity with the TSP of phage N4 (gp65); the latter functions as a receptor binding protein and interacts with the outer membrane receptor NfrA of its host bacterium. Thus, we hypothesized that ORF69 is the receptor-binding protein of phiAxp-3. In the present study, a series of ORF69 truncation variants was constructed to identify the part(s) of this protein essential for binding to A. xylosoxidans LPS. Phage adsorption and enzyme-linked immunosorbent assay showed that amino acids 795-1195 of the TSP, i.e., ORF69(795-1195), are sufficient and essential for receptor and binding. The optimum temperature and pH for the functions of ORF69 and ORF69(795-1195) are 4/25°C and 7, respectively. In vitro cytotoxicity assays showed that ORF69 and ORF69(795-1195) were respectively toxic and non-toxic to a human immortalized normal hepatocyte cell line (LO2; doses: 0.375-12 μg). The potential of this non-toxic truncated version of phiASP-3 TSP for clinical applications is discussed.