Some phages have evolved specifically to recognize and cleave bacterial exopolysaccharide structures (EPS) like capsule, LPS or slime. Recognition of a specific EPS structure generally occurs by a tail spike or tail fiber which binds the EPS receptor and subsequently cleave it by its associated enzymatic activity. Bacteriophages possessing virion-associated depolymerases are able to freely diffuse in the biofilm matrix.
Pseudomonas LKA1 phage belonging to phiKMV-like viruses encodes one tail spike protein (Gp49) which is essential for recognition of the B-band O-antigen and for the formation of halo zones on bacterial lawn. The LKA1 (Gp49), as potential LPS depolymerase has been prepared as recombinant protein. LKA1-Gp49 forms SDS resistant homotrimers of 240.0 kDa as was also observed for other polysaccharide degrading tail spike proteins interacting with LPS-structures.
It was confirmed that both LKA1 particles and its recombinantly purified tail spike protein are not able to form halo-zones on a B-band deficient PAO1 strains, indicating that for LKA1 degradation of B-band LPS and halo formation are coupled. SDS-PAGE analysis with the Marolda protocol has been used to visualize the LPS degrading activity of obtain enzyme on PAO1 host strain and its rmd (GDP-4-keto-6-deoxy-D-mannose reductase) knock out mutant deficient in A-band LPS synthesis, but producing smooth B-band LPS. The stability of enzyme in different pH and temperature has been determined, as well. The ability of LKA1-Gp49 and the LKA1 phage to degrade biofilm matrix was evaluated by laser interferometry method. The in vivo Galleria mellonella model and the efficiency uptake of phagocytic cells were applied to assess the influence of LKA1-Gp49 depolymerase on the virulence properties of PAO1 culture.