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. 2024 Nov 21;25(23):12497. doi: 10.3390/ijms252312497

Figure 6.

Figure 6

Characterization and functional evaluation of ENgase (ORF40). (A) Domain prediction of ENgase via NCBI BLASTp and conserved domain database searches revealed the different functional domains within the protein, indicated by their respective Pfam numbers above. The domains are as follows: the N-terminal extension of bacteriophage endosialidase (Pfam 12218), the catalytic beta propeller domain of bacteriophage endosialidase (Pfam 12217), the unspecified catalytic domain of bacteriophage endosialidase (Pfam 12219), and the peptide S74 family C-terminal domain (Pfam 13884). The tail spike is distinctly marked to denote its unique role. (B) Expression of recombinant ENgase in BL21 host cells, where lane M represents the protein marker, lane 1 represents non-induction, lane 2 represents the induction of protein expression, and lane 3 represents the recombinant protein with a predicted size of approximately 113 kDa, as indicated by arrow. (C) TEM revealed the localization of ORF40 on the tail of the phage particle, as indicated by arrows. (D) Capsule deprivation by ENgase was observed after treatment with recombinant ENgase (middle panels) and phage JSSK01 (right panels) compared with that in untreated cells (left panels). The red boxes have been magnified to observe the effects of the enzyme (bottom panels). (E) Recombinant ENgase caused dose-dependent cell lysis, as indicated by a “halo zone” on the host cell lawn. The purified ENgase was serially diluted two-fold to 0.00975 μg. The halo zone could still be observed at 0.039 μg of the enzyme. (F) A single colony agglutination test demonstrated the specific recognition of the E. coli K1-type capsule by ENgase. 70751 denotes the host strain for JSSK01; BCRC10675 (Bioresource Collection and Research Center, Taiwan) is an O1:K1:H7 for the K1 capsule control; 78030 is a strain containing both K1-type capsules and R1 lipopolysaccharides and is sensitive to JSSK01; and 71464 represents a non-K1 strain used as a negative control. All tests utilized BAS as the agglutination control.