Backgrounds <p>Endolysins are promising alternatives to antibiotics because they can lyse bacterial cells rapidly with a low risk of resistance development, however, their effectiveness against Gram-negative bacteria is hindered by the presence of the outer membrane present in Gram-negative bacteria. Several endolysins with amphipathic helices at the C-terminus have been reported to have intrinsic antibacterial activity against Gram-negative bacteria but their action mechanism is not fully elucidated.</p> Methods <p>The sequence alignment analysis was assessed with the CLC Main workbench 7, and His-tagged endolysins were purified with affinity chromatography. Site-directed mutagenesis was used to generate mutations in the endolysin to make various endolysin mutants. The muralytic activity of the endolysin against Gram-negative bacteria was analyzed using a turbidity reduction assay and the antibacterial activities of the endolysins were assessed through a viable cell counting assay.</p> Results <p>We identified two endolysins, LysTS3 and LysTS6, both of which have similar sequences and structures including the amphipathic helices at their C-terminus. LysTS6 exhibited significantly higher antibacterial activity against Gram-negative bacteria compared to LysTS3 even though both enzymes have similar muralytic activity against the outer membrane-permeabilized Gram-negative bacteria. Systematic truncation and bioinformatic analysis of these two endolysins revealed a major difference in the charge on the surface of their C-terminal helices, suggesting the possibility that the charge on this helix can determine the antibacterial activity of the endolysins against Gram-negative bacteria. We could enhance the activity of LysTS3 against Gram-negative bacteria by replacing Ala₁₅₆ and Glu₁₆₀ with lysine and alanine, respectively, the amino acid residues at the structurally equivalent positions in LysTS6. A similar activity boost was also seen in LysSPN1S and LysJEP4 when the surface charge of the C-terminal amphipathic helix was altered to be more positive through the modification of the surface-exposed amino acid residues.</p> Conclusions <p>The antibacterial activity of endolysin against Gram-negative bacteria could be enhanced by adjusting the surface charge on the C-terminal amphipathic helix to more positive, suggesting that the positive surface charge on the C-terminal amphipathic helix of endolysin is crucial for its penetration of outer membrane to reach peptidoglycan layer of Gram-negative bacteria.</p>