Novel amino acid residues in listerolysin O drive phagosome escape and pathogenicity in <i>Listeria monocytogenes</i>

<i>Listeria monocytogenes</i>, a significant zoonotic foodborne intracellular pathogen, relies on its principal virulence factor, listeriolysin O (LLO), to escape from host cell phagosomes-a critical step for establishing intracellular infection and replication. LLO, composed of four structural domains with amino acids essential for its pore-forming abilities, still harbors many sites with undefined roles. We identified a critical domain within LLO (amino acids 251-255) crucial for bacterial pathogenesis and systematically investigated the individual roles of five amino acid sites within this domain. Our findings demonstrate that LLO K252 is dispensable for LLO functionality, residues F251, Q253, and Y255 are critical for LLO's hemolytic activity and cytotoxicity. Mutation of I254 to alanine alters hemolytic activity, with effects dependent on environmental pH. Notably, alanine mutations at F251, Q253, and Y255A significantly impair bacterial proliferation in mouse organs and disruption of late phagosome membranes. In particular, mutations F251A and Y255A abolish late phagosome escape, underscoring their importance in host cell infection. This study has expanded our understanding of LLO's role in <i>Listeria</i> pathogenicity and phagosome escape, offering new insights into the molecular mechanisms driving this pathogens' virulence.