Critical Roles of Ala211 and Tyr212 in the Hemolytic Activity and Infectivity of Listeriolysin O in <i>Listeria monocytogenes</i>

<i>Listeria monocytogenes</i> employs the pore-forming toxin listeriolysin O (LLO) for virulence. This study investigates the poorly defined residues 211-215 (AYSES) within its intertransmembrane α-helical region. While deletion of the entire motif abolished hemolytic activity, single-residue mutagenesis revealed a critical functional divergence: A211V and Y212A mutants exhibited severe (70-80%), pH-independent reductions in hemolysis and pore formation, whereas S213A, E214A, and S215A mutants remained near wild type. This attenuation directly correlated with impaired epithelial invasion, intracellular replication, and membrane disruption. Mechanistically, A211V and Y212A mutations disrupted LLO oligomerization on membranes without altering secondary structure. Consequently, these mutants were severely attenuated <i>in vivo</i>, resulting in 100% survival in mice and a dramatic reduction in the bacterial organ burden. As these mutations do not affect bacterial growth or LLO secretion, A211 and Y212 represent promising targets for novel antivirulence strategies.