BPS2026 – How Listeria monocytogenes manipulates host cell membranes to promote infection

Listeria monocytogenes (Lm), a Gram-positive pathogen responsible for food poisoning, meningitis, and spontaneous abortions, depends on the combined actions of Listeriolysin O (LLO) and phospholipase B (PLCB) to disrupt host cell membranes during cell-to-cell spread. While these factors are crucial for breaching the complex double-membrane compartments of the secondary cell during infection process, the exact mechanisms underlying membrane rupture and lipid remodeling remain poorly understood. To explore PLCB’s role, we generated a PLCB deletion mutant strain (ΔPLCB) and observed a significant impairment in bacterial spread and infection. Biochemical analyses, including mass spectrometry, hemolysis, pore formation assays, and membrane-binding studies with toxin sensors (OlyA and ALOD4), demonstrated that PLCB hydrolyzes phosphatidylcholine (PC), generating diacylglycerol (DAG) and liberating cholesterol that is otherwise sequestered by PC. This liberated cholesterol enhances LLO binding, pore formation, and membrane disruption. Importantly, PLCB also binds to negatively charged phosphatidylserine (PS), which boosts the PC hydrolysis activity of PLCB and further promotes membrane remodeling, facilitating cholesterol release and supporting LLO-driven pore formation. Mutational analysis showed distinct roles for PLCB’s PS binding activity and PC hydrolysis activity: the D55N mutant retains PS binding but loses PC hydrolysis, whereas the R76A/D76A mutant preserves PC hydrolysis but loses PS binding. These results reveal a novel membrane manipulation mechanism employed by Listeria and point to potential therapeutic targets against listeriosis.