LAP Drives Monocyte-Mediated and Extracellular Translocation of Listeria monocytogenes Across the Placental Barrier

Abstract

Listeria monocytogenes (Lm) is a major cause of fetal infection, yet the mechanisms by which it traverses the placental barrier remain incompletely defined. We established and validated a physiologically relevant in vitro human placental co-culture barrier model using BeWo trophoblasts and primary human placental vascular endothelial cells (HPVECs), which exhibited robust barrier integrity and organized tight junctions. Using this model, we demonstrate that Lm exploits a Trojan-horse transmigration mechanism via infected monocytes. Lm-infected THP-1 monocytes displayed a [~]4.8-fold higher monocyte transmigration and [~]8.12-fold greater intracellular bacterial delivery across the placental barrier compared with non-pathogenic Listeria innocua (Li). Monocyte transmigration was associated with claudin-1 and occludin disruption, increased paracellular permeability, and localization of infected monocytes at junctional breach sites. Monocyte-mediated placental traversal required the Lm virulence factors Listeria adhesion protein (LAP) and internalin B (InlB), as lap- and{Delta} inlB mutants exhibited Li-like transmigration and an [~]90% reduction in intracellular bacterial delivery, despite normal monocyte uptake. Lm-infected monocytes induced a [~]1.86-fold increase in sVCAM-1 secretion, which was LAP- and InlB-dependent, consistent with pro-transmigration endothelial phenotype. In contrast, extracellular traversal required LAP and internalin A (InlA), as lap- and{Delta} inlA mutants exhibited severe defects comparable to Li, whereas{Delta} inlB and{Delta} hly were minimally impaired. Exposure of monocytes to extracellular Lm further amplified transmigration and barrier permeability. Together, these data define two distinct placental invasion routes: a monocyte-associated LAP/InlB-dependent pathway and an extracellular LAP/InlA-dependent pathway, identifying LAP as a critical noncanonical virulence factor in placental infection and mechanisms of vertical transmission.