Listeria monocytogenes favors the vacuolar compartment during ex vivo human placental infections

Listeria monocytogenes poses a serious concern during pregnancy, as these microbes can cross the placental barrier to infect the developing fetus. The pathogen’s intracellular lifecycle has been well characterized in cultured cells, where the internalized bacteria escape short-lived Listeria -containing vacuoles (LCVs), replicate in the cytoplasm, and recruit host actin to generate comet tails that enable their intra- and inter-cellular movement. Whether these events occur during intact human placental infections remains unknown. Through ex vivo placental infections, we characterized the spatiotemporal progression of L. monocytogenes in a physiologically relevant system. We found that the actin-rich structures, which are hallmarks of cultured cell infections, were essentially absent (<1% of L. monocytogenes were associated with actin-rich structures). Instead, the bacteria remained predominantly restricted within LCVs, where they also replicated. Quantitative histological and immunofluorescence analyses showed that 39% of wild-type L. monocytogenes remained within Rab5-positive vacuoles and 44% in Lamp1-positive vacuoles, whereas only 1%–4% were observed in cultured placental cells. Most vacuoles lacked the vATPase and cathepsin B, indicating that placental LCV vacuoles were non-acidified, non-degradative endosomal niches. These results closely phenocopied infections using Δ hly-L. monocytogenes , which do not generate listeriolysin O (LLO), a bacterial toxin crucial for LCV escape. Together, our findings reveal that L. monocytogenes primarily reside within LCVs during ex vivo human placental infections, contrasting the classical in vitro models and emphasizing the importance of studying bacterial pathogenesis in intact human tissues.