Repurposing Etravirine as a PlcA-Targeting inhibitor to block autophagic escape of Listeria monocytogenes

The rapid rise of antibiotic resistance underscores the urgent need for anti-infective strategies that circumvent classical bactericidal approaches. Host-directed antivirulence therapies, which target host defense pathways or pathogen virulence rather than essential viability, may reduce selection pressure and delay resistance, but potent small-molecule examples remain limited. Here we identified a drug repurposing candidate against the intracellular pathogen Listeria monocytogenes (L. monocytogenes) by targeting its phosphatidylinositol-specific phospholipase C PlcA, a key virulence factor involved in autophagy escape. Using a compound-microarray high-throughput binding platform, we discovered that the approved HIV non-nucleoside reverse transcriptase inhibitor Etravirine directly bound PlcA and inhibited its phosphatidylinositol-hydrolyzing activity without affecting bacterial growth or plcA transcription. In infected cells, Etravirine restored PtdIns3P levels, increased MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) recruitment and autophagic flux, promoted lysosomal delivery, and reduced infection-induced cellular damage. Etravirine also enhanced basal autophagic flux and induced an autophagy-associated transcriptional program. These effects were reproduced in RAW264.7 macrophages and were attenuated by 3-MA. In mouse infection models, Etravirine reduced bacterial burdens and inflammatory pathology and improved survival. Biophysical, hydrogen-deuterium exchange mass spectrometry (HDX-MS), docking, mutational, and preliminary analogue analyses supported a putative Etravirine-binding region near the PlcA myo-inositol-binding pocket. Together, these findings establish Etravirine as a PlcA-targeting antivirulence candidate that also strengthens host autophagy-associated restriction of L. monocytogenes.