Deciphering the Bacterial and Host Interactions Driving Listeria monocytogenes Paracellular Translocation Across the Blood-Brain Barrier - PROJECT SUMMARY/ABSTRACT Listeria monocytogenes (Lm) is a highly invasive foodborne pathogen that results in neurolisteriosis in ~31% of listeriosis cases. This severe central nervous system (CNS) infection typically presents as meningitis (~90% of cases) and has a fatality rate approaching 30% despite antibiotic therapies. Although host immunosuppression is a known risk factor, the molecular mechanisms that enable Lm to breach the blood-brain barrier (BBB) remain poorly defined. The canonical invasion factors internalins (InlA/B) are essential for intestinal and placental entry but are insufficient to explain CNS infection because clinical neuroinvasive isolates lacking these factors still cause disease. Compounding the challenge, multidrug-resistant (MDR) Lm strains are emerging, underscoring the urgent need for novel mechanism-driven interventions. Our long-term goals are to elucidate how Lm invades the CNS and to develop targeted interventions for neurolisteriosis. Our preliminary data in vitro and in vivoidentify Listeria Adhesion Protein (LAP) as a critical virulence factor that promotes paracellular BBB crossing. The overall objective is to define the mechanism by which LAP enables Lm translocation across the BBB, while providing a rigorous, hypothesis-driven research training experience for graduate and undergraduate students in molecular microbiology, neurobiology, and proteomics. We hypothesize that LAP promotes paracellular neuroinvasion by disrupting endothelial tight junctions through a novel non-canonical, receptor-mediated pathway, independent of internalins and the Hsp60/MLCK axis. We will test this hypothesis in physiologically relevant in vivo and in vitro models through two Specific Aims: Aim 1: Define the role of LAP in promoting Lm neuroinvasion. We’ll define how LAP promotes Lm neuroinvasion by evaluating BBB disruption and bacterial translocation in mice infected with clinically neuroinvasive wild-type (F4244, Lineage 1, serovar 4b), isogenic modified murinized InlA (InlAm) strain with enhanced InlA binding to mouse E-cadherin, InlA-mutant (ΔinlA), LAP-mutant (InlAmlap─), and LAP- complemented (InlAmlap─lap+) strains. In vivo bacterial tissue burdens, BBB permeability, junctional protein expression, and inflammatory responses will be quantified. In parallel, we’ll use a human brain endothelial cell (HBEC-5i) triculture BBB model to assess LAP-driven bacterial adhesion, translocation, and junctional integrity. Aim 2: Identify and validate LAP-binding endothelial receptor(s) mediating BBB disruption. Candidate receptors will be identified via affinity purification, LC-MS/MS, and co-immunoprecipitation. Functional validation will include receptor knockdown, blocking antibody assays, and colocalization studies by confocal microscopy to assess their role in LAP-mediated barrier disruption and Lm translocation. This research is innovative in targeting non-canonical, internalin-independent mechanisms of CNS invasion using clinical neuroinvasive strains and advanced BBB models. It is significant because it will uncover novel host-pathogen interactions essential for Lm neuroinvasion, providing new molecular targets for therapeutic intervention and enhancing the biomedical research capacity at Old Dominion University, preparing the next generation of biomedical scientists.
