Deciphering the bacterial and host interactions driving listeria monocytogenes paracellular translocation across the blood-brain barrier

The National Institute of Allergy and Infectious Diseases awarded Old Dominion University $157,982 on August 1, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to support research deciphering how Listeria monocytogenes breaches the blood-brain barrier and causes neurolisteriosis.

The research focuses on defining the mechanism by which Listeria adhesion protein (LAP) enables bacterial translocation across the blood-brain barrier through paracellular pathways, while providing hypothesis-driven research training for graduate and undergraduate students in molecular microbiology, neurobiology, and proteomics. The study will evaluate LAP's role in promoting neuroinvasion in physiologically relevant in vivo and in vitro models. The project addresses the clinical challenge that neurolisteriosis presents as meningitis in approximately 90 percent of cases with a fatality rate near 30 percent despite antibiotic therapy, and that emerging multidrug-resistant strains require novel mechanism-driven interventions.

The work is performed in Norfolk, Virginia. The period of performance runs from August 1, 2026, through July 31, 2030. Old Dominion University Research Foundation administers the award on behalf of Old Dominion University as the receiving institution.

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.