Date of Award

Spring 2026

Document Type

Thesis

Degree Name

Master of Science (MS)

Program/Concentration

Biology

Committee Director

Rishi Drolia

Committee Member

Wayne Hynes

Committee Member

Lisa Shollenberger

Committee Member

Isaura Simões

Abstract

Listeria monocytogenes is a facultative intracellular foodborne pathogen capable of crossing the placental barrier, leading to miscarriage, stillbirth, and neonatal sepsis, and has a fetal/neonatal fatality rate of ~20–30%. Although the importance of canonical virulence factors such as Internalin A (InlA) and Internalin B (InlB) in L. monocytogenes crossing of the placental barrier has been demonstrated, clinical strains lacking functional canonical internalins have been reported to cause severe neonatal disease, suggesting additional mechanisms of placental translocation.

To investigate these virulence mechanisms, we adapted a physiologically relevant co-culture human placental barrier Transwell model composed of BeWo trophoblasts and primary human placental vascular endothelial cells (HPVECs). Co-culture significantly enhanced barrier integrity, as demonstrated by increased transepithelial electrical resistance (TEER), with resistance values increasing by ~92% compared with BeWo monocultures and by ~302% compared with HPVEC-only cultures.

Extracellular translocation assays demonstrated that the lap⁻ and ΔinlA mutants exhibited ~75% and ~94% reductions in bacterial translocation, respectively, compared with the wild-type (WT) strain F4244. In contrast, the ΔinlB mutant translocated at levels comparable to WT, whereas the Δhly mutant showed a modest non-significant reduction relative to its respective WT strain 10403S.

To examine monocyte–mediated translocation, THP-1 monocytes infected with WT or mutant strains were applied to the apical compartment under gentamicin protection. Monocytes infected with the lap⁻ and ΔinlB mutants exhibited ~76% and ~57% reductions in transmigration compared with WT, with intracellular bacterial recovery from transmigrated monocytes reduced by 89–92%. Infected monocytes also induced increased secretion of adhesion molecules, including P-selectin (2.25-fold), BCAM (2.38-fold), and VCAM-1 (1.73 fold). Consistent with reduced transmigration, lap⁻ and ΔinlB-infected monocytes elicited diminished soluble VCAM-1 secretion, comparable to that of L. innocua–infected monocytes.

Collectively, these findings support that LAP and InlA contribute to extracellular bacterial translocation, while LAP and InlB contribute to monocyte-mediated transmigration across the in vitro human co-culture placental barrier model.

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DOI

https://doi.org/10.25777/42fg-5s77

ISBN

9798197810649

ORCID

0009-0009-4869-667X

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