Author ORCID Identifier
Date Available
5-1-2024
Year of Publication
2024
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Medicine
Department/School/Program
Microbiology, Immunology, and Molecular Genetics
First Advisor
Dr. Sarah D'Orazio
Abstract
Listeria monocytogenes is a facultative intracellular pathogen transmitted through the consumption of contaminated food products. Typically, infections range from mild, self-limiting gastroenteritis to life-threatening systemic infections; however, the events that occur in the gut to allow for this spread are unclear.
The focus of my thesis aims to determine how L. monocytogenes escape the mesenteric lymph nodes (MLN), the final barrier to systemic spread for both commensal and pathogenic bacteria in the gut. I have shown that intracellular replication of L. monocytogenes in an as-yet-unidentified cell type is essential for the colonization and dissemination of the bacteria from the MLN. Intracellular replication protected L. monocytogenes from clearance by monocytes and neutrophils in the intestinal tissue, thereby promoting colonization of the MLN. I developed an in vitro assay to measure free lipoate concentration and determined that intestinal tissue had enough lipoate to support LplA2-dependent extracellular growth of L. monocytogenes, but exogenous lipoate in the MLN was severely limited. Thus, the bacteria could replicate only inside cells in the MLN, where they used LplA1 to scavenge lipoate from host peptides. I also found that intracellular replication is required for actin-based motility and cell-to-cell spread and showed that this intracellular function is vital for rapid exit from the MLN. Given these results, I developed a model of systemic spread in which L. monocytogenes must invade, escape the phagocytic vacuole, replicate, and undergo actin-based motility in the cytosol, in a critical cell type in the MLN that provides access to the bloodstream.
I focused my attention on lymph node stromal cells, specifically fibroblastic reticular cells (FRC) and blood endothelial cells (BEC), which make up the high endothelial venules within the MLN and could allow L. monocytogenes direct access into the blood. These cells comprise less than 1% of the lymph node cellularity, but I sort-purified these tiny subsets and developed ex vivo assays to show that L. monocytogenes could replicate exponentially, undergo actin-based motility, and induce an IFN-beta response within the cytosol of both FRC and BEC in vitro. Infected FRC and BEC also produced a robust chemokine and pro-inflammatory cytokine response during in vitro infection. Flow cytometric analysis confirmed that GFP+ Lm were associated with stromal subsets in vivo following foodborne infection of mice, and ex vivo cultures revealed that the L. monocytogenes associated with these cells were viable, replicating bacteria. In BEC in particular, we found using fluorescence microscopy that the number of intracellular bacteria increased over the course of a three-day infection. These results suggest that FRC and BEC could be an essential growth niche for L. monocytogenes in the MLN.
These additional data have refined my model. I predict that Lm invades perivascular FRC that surround the BEC in the T cell zone of the lymph node. Once taken up, L. monocytogenes replicate in the cytosol and use actin-based motility to spread into the underlying BEC which they cannot otherwise invade efficiently. Despite inefficient invasion, L. monocytogenes replicate much faster within BEC compared to FRC. I hypothesize that exponential replication of L. monocytogenes within these cells results in lysis or membrane damage that allows L. monocytogenes to escape extracellularly into the blood. Overall, this work lays the groundwork for future studies aimed at determining the key events that must occur in the MLN that permit the systemic spread of L. monocytogenes.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2024.73
Recommended Citation
Tucker, Jamila S., "THE ROLE OF INTRACELLULAR GROWTH DURING THE SYSTEMIC SPREAD OF LISTERIA MONOCYTOGENES" (2024). Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics. 31.
https://uknowledge.uky.edu/microbio_etds/31
