As of 2024, more than 50,000 people suffer from Lyme arthritis — a debilitating late-stage symptom of Lyme disease. Symptoms remain even after the completion of antibiotic therapy and when there is no longer any indication of an active infection. Studies have found that a portion of the bacterial cell wall from the causative agent, Borrelia burgdorferi, is a persistent antigen in Lyme arthritis patients, lingering within the synovial fluid. This antigen, peptidoglycan, is recognized by the immune system in numerous ways. Multiple publications have shown that peptidoglycan is proinflammatory and can cause arthritis when injected in vivo. The same was found to be true for B. burgdorferi peptidoglycan. Studies focused on the structure of peptidoglycan from B. burgdorferi have shown atypical differences in both glycan and peptide chemistry that likely alter immune recognition. Due to a lack of necessary enzymes and transporters B. burgdorferi are unable to recycle their peptidoglycan as they elongate and produce daughter cells. This leads to a 45% reduction of their total cell wall that is released into the environment. The work detailed below focuses on this antigen to further our knowledge as to its in vivo biodistribution pattern, half-life, and ability to induce arthritis. For these experiments B. burgdorferi peptidoglycan (pBb PG) was purified, fluorescently labeled, and tracked in vivo to study its clearance pattern and rate. Three different mouse models for Lyme arthritis were utilized in these studies and all experienced persistence of B. burgdorferi peptidoglycan in their liver for upward of 20 days. There were differences in the rate of clearance between types of mice, suggesting the involvement of host genetics. Serum collected weekly throughout this experiment showed over a log fold change in the abundance of ALT and AST levels, which indicates liver dysfunction. Proteomic analysis of the livers of mice post pBb PG injection showed altered levels of proteins important for mitochondrial function and iron homeostasis. When human PBMCs were stimulated with PG from various bacteria it was found that at 12 h pBb PG differentially expressed genes involved in energy metabolic pathways, including oxidative phosphorylation and the citric acid cycle. A subset of Lyme disease patients continue to experience symptomology even after completion of multiple rounds of antibiotics. These patients are termed to have post treatment Lyme disease syndrome and typically experience fatigue as their most common symptom. This symptom in combination with the findings of this dissertation regarding the link between pBb PG and energy metabolism warrants further investigation. Especially since this biopolymer has been found to persist in the synovial fluid of Lyme arthritis patients. Better understanding how these processes are connected could allow for the eventual development of a way to target this material for clearance, or ways to inactivate it. Both options have the potential to help alleviate the devastating symptomology experienced by patients. / Doctor of Philosophy / Lyme disease is the most common human disease originating from a nonhuman host in the United States, with the estimated number of cases ~500,000 each year. This disease is caused by the bacterium Borrelia burgdorferi, that is transmitted by the black legged tick. This disease usually causes flu-like symptoms and if left untreated can cause more severe symptomology like arthritis, carditis, and neurological symptoms. Lyme arthritis is the most common late-stage symptom of this disease. Current areas of weakness within the field include ways to diagnose this disease, the treatment options, and our understanding of how these bacteria cause the symptoms they do. Recent work has made strides in studying Lyme arthritis, suggesting a major contributing factor to be a specific component of the bacterial cell wall that continues to persist. This component is called peptidoglycan and has been found in Lyme arthritis patients even after they've finished antibiotic therapy. Studies have also shown that the structure of this cell wall component is unique in comparison to other well-known bacteria. The research conducted as a part of this dissertation aims to investigate how this bacterial peptidoglycan is able to persist within patients for so long. To study this we utilized three mouse models of Lyme disease that all develop different severities of Lyme arthritis. By isolating the peptidoglycan from B. burgdorferi and labeling it with a molecule that fluoresces, we were able to track it over time in mice. We found that in all three mouse backgrounds peptidoglycan from B. burgdorferi persists for extended periods of times in the liver. We tested peptidoglycan from other common bacteria and found that they rapidly clear the mice. This suggests that there is something about the structure of B. burgdorferi's peptidoglycan that allows it to go unnoticed by the body for so long. Since this material is persisting within the liver we wanted to test if these mice had altered liver function. We found increased serum levels of enzymes that are indicators of overall liver health, suggesting some form of dysregulation. We also measured the total abundance of proteins in the livers of these mice in comparison to healthy controls. The mice injected with B. burgdorferi peptidoglycan had changes in the level of proteins involved in energy production and iron utilization. By measuring changes in gene expression, we confirm the specificity of these results to peptidoglycan from B. burgdorferi, even when using cells isolated from humans. One of the major conundrums of Lyme disease are the patients who continue to experience symptomology even after treatment, who are referred to as having post treatment Lyme disease syndrome. The primary symptom affecting these patients is fatigue, drawing an interesting parallel to our recent studies showing that B. burgdorferi peptidoglycan seems to be impacting energy metabolism. These findings warrant further investigation into the exact way in which B. burgdorferi peptidoglycan is affecting this process, which will hopefully lead to the generation of more targeted therapies to help alleviate this symptomology.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/119087 |
| Date | 23 May 2024 |
| Creators | McClune, Mecaila Elizabeth |
| Contributors | Biochemistry, Jutras, Brandon L., Helm, Richard F., Allen, Irving Coy, Tu, Zhijian |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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