Peptidoglycan (PG) is an essential cell-wall biopolymer in virtually all bacteria. It is composed of glycan strands of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) crosslinked by peptide chains of alternating D- and L- amino acids and diamines. PG plays an important role in 1) cell elongation and division, 2) cell strength and morphology, 3) antibiotic susceptibility, and 4) host immune detection and modulation. While differences in peptide chains are common, deviations in glycan strand composition were not previously known to occur. Here, we provide characterization of the first known deviation to bacterial glycan strand composition — GlcNAc-GlcNAc-anhMurNAc (G-G- anhM) in Borrelia burgdorferi, the causative agent of Lyme disease. B. burgdorferi with less G-G-anhM were found to be significantly less motile, flexible, and stress-tolerant while possessing gross morphological defects and less overall PG. Our studies also characterized the muropeptide profile of Borrelia afzelii, Borrelia garinii, and Borrelia hermsii — species of Borrelia associated with causing different disease manifestations of Lyme disease, and relapsing fever, respectively. These species were found to incorporate appreciable amounts of G-G-anhM into their PG, suggesting an evolutionary adaptation to life inside a tick that predates the differentiation of Lyme disease and relapsing fever Borrelia. Finally, we provide partial characterization of a putative penicillin-binding protein in B. burgdorferi — a class of highly conserved PG synthesis enzymes present in the vast majority of bacteria. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia. / Master of Science in Life Sciences / Peptidoglycan (PG) is the main cell-wall component in the vast majority of bacteria. PG is composed of strong, rigid sugars linked together by short, flexible amino acid chains, and resembles a mesh-like bag that surrounds the cell. In nearly all bacteria that have PG, it plays an important role in how 1) the cell grows and divides, 2) the cell dictates its shape, 3) antibiotics treat bacterial infections, and 4) the human body detects and responds to a bacterial infection. While the amino acids that make up PG are known to differ between bacterial species, deviations in sugar organization are not known to occur. Here, we characterize the first known deviation to sugar organization in bacterial PG in Borrelia burgdorferi — the bacteria that causes Lyme disease. B. burgdorferi with less of this deviation possess defects absent in their normal counterparts. In addition, we show that other Borrelia species that cause a variety of different diseases around the world mimic this sugar deviation, suggesting the majority, if not all, of Borrelia may do so. Finally, we provide partial characterization of the function of an enzyme thought to synthesize PG in B. burgdorferi. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia.

CwlD has sequence similarities to N-acetyl muramoyl-L-alanine amidases, a class of enzymes known to cleave the bond between the peptide side chain and the N-acetyl muramic acid residue in cortex peptidoglycan formation during sporulation. A major difference between vegetative peptidoglycan and spore peptidoglycan is the presence of muramic-<FONT FACE="Symbol">d</FONT> -lactam (MAL) in spore peptidoglycan. It was previously determined that a <I>cwlD</I> null mutant does not contain muramic-<FONT FACE="Symbol">d</FONT> -lactam in the spore cortex peptidoglycan and the mutant spores were unable to complete germination. Therefore, it is believed that CwlD plays a role in MAL formation during sporulation. However, the specific role of the protein had not been demonstrated. It was also previously found that <I>cwlD</I> is in a two-gene operon with <I>orf1</I>. Orf1 is produced within the forespore with CwlD. The hypothesized role of Orf1 is to inhibit CwlD activity from within the forespore. Muramoyl-L-alanine amidase activity was demonstrated by CwlD <I>in vivo</I>. Therefore, CwlD is carrying out the first step of MAL synthesis, cleaving the peptide side chain while other enzymes are needed to complete MAL formation. Two different forms of CwlD were over-expressed, with and without the protein's signal peptide sequence. Both forms of the protein were purified and in both cases activity was undetectable. Antibodies specific for CwlD were obtained which can be used in future research as a tool to further characterize CwlD activity. A series of <I>B. subtilis</I> <I>cwlD</I> operon mutants were constructed altering the expression patterns of Orf1 and CwlD within the mother cell and forespore compartments. Various resistance properties and the germination ability of the mutant dormant spores were analyzed. It was determined that the absence of just Orf1 or Orf1 and CwlD from within the forespore has no effect on the phenotypes tested. Peptidoglycan from developing mutant forespores was extracted and analyzed throughout sporulation. Evidence was obtained demonstrating that the role of Orf1 is not to inhibit CwlD from within the forespore as hypothesized. / Master of Science

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton. The PG synthases that build this structure are called penicillin-binding proteins (PBPs). Since they are the targets of penicillin and related antibiotics, the structures and in vitro biochemical functions of the PBPs have been extensively studied. However, the in vivo functions of the PBPs and the factors they work with to build the PG meshwork remain poorly understood. PBPs work in the context of multicomponent complexes organized by cytoskeletal elements. A major outstanding question has been whether or not these complexes contain factors required for PBP function. I addressed this using Escherichia coli as a model system by taking advantage of the synthetic lethal phenotype resulting from simultaneous inactivation of the major PG synthases: PBP1a and PBP1b. Using a screen for mutants synthetically lethal with the inactivation of PBP1b, I identified LpoA as a factor required for PBP1a function. A colleague in the lab performed the analogous screen for mutants synthetically lethal with the inactivation of PBP1a and identified LpoB as a factor required for PBP1b function. We showed that the Lpo factors are outer membrane lipoproteins that form specific trans-envelope complexes with their cognate PBPs in the inner membrane and that LpoB can stimulate the activity of PBP1b in vitro. Our results reveal unexpected complexity in the control of PBP activity and indicate that they likely receive regulatory input from the outer membrane in addition to cytoskeletal elements in the cytoplasm. To investigate the role of LpoB in morphogenesis further, I took a genetic approach that has identified PBP1b* variants capable of functioning in vivo in the absence of LpoB. Preliminary characterization of these variants indicates that LpoB has cellular functions in addition to PBP1b activation and that LpoB may be important for coordinating the two different catalytic activities of PBP1b. Future study of these mutants is likely to uncover important insights into PBP function and their control by the Lpo factors. These insights may open new avenues for the development of novel therapeutics that target the PBPs.

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

Peptidoglycan glycosyltransferases (PGTs) are highly conserved bacterial enzymes that catalyze the polymerization of the lipidic disaccharide, Lipid II, to form individual peptidoglycan (PG) strands which are subsequently cross-linked to form mature PG, the major skeletal component of the bacterial cell wall. Recent advances in the preparation of well-defined PGT substrates have enabled the biochemical characterization of Lipid II polymerization by the PGTs. In the course of these studies, we have observed that a distinctive lag phase in the initial rate of PG synthesis by the PGTs can be abrogated if the enzyme is preincubated with Lipid IV, the shortest PG fragment. The origins of this lag phase are intriguing because the chemical transformation involved in coupling Lipid II to yield Lipid IV is identical to the transformation involved in the synthesis of longer PG fragments from Lipid II. Crystallographic structures of the PGTs with Moenomycin A, an inhibitor that is believed to bind to the same site as Lipid IV, suggest that the PGTs possess flexible regions near the putative active site that can undergo substrate-induced conformational changes to accelerate PG synthesis. However, there is currently no structural evidence on how the PGTs interact with its substrates. The work in this thesis lays the foundation for pursuing a solution structure of a Lipid IV bound PGT complex by Nuclear Magnetic Resonance (NMR) spectroscopy, enabling the study of important enzyme conformational states and structural dynamics involved in PG synthesis. Specifically, Chapter 2 of this thesis presents the biochemical evidence that the preincubation of the PGTs with a Lipid IV derivative, Gal-Lipid IV abrogates the lag phase and accelerates the initial rate of PG synthesis. Chapter 3 presents a robust methodology for obtaining multimilligram quantities of isotope labeled, monodisperse and monomeric SgtB, a PGT from a clinically relevant pathogen, Staphylococcus aureus for solution structural studies. Chapter 4 describes the systematic development of a methodology for producing a well-behaved, stable sample of Moenomycin A bound SgtB for NMR spectroscopy. Chapter 5 delineates the adaptation of the methodology described in Chapter 4 for pursuing the solution structure of Lipid IV bound SgtB. / Chemistry and Chemical Biology

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

Antibiotics are small molecules that kill bacteria by inhibiting essential processes. However, the concentrations used to kill bacteria in a clinical setting are typically much higher than the concentrations generated in nature, where most antibiotics are secreted by microbes. This discrepancy in concentrations, combined with a recognition that the human use of antibiotics bears little resemblance to the role of antibiotics in nature, prompted questions about whether growth inhibition was the primary function of antibiotics. Studying the effects of antibiotics at sub-lethal concentrations on bacteria could provide new insights into the natural role of antibiotics. One striking effect of bacterial encounters with sub-lethal antibiotics is the stimulation of biofilm formation. Biofilms are surface-adhered communities of bacteria. The biofilm lifestyle confers many benefits for bacteria and is a major mode of bacterial growth. Therefore, the ability of sub-lethal antibiotics to cause a transition from planktonic to biofilm growth indicates that antibiotics could be a driving force behind the assembly and abundance of bacterial communities in nature. Chapters Two and Three investigate the underlying mechanisms of this response in Escherichia coli and Pseudomonas aeruginosa, and suggest that sub-lethal antibiotics perturb central metabolism and respiration, changes that are sensed and relayed into increased biofilm formation to provide population-level protection. Chapters Four and Five investigate the effects of sub-lethal antibiotics on peptidoglycan metabolism in P. aeruginosa and E. coli. Peptidoglycan is an essential macromolecule for bacterial survival and is deeply integrated into their physiology. Furthermore, peptidoglycan synthesis is among the most favoured targets of antibiotics. Chapter Four investigates interactions between peptidoglycan-targeting antibiotics and folate metabolism-targeting antibiotics, and characterizes an overlooked connection between folate and peptidoglycan metabolism. Based on this work, we rationally designed a new inhibitor that potentiates folate and peptidoglycan-targeting antibiotics. Chapter Five sheds new light on peptidoglycan recycling by leveraging a pathway in P. aeruginosa for sensing and responding to sub-lethal doses of PG-targeting antibiotics. Finally, Chapter Six summarizes the understanding gained from Chapters Two through Five and synthesizes this information for broader insights on the possible roles of antibiotics in nature. / Thesis / Doctor of Philosophy (PhD) / The ability to cure infections with antibiotics revolutionized modern medicine and kick-started decades of research into the growth inhibitory properties of antibiotics. Although the therapeutic role of antibiotics as anti-bacterials is clear, the natural role of antibiotics is not. In nature, microbes export antibiotics, allowing them to interact with surrounding microbes that import those antibiotics, changing the physiology of the recipient. Understanding how antibiotics affect bacterial physiology at concentrations below the lethal dose can provide information about the natural role of antibiotics, which in turn can inform future antibiotic discovery. This thesis investigates two major effects of sub-lethal antibiotics on pathogenic bacteria. The first is the ability of antibiotics to induce the formation of surface-attached clusters of bacteria called biofilms. We showed that sub-lethal antibiotics have a common effect of disrupting cell metabolism, and this effect is translated into a signal for increased biofilm formation. The second is the effects of antibiotics on bacterial cell wall metabolism. We discovered that sub-lethal antifolate antibiotics impact cell wall metabolism in at least two different ways, and used this information to rationally design an inhibitor that overcomes antibiotic resistance. Further, sub-lethal antibiotics were used to identify new features of a cell wall recycling pathway. Overall, this work furthers our understanding of bacterial physiology at scales ranging from sub-cellular to multi-cellular, and reveals new impacts of sub-lethal antibiotics.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses, such as acidic conditions and reactive oxygen species, as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation through the use of small regulatory RNAs (sRNAs). sRNAs bind to messenger RNA (mRNA) targets via complementary base pairing. sRNAs are a class of regulatory molecules in bacteria that elicit rapid post-transcriptional regulation. sRNA-mRNA binding can positively or negatively influence gene expression. Positive regulation can occur through sRNA binding to protect the mRNA from RNases. sRNA binding can also alleviate the secondary structure and reveal the ribosomal binding site. Alternatively, sRNA-mRNA interactions can have negative consequences on gene expression through degradation via RNases or sRNA binding can occlude the ribosomal binding site. Although some sRNAs have been discovered in B. abortus, few have been characterized in regards to virulence. In this study, B. abortus was stressed in conditions relevant to the macrophage, including, including low pH, oxidative stress, and nutrient limitation. Transcriptomic analysis revealed high levels of transcripts in intergenic regions, a hallmark of sRNAs, which led to the discovery of VcrS for virulence and cell wall regulating sRNA. A ΔvcrS was engineered and this mutant was used to infect both naïve murine macrophages, as well as BALB/c mice. Both virulence studies demonstrated significantly decreased bacterial recovery of ΔvcrS compared to the wildtype strain. Quantitative proteomics revealed that one protein, BAB1_1454, is 30-fold over-produced in ΔvcrS compared to wildtype. This essential protein encodes MurF, which catalyzes the final cytoplasmic step of generating the mura-pentapeptide precursor for peptidoglycan synthesis. VcrS is hypothesized to interact with murF mRNA and interfere with translation initiation. Sequence data indicates a putative 6 nucleotide motif in VcrS that has complementarity to the ribosomal binding site of murF. Identification of the binding site and further characterization of VcrS will showcase the importance of sRNA regulation in the virulence of B. abortus. / Master of Science / Brucella abortus is a bacterial pathogen that primarily infects cattle but is also transmitted to humans. Human disease most commonly results from the consumption of unpasteurized milk and milk products. Human brucellosis has very limited treatment options, with a high incidence of disease relapse. B. abortus survives and replicates within immune cells, which create a harsh environment. However, the bacteria are able to sense and adapt to survive and replicate within these immune cells, maintaining a chronic infection. A better understanding of the adaptation process B. abortus utilizes to survive within the human host can lead to improvement of treatment options. The present work characterizes a novel regulatory small RNA- VcrS, which was found required for survival and replication inside immune cells

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.