Borrelia burgdorferi — the causative agent of Lyme borreliosis — accounts for ~500,000 infections in the United States per year. Relative to other bacteria, B. burgdorferi is highly unusual in many regards. For instance, the synthesis and composition of B. burgdorferi cell wall is extremely unique and plays a critical role in Lyme pathogenesis. The cell wall is made up of peptidoglycan (PG) - a mesh-like structure, composed of long rigid glycan strands of repeating sugars GlcNAc and MurNAc, and flexible peptide stems, interlinked by amino acid cross-bridges. PG is an essential component for survival of the bacterial cell, protecting it from the osmotic stress and environmental threats, as well as defining the shape of the bacterium and aiding in the motility. One unique feature of the B. burgdorferi PG is the chemical composition of stem peptide, which involves the atypical cross-link between Ornithine and Glycine. We identified gene bb0586 as a femX homologue in borrelial genome and hypothesize that it encodes a glycyl transferase enzyme responsible for synthesis of glycine cross-bridges, that hold together glycan strands in the peptidoglycan cell wall. Here, we predicted the structure of FemXBb, identified and characterized the substrate-binding site, and proposed a novel mechanism for substrate recognition and recruitment, involving previously uncharacterized elements of the structure. We have also determined the ability of recombinant FemXBb to add Glycine bridges to mDAP in E. coli and investigated the effect that femX knock-out can have on the B. burgdorferi. In addition, we have investigated the steps of PG biosynthesis in B. burgdorferi. The results of our research suggest the existence of a highly unusual mechanism of PG synthesis in Lyme disease spirochete, which has a potential to be used for development of targeted antibacterial therapies. / Master of Science in Life Sciences / Borrelia burgdorferi — the causative agent of Lyme borreliosis — accounts for ~500,000 infections in the United States per year. Relative to other bacteria, B. burgdorferi is highly unusual in many regards. For instance, the synthesis and composition of B. burgdorferi cell wall is extremely unique and plays a critical role in Lyme pathogenesis. The cell wall is a mesh-like structure, a sacculus, enclosing the vulnerable inside contents of a bacterial cell. It is composed of long rigid glycan strands of repeating sugars, and flexible peptide stems, interlinked by cross-bridges, holding the whole structure together. PG is an essential component for survival of the bacterial cell, protecting it from the outside stress and environmental threats, as well as defining the shape of the bacterium and aiding in the motility. B. burgdorferi PG is known to be highly atypical compared to other bacteria. One of its features is the unusual cross-link between peptides, made up of single Glycine amino acid. We identified a gene encoding a protein responsible for the addition of this amino acid during the cell wall biosynthesis. Here, we predicted the structure of this protein, its substrate-binding site, and proposed a novel mechanism for substrate recognition and recruitment. We have also expressed the borrelial protein in E. coli and confirmed its activity and the impact it has on the bacterium and investigated the effect that gene knock-out can have on the B. burgdorferi. In addition, we have investigated the steps of PG biosynthesis in B. burgdorferi. The results of our research suggest the existence of a highly unusual mechanism of PG synthesis in Lyme disease spirochete, which has a potential to be used for development of targeted antibacterial therapies.

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.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, a multisystem disorder that can cause a variety of disorders in susceptible mammalian hosts. The immune response of infected mammals, including humans, is ineffective in clearing B. burgdorferi as demonstrated by the ability to reisolate the spirochete from naturally and experimentally infected hosts after extended periods of time. Recent evidence suggests that this pathogen evades the immune response in part through changes in antigenic reactivity.The purpose of this study was to determine if outer surface protein A (OspA) of B. burqdorferi varies in the course of infection in Syrian hamsters and thus potentially plays a role in evading the host immune response. To assess the degree of change, differences in the binding of a murine monoclonal antibody (H5332) were measured using IFA and ELISA techniques over a 9-week period of time.Results of this study suggest that OspA is persistently expressed in infected Syrian hamsters for at least 9-weeks. Moreover, this protein, or at least the epitope that H5332 binds with, is stably expressed. These results indicate OspA, or at least the epitope of OspA that I probed, does not appear to contribute to the evasive mechanisms of 8. burgdorferi in Syrian hamsters. / Department of Biology

Thesis (Ph. D.)--University of Chicago, Dept. of Ecology and Evolution. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.) -- University of Rhode Island, 2009. / Typescript. Includes bibliographical references (leaves 188-202).

La maladie de lyme est une maladie infectieuse transmise pas les tiques. L'EUCALB (European Concerted Action on Lyme Borreliosis) et la FFI (fédération française d'infectiologie) ont mis en place des directives pour le diagnostic et le traitement de cette maladie. Mais, en France comme internationalement, il existe des oppositions et des ambiguïtés révélées par les différents discours de professionnels, de malades et d'associations de malades, essentiellement concernant la définition de la maladie, son diagnostic et la cure des symptômes persistants associés.Le travail consiste à suivre, à l'échelle nationale, la controverse et le débat public en soulignant les forces et les faiblesses de chaque camp en opposition. Il s'agit de mettre en lumière la contestation grandissante et la manière dont les idées, les pratiques et les théories évoluent. / Lyme disease is an infectious disease transmitted by ticks. EUCALB (European Concerted Action on Lyme Borreliosis) and FFI (French Infectiology Federation) have set up recommadations for this disease’s diagnosis and treatment. But, in France and internationally, oppositions and ambiguities exist, revealed by professionals, sufferers and patients groups discourses, essentially regarding the definition of the disease, his diagnosis and his persistant associated symptoms treatment.The work consists to closely follow, on a national scale, the controversy and the public discussion, emphasizing strengths and weaknesses of each side. The aim is to highlight the growing dispute and the way ideas, practices and theories change.

Lyme disease is the most common vector-borne disease in the United States with over 20,000 cases reported yearly. A common and sometimes severe symptom of Lyme disease is Lyme arthritis, which has clinical and etiological similarities to rheumatoid arthritis. While risk factors for Lyme disease are established, there have been no studies exploring risk factors for Lyme arthritis. To assess this relationship a cross-sectional study was conducted, using data from confirmed cases of Lyme disease reported to the Massachusetts Department of Public Health (MDPH) from 2000 to 2006. Bivariate analyses and ANOVA tests were conducted to assess the relationship between age, sex and Lyme arthritis, as well as other symptoms of Lyme disease. Results showed that those in the lowest quartile of age were more likely to be diagnosed with Lyme arthritis alone than those in higher age quartiles (p <0.001). No significant difference was seen in the proportion of Lyme arthritis diagnosis between males and females (p = 0.61). By recognizing that younger patients with Lyme disease are more likely to be diagnosed with Lyme arthritis, measures may be taken to improve early identification and treatment of Lyme disease in this group. We recommend that a future prospective study be conducted to further elucidate the true relationships between age, sex, and Lyme arthritis.

Despite the fact that eight cases of Lyme disease were diagnosed in San Luis Obispo County between 2005-2013, the identity of wildlife hosts serving as sources for tick infection in this region remained unidentified. The primary cause of Lyme disease in the U.S. is the spirochetal bacterium Borrelia burgdorferi sensu stricto, and this agent had not been previously isolated from the region. Borrelia bissettii, a related species that has not been implicated as a common causative agent of Lyme disease, was isolated in small rodents inhabiting coastal scrub and chaparral habitats in a previous San Luis Obispo County study. However, B. burgdorferi was not detected. In northwestern California, B. burgdorferi has been primarily associated with high populations of the tick vector Ixodes pacificus in dense woodlands or hardwood-conifer habitats, particularly in the western gray squirrel reservoir host, Sciurus griseus. My study investigated the role of S. griseus and other associated rodents as potential reservoirs for B. burgdorferi in central coastal California woodland habitats. Rodents were live-trapped at four sites in San Luis Obispo County in oak and mixed woodland. Rodent ear samples were tested for B. burgdorferi genospecies by bacterial culture and PCR. Ticks were collected from captured rodents and surrounding environments and tested by PCR for the presence of Borrelia. Of 119 captured rodents, seven were positive for Borrelia infection (5.9%) and of these, six were positive for B. burgdorferi (5.0%). There were multiple infected rodent species that included two western gray squirrels, three deer mice (Peromyscus maniculatus), and one brush mouse (P. boylii). Borrelia spp. were not detected by PCR from the 81 ticks recovered from the environment and rodents. Here, for the first time, we verify the presence of B. burgdorferi sensu stricto in San Luis Obispo county rodents. However, in contrast to previous Northern California studies, the western gray squirrel may not be the primary reservoir host for B. burgdorferi in this region. Multiple rodent species in oak woodlands may be involved in spirochete maintenance in San Luis Obispo County.

Since its initial identification in 1975, Lyme disease has become a public health concern in the U.S.  Increased concern is sparked by the rapid rate at which the disease is emerging into new areas.  One area of disease emergence is the state of Virginia which has been experiencing exponentially increasing rates of the disease.  This research studies Virginia's landscape-level habitats to explore demographic and environmental variables related to the spread of Lyme disease. The land cover data came from the National Land Cover Database (2006), demographic data came from the U.S. census (2010), and Lyme disease case data came from the Virginia Department of Health (2006-2010).  Key variables examined in this statewide study include the percentages of landscape types measured inside each census tract, measures of forest fragmentation, and measures of land cover interspersion inside state census tracts. Analysis was carried out using a spatial Poisson regression model.  Of the original 15 variables, 10 were significantly correlated to Lyme disease.  The six that were positively correlated with disease incidence include percent herbaceous land, percent water, two edge contrast measurements of herbaceous-forest land, median age, and average income.  The four that were negatively correlated were percent developed, population density, and two edge contrast measurements of developed-herbaceous land. Overall results indicate that specific environmental and demographic variables are associated with increased disease incidence as Lyme disease emerges in Virginia.  Results from this study could help create a predictive statewide map for Lyme disease incidence and aid in disease awareness and resource allocation. / Master of Science

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.