Theses and Dissertations from UMD
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Item STRUCTURE-FUNCTION ANALYSES OF AN ESSENTIAL VIRULENCE DETERMINANT OF THE LYME DISEASE PATHOGEN(2022) Foor, Shelby Dimity; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme Disease (LD) is a tick-borne disease caused by a group of gram-negative-like spirochetal pathogens called Borrelia burgdorferi sensu lato. The number of cases reported in the United States have dramatically increased with CDC estimating 476,000 cases annually. This multifaceted infection can spread throughout the entire body, causing clinical complications of the central nervous system, joint and heart. Early antibiotic treatment is available and effective; however, untreated patients can develop chronic symptoms, and even after antibiotics, symptoms of unknown etiology and pathogenesis can develop into post-treatment Lyme disease syndrome (PTLDS). The enzootic life cycle of B. burgdorferi is maintained typically between a small rodent and the Ixodes tick vector, where transmission occurs during tick feeding on a host. Infection establishes after B. burgdorferi is deposited in the dermis and undergoes the required shift in its protein expression profile necessary to support spirochete persistence and pathology, often highlighting protein targets for development of diagnostic, therapeutic, and preventative measures. Two such proteins identified, BB0238 and BB0323, serve as novel virulence determinants and are essential for mammalian infection. These two proteins directly interact, mutually stabilize each other post-translationally, and form an essential complex required for infection; however, their precise functions remain undetermined. In collaborative efforts, we predicted a two-domain structure of BB0238. The N-terminal domain was predicted by AI methods to harbor an antiparallel helix-turn-helix motif (HTH) followed by a third helix and a low-confidence predicted meandering segment. The C-terminal domain structure was determined by X-ray crystallography as well as predicted with high confidence to adopt an α+β fold that resembles closely that of the nuclear transport factor 2 (NTF2) superfamily. While full-length BB0238 lacks homology to singular proteins of known functions, the individual N- and C-terminal regions display structural homology to non-bacterial proteins, particularly to eukaryotic sorting, or transport proteins, suggesting that BB0238 supports an unconventional function in spirochetes. We discovered that BB0238 binds another borrelial protein annotated as BB0108, orthologs of two bacterial chaperones and foldases, the extracellular membrane anchored PrsA, and the periplasmic SurA. This identified interaction requires further investigation, however, may be important for BB0238 protein stability or assist with the novel BB0238 function discovered herein, which regulates proteolytic processing of BB0323. Furthermore, We show that key amino acid residues within the HTH stabilize BB0238 in an environment-specific manner, influence its oligomerization properties, and facilitate tick-to-mouse transmission by aiding spirochete evasion of host cellular immunity, underscoring BB0238’s ability to support microbial establishment during early mammalian infection. Together, these studies highlight the divergent evolution of multidomain spirochete proteins involved in multiplex protein-protein interactions, possibly facilitating multiple functions, which support pathogen survival and thus, represent novel targets for vaccine and therapeutic development against Lyme disease.Item Novel surface proteins in the pathogenesis and diagnosis of Lyme disease(2010) Coleman, Adam Steven; Pal, Utpal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease is caused by an infection with the spirochete Borrelia burgdorferi. Transmitted between mammal reservoirs by the bite of an Ixodes tick, the pathogen exists in a complex life cycle that requires long-term persistence in arthropod and mammal hosts. The mechanisms responsible for persistence and the pathogenesis of Lyme disease are not well understood, but may involve interactions between bacterial surface proteins and the host. Previous experiments have shown that differential gene expression of surface proteins assists the pathogen in adaptation and persistence in a new host. Most B. burgdorferi surface proteins have no homology to known proteins, making the identification of virulence factors difficult. Gene expression analyses can be used to identify potentially important gene products for further study, based on the conditions under which they are expressed. To this end, the B. burgdorferi in vivo transcriptome of selected potential surface proteins was analyzed to identify promising targets for further study. Based on these analyses and other observations from the literature, the lipoproteins BbCRASP-2 and BBK07 were selected for further characterization. My hypothesis is that these proteins are important for B. burgdorferi virulence and persistence in the murine host. The surface exposure of each protein was assessed, as well as a detailed transcriptional profile of each gene. Using specific antibody-mediated interference and gene inactivation, I show that neither BbCRASP-2 nor BBK07 is essential for infectivity or pathogenesis in the murine model of Lyme disease. My results also indicate that BBK07 is a novel immunodominant antigen of B. burgdorferi that could be used as a serodiagnostic marker for human Lyme disease. Using a peptide library, the most immunodominant epitopes of BBK07 were identified, and shown to improve the diagnostic accuracy over that of the full-length recombinant BBK07. Finally, I show that BBK07-based diagnosis was sensitive even in the early stages of Lyme disease, and that the addition of BBK07 epitopes to current serodiagnostic assays could improve their sensitivity.