Department of Veterinary Medicine Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2762
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Item METABOLIC VIRULENCE DETERMINANTS AND RAPID MOLECULAR DIAGNOSTICS OF PATHOGENIC SPIROCHETES(2016) Backstedt, Brian; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Borrelia burgdorferi and Leptospira interrogans are pathogenic spirochetes that elicit serious health threats, termed as Lyme disease and leptospirosis. Key areas of spirochete research involve a better understanding of their intriguing biology and infection, including identification of novel virulence factors and improvements in diagnostic and preventive methods. Notably, certain bacterial metabolic enzymes are surface-exposed, having evolved to acquire additional functions referred to as protein moonlighting that contributes in microbial virulence. Comparative genome analysis revealed that certain components of sugar metabolism pathways are either absent or seemingly inactive in pathogenic spirochetes, which were studied herein for their potential roles as metabolic virulence factors. Of nine borrelial enzymes investigated, only phosphomannose isomerase (PMI) was found to be surface-exposed and remained enzymatically active in the spirochete outer membrane. PMI is critical for mannose metabolism and facilitates the interconversion of fructose 6-phosphate and mannose-6-phosphate, although its occurrence in borrelial surface remains enigmatic. PMI may provide a critical function for B. burgdorferi viability as it is constitutively expressed and all attempts to create genetic mutants remained unsuccessful. Active immunization studies using recombinant PMI did not influence the outcome of infection within tick or murine hosts, although a significant reduction in bacterial levels within the joints of mice was recorded, suggesting its involvement in spirochete persistence in a tissue-specific manner. Despite substantial advancement, the development of more effective diagnostics for leptospirosis and Lyme disease still remains a critical need since human vaccines are unavailable. Antibiotic treatment can resolve these infections but is most effective when administered early during infection, prior to pathogen dissemination to distant organs. As diagnostic methods for spirochete infection still depends on ineffective and antiquated technologies, we sought to develop novel RNA-based assays for better detection of early spirochete infection. Results indicated that targeting specific regions of 16S and 23S ribosomal RNA targets provided the highest possible sensitivity and specificity of detection, which was far superior to current serological, microbiological or molecular methods used to detect presence of invading pathogens.Item ROLE OF SELECT BORRELIA BURGDORFERI-INDUCIBLE TICK GENE-PRODUCTS IN PATHOGEN PERSISTENCE WITHIN THE VECTOR(2015) Smith, Alexis Ayn; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease, also known as Lyme borreliosis, a common vector-transmitted illness across the Unites States and Europe, is caused by the pathogen Borrelia burgdorferi, which is transmitted by Ixodes scapularis ticks. While ticks are known to transmit a diverse set of bacterial, protozoan and viral disease agents, there are only limited investigations addressing how Ixodes immune responses influence the survival or persistence of specific pathogens within the tick. In North America, I. scapularis transmits a wide array of human and animal pathogens including a group of pathogenic bacteria, known as Borrelia burgdorferi sensu lato complex. Due to the evolutionary divergence from other bacteria, and the possession of a unique cellular structure, B. burgdorferi cannot be classified as a conventional Gram-positive or Gram-negative bacteria, instead they are classified as a spirochete. Additionally, key pattern recognition molecules or PAMPs, such as lipopolysaccharides and peptidoglycans, are absent or structurally distinct in B. burgdorferi. Thus, the wealth of knowledge generated in other model arthropods, regarding the genesis of host immune responses against classical bacterial pathogens, might not be applicable to B. burgdorferi. The primary goal for this dissertation is to characterize components of the tick immune responses that modulate B. burgdorferi infection and use this information to better understand specific aspects of tick immunity as well as to contribute to the development of new strategies that interfere with pathogen persistence and transmission. The following aims were addressed: assessment of the expression profile of the I. scapularis innate immune transcriptome to identify genes that are induced in the B. burgdorferi-infected vector. Next, a select set of pathogen-inducible gene-products was further studied for their possible harmful or beneficial roles in pathogen persistence in the vector. Based on recent findings in other disease vectors as well data generated within this thesis, I particularly focused on characterization of two select sets of B. burgdorferi-inducible tick gene-products that are potentially involved in maintenance of gut microbe homeostasis (Dual oxidase and peroxidase) and events linked to phagocytosis (Rho GTPase).