Department of Veterinary Medicine Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2762
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Item IDENTIFICATION AND ENGINEERING BACTERIOPHAGE ENDOLYSINS FOR INACTIVATION OF GRAM-POSITIVE SPORE-FORMING BACILLI(2018) Etobayeva, Irina V.; Nelson, Daniel C.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation concentrates on the study of the antibacterial potential of bacteriophage-encoded endolysins derived from phages that infect the Gram-positive Bacillus cereus sensu lato group. Bacteriophage-encoded endolysins are peptidoglycan hydrolases that have been identified as important factors in the phage life cycle. Endolysins are encoded by phage late genes during an intracellular infection cycle to lyse the bacterial cell wall from within and allow phage progeny release. Endolysins derived from phages of Gram-positive bacterial hosts are equipped with an enzymatic domain that hydrolyzes conserved covalent bonds in bacterial peptidoglycan, and a cell wall binding domain that ensures proper attachment of endolysins to bacilli. In this study three novel endolysins, PlyP56, PlyN74, and PlyTB40 have been discovered and identified. The biochemical analysis shows that all three endolysins have relatively broad antimicrobial activity against organisms of the B. cereus group with the optimal lytic activity at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55°C), and at low concentrations of NaCl (<50 mM). The domain shuffling engineering studies were undertaken to observe enhancements of bacteriolytic properties of chimeric lysins that retained their specificity to B. cereus species. Finally, these studies have identified a new development in lysis of peptidoglycan of Gram-positive B. cereus group of organisms by phage-encoded endolysins. When grown to stationary phase, bacilli, especially, in overnight cultures become more resistant to lysis despite the evidence that the cell wall domains bind the bacterial surface. In light of these findings, I hypothesize that B. cereus group of species have evolved complex behaviors to interact with phage by modulating surface associated secondary polymers throughout the maturation of the bacilli in order to render them more resistant to the lytic action of phage encoded endolysins, which, contributes to bacterial survival from phage infection.Item Antagonizing JAK-STAT signaling by porcine reproductive and respiratory syndrome virus(2018) Yang, Liping; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is activated by numerous cytokines. JAK-STAT pathways involve in regulation of cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. Because of their significance in immune response, they are often targeted by pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes reproductive failure in sows and severe respiratory disease in pigs of all ages. A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response. One possible reason for the weak protective immune response is that PRRSV interferes with innate immunity and modulates cytokine signaling, including JAK-STAT pathways. The objective of this project was to elucidate the mechanisms of PRRSV interference with JAK-STAT2 and JAK-STAT3 signaling. This study demonstrates that PRRSV antagonizes interferon (IFN)-activated JAK-STAT2 signaling and oncostatin M (OSM)-activated JAK-STAT3 pathway via inducing STAT2 and STAT3 degradation. Mechanistically, PRRSV non-structural protein 11 (nsp11) and nsp5 induce the degradation of STAT2 and STAT3, respectively, via the ubiquitin-proteasome pathway. Notably, PRRSV manipulates karyopherin alpha 6 (KPNA6), an importin that is responsible for STAT3 nuclear translocation in the JAK-STAT signaling, to facilitate viral replication. Knockdown of KPNA6 expression led to significant reduction in PRRSV replication. These data demonstrate that PRRSV interferes with different JAK-STAT pathways to evade host antiviral response while harnessing cellular factors for its own replication. These findings provide new insights into PRRSV-cell interactions and its molecular pathogenesis in interference with the host immune response, and facilitate the development of novel antiviral therapeutics.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).Item Epidemiological Analysis of Biosecurity Practices and Associated Prevalence of Diseases in Non-Commercial Poultry Flocks(2012) Madsen, Jennifer Marie; Tablante, Nathaniel L.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A cross-sectional study was conducted in backyard poultry flocks among nine counties of Maryland from May 2011 to August 2011. The objective of this study was to obtain baseline data from a survey on biosecurity practices and investigate risk factors associated with positive findings of avian influenza (AI), Newcastle disease (ND), infectious laryngotracheitis (ILT), Mycoplasma gallisepticum (MG), and Salmonella Enteritidis (SE). Serum, tracheal, and cloacal swabs were randomly collected from 262 birds among 39 registered premises. Analysis revealed flock prevalence and seroprevalence respectively for the following: AI (0%, 23%), ND (0%, 23%), ILT (26%, 49%), MG (3%, 13%), SE (0%, ND). Vaccine status could not be confirmed for ND, ILT, or MG. Premises positives were identified by partial nucleotide sequencing. No statistically significant associations were identified, however, AI seroprevalence was positively associated with exposure to waterfowl (Relative Risk [RR] = 3.14, 95% confidence interval [CI] 1.1-8.9) and absence of pest control (RR=2.5; 95% CI, 0.6-10.4).Item REGULATION OF MACROAUTOPHAGY BY VITAMIN A/ RETINOIDS(2013) Rajawat, Yogendra Singh; Bossis, Ioannis; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Retinoic acids (RAs) have diverse biologic effects and regulate several cellular functions. Here, we investigated the role of RA on autophagy by studying its effects on autophagosome (AUT) maturation, as well as on upstream regulators of autophagosome biogenesis. Our studies, based on the use of pH-sensitive fluorescent reporter markers, suggest that RA promotes AUT acidification and maturation. By using competitive inhibitors and specific agonists, we demonstrated that this effect is not mediated by the classic Retinoic Acid Receptor (RAR) and Retinoid X Receptors (RXR). RA did not affect the protein expression levels of upstream regulators of autophagy, such as Beclin-1, phospho-mTOR, and phospho-Akt1, but induced redistribution of both endogenous cation-independent mannose-6-phosphate receptor CI-MPR and transiently transfected GFP and RFP full-length CI-MPR fusion proteins from the trans-Golgi region to acidified AUT structures. Those structures were found to be amphisomes (acidified AUTs) and not autophagolysosomes. The critical role of CI-MPR in AUT maturation was further demonstrated by siRNA-mediated silencing of endogenous CI-MPR. Transient CI-MPR knockdown resulted in remarkable accumulation of nonacidified AUTs, a process that could not be reversed with RA.These results suggest that RA induces AUT acidification and maturation by regulating CI-MPR subcellular location, a process critical in the cellular autophagic mechanism.