Department of Veterinary Medicine Theses and Dissertations
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Item MOLECULAR DISSECTION OF BORRELIA BURGDORFERI BB0323 PROTEIN COMPLEX SUPPORTING MICROBIAL BIOLOGY, INFECTIVITY, AND AS A NOVEL THERAPEUTIC TARGET(2023) Bista, Sandhya; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme disease (LD), also known as Lyme borreliosis, is the most common vector-borne disease in the United States, caused by the gram-negative bacteria of the Borrelia burgdorferi sensu lato group. This atypical bacterial group features distinct genomic and antigenic elements, does not possess any classical toxins, and the pathogenesis of LD is primarily due to the immune activity of the host. These multi-organotrophic spirochetes can elicit severe clinical complications in susceptible hosts, including neuroborreliosis, carditis, and arthritis. If diagnosed early, the disease can be treated with a conventional antibiotic regimen; however, persistent, or relapsing symptoms later develop in a subset of patients. Six months to a year after the antibiotic treatment, up to 20% of the patients can experience various subjective symptoms pertaining to pain, cognitive dysfunction, or other neurological complications, collectively termed Post Treatment Lyme Disease Syndrome (PTLDS). The diagnosis, etiology, and treatment of PTLDS remain currently unknown. To better understand microbial pathogenesis, we have characterized a select set of structurally unique spirochete gene products that act as novel virulence determinants and support microbial infection in mammals. The current study focused on the BB0323 protein of B. burgdorferi, a unique and multifunctional virulence determinant undergoing a complex post-translational maturation process. The maturation, stability, and functions of BB0323 require multifaceted protein-protein interaction (PPI) events involving specific B. burgdorferi proteins, such as a protease-chaperone called BbHtrA, and a membrane-associated protein of unknown function annotated as BB0238. In our current study, we have further dissected the biological significances of the protein-protein interaction complex (PPI), either involving BbHtrA: BB0323 and BB0323:BB0238. The latter PPI event was more thoroughly investigated for its role in spirochete biology and infection and as a novel target for therapeutic intervention against B. burgdorferi infection. We identified a cleavage site where BB0323 full-length protein cleaves into N and C termini by BbHtrA. Subsequently, we have introduced point mutations in the recombinant BB0323 (at the cleavage site for BbHtrA- NL residues replaced with AA), as well as generated an isogenic B. burgdorferi isolates (Bbbb0323NL) with the point mutations in native BB0323. Further analyses show that the cleavage site mutated BB0323 protein could not be processed by the recombinant BbHtrA. Notably, despite the inability of BbHtrA to process BB0323 in vitro, within Bbbb0323NL, BB0323 could indeed be processed to some degree, which yields a basal level of mature N-terminal protein. Notably, in these B. burgdorferi cells, at least two other BB0323 polypeptides of lower molecular weight (less than 27 kDa of mature N-term BB0323) were also produced, possibly due to the action of BbHtrA on non-specific sites. However, the Bbbb0323NL mutants were non-infectious in the murine host, demonstrating the importance of precise cleavage of BB0323 full-length protein and optimal production of N-terminal, which needed to form a complex with another PPI partner, BB0238. Overall, these results further underscored the event of BbHtrA and BB0323 interaction for processing the latter protein as an essential prerequisite for spirochete infection in mammals. Our previous studies have shown that BB0323 N-terminal and BB0238 interact and post-translationally stabilize each other. We used an interaction-deficient borrelial mutant, replacing the BB0323 interaction motif in BB238 (termed as bb0238 Delta Interaction Motif, or bb0238∆IM), which despite showing no growth defects in vitro or other abnormalities, is unable to infect mammalian host. We, therefore, explored the possibility of using the BB0323:BB0238 complex as a novel therapeutic target to combat B. burgdorferi infection in mammals. We first examined whether bb0238∆IM mutants (without interaction motifs) can persist in mice for a long term or could be acquired by naïve ticks. The results show that, unlike the wild type or another B. burgdorferi mutant, The bb0238∆IM could not establish the infection in mice and, as a result, could not be acquired by the ticks, suggesting blockade of BB0323:BB0238 interaction by small molecules could be a novel therapeutic approach to combat incidence of LD. An AlphaLisa assay platform was developed in our lab to monitor BB0323-BB0238 PPI on a high-throughput basis using 384-well microtiter plates, which was then miniaturized to 1536 well at the National Center for Advancing Translational Sciences (NCATS) in a collaborative effort. An AlphaLisa quantitative HTS later screened several small molecule libraries available at NCATS, which were further filtered by counter assays, and a selected set of 84 compounds was tested in a secondary, cell-based assay for cell-permeable compounds that impair BB0323-BB0238 interaction with spirochete cells. A B. burgdorferi cell-based assay comprising a dot-blot assay and regrowth assay was developed to examine the PPI inhibitory activities of the molecules inside the cells. We finally selected one of the compounds, Lomibuvir, for the in vivo studies and demonstrated its PPI inhibitory activity in an in vitro experiment. A pharmacokinetic study in mice showed an increase in the level of the compound in plasma and liver over 21 days. Additional in vivo efficacy studies of Lomibuvir to reduce B. burgdorferi infection in mice were performed using vehicle and ceftriaxone as negative and positive controls, respectively. The results showed that the bacterial load in the skin and heart of the mice was significantly lower in the Lomibuvir-treated group, as compared to the vehicle-treated animals; however, the effect was not as dramatically effective as the antibiotic (ceftriaxone) treatment groups. While future medicinal chemistry approaches could be adopted to further enhance the impact of Lomibuvir as an anti-B. burgdorferi agent, to the best of knowledge, is the first proof-of-concept study that highlights the utility of targeting borrelial PPI events as a possible therapeutic target of Lyme disease.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 A NOVEL INTERFERON-INDUCING PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS STRAIN: CHARACTERIZATION AND VACCINE DEVELOPMENT(2018) Ma, Zexu; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Porcine reproductive and respiratory syndrome virus (PRRSV) causes a swine infectious disease characterized by severe reproductive failure in sows and respiratory disease in pigs of all ages. Despite substantial efforts to control PRRS, no production or vaccination regimen has demonstrated sustaining success. Type I interferons (IFNs) are critical to the innate immunity against viral infections and play an important role in activation of the adaptive immune response. PRRSV appears to antagonize induction of type I IFNs. Fortunately, we discovered an atypical PRRSV strain, A2MC2, which induces type I IFNs in cultured cells. A2MC2 elicits earlier onset and higher levels of virus-neutralizing antibodies than the Ingelvac PRRS® MLV in pigs. However, moderate virulence of A2MC2 was observed in infected piglets. The objective of this project was to characterize A2MC2 and explore this unique strain for the development of an improved vaccine against PRRS. First, I attenuated this strain by serial passaging in MARC-145 cells for 90 consecutive passages. The passage 90 virus (A2MC2-P90) was avirulent and retained the capability of IFN induction. The A2MC2-P90 virus induced higher level virus-neutralizing antibodies in pigs. Secondly, I constructed an infectious cDNA clone of A2MC2. The recovered virus from the infectious clone was similar to the parental strain in growth properties and IFN induction. Gene fragment swapping demonstrated that the middle half genome of A2MC2 was essential for its IFN induction. Thirdly, I conducted studies to exam the genetic source of A2MC2 in IFN induction. Comparison of A2MC2 and other closely relevant PRRSV strain identifies five unique non-synonymous nucleotides. These five nucleotides remained unchanged in the A2MC2-P90 virus. Site-directed mutagenesis indicated that one unique nucleotide in A2MC2 genome was critical in the IFN induction as mutation of this nucleotide led to the loss of IFN induction. Together, our data demonstrate that A2MC2 is a novel strain that is worth further exploration for an improved vaccine against PRRS. The infectious clone of A2MC2 will be useful for the development of a marker vaccine by insertion of a marker sequence into the A2MC2 genome.Item ROLE OF B. BURGDORFERI HIGH TEMPERATURE REQUIREMENT PROTEASE A, BBHTRA IN BIOLOGY AND PATHOGENESIS(2017) Sharma, Kavita; 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, caused throughout the globe by the pathogen Borrelia burgdorferi, which is transmitted by Ixodes scapularis ticks. In North America, I. scapularis transmits a wide array of human and animal pathogens including a group of pathogenic bacteria, known as B. burgdorferi sensu lato complex. Life cycle of B. burgdorferi primarily involves an intricate tick-mammal infection cycle. It transits between different hosts, an arthropod vector and a variety of vertebrate hosts. Maintenance of B. burgdorferi in the enzootic cycle requires successful persistence in the arthropod and reservoir hosts, as well as efficient transmission between tick and mammalian host. Therefore, in order to survive transitions between diverse host-vector environments, B. burgdorferi must not only be able to detect changes in its environment, but also generate suitable response to these changes. As a result, gene-products playing roles in adaptation to stress, including temperature, oxidative stress, pH etc. must be critical for the maintenance of life cycle of the pathogen. One such gene product, which is very important for the bacterial adaptation of stress, is the High temperature requirement protease A (HtrA). HtrAs in different bacteria primarily function in protein homeostasis and quality control, acting as protease and chaperone for stabilizing specific proteins and modulate signaling pathways. While other bacteria like Escherichia coli or other spirochetes like Leptospira possess multiple homologs of HtrA, B. burgdorferi genome harbors a single HtrA gene; which was first described from our laboratory and termed as BbHtrA. The primary goal for this dissertation is to characterize the function of BbHtrA and to study the physiological relevance of this protease during pathogenesis of Lyme disease. Utilizing BbHtrA mutant we studied the biological relevance of this protease on B. burgdorferi survival at higher temperatures and the effects of its deletion on different virulence determinants. Key areas of this research involves a better understanding of intriguing biology and infection of B. burgdorferi, including identification of novel virulence factors which will help and contribute to the development of new strategies that interfere with pathogen persistence and transmission.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 CHARACTERIZATION AND DEVELOPMENT OF REVERSE GENETICS SYSTEM FOR AVIAN PARAMYXOVIRUS TYPE-3 AND ITS EVALUATION AS A LIVE VIRAL VECTOR(2010) Kumar, Sachin; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian Paramyxovirus (APMV) serotype 3 is one of the nine serotypes of APMV that infect a variety of avian species around the world. In chickens and turkeys, APMV-3 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-3, the complete genome sequences of prototype strain Netherlands and strain Wisconsin were determined. The genome length of APMV-3 strain Netherlands is 16,272 and for strain Wisconsin is 16,181 nucleotides (nt). Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Comparison of the APMV-3 strain Wisconsin nt and the aggregate predicted amino acid (aa) sequences with those of APMV-3 strain Netherlands revealed 67 and 78%, identity, respectively. The phylogenetic and serological analyses of APMV-3 strains Netherlands and Wisconsin indicated the existence of two subgroups within the same serotype. Both the strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenesis of APMV-3, a reverse genetics system for strain Netherlands was established in which infectious recombinant APMV-3 was recovered from a cloned APMV-3 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to parental virus. A recombinant APMV-3 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of recombinant APMV-3 expressing Newcastle disease virus (NDV) F and HN proteins demonstrated that the F protein plays a major role in protection against virulent NDV challenge. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-3 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-3 would be of considerable utility for introducing defined mutations into the genome of this virus and developing a vaccine vector for animal and human pathogens.