Department of Veterinary Medicine Theses and Dissertations
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Item AVIAN PARAMYXOVIRUS-VECTORED VACCINES AGAINST INFECTIOUS BRONCHITIS VIRUS AND HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS(2019) Shirvani, Edris; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Highly pathogenic avian influenza (HPAI), infectious bronchitis (IB), and Newcastle disease (ND) are highly contagious and economically important diseases in poultry. Vaccination is the major strategy which is implemented to combat highly pathogenic avian influenza virus (HPAIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV), worldwide. However, among these viruses, some NDV strains are naturally avirulent and have been used as highly safe vaccines for more than 60 years. Live attenuated IBV vaccines that are produced by passaging virulent strains in eggs have safety concerns and are genetically unstable. Inactivated IBV and HPAIV vaccines also are less efficacious and affordable. Therefore, development of alternative vaccines against IBV and HPAIV is highly needed. In this multistep study, we have employed NDV vector and other novel avian paramyxovirus (APMV) vectors to develop improved IBV and HPAIV vaccines. Firstly, we conducted a study to investigate the contributions of the S1, S2, and S proteins of IBV in protection against virulent IBV, and to develop a safe and efficacious recombinant NDV-vectored IBV vaccine. We generated recombinant (rNDV) strain LaSota viruses expressing S1, S2 or S protein of IBV using reverse genetics. We evaluated the protective efficacies of rNDVs against virulent IBV and NDV challenges. Our results showed that the S protein, which contains the S1 and S2 neutralizing epitopes in correct confirmation is the best protective antigen of IBV. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV. Secondly, besides rNDV strain LaSota vector, we employed a novel chimeric rNDV/avian paramyxovirus serotype-2 (rNDV/APMV-2) vector that replicates less efficiently and a modified NDV strain LaSota (rLaSota-527) vector that replicates more efficiently to develop a likely improved viral vectored vaccine against IBV. We generated rNDV/APMV-2 or rLaSota-527 virus expressing the best protective protein of IBV (S protein), which was found in the first study. The protective efficacies of rNDV/APMV-2 or rLaSota-527 virus expressing the S protein was evaluated against IBV in chickens. Our results showed that immunization of chickens with either chimeric rNDV/APMV-2 expressing the S protein, which is a better candidate for in ovo vaccination, or rLaSota virus expressing the S protein provided protection against IBV. Most importantly, compared to prime-boost vaccination or vaccination with rLaSota-527 virus expressing the S protein, single immunization of chickens with rLaSota virus expressing the S protein induced better immune responses against IBV. Thirdly, we conducted a study to evaluate the contributions of HA1 and HA2 subunits of HPAIV HA protein in the induction of neutralizing antibodies and protection in chickens, using rNDV strain LaSota vector. Our results showed that the HA1 and HA2 subunits when expressed separately, neither provided protection nor induced neutralizing antibodies. To be effective the HA protein must be incorporated into a vaccine as an intact protein. These results also highlight the importance of using chickens in HPAIV vaccine studies as they are susceptible natural hosts. Finally, we employed APMV-3 strain Netherlands as a vaccine vector, for its high efficiency replication in multiorgans of host, to generate an improved vaccine against HPAIV. Our results showed that immunization of chickens with either rAPMV-3 expressing the HA protein (rAPMV-3/HA) or rNDV expressing the HA protein (rNDV/HA) provided complete protection against HPAIV challenge. However, the immunization of chickens with rAPMV-3/HA induced higher levels of neutralizing antibodies than that induced by rNDV/HA. These results suggest that mass-vaccination with a rAPMV-3/HA might provide better protection against H5N1 HPAIV in field conditions. In conclusion, the individual subunits of the S protein of IBV or the HA protein of HPAIV when expressed separately, neither provided protection nor induced neutralizing antibodies. To provide protective efficacy, the intact HA or S protein must be incorporated into vaccine. The rNDV expressing the S protein is a safe and efficacious bivalent vaccine against IBV and NDV. Other than rNDV strain LaSota, rNDV/APMV-2 and rAPMV-3 are promising vaccine vectors for development vaccines against IBV and HPAIV, respectively.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 INITIATION AND PROGRESSION OF BRAF/NRAS WILDTYPE MELANOMA IN UV-INDUCED MOUSE MODELS OF CUTANEOUS MELANOMA(2018) Michael, Helen Thompson; Samal, Siba; Merlino, Glenn; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Melanoma is the deadliest skin cancer and is responsible for nearly 60,000 deaths worldwide each year. At least some melanomas are believed to arise from stepwise progression from normal melanocytes through a benign nevus stage to malignant melanoma and finally metastatic disease. Approximately 20-50% of melanomas have evidence of a pre-existing nevus, indicating that progression is an important route of melanomagenesis. Ultraviolet radiation exposure is believed to play an important role in nevus and melanoma formation, although the mechanisms of this remain unclear. Childhood sunburn and intermittent sun exposure are epidemiologically linked to increased melanoma risk. While most melanomas have activation of the mitogen activated protein kinase pathway, often due to mutations in BRAF or RAS genes, nearly 15% of cutaneous melanomas do not have an identified strong driver. Despite targeted therapies and immunotherapy, the death rate from melanoma has remained nearly static for several decades, so there is a need to identify additional genes and pathways to provide novel therapeutic targets. We hypothesized that progression of melanocytic lesions from benign to malignant is associated with the acquisition of additional genomic mutations. Unlike wildtype mice, hepatocyte growth factor (HGF) transgenic mice have “humanized” distribution of melanocytes along the dermal-epidermal junction. Following a single dose of UV at 3 days of age, HGF mice develop melanocytic nevi and melanomas. In this project, two HGF models were used to generate melanocytic lesions. The first model, on an albino FVB background had a tumor incidence of only 10% and used melanocyte-specific green fluorescent protein expression to identify early nevi and melanomas. The second model, on a C57BL/6 had a high tumor incidence (80%), and 60% of tumor-bearing mice have metastatic lesions. Sequencing of melanocytic lesions at different stages revealed a variety of driver mutations, including Nf1, Gnaq, and Gna11, as well as genes and pathways with less established roles in melanoma development. Our data provide a broad overview of genes and pathways involved in progression of non-BRAF, non-NRAS melanoma. Additionally, we present the first potential germline variants that may increase metastatic susceptibility for melanoma patients. These genes suggest potential biomarkers for progression of melanocytic lesions.Item The immunoregulation of interleukin-27 in African trypanosome infection(2018) Liu, Gongguan; Shi, Meiqing; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Interleukin (IL)-27 is a cytokine with diverse impacts on regulation of vertebrate T helper Type 1 (Th1) responses. Initially, it was predicted as a promoter of Th1 responses. However, it was lately identified as a potent negative regulator of T cell responses in a variety of disease models, including infection with viruses, bacteria, and intracellular parasites. The extracellular protozoan parasites, African trypanosomes, cause a chronic debilitating disease associated with persistent inflammation. Using this infection model, we aim to identify novel immunoregulatory functions of IL-27 on innate and adaptive immunity. Here we demonstrate that IL-27 receptor deficient (IL-27R-/-) mice infected with African trypanosomes display excessive production of IFN-γ by CD4+ T cells, exacerbated liver pathology, and dramatically shortened survival as compared with infected wild-type mice. Depletion of CD4+ T cells or neutralization of IFN-γ ameliorates the liver pathology and extends the survival of infected IL-27R-/- mice. Our further interest is in deciphering the mechanisms of how CD4+ T cells and IFN-γ shape the monocyte-featured innate immunity in African trypanosome infected IL-27R-/- mice. Blood monocytes typically consist of a heterogenous population of Ly6C+ and Ly6C- monocytes. Ly6C+ monocytes can give rise to inflammatory TNF-α/iNOS producing dendritic cells (Tip-DCs) and anti-inflammatory macrophages. Here we find that IL-27R-/- mice exhibit a higher frequency of Ly6C+ monocytes recruitment to the liver, where they preferentially differentiate into Tip-DCs. This is coincided with impaired development of Ly6C- monocytes and macrophages in the liver. Depletion of CD4+ T cells or neutralization of IFN-γ in infected IL-27R-/- mice diminishes the recruitment of Ly6C+ monocytes, and their differentiation into Tip-DCs in the liver. This is accompanied by the greatly enhanced counts of Ly6C- monocytes and macrophages following antibody treatments. Further evidences show that 1) IFN-γ produced by CD4+ T cells induces cell death of Ly6C- monocytes which perturb the development of Tip-DCs in infected IL-27R-/- mice and 2) cell intrinsic IFN-γ signaling drives Ly6C+ monocytes to differentiate into Tip-DCs in infected IL-27R-/- mice. Thus, our data identify IL-27 signaling as a novel immunoregulator to prevent Ly6C+ monocytes from differentiation into Tip-DCs through suppressing CD4+ T cells to secrete IFN-γ.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 INTRAVASCULAR CLEARANCE OF DISSEMINATING CRYPTOCOCCUS NEOFORMANS(2018) Sun, Donglei; Shi, Meiqing; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen causing fatal cryptococcal meningoencephalitis in humans. The initial infection organ of C. neoformans is the lung; however, lung infection is commonly seen in healthy individuals and does not always have symptoms. Under certain conditions, the fungi may disseminate out of the lung and enter blood circulation. The fungi, once trapped in the brain vasculature, will breach the blood-brain barrier and proliferate rapidly leading to fatal outcomes. Considering the weak immune responses in the brain, it is important to limit the possibility for fungi to arrive at the brain. The current dissertation focused on the interactions of C. neoformans with host immune cells after pulmonary dissemination, seeking mechanisms that can clear the fungi from circulation. It is identified that neutrophils have the ability to remove fungi from the brain vasculature. This effective fungicidal cell kills C. neoformans via C5a-C5aR and CD11b axis. The C5aR signaling provides navigation and alerts neutrophils by up-regulating CD11b surface expression, CD11b as complement receptor is essential for subsequent uptake and killing of fungi by neutrophils. In vivo, neutrophil clearance of C. neoformans occurs in the brain vasculature but the effect is more efficient in the lung vasculature because lung is able to recruit massive amount of neutrophils into the vasculature through complement activation and C5aR signaling, which induce neutrophil actin polymerization and increase their retention. Neutrophils once activated by C. neoformans can further augment self-recruitment through the release of leukotriene B4. Directing neutrophils to the brain can help with fungal clearance in the brain. Moreover, using intravital microscopy, we reported that liver Kupffer cells can filter disseminating C. neoformans. This filtering requires C3b deposition on the fungal surface to provide the holding force and CRIg on the Kupffer cells. Without the holding force, fungi in the liver have higher tendency to be flushed back into circulation. The Kupffer cells rapidly phagocytize the captured fungi and suppress their proliferation in an IFN-γ independent way. Collectively, the results suggest that neutrophils and liver Kupffer cells are critically involved during intravascular clearance of C. neoformans.Item VIRAL IMMUNE EVASION OF FCRN FUNCTIONS(2018) LIU, XIAOYANG; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human Cytomegalovirus (HCMV) is known to evade host immunity, allowing it to persistently infect humans. Although the strategies of HCMV to evade cellular immunity is well studied, there is limited understanding on how HCMV antagonizes humoral immunity. The neonatal Fc receptor (FcRn), an MHC class I-related FcγR, plays a critical role in IgG-mediated humoral immunity. Through screening the HCMV proteome, we discovered that US11 specifically captured FcRn in both virally-infected and US11-expressing cells. US11 selectively inhibited the assembly of FcRn with β2m, impaired FcRn IgG binding capacity and blocked FcRn trafficking to the endosome by retention of FcRn in ER. Furthermore, US11 recruited Derlin-1 and E3 ubiquitin ligase TMEM129, to induce degradation of FcRn in US11+ or HCMV-infected cells. This complex led to the dislocation of FcRn from the ER to the cytosol and facilitated its degradation in an ubiquitination and proteasome-dependent manner. The cytosolic interaction between FcRn and Derlin-1 was shown necessary for degrading FcRn. FcRn is widely expressed in most cell susceptible to HCMV infection, including epithelial, endothelial and macrophage. Our data showed that either HCMV infection or recombinant US11 expression significantly inhibited human IgG transcytosis across polarized human primary intestinal epithelial Caco-2 cells, Vascular endothelial HMEC-1 cells and placental trophoblast BeWo cells, and facilitated considerable IgG degradation inside endothelial HMEC-1 cells. Hence, our results show that HCMV exploits the Derlin-1/TMEM129 pathway through US11 to disable FcRn, revealing a novel strategy for viral evasion from antibody mediated-immunity. We also studied whether HCMV viral FcγRs (gp34 and gp68) and US11 work together to facilitate IgG degradation. HCMV vFcγRs has been reported to internalize IgG via endocytosis. Interestingly, we found that in acidic pH (6.0) condition, the IgG binding capacity of gp34 was largely impaired while the IgG binding capacity of gp68 had minimal change. Consequently, in the presence of FcRn, gp34 did not enhance IgG degradation whereas gp68 significantly promoted the IgG degradation. Furthermore, the presence of US11 induced more gp34 and gp68-mediated IgG degradation in FcRn+ cells.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 IMPROVED NEWCASTLE DISEASE VIRUS VACCINES AND VECTORS(2017) Manoharan, Vinoth Kumar; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Newcastle disease (ND) is an economically important disease of poultry worldwide. The use of vaccines to control ND is necessary because of frequent outbreaks of the disease in enzootic countries. The fusion (F) and hemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are multifunctional proteins that play critical roles during infection. The F protein of NDV is a type I membrane glycoprotein that mediates the fusion of viral envelope to the host cell membrane. The F protein activation initiates a series of conformational changes in the F protein leading to virus-cell membrane fusion, which occurs at the cell surface at neutral pH thus modulating NDV entry and spread. In the present study, we investigated the role of tyrosine to alanine mutation at amino acid position 524 and 527 in the F protein cytoplasmic tail (CT) of NDV strain LaSota by using reverse genetic techniques. Our results suggest that tyrosine residues at 524 and 527 position of F protein CT domain play a major role in fusogenicity and in replication thus modulating NDV infectivity. The F protein is synthesized as an inactive precursor, F0, which is functionally activated after cleavage by host cell proteases into F1 and F2 polypeptides, linked by disulfide bonds. The amino acid sequence surrounding the F protein cleavage site determines the virulence of NDV. We also studied the role of other avian paramyxovirus fusion protein cleavage site sequences in F protein cleavage of NDV strain Banjarmasin. This study has helped us to understand the requirement of F protein cleavage site in proteolytic processing, plaque formation and virus infectivity. Further, the role of these F cleavage site mutant viruses as genotype-matched vaccines for virulent NDV infection has been explored. Reverse genetics has also been used to develop NDV strains as a potential vaccine vectors for various human and animal pathogens, such as highly pathogenic avian influenza (H5N1), human immunodeficiency virus, severe acute respiratory syndrome coronavirus, ebola virus, respiratory syncytial virus and human parainfluenza virus type 3. NDV has several characteristics that makes it a suitable candidate for vaccine vector development. It is safe in humans and animals due to natural host range restriction, expresses foreign protein abundantly, infects via intranasal route, produces both humoral and mucosal immune responses, is antigenically distinct from human and animal pathogens, and lack of preexisting immunity to NDV in humans and animals. In one vaccine trial with non-human primates, the mesogenic NDV strain Beaudette C (BC) replicated to a high titer and induced a substantially higher antibody response compared to the lentogenic strain LaSota, and thus appeared to be more effective. However, NDV strains that have a polybasic cleavage site in the F protein and an intracerebral pathogenicity index (ICPI) >0.7 have been classified as Select Agents. Most mesogenic strains, including strain BC, fall into this category and therefore cannot be handled in BSL-2 conditions. In this study, we constructed a series of recombinant (rNDV) vectors containing the cleavage site sequence of avirulent strain LaSota and other avian paramyxoviruses, together with various regions of the F protein exchanged between NDV strains AKO-18 and BC. We used these modified rNDV vectors to express SIV gp160 envelope protein and immunized guinea pigs. Our results showed that rNDV/SIV vaccines were immunogenic and effectively neutralized SIV mac251 strain in vitro. These results support the idea of the use of NDV as a vaccine vector for expression of SIV immunogens capable of inducing neutralizing antibodies against diverse SIV strains, thus providing an improved vaccine vector platform for ultimately testing the NDV vectored vaccines in non-human primates and humans.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.