Department of Veterinary Medicine Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2762
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Item HOST-PATHOGEN INTERACTION DURING CRYPTOCOCCUS NEOFORMANS CNS INFECTION(2024) Chen, Yanli; Shi, Meiqing MS; 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 widely distributed in the environment globally. C. neoformans infection initiates from the lung through inhaling the spores. While most healthy individuals can clear the fungus or contain the fungus in the granuloma, immunosuppressed patients and a small group of healthy populations fail in controlling the cryptococcal fungal pulmonary infection. In those cases, C. neoformans transmigrates from the lung to the central nervous system (CNS) and causes fatal meningoencephalitis, which accounts for 112,000 deaths each year worldwide. However, we have a very limited understanding of the transmigration of C. neoformans from the bloodstream to the brain in vivo, and the mechanism involved in the clearance of the organism in the brain remains poorly understood. In this study, we first report a novel approach to quantitatively analyze the interactions between C. neoformans and brain endothelial cells in a mouse model using flow cytometry. Using this system, we show that C. neoformans was internalized by brain endothelial cells in vivo and that mice infected with acapsular or heat-killed C. neoformans yeast cells displayed a lower frequency of brain endothelial cells containing the yeast cell compared to mice infected with wild-type or viable yeast cells, respectively. We further demonstrate that brain endothelial cells were invaded by the serotype A strain (H99 strain) at a higher rate compared to the serotype D strain (52D strain). Moreover, we found that clearance of C. neoformans in the brain correlates with accumulation and pro-inflammatory M1 polarization of Ly6Chi mononuclear phagocytes and that these phagocytes play a critical role in the clearance of C. neoformans in the brain. Notably, the accumulation of Ly6Chi mononuclear phagocytes coincides with enhanced secretions of TNF and IFN-γ in the brain. TNF receptor (TNFR) signaling, but not IFN-γ receptor (IFN-γR) signaling, mediates the recruitment of Ly6Chi mononuclear phagocytes to the brain in a cell-intrinsic manner. By contrast, IFN-γ induces M1 polarization of Ly6Chi mononuclear phagocytes. Disruption of TNFR or IFN-γR signaling enhances cryptococcal growth in the brain. Thus, Ly6Chi mononuclear phagocytes act as effector cells for cryptococcal clearance in the brain, involving TNFR as well as IFN-γR signaling. Collectively, our study established that 1) internalization of C. neoformans by brain endothelial cells occurred in vivo and offered a powerful approach to quantitatively analyze fungal migration into the brain; 2) Ly6Chi mononuclear phagocytes accumulate in the brain during brain infection with C. neoformans and function as effector cells for clearance of C. neoformans in the brain involving TNFR signaling and IFN-γ signaling.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 Immune Modulations of a Helminth Derived Protein(2023) Mekhemadhom Rajendrakumar, Arunraj; Zhu, Xiaoping XZ; Tuo, Wenbin WT; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune responses at the gastrointestinal mucosa modulate nematode parasite infection, initially characterized by the production of epithelium-derived, robust T helper 2 (Th2) type alarmin cytokines, such as interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Subsequently, the immune responses are mediated by releasing the lymphoid cell-derived Th2 immune cytokines, such as IL-13, IL-4, IL-5, IL-9, and parasite-specific antibodies. Studies have shown that parasitic nematode worms can establish a chronic infection in the intestine, even in a robust immune response. This evidence leads us to hypothesize that the nematode evolves to evade or regulate intestinal immunity through specific modulatory mechanisms that interfere with initial intestinal immune responses, allowing the nematode to survive. We used a model nematode, Heligmosomoides polygyrus bakeri (Hpb), to identify nematode-derived proteins with regulatory effects on Th2 immune cytokines during chronic infection. Through high throughput analysis, we found that a Hpb-derived protein could precisely modulate mouse immune response. The presence of the Hpb-derived protein was essential for the parasite's survival as the vaccine conferred a sterilizing immunity. As Th2 cytokines are directly associated with the pathogenesis of several inflammatory and autoimmune diseases, we are understanding how this protein regulates the function of the Th2 cytokines in vitro and in vivo and explore whether the protein could use to treat inflammatory diseases and serve as a vaccine target to control nematode infections.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 Host Immune Responses to Fungal Pathogens(2022) Strickland, Ashley Brenda; Shi, Meiqing; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal infections are an increasing threat to global public health. Worldwide, more than one billion fungal infections are diagnosed each year and result in 1.5 million deaths annually. This number is expected to rise as advances in medical care for immunocompromised patients continue to be made. Furthermore, limited antifungal treatment options, coupled with the emergence of new and resistant fungal species, is anticipated to exacerbate this issue. For these reasons, it is essential to understand the dynamic interactions between host and fungus that contribute to fungal pathogenesis. The following work examined the role of IL-27 on T cell regulation in the lungs during infection with Aspergillus fumigatus, and in the brain during infection with Cryptococcus neoformans. In contrast to most infection settings demonstrating that IL-27 is anti-inflammatory, we report that this cytokine is proinflammatory in both the lung and the brain during infection with A. fumigatus and C. neoformans respectively. Genetic ablation of IL-27Rα in mice resulted in higher fungal burdens in the lung during Aspergillus infection and in the brain during Cryptococcus infection. These were associated with reduced IFN-γ production, as well as a fewer CD4+ T cells in target organs. In the case of C. neoformans infection, IL-27 signaling enhanced T cell accumulation by promoting T cell proliferation early during infection, and inhibiting T cell death at later time points. These suggest that IL-27 promotes T cell responses required for fungal clearance during infection with A. fumigatus or C. neoformans. In addition, the work presented here also characterized lung resident macrophage responses during infection with C. neoformans. Here, we report that cryptococcal infection induced the accumulation of CD68hi macrophages in the lungs of infected mice. Approximately 10% of these cells were alveolar macrophages, while nearly 90% were interstitial macrophages. Both of these populations were observed to interact with fungi and upregulated their expression of arginase 1. The absence of either macrophage in transgenic mice, or following pharmacological depletion, significantly reduced fungal burdens in the lungs of mice, indicating that these cells enhance fungal growth and may represent attractive targets aimed at limiting the pathogenesis of C. neoformans.Item HEPATITIS E VIRUS MODULATES HOST FACTORS TO GENERATE A CONDUCIVE ENVIRONMENT FOR REPLICATION(2020) lin, shaoli; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV infection mainly presents as acute and self-limiting hepatitis in young adults. However, it can be exacerbated to fulminant hepatitis in HEV-infected pregnant women, resulting in up to 30% case fatality. Besides, chronic HEV infection with rapid progression in immunocompromised patients has been a challenge in many countries since it was reported years ago. HEV infection is zoonotic, and human HEV strains are grouped into four major genotypes in the genus Orthohepevirus A, the family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. Due to the lack of an effective cell culture system and a proper animal model, HEV biology, virus-cell interactions, and pathogenesis are understudied. HEV is known to inhibit the innate immune response by targeting type I interferon (IFN) signaling pathway via its ORF1 products. Nevertheless, it remains largely unknown how the virus manipulates host factors to facilitate its replication. The objective of these studies was to elucidate the mechanism of HEV manipulation of host factors to generate a conducive environment for replication. Our results show that the capsid protein of HEV inhibits the IFN production to dampen the antiviral response through its N-terminal arginine-rich motif. In addition to the impairment of innate immunity, HEV proliferation requires the presence of other host factors: DDX3, an RNA helicase, and oxysterol-binding protein (OSBP), a lipid transporter. The knockdown of these two factors led to a significant reduction of HEV replication, whereas the reconstitution of these two genes restores the HEV proliferation level. The capsid protein was found to interact with the C-terminal domain of DDX3. The HEV helicase was shown to interact with OSBP and block its translocation to the Golgi apparatus. These results indicate that HEV employs multiple strategies including blocking antiviral response and recruiting host factors for its invasion and proliferation. Our data provide insights into the HEV-cell interactions and may facilitate the development of novel antiviral strategies.