Department of Veterinary Medicine Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2762
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Item Characterization of the GBF1-Arf1 axis in enterovirus RNA replication(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.Item A NOVEL IXODES SCAPULARIS PROTEIN DICTATES TICK HEMATOPHAGY AND CUTICLE INTEGRITY, IMPACTING TICK DEVELOPMENT(2023) DUTTA, SHRABONI; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ticks are prevalent throughout the world and are capable of transmitting a variety of pathogens (e.g., bacteria, protozoa, and viruses) to humans. Incidence rates for tick-borne diseases (TBD) are also increasing globally, and effective vaccinations to combat tick infestations and TBD transmission remain a critical unmet need. Of the six major tick genera that spread human illnesses worldwide, Ixodes ticks are the most prevalent. Specifically, Ixodes scapularis (also known as the blacklegged or deer tick) is an obligate blood-feeding arthropod that transmits several human and animal pathogens that include Borrelia burgdorferi sensu lato complex – the causative agent for Lyme disease. Unlike many hematophagous insects and soft ticks, I. scapularis (hard ticks) remain attached to their hosts for several days and are capable of uptaking bloodmeals that are 100 times greater than their initial body weight. A large and nutrient-dense bloodmeal is essential for their sub-adult and adult development processes and fecundity. However, the molecular and cellular processes that regulate tick blood feeding (hematophagy) and development have not been extensively elucidated. Therefore, our major objective is to characterize tick molecular components that are critical in the tick parasitism and life cycle in order to develop new strategies to combat tick infestations and spread of tick-borne diseases. Herein, we describe the structural and functional properties of a newly identified I. scapularis protein isolated from the partially fed nymphal ticks. Although the protein displays minor homology to proteins of known functions, structurally, it resembles some features of arthropod Odorant Binding Proteins (OBP). Therefore, we refer to this protein as, Ixodes Gut OBP (IGOBP). We show that the knockdown of IGOBP via RNA interference in ticks results in impaired blood feeding (hematophagy) and significantly decreases their post-fed weights. In addition, systemic IGOBP knockdown gives rise to aberrant phenotypes, significantly reduces tick molting rate, and compromises the structural integrity of the cuticle, specifically the flexible alloscutum components. Notably, IGOBP knockdown has profound effects on the molting efficacy and fitness of females than males. This is likely due to the fact that female adults consume a greater volume of bloodmeal than male adults, necessitating a more pronounced expansion of the alloscutum. Subsequently, our RNA sequencing data identifies multiple genes whose expressions are regulated by IGOBP. The underlying mechanism of possible IGOBP or associated gene functions may aid in identifying future targets for anti-tick vaccines. In summary, our studies characterized a novel I. scapularis protein revealing that the protein is essential for tick hematophagy and development. To the best of our knowledge, this is the first characterization of a tick odorant-binding protein (OBP), using structural and functional genomic tools that unearthed the unique and possibly multifunctional role of IGOBP in vector biology and parasitism. We anticipate that the presented data will enhance our fundamental understanding of tick biology and contribute to the development of potential anti-tick measures.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.Item CD23 MEDIATED IGE TRANSCYTOSIS IN AIRWAY INFLAMMATION(2012) Palaniyandi, Senthilkumar; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)CD23 (FceRII), a C-type lectin type II membrane glycoprotein, plays an important role in IgE homeostasis and development of allergic inflammation. I showed that CD23 was constitutively expressed in the established or primary human airway epithelial cells and its expression was significantly up-regulated by IL-4 stimulation. In a transcytosis assay, human IgE or IgE derived immune complex was transported and enhanced by IL-4 stimulation across a polarized Calu-3 monolayer. A CD23 specific antibody or soluble CD23 significantly reduced the transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the immune complex was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed antigen-IgE complexes were competent in inducing degranulation of the cultured human mast cells. This study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation. To verify the above results in a mouse model, CD23 expression was detected in epithelial cells lining mouse airway and enhanced by IL-4 exposure as well as in ovalbumin (OVA) sensitized mouse. I showed that CD23 transported IgE and OVA-IgE derived immune complex across airway epithelial cells in wild-type, but not CD23 knockout (KO), mice. The chimeric CD23KO mice repopulated with wild-type myeloid cells, sensitized and challenged with OVA showed significant reduction in siglec-F+ cells, eosinophils, macrophages and IL-4 in bronchoalveolar lavage fluid recovered 24 hours later compared to the wild-type mice. Our finding of CD23-mediated IgE transport in airway epithelial cells suggest a possibility of CD23 transporting an IgE Fc-fused protein for immunotherapy. CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) which competitively binds CD80 and CD86 expressed on antigen presenting cells and inhibits CD28 mediated co-stimulation of T cell activation. A CTLA4-Fc (IgE) fusion protein produced in Chinese hamster ovary cells was intranasally administrated into mouse airway for assessing its specific transport by CD23. The effect of this fusion protein on the development of allergic inflammation is being fully investigated in wild-type, CD23-KO, and chimeric mouse model.