Saudi Cultural Missions Theses & Dissertations
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Item Restricted Legionella pneumophila Infection and The Host Unfolded Protein Response(Monash University, 2024) Alshareef, Manal Hashim; Hartland, Elizabeth; McCaffrey, KathleenLegionella pneumophila is a Gram-negative bacterium that survives in the environment by replicating within free-living amoebae. When transmitted to humans through contaminated aerosols, the pathogen infects phagocytic immune cells within the lung, such as macrophages and monocytes, to cause disease. This respiratory disease features either severe pneumonia, known as Legionnaires’ disease, or a milder infection called Pontiac fever. To survive and replicate within a eukaryotic cell, Legionella species use a type-IVB secretion system, termed Dot/Icm, to secrete >330 “effector” proteins into the host cell. Dot/Icm effectors manipulate various host processes to evade elimination by phago-lysosomal degradation and establish an intracellular replication vacuole, termed the Legionella-containing vacuole (LCV). A key feature of the LCV is its similarity to rough endoplasmic reticulum (ER) membranes raising the possibility that Legionella induces ER stress and the unfolded protein response (UPR). The UPR is a homeostatic response to ER stress that can play an important role in infection and immunity. L. pneumophila Dot/Icm effectors, including Lgt1-3, SidI, and SidL, have been previously shown to inhibit UPR signalling by blocking host cell protein synthesis. However, whether the UPR restricts L. pneumophila replication or modulates the host immune response to Legionella infection remains unknown. Here we demonstrated that L. pneumophila infection of a macrophage THP-1 cell line induces host ER stress and activates canonical UPR signalling via IRE1, PERK, and ATF6. This activation is a Dot/Icm-dependent. Using pharmacological inhibitors of UPR signalling, we also demonstrated that IRE1 RNase activity supports L. pneumophila intracellular replication in THP-1 macrophages. In contrast, pre-treatment of THP-1 macrophages with pharmacological inducers of ER stress, tunicamycin and thapsigargin prior to infection reduced L. pneumophila intracellular replication. Drug pre-treatment did not inhibit L. pneumophila growth in vitro, phagocytic uptake of the bacterium or Dot/Icm effector translocation. Although tunicamycin enhanced cell death resulting in reduced bacterial load, thapsigargin pre-treatment instead protected macrophages from L. pneumophila-induced cytotoxicity. Thapsigargin induced restriction of L. pneumophila intracellular replication relied on IRE1-kinase activity and STAT1 activation, and hence was linked to UPR-mediated immunity during ER stress. How this restriction is orchestrated needs to be further investigated. Finally, we successfully constructed a L. pneumophila mutant of strain 130b lacking Lgt1, Lgt3, SidI, and SidL, which we termed delta4. The delta4 mutant exhibited normal in vitro growth and was not different from the wild-type parent strain in terms of intracellular replication withinTHP-1 macrophages and loss of Lgt1, Lgt3, SidI, and SidL partially restored host protein synthesis and IRE1-dependent XBP1 mRNA splicing, similar to previous studies with L. pneumophila strain Philadelphia. The delta4 mutant inhibited UPR signalling in THP-1 cells early during the infection (~6 h) but not later in the replicative phase of the infection. Interestingly, wild-type L. pneumophila 130b inhibited STAT1 signalling compared to the delta4 mutant, which induced ER stress in THP-1 macrophages suggesting that Dot/Icm effectors play a role in modulating the host immune response induced by ER stress during L. pneumophila infection.17 0Item Restricted HCV and SARS-CoV-2 Envelope Glycoproteins Down-Modulate NF-κB Activity in Association with Induction of ER Stress(University of Liverpool, 2024) Albalawi, Wejdan; Paxton, William AViruses, as highly adaptable pathogens, possess the remarkable capability to modulate host immune responses and influence disease pathogenesis. Within this context, our research endeavors focused on studying the intricate interplay between HCV E1E2 Env glycoprotein on downmodulating HIV-1 long terminal repeat (LTR) activation, as well as host gene expression patterns. Measuring the HIV-1 LTR activity in hepatic cells co-transfected with HCV E1E2 Env protein and HIV-1 promoter driving expression of a Luciferase reporter molecule demonstrated that the protein downmodulated LTR activity. Using different reporter constructs we demonstrated that the mechanism is mediated specifically via NF-κB. Notably, within this investigation, it is important to highlight that the use of the HIV-1 LTR as a tool is specifically designed for monitoring NF-κB activation and does not imply co-infection of HCV and HIV cells in vivo. Cells expressing the HCV E1E2 Env glycoprotein, NF-κB relinquished its binding affinity to the HIV-1 LTR promoter, while other transcription factors exhibited augmented interactions. Furthermore, transcriptomic analysis of cells expressing the HCV E1E2 Env glycoprotein identified the stimulation of the endoplasmic reticulum (ER) stress response pathway, concomitant with the upregulation of responsive genes, including ATF3. Through shRNA mediated inhibition of ATF3 expression, we identified that the E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Additionally, we observe that ATF3 knock-down raises the basal level of HIV-1 LTR activity in the absence of E1E2 Env expression. The result suggested that the HCV Env glycoprotein inhibits NF-κB signalling via upregulation of the ER stress pathway. In parallel to our investigation on HCV E1E2 Env, we extended our molecular approach to explore the impact of SARS-CoV-2 structural proteins, spike (S), membrane (M), envelope (E), and nucleocapsid (N), on HIV-1 LTR activity. We found that Env glycoproteins of SARS-CoV-2 downmodulate LTR activity, while the N protein did not exhibit any discernible impact on LTR activity. Comparative transcriptomic analysis of stable cell lines expressing individual SARS-CoV-2 structural proteins revealed that the viral proteins S, M, and E have the capacity to influence key transcriptional factors, including NF-κB and SP1. Additionally, the expression of these Env glycoproteins stimulated ER stress and activation of the unfolded protein response (UPR). Furthermore, there was an upregulation of HSF-1, a transcription factor involved in regulating heat shock proteins (HSPs) and early gene response. The activation of HSF-1 may play a significant role in counteracting acute inflammation by inhibiting NF-κB and downstream pro-inflammatory signals. Importantly, this response was absent in the context of the N protein, possibly indicating that the N protein may not stimulate HSPs to the same extent as the other SARS-CoV-2 structural proteins, possibly insufficient to activate HSF-1. Together, these findings suggest a potential link between the two viral systems, highlighting common mechanisms by which viral proteins modulate host cellular processes. Both the HCV E1E2 and SARS-CoV-2 Env glycoproteins can perturb NF-κB signalling, leading to alterations in gene expression patterns. This indicates that these viral proteins may exploit similar strategies to evade host immune responses and promote viral replication. This study adds to the growing body of knowledge by providing novel insights into the dynamic interaction between HCV and SARS-CoV-2 viral systems and their impact on host cellular processes. By revealing shared mechanisms of viral protein modulation and their implications for viral pathogenesis and immune responses, this work presents a novel contribution to our understanding of viral infections and paves the way for future investigations and targeted interventions.14 0Item Restricted Evaluating the Ferroptosis Potential and Mechanistic Variability of the Ferronucleoside TUC1 in the MIAPaCa2 Cell Line(University of Birmingham, 2023-12-04) Alzahrani, Asma; Hodges, NikolasAddressing the therapeutic challenges of pancreatic ductal adenocarcinoma (PDAC), known for its rapid resistance to treatments like gemcitabine, we examined the capabilities of ferronucleoside 1-(S,Rp) TUC1. Notably, TUC1 demonstrated compelling efficacy against the gemcitabine-resistant MIAPaCa2 cells, closely matching cisplatin. Using MTT assays and accounting for variables like cell confluency and treatment duration, we assessed TUC1's cytotoxic impact. While the outcomes were on par with cisplatin, they varied across experimental conditions. Interestingly, based on cell confluency, we identified apoptosis as the primary cell death pathway, a departure from the initially hypothesized ferroptosis. This study highlights TUC1's complex mechanisms, underlining the need for more comprehensive research to further understand its effects.10 0