Saudi Cultural Missions Theses & Dissertations
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Item Restricted The transcriptional landscape and small RNAs in Acinetobacter baumannii AB5075 during antibiotic and environmental stress(University of Dublin, 2024-06-28) Bakheet, Ali Ahmad; Kröger, CarstenMulti-drug resistant Acinetobacter baumannii is considered a major challenge because it causes a variety of serious infections. Studying these bacteria at the level of their genetic response could facilitate finding a way to treat them. Tracking gene expression throughout the growth phases or when affected by any environmental stresses can deepen our understanding of how genes work and how cells function. Here, we studied global gene expression of A. baumannii AB5075 using RNA-seq under the influence of 12 different stresses divided into three groups. The first group includes five growth stages in rich medium, the second group includes four environmental shocks (osmotic, iron limitation and two antibiotic shocks), and the third group contains temperatures shift experiments (between 22°C and 37°C). An important finding is the dramatic up-regulation of the phenyl-acetic acid (paa) pathway including paaA, paaB, paaC, paaJ, paaK and paaZ in late exponential and early stationary phase in rich medium which influences virulence, antibiotic resistance, and biofilm formation. In the environmental shock conditions, we reveal that the transcriptional responses were similar between osmotic shock and iron limiting conditions, and the effect of antibiotics (exposure to chloramphenicol and tetracycline), respectively, indicating that similar pathways are involved in these stress responses. These included genes encoding siderophore Acinetobactin encoding genes that were up-regulated in both osmotic shock and iron limiting conditions. The two antibiotic shocks showed gene expression changes to a much larger than osmotic shock and iron limiting conditions suggesting that protein synthesis stress require a much larger transcriptomic response to cope with the stress compared to the osmotic and iron limitation stress. A response to the antibiotic stress was observed to be a down-regulation of the NADH-quinone oxidoreductase complex, a central protein complex in the respiratory chain, indicating the antibiotics could influence the proton gradient across the membrane or that further growth and ATP synthesis should be immediately stopped. When growing at different temperatures, there were large changes to the transcriptome in all temperature comparisons. The virulence-associated otsAB operon and paa genes were strongly up-regulated upon temperature shift. Type IV pilus encoding genes were down-regulated genes when cells were experiencing a temperature upshift (TS) which could mean that DNA uptake is at least transiently down-regulated when A. baumannii enters a warm-blooded host. In contrast, a temperature downshift induced expression of the Type 6 Secretion System and Csu pili. We also provide a first expression landscape of sRNAs in the suite of 12 conditions. We observed dynamic expression of many small RNAs across the 12 conditions. The sRNAs sRNA99 and sRNA100 very quickly dropped in abundance when the cells experienced antibiotic shocks which was independently validated by Northern blotting for sRNA99 and sRNA100. Deletion of sRNA99 and sRNA100, resulted in large expression changes in MEP in the mutants compared to the WT, which will require further analysis. The sRNA44 was investigated mechanistically in more detail in this study because previous data in A. baumannii AB5075 suggested that sRNA44 was ligated to the mRNA of the biofilm-associated bap gene. We show that sRNA44 translationally represses bap translation in E. coli and that overexpression of sRNA44 reduces bap mRNA levels, likely induced by a base-pairing mechanism. Deletion of sRNA44 and bap showed that bap is required for full biofilm formation after 24h, while deletion of sRNA44 increased biofilm formation suggesting that sRNA44 is a regulator of A. baumannii biofilm formation.46 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.20 0