Saudi Cultural Missions Theses & Dissertations
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Item Restricted Functional characterization of gonococcal toxin-antitoxin systems(University of Nottingham, 2024-03) Bagabs, Salwa; Oldfield, Neil; Turner, David; Stocks, MichaelNeisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Gonorrhoea is a global health problem and one of the most common sexually transmitted infections in the world. Antibiotic resistance in N. gonorrhoeae is a growing problem, and in order to overcome this, it is necessary to develop preventative vaccines or novel treatments. Bacteria have evolved toxin-antitoxin (TA) systems to protect themselves from stressful conditions. TA systems have been linked to various functions including persister cell formation, biofilm formation, phage resistance and stabilisation of genetic elements. Previous work using the Toxin-Antitoxin DataBase (TADB) revealed the presence of three chromosomally-encoded type II TA systems (HicAB, MazEF, VapBC/FitAB) in N. gonorrhoeae strain FA1090. These were hypothesised to play an important role in the lifestyle of this human-restricted pathogen. In addition, exploitation of one or more of gonococcus TA systems could lead to novel treatment interventions. In this study, further bioinformatics analysis using PubMLST.org examined the prevalence and conservation of the TA systems across isolates. The FitAB, MazEF (and to a lesser extent the HicAB) TA systems were very highly prevalent and conserved across over 5000 N. gonorrhoeae isolates examined. To provide experimental confirmation that the predicted TA systems could influence bacterial growth, DNA fragments corresponding to fitA, fitB and both genes were amplified from N. gonorrhoeae FA1090, ligated into an arabinose-inducible expression plasmid and transformed into E. coli. Qualitative and quantitative growth assays utilizing these strains, along with previously engineered strains harbouring hicAB and mazEF constructs, revealed a lack of FitB or MazF-mediated toxicity in E. coli. In contrast, a HicA-mediated growth arrest effect, which could be abolished by co-expression of HicB, was detected, thus providing confirmation that the N. gonorrhoeae FA1090 genes, NGO1627/1628, encode a functional HicAB TA system. Subsequently, the HicA residues His24, His29, or His40, respectively, were changed to alanine residues by site-directed mutagenesis. Quantitative growth analysis confirmed that the His24 and His29 residues, but not His40 of gonococcal HicA are required for toxicity. Mutagenesis of the three TA systems was undertaken where both toxin and antitoxin genes were deleted and replaced by a kanamycin cassette in N. gonorrhoeae FA1090. Furthermore, complemented derivatives of FA1090ΔhicAB that expressed IPTG-inducible HicA, HicB or HicAB, respectively, were also generated. The growth characteristics of the complemented strains (FA1090ΔhicAB:hicA, FA1090ΔhicAB:hicB and FA1090ΔhicAB:hicAB) were examined in comparison to the wild-type and mutant strain. The results confirmed that all strains grew as the wild-type, either with induction with IPTG or not, with the exception of FA1090ΔhicAB:hicA induced with IPTG which exhibited growth arrest as judged by OD measurements. Another study finding was confirmation of hicAB gene expression in wild-type FA1090 during in vitro growth by extraction of total RNA and reverse transcription polymerase chain reaction (RT-PCR) analysis to detect specific mRNA transcripts. The use of specific hicA and hicB primers confirmed expression of both genes, and the combination of a hicA forward and hicB reverse primer provided evidence that both genes are co-transcribed. A final finding came when the total RNA preps were examined using an Agilent Bioanalyser. Notably, a doublet 16S rRNA peak was apparent in total RNA prepared from FA1090ΔhicAB:hicA strain induced with IPTG, but not the uninduced strain, suggesting interaction (or cleavage) of 16S rRNA by HicA, and potentially giving an insight into the mechanism by which gonococcal HicA influences bacterial growth and viability.18 0Item 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 Altering the Morphological Properties of Nano-Scale Hydroxyapatite Via Sol-Gel Synthesis.(Saudi Digital Library, 2023-11-03) Alnasr Allah, Fahad; Miller, Cheryl; Harrison, Caroline; Joshi, ShivaniBackground: Bone defects and infections are significant clinical challenges facing maxillofacial and orthopaedic surgeons. These conditions can arise from trauma, cancer or infections, leading to bone tissue loss and structural changes requiring intervention and treatment. Traditional approaches to bone regeneration and infection management have limitations (e.g., immunological rejection by the host, transmission of diseases and costs); this highlights the need for innovative solutions to overcome the clinical obstacles associated with traditional treatment of bone defects. Nano-hydroxyapatite biomaterials have shown promising effects when used as bone graft substitutes because they promote bone tissue growth, making them candidates for addressing bone defects. Materials and Methods: Nano-scale hydroxyapatite was synthesised via the sol-gel method, three with different stirring speeds overnight and the fourth batch with 5 g of 3-Aminopropyltriethoxysilane (APTES) stirred at medium stir speed overnight. After that, the supernatant was poured off, and the nHA was washed until the conductivity was stable. The suspensions were dried to a powder (for characterisation) in the oven at 60°C overnight. The samples were characterised using x-ray diffraction (XRD) to identify the crystal phases, transmission electron microscopy (TEM) to image the particle shapes and zeta potential to analyse the surface charge. Results: All samples were successfully crystallised based on the XRD results. The main crystal phase of all the experimental samples was identified precisely and matched those specified in (pure hydroxyapatite, JCPDS card #09-0432), but the samples prepared had a lower degree of crystallinity than the ReproBone® novo. In addition, the stirring speed and/or the addition of APTES affected the size, morphology, particle aggregate and surface charge. Conclusion: Within the study's limits, it was concluded that the difference in the stirring speeds and/or the addition of APTES affect intense crystallisation. In addition, that affects the size, morphology, aggregate of particles and the surface charge of the particles. Thus, knowing the causes and effects of these changes may contribute to the synthesis of HA with better biocompatible and mechanical properties.24 0Item Restricted The interactive effects of juvenile hormone analogs and environmental factors on mosquito phenotypic traits and susceptibility to arbovirus infection(Saudi Digital Library, 2023-09-10) Alomar, Abdullah Abdulaziz; Alto, Barry WilmerEmerging arthropod-borne viruses (arboviruses) are among the most important global public health concerns. Arboviruses, such as Zika virus (ZIKV) have caused explosive epidemics affecting thousands of people worldwide. In the absence of effective antiviral medications, prevention measures rely largely on reducing the number of adult mosquito vectors by targeting juvenile stages. However, a full understanding of the interactive effects of these measures and environmental factors in determining mosquito phenotypic traits and interactions between arboviruses and mosquito vectors is poorly understood. Pyriproxyfen is a juvenile hormone analog (JHA) that primarily blocks adult emergence, by mimicking the natural insect juvenile hormone, and minimally affects larval growth and development. This mechanism of JHA has the potential to act in combination with other larval sources of mortality in nature to affect mosquito populations. Here, we designed a series of experimental manipulations to determine the influence of juvenile exposure to JHA on the Ae. aegypti life-history traits and susceptibility to infection with ZIKV under different environmental factors, including predation and variation in temperature. Concentrations of JHA that cause over 50% inhibition in adult emergence of Ae. aegypti had no effect on adult emergence and lifespan of predatory mosquito Tx. rutilus. Weights of adult Ae. aegypti and Tx. rutilus were not influenced by JHA exposure. The combination of the presence of JHA and Tx. rutilus heavily lowered Ae. aegypti emergence to adulthood more than the independent effects of JHA or Tx. rutilus. Lifespan of adult Ae. aegypti was shortened by exposure to JHA and Tx. rutilus. The effects of JHA on mosquitoes were modulated by temperature. Phenotypic traits (development time, wing size, and adult emergence) and susceptibility to ZIKV infection, dissemination, and transmission were differently influenced by JHA and temperature interactions. These findings suggest that the use of JHA to control mosquito vectors may have low effects on mosquito biocontrol agents, but it may influence adult susceptibility to arboviruses under different environmental conditions. Understanding the ultimate consequences of juvenile mosquito control measures on subsequent adults’ ability to transmit viral pathogens is critical to fully understanding their overall impacts on the epidemiology of arboviruses.22 0