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  • (2022) Bartolec-Criss, Tara
    Thesis
    The function of a protein is mediated by its ability to form precise three-dimensional structures or specific protein-protein interaction (PPI) interfaces. However, it has been challenging to study these aspects of the proteome at scale, in sufficient detail, and in a generalisable way. Cross-linking mass spectrometry (XL-MS) performed on high complexity samples (such as entire organelles or cells) theoretically enables the large scale mapping and monitoring of protein structural conformations and PPI interfaces in native proteoforms. However, as the large scale XL-MS field is young and historically driven by the reporting of technological innovations, assessment of the biological accuracy (and overall utility) of large scale XL-MS studies is still largely underexplored. This thesis applies large scale XL-MS to two eukaryotic systems (Saccharomyces cerevisiae and cultured human cells) and is presented as three independent studies. Firstly, it reports the first budding yeast XL-MS interactome, generated by cross-linking intact nuclei isolated from wild-type cells with DSSO. The wealth of yeast interactome data enabled identification of an inflated false discovery rate for cross-links representing PPIs, and development of a novel quality control approach. Surprisingly, the high confidence PPIs were substantially orthogonal to historical binary interactome mapping efforts. Subsequently, it reports the largest XL-MS dataset currently available for any species, generated by diversifying analyses (cross-linkers, mass spectrometry analyses, software) performed on organelles isolated from human HEK293 cells. Here, we show how the ~30,000 low-resolution distance constraints generated in near-native proteoforms can be used to uniquely (1) contextualise regions within existing experimental protein structures, (2) annotate interfaces in PPIs and multi-protein complexes, and (3) validate structures predicted by computational modellers like AlphaFold. Finally, this thesis reports the use of quantitative XL-MS to explore links between gene function and cellular phenotype, comparing wild-type yeast with a highly pleiotropic deletion strain (Δhpm1) using SILAC multiplexing and PIR cross-linking of intact cells. The untargeted comparison of structural conformations and PPIs uniquely enabled insights into phenotypes entirely invisible to investigation of protein abundance alone. This thesis will therefore argue that XL-MS is the missing link in the systems biology toolbox.

  • (2022) Gutierrez Chavez, Carolina
    Thesis
    Microbial motility is a survival strategy. Bacteria utilise motility in growth and reproduction to protect against desiccation and antibiotics, defend against competitors, colonise new habitats, and find nutrient-rich environments. Antarctica is the most extreme and pristine region on Earth. Here, soils are exposed to low temperatures, considerable levels of UV radiation, strong winds, extreme drought, and low availability of nutrients. Currently, there are no reports of motility in polar soil bacteria. Due to the extreme conditions in the Antarctic continent, we used these polar desert soils as a study model. We hypothesise that Antarctic soils are an ideal environment for bacteria to perform motility to find more favourable conditions. In this thesis, we describe for the first-time mechanisms of motility by Antarctic soil bacteria. As model microorganisms, we selected bacteria from the genera Streptomyces and Arthrobacter, belonging to the phylum Actinomycetota, the most abundant in East Antarctic soils. The first aim was to investigate the incidence and behaviour of Antarctic Streptomyces performing exploration. The genus Streptomyces had been described as a group of sessile bacteria. In 2017, the phenomenon of Streptomyces exploration was described for the first-time as a mechanism of translocation performed by Streptomyces cells through biotic and abiotic surfaces to facilitate access to distant nutrients. It was reported that the incidence of wild-type Streptomyces strains exhibiting exploration was ~10%. In addition, two critical conditions involved in the exploration process were described, glucose depletion and alkaline pH in the medium produced by the synthesis of volatile organic compounds (VOCs). Here, we screened 57 Streptomyces strains previously isolated from four sites in Eastern Antarctica. We found that 22.8% did not exhibit exploration capabilities, and 19.4% showed exploration behaviour in glucose depletion and alkaline environments. In contrast, 57.8% exhibited exploration at neutral pH or in the presence of glucose. These findings about the incidence of glucose-independent exploration suggest that the exploratory behaviour might be encouraged by searching for alternative carbon sources due to the low nutrient availability in East Antarctic soils. The second aim was to provide insight into potential compounds involved in Streptomyces exploration. Here, we obtained a fraction with emulsifying, antimicrobial, and haemolytic activities from the secretome of Streptomyces INR7 explorer cells. LC-MS analysis of the fraction and bioactive predictors revealed that a LysM-containing peptide with amphipathic properties is the responsible for the antibacterial and haemolytic activities. Furthermore, genomic annotation and sequence alignment indicate that the peptide is part of a protein belonging to the resuscitation-promoting factors family, contributing to physiological processes of cell wall remodelling and metabolic activation of dormant cells. The third aim was to describe whether Antarctic Arthrobacter isolates can use microbial hitchhiking as a motility strategy to migrate across the fungal hyphae of Antarctomyces psychrotrophicus. So far, Arthrobacter bacteria have been associated with active motility, also known as flagella-dependent motility. Flagella-assisted displacement is typical in aqueous environments; however, East Antarctic soils are exposed to arid and hyperarid conditions, suggesting that the motility of Arthrobacter isolates from these polar desert soils might be challenging. Here, we found that microbial hitchhiking facilitates the migration of Arthrobacter strains previously isolated from Antarctic soils. Hitchhiking is a form of transportation where microorganisms with null or limited motility, known as 'hitchhikers' or 'riders', use the motility of nearby motile organisms known as 'carriers' or 'host' to move. In soils, fungi facilitate the migration of bacteria over short and long distances by forming 'networks' or 'highways' on which bacteria can travel actively, passively or by direct attachment to the fungal hyphae. By using SEM, motility screening, flagella identification and hitchhiking assays, we found that of 17 Arthrobacter strains examined, 14 migrated through the fungus A. psychrotrophicus. The results suggest that Arthrobacter isolates migrated by three mechanisms: two strains travelled flagella-dependent motility, seven isolates translocated by flagella-independent motility and five strains migrated by direct attachment to the fungal surface. These findings provide evidence that Antarctic soil bacteria, specifically members of the genera Streptomyces and Arthrobacter, exhibit a high incidence of motility. We propose that motility could be a survival strategy used by bacteria to cope with the extreme conditions in Antarctica.

  • (2023) Wu, Winton
    Thesis
    Staphylococcus aureus is a major opportunistic human pathogen and a leading cause of bacteraemia, infective endocarditis, and medical device-related infections. The emergence of multidrug-resistant S. aureus (MRSA) is a global public health concern as these S. aureus isolates are resistant to several antibiotics. Current treatment is dependent on the efficacy of last line antibiotics like vancomycin. However, isolates with intermediate resistance (MIC 4-8 µg/mL) to vancomycin are increasingly detected worldwide and are associated with treatment failure. These vancomycin-intermediate S. aureus (VISA) isolates appear to arise from the acquisition of a disparate series of point mutations that commonly lead to physiological changes including cell wall thickening and reduced autolysis. Transcriptional profiling has revealed that antibiotic treatment drives conserved changes in small RNA (sRNA) expression in S. aureus and may contribute to the VISA phenotype. However, the function of hundreds of sRNAs in S. aureus are still poorly understood. To address this, the endoribonuclease RNase III, which processes sRNA-RNA duplexes, was used as a scaffold to capture sRNA-RNA interactions in VISA using a proximity-dependant ligation and sequencing technique termed CLASH. RNase III-CLASH recovered 215 unique sRNA-RNA interactions in vivo and 135 (63%) of these interactions are novel when compared to the vancomycin-susceptible S. aureus (VSSA) sRNA interactome. Gene ontological analyses revealed that these sRNA-RNA interactions are enriched for functions associated with reproduction, citrate transport, and cellular responses to oxidative stress. To determine whether some of these sRNAs possess coding potential, ribosome profiling was performed to delineate novel ORFs in VISA. Fifty-two potential ORF-containing sRNAs were discovered, seven of which have been confirmed in past literature, and a potential dual-function RNA Sau6072 was revealed. In addition, we identified two novel ribosome-associated noncoding RNAs PemZ1 and PemZ2 which are derived from the 3’ UTR of two different type II toxin-antitoxin operons. By mapping the translational landscape of the VISA genome with ribosome profiling, it can provide insight into the additional uncharacterised functions of these regulatory RNAs. Small RNA interactome techniques have enriched our understanding of sRNA-mRNA interactions and their roles in several physiological functions to conferring antibiotic tolerance, nutrient adaptation, and oligopeptide transport. However, one major challenge is determining how these sRNAs affect the expression of their target mRNAs. To address this, we examined the correlation between gene transcript abundance (RNA-seq), ribosome occupancy (Ribo-seq), and protein levels (proteomics) to identify sRNA-mRNA interactions that post-transcriptionally regulate mRNA translation. We used the machine learning technique Self-Organising Maps to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By overlaying our sRNA-mRNA interaction network on these clusters we identified sRNAs that may be mediating this post-transcriptional repression. Two of these sRNA-mRNA interactions are mediated by RsaOI, a sRNA that is highly upregulated in the presence of vancomycin. CRISPRi knockdown of RsaOI resulted in increased vancomycin sensitivity in two different VISA strains and we discovered that RsaOI post-transcriptionally represses the expression of the major autolysin Atl. This autolysin has been previously implicated in vancomycin tolerance whereby its expression is reduced in several VISA isolates compared to their VSSA parent strains. We postulate that this interaction contributes to vancomycin tolerance in VISA as this could result in reduced autolytic activity, which is a common phenotypic hallmark of VISA. Further, we have confirmed that RsaOI post-transcriptionally represses the sugar phospotransferase component PtsH and Arginase. These results collectively suggest that RsaOI is a global regulator of several biological functions including arginase catabolism, sugar transport and cell wall turnover. Taken together, the work in this thesis has contributed to a greater understanding of the functions of regulatory RNAs in S. aureus. Our multi-omics analyses have provided insight into how sRNA-responsive networks induce changes in response to vancomycin treatment and adapt to antibiotic stress. This research supports the development of novel and robust antimicrobial therapies by developing an enhanced understanding of post-transcriptional regulatory mechanisms in S. aureus and their roles in antibiotic tolerance.

  • (2022) Sah, Saroj
    Thesis
    Interactions between neurons are mediated by cell adhesion molecules (CAMs). NCAM2 and NEGR1 are two CAMs, which accumulate in synapses. Abnormal expression of these synaptic CAMs is associated with behavioral abnormalities and neurodevelopmental disorders in humans. The effects caused by deficiency in these CAMs in the brain remain, however, unclear. To better understand human conditions associated with the loss of NCAM2 and NEGR1, in this thesis, we analyzed the behavior of NCAM2 and NEGR1 deficient mice and performed immunohistochemical analyses of the brain to study the underlying mechanisms of these effects. We found that NCAM2 deficiency in mice leads to the impairment in short-term memory, mild cognitive deficit, and causes an increase in olfactory acuity and repetitive and self-grooming behaviors. Our immunohistochemical studies showed shortening of the infrapyramidal bundle of the mossy fibers in the hippocampus of the NCAM2 knockout mice suggesting axonal guidance defects. Similar axon guidance defects and changes in behavior were also observed in mice deficient in BACE1, an enzyme involved in processing of NCAM2. Further analysis showed changes in distribution and a reduction in levels of BACE1 protein in the hippocampus in NCAM2 knockout mice. Additionally, cell culture studies showed that NCAM2 knockout neurons were characterized by altered organization of the Golgi body organelles and lower levels of the microtubule-associated proteins tau and CRMP2, which are required for transporting protein cargoes and maintaining the cytoskeleton. We observed spatial and short-term memory impairment, hyposmia, and a reduction in motivation towards highly palatable food rewards in NEGR1 knockout mice. These mice had lower densities of synapses in the hippocampus, particularly of those formed by mossy fibers. The overall size of the suprapyramidal bundle of mossy fibers was reduced in NEGR1 knockout mice. These effects were accompanied by the deposition of alpha-synuclein aggregates in the hippocampus. Similarly, the density of inhibitory synapses was reduced in the CA3 pyramidal cell layer of the hippocampus and arcuate nucleus of hypothalamus, which is involved in regulating the feeding behavior. In conclusion, we found that NCAM2 and NEGR1 deficiency causes synaptic changes in the brain, which are accompanied by abnormalities in learning, suggesting that both proteins are involved in regulating neuronal development and plasticity. Our data indicate that NCAM2 and NEGR1 deficiency can directly contribute to neurodevelopmental and other types of disorders in people with variations in the genes coding for NCAM2 and NEGR1.

  • (2022) Ray, Angelique
    Thesis
    Cold desert soils are one of the most hostile environments on Earth, notoriously scarce in liquid water and nutrients, and exposed to highly variable levels of sunlight. Whilst higher taxa are frequently out selected by the harsh conditions, microorganisms have adapted a breath of unique strategies to survive and become widely established within this niche. Despite this, photoautotrophs are reported as scarce within a growing number of cold desert environments, and soil oligotrophy frequently limits geochemical nutrient oxidation by chemoautotrophs. The first-order processes that supply carbon and energy to the broader trophic webs remain an elusive gap in our understanding of cold desert soil ecology. In 2017, a primary production strategy reliant upon the RuBisCO form IE driven Calvin-Benson-Bassham (CBB) cycle and the oxidation of atmospheric H2 and CO by high-affinity enzymes, since coined as ‘atmospheric chemosynthesis’, was identified in soil microbiomes from two Eastern Antarctic sites; Robinson Ridge and Adams Flat. This thesis builds on this initial discovery, revealing that atmospheric chemosynthesis is a significant form of bacterial primary production occurring throughout cold deserts globally. Moreover, we investigate atmospheric chemosynthesis in two cultured bacterial isolates, Rhodococcus opacus (DSM 43205) and Mycobacterium agri (DSM 44515). First, we aimed to investigate how widely dispersed the genetic determinates of atmospheric chemosynthesis are and identify their environmental drivers within oligotrophic cold edaphic deserts. We hypothesised that trace gas chemosynthetic marker genes would be widespread throughout polar communities, and that their abundance relative to community size would increase in drier, more nutrient-poor soils. Using qPCR, we quantified the 16S rRNA gene alongside the RuBisCO form IE (rbcL1E) and high-affinity 1h [NiFe]-hydrogenase large subunit (hhyL) genes in 122 soil microbiomes from 14 cold deserts spanning the Antarctic, Arctic and Tibetan Plateau. Both genes were ubiquitous (rbcL1E, 6.25 × 103–1.66 × 109 copies/g soil; hhyL, 6.84 × 103–5.07 × 108 copies/g soil), indicating that microbiomes in numerous globally distributed cold deserts have a genetic capacity to oxidise atmospheric H2 and fix CO2 through the RuBisCO IE driven CBB cycle. Relative rbcL1E and hhyL gene abundances in the Antarctic and Arctic soils were analysed against 26 measured physicochemical parameters using Random Forest and Spearman correlation strategies, with the relative abundances significantly explained by soil moisture (IncMSE = 69.35 and 24.38, respectively) and total carbon (IncMSE = 16.45 and 17.48, respectively). Additionally, significant increases (p < 0.05) in both gene abundances were observed in soils where moisture and carbon were more limited, implicating these physicochemical parameters as the most prominent selective pressures for this novel form of chemoautotrophy. Next, we aimed to evaluate the significance of atmospheric chemosynthesis as a primary production strategy within cold desert soils. By quantifying trace gas chemosynthetic activity alongside photosynthesis in soils from six sites that encompass the Antarctic, Arctic and Tibetan Plateau, we show that atmospheric chemosynthesis is a global phenomenon, extending throughout cold deserts, with significant implications for the global carbon cycle and bacterial survival within environmental reservoirs. Gas chromatography analysis showed that all 18 soil microcosms examined rapidly oxidised trace H2 (~10 ppmv) to sub-atmospheric levels (6.3 - 623.9 nmol/mol/h/g), with slower and highly variable rates of trace CO (~10 ppmv) oxidation detected both across and within individual sites (0 - 2.6 nmol/mol/h/g). These oxidation rates exceeded levels that are reported to be required to support microbial persistence in similarly structured environmental microbiomes. Carbon assimilation assays revealed that in the McMurdo Dry Valleys and in the high Arctic, trace H2 oxidation significantly increased microbial carbon fixation and, in the high Arctic, so did light exposure (p < 0.05), confirming that atmospheric chemosynthesis occurs broadly within cold deserts and can co-occur alongside photosynthesis. In this chapter, we also aimed to use genomic analysis to identify additional taxa potentially capable of trace gas chemosynthesis and further characterise predicted proteins involved in this process. Analysis of 18 metagenomes, 230 dereplicated medium-to-high quality metagenome-assembled-genomes (MAGs) and 24,080 publicly available genomes revealed substantial genetic diversity within characterised groups of RuBisCO form I and [NiFe]-hydrogenases, including uncovering a new form of high-affinity [NiFe]-hydrogenase not previously identified; group 1m. Genes encoding key trace gas chemosynthetic enzymes, including high-affinity hydrogenases, aerobic carbon monoxide dehydrogenase and RuBisCO form IE, were widespread in bacterial taxa that dominated all six cold desert environments examined. Trace gas chemosynthesis genes co-occurred within representative genomes from four phyla not previously known to harbor both genes: Chloroflexota, Firmicutes, Deinococcota and Verrucomicrobiota. Furthermore, key marker genes co-occurred within 92 already isolated bacterial taxa, 60 of which are available from the Leibniz-Institut culture collection, revealing a potential pathway for future pure culture characterisation studies. Finally, we aimed to validate atmospheric chemosynthesis as a carbon fixation strategy and characterise the underlying pathway in two strains found to harbor 1h [NiFe]-hydrogenase, aerobic carbon monoxide dehydrogenase, and RuBisCO form IE; R. opacus and M. agri. Both strains were originally isolated under heterotrophic conditions. We cultivated both taxa in organic carbon-limited and organic carbon-rich media, in the absence of light and under conditions that allowed continuous gas exchange with the atmosphere. Subsequently, cultures were sealed with trace levels of H2 (~10 ppmv) and CO (~0.9 ppmv). When inoculated in organic carbon-free media, R. opacus and M. Agri rapidly consumed H2 and CO to sub-atmospheric or atmospheric levels (R. opacus; H2 68.6 nmol/mol/h and CO 9.1 nmol/mol/h. M. agri; H2 35.9 nmol/mol/h and CO 2.3 nmol/mol/h). When grown in organic carbon-rich media, M. agri rapidly oxidised H2 within 24 hrs and produced carbon monoxide throughout the incubation period. In contrast, R. opacus gradually oxidised H2 and rapidly produced CO for the first 24 hrs prior to organic carbon starvation, after which H2 and CO were both oxidised to sub-atmospheric levels within a further 48 hrs. Transcriptomic analysis was performed in parallel to activity studies of R. opacus in organic carbon-rich media and with H2 stimulation, to investigate the metabolic expression underlying the observed activities and the concurrent growth that was observed. Aerobic carbon monoxide dehydrogenase and forms 1h and 3b [NiFe]-hydrogenase drove the observed trace gas oxidation and, throughout growth, were upregulated alongside the RuBisCO form IE small subunit and heterotrophic pathways. Therefore, in R. opacus, atmospheric CO and H2 oxidation provides critical energy sources that support carbon limitation and microbial growth. We now know that microbial taxa throughout Earth’s cold desert soils use trace gas oxidation, not only to support persistence during starvation, but also to support carbon fixation and growth. Research presented here characterises trace gas driven growth for the first time within pure culture. Trace gas chemosynthesis could have implications upon how we culture and isolate extremophiles for characterisation and biotechnological applications, the survival strategies of airborne and aeolian dispersed microbial taxa, and the redefinition of planetary habitable zones.To explore this further, it is recommended that future research focuses upon characterising atmospheric chemosynthetic taxa and investigating the distribution and significance of trace gas driven carbon fixation in additional microbiomes throughout other terrestrial ecosystems, aquatic bodies, and the atmosphere.

  • (2021) Luo, Lijuan
    Thesis
    Bacterial foodborne pathogens are significant public health and economic burdens. Whole-genome sequencing (WGS) offers unprecedented power to characterise the molecular epidemiology of both epidemic and emerging foodborne pathogens. The objectives of this thesis were to elucidate the genomic epidemiology of an epidemic foodborne pathogen Salmonella enterica serovar Enteritidis and an emerging foodborne pathogen Escherichia albertii, and to develop a multilevel genome typing (MGT) scheme and database for genomic surveillance of S. Enteritidis. S. Enteritidis is a global epidemic foodborne pathogen. Multi-country outbreaks and multidrug resistance (MDR) are of increasing concern. In this thesis, the core genome of S. Enteritidis was defined and used to develop a global publicly available MGT database for S. Enteritidis (https://mgtdb.unsw.edu.au/enteritidis/). By analysing all publicly available genomes using MGT, the global genomic epidemiological characteristics of S. Enteritidis were elucidated in detail. Further, the application of MGT was evaluated for the local public health surveillance of S. Enteritidis in Australia. The S. Enteritidis MGT database was found to be powerful in the rapid determination of the population structure, comparison of isolates from different regions, sources and time periods, investigation of international outbreak clusters, and identification of isolates related to MDR and invasive infections. E. albertii is an emerging diarrhea-causing pathogen. The genomic epidemiological characteristics of E. albertii remain unclear. This thesis analysed 169 newly sequenced E. albertii genomes from China and 312 publicly available genomes. The E. albertii population was divided into two clades and eight lineages, with 3 lineages more common in China. Virulence genes were found to be distributed differently among lineages. Seven new subtypes of the intimin encoding gene eae and one new subtype of the cytolethal distending toxin gene cdtB were identified. Alarmingly, 85.9% of the Chinese E. albertii isolates were predicted to be MDR with 35.9% harbouring genes capable of conferring resistance to 10 to 14 different drug classes. The findings in this thesis provide fundamental and new insights into the genomic epidemiology of S. Enteritidis and E. albertii. The open MGT database was shown to be a good candidate in global and local public health surveillance of foodborne pathogens.

  • (2022) Bello, Idris
    Thesis
    The inflammatory artery diseases atherosclerosis and abdominal aortic aneurysm (AAA) are major causes of morbidity and mortality and there is significant attention towards identifying and targeting prominent inflammatory mediators underpinning these cardiovascular diseases. In the first chapter, the role of the pro-inflammatory and pro-oxidant enzyme myeloperoxidase (MPO) in inflammatory artery disease was studied. A clinical study showed that while circulating plasma MPO levels were not different in AAA patients versus healthy controls, immunohistochemistry showed that the MPO protein was prevalent in human AAA tissue. In the angiotensin II (AngII)-infusion model of AAA and atherosclerosis in apolipoprotein-E gene-deficient (ApoE–/–) mice, administration of 2-thioxanthines (2-TX), a clinically-trialled MPO inhibitor, significantly inhibited AAA but not atherosclerosis. Paradoxically, MPO gene-deficiency did not affect AngII-induced AAA but attenuated atherosclerosis. Notably, 2-TX significantly inhibited AAA in ApoE–/–MPO–/– mice, indicating 2-TX protects against aortic disease in the absence of MPO. The role of MPO in the diabetes-accelerated atherosclerosis in ApoE–/– mice was also examined. While MPO gene-deficiency did not impact on the degree of diabetes it significantly reduced diabetes-accelerated atherosclerosis at the brachiocephalic artery and aortic sinus, but not aortic arch, indicating that MPO exhibits site-specific effect on atherosclerosis. A second chapter focused on semicarbizide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1), a pro-inflammatory enzyme that facilitates the vascular recruitment of activated leukocytes. Treatment of AngII-infused ApoE–/– mice with a clinically-viable SSAO/VAP-1 inhibitor significantly protected against AAA and atherosclerosis, independent of alterations to plasma lipid levels. A third chapter tested the therapeutic efficacy of apolipoprotein A-I (ApoA-I), the major cardioprotective protein in high-density lipoproteins, and a class of immunomodulatory nanoparticles (INPs), which selectively target and disable a pro-inflammatory monocyte subset. Although ApoA-I treatment did not impact on the development of arterial disease in AngII-infused, aged ApoE–/– mice, INPs provided significant protection against AAA and atherosclerosis in these mice. This novel research provides new insights on the roles of MPO and SSAO in AAA and atherosclerosis and identified clinically-viable inhibitors of MPO and SSAO and a class of biodegradable immunomodulatory nanoparticles as potential new therapeutics for treating inflammatory artery disease.

  • (2023) Suyama, Hiroki
    Thesis
    Pertussis (whooping cough) is a respiratory disease caused by Bordetella pertussis. The currently used vaccine provides a relatively short duration of immunity and pathogen adaption against the vaccine has been linked to a resurgence of pertussis globally. Past research and vaccine development has been based on planktonic cells, however, recent studies have shown that B. pertussis readily forms biofilms. Biofilms are a community of cells encased within a matrix and these cells have been shown to be more resilient against antimicrobials. Studies of the biofilm lifestyle may provide further insight into B. pertussis evolution and pathogenesis. A comparative study of biofilm and planktonic cells was performed by combining mass spectrometry (MS) and metabolic modelling. It was found that the glyoxylate shunt was utilised in planktonic cells while the biofilm cells completed the tricarboxylic acid cycle providing novel targets for biofilm disruption. Single nucleotide polymorphism (SNP) cluster I (ptxP3/prn2) strains that have overtaken the previously dominant SNP cluster II (ptxP1/prn3) strains were compared in biofilm conditions. Denser biofilm structures for a cluster I strain were identified using confocal laser scanning microscopy. MS analysis revealed cytochrome proteins were strongly upregulated in the SNP cluster I strain, however, this change was specific to the individual strain. Interestingly, increased biofilm formation was found when the strains were grown in low oxygen, which are conditions found during infection. The cluster II strains had increased levels of cells dispersed from the biofilm compared to the cluster I strains in these conditions. These results show that there is an underlying difference in the way the clusters respond to changes to oxygen concentrations when grown in biofilm conditions and may relate to the dominance of the cluster I strains. Finally, an immunoproteomic method for identifying novel vaccine antigens was optimised. This method utilised immortalised mouse dendritic cells (MutuDC) infected with B. pertussis to identify T-cell epitopes naturally presented to the immune system. There were 16 proteins identified that provide the basis for further testing as prospective vaccine antigens. Taken together, these findings develop our knowledge of B. pertussis biofilms and presents potential new targets for an improved vaccine.

  • (2023) Keable, Ryan
    Thesis
    NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules, a family of proteins which engage in homophilic trans-interactions at contacts between cells. NCAM2 accumulates at excitatory synapses in the brain and plays established roles in synapse maturation and maintenance. Deletions and single nucleotide polymorphisms within the NCAM2 gene are associated with neurodevelopmental disorders, while elevated levels of NCAM2 and increased cleavage of NCAM2 are associated with Alzheimer’s disease, a neurodegenerative disorder. In this thesis, we study the role of NCAM2 in regulating the transport of AMPA receptors: excitatory neurotransmitter receptors which accumulate at the postsynaptic density of neurons, and BACE1: an enzyme involved in the generation of Aβ: a toxic peptide which accumulates in the brains of people with Alzheimer’s disease. We show that NCAM2 is a BACE1 substrate and uncover a pathway whereby the BACE1-generated C-terminal fragment of NCAM2 promotes the accumulation of BACE1 in recycling endosomes. NCAM2 deficiency results in defects in the targeting of BACE1 to presynaptic boutons, impaired activity dependent transport of BACE1 to axons, and lower levels of Aβ. In related work, we show that NCAM2 interacts with the GluA1 subunit of AMPA receptors via the extracellular domain, regulates the endocytic recycling of GluA1 and promotes the accumulation of GluA1 at the cell surface. Complexes of NCAM2 and GluA1 are enriched at contact sites between heterologous cells expressing NCAM2, suggesting that NCAM2 can simultaneously bind GluA1 and other NCAM2 molecules on adjacent cells. The targeting of GluA1 to synaptic contacts between neurons is reduced in NCAM2 deficient mouse brain, suggesting that NCAM2 regulates synaptic strength by controlling the levels of AMPA receptors at synapses. Our results indicate that NCAM2 plays an important role in synapse remodeling by targeting BACE1 to recycling endosomes and presynaptic boutons, and AMPA receptors to the post-synapse. Future research will investigate the role that NCAM2 plays in regulating the activity of AMPA receptors, delineating the mechanisms by which NCAM2 controls the endocytosis and recycling of synaptic proteins, and improving our understanding of how changes in NCAM2 expression and cleavage contribute to the etiology of neurodevelopmental and neurodegenerative disorders.

  • (2023) Bell, Henry
    Thesis
    Methylation of CpG dinucleotides in the human genome can significantly alter the profile of genes we express in our cells. In human erythrocytes, removal of CpG methylation from the genome reactivates the fetal gamma-globin subunit of haemoglobin (which is normally repressed in adult cells) through an unknown mechanism. Expression of gamma-globin in patients with sickle cell disease or beta-thalassaemia has an ameliorating effect on disease outcomes by interfering with the mechanisms of these diseases. Understanding the mechanism of gamma-globin induction by CpG demethylation is therefore of great interest for the development of therapeutic strategies to treat patients of these diseases. We identified the affinity of the MBD2 component of the NuRD complex to methylated CpG sites as an important factor in repression of the HBG1/2 genes that encode gamma-globin. This appears to be a direct mechanism of HBG1/2 repression, as impairment of this affinity did not appear to affect expression of other regulators of HBG1/2 but displayed a trend towards decreased localisation and activity of MBD2-NuRD at these genes. Direct involvement of local CpG methylation in HBG1/2 gene repression was further evidenced by substantial reactivation of the genes following treatment targeting a demethylase domain to the promoters. These findings advance our understanding of the mechanism by which CpG methylation affects expression of the HBG1/2 genes, demonstrating that this epigenetic mark acts locally at the promoters and is involved in localisation of the NuRD complex. Targeting of CpG methylation directly at the HBG1/2 promoters shows promise for the development of effective therapeutic strategies for induction of gamma-globin that may improve on the safety of existing gene therapy approaches.