This thesis tested the hypothesis that immunosenescence contributes to reduced immunity to \(Streptococcus\) \(pneumoniae\). The effect of age on neutrophil and monocyte responses to \(S.\) \(pneumoniae\) and on CD4+ T cell polarisation during health, pneumococcal carriage and clinical pneumonia infection were determined. Older adult’s neutrophils produced less ROS in response to serotypes 19A and 23F, but not 4, and increased NETs towards 23F. However, neutrophils of older pneumonia patients produced high levels of ROS to all three serotypes but had impaired NET release. Older patients also had immature granulocytes and CD16\(^h\)\(^i\)\(^g\)\(^h\)CD62L\(^d\)\(^i\)\(^m\) neutrophils in blood. CCR2 and CD11b expression, TNF-α and IL-6 production by monocytes were unaffected by age. Pneumococcal colonisation of the nasopharynx is an immunising event. The effect of age on carriage was tested using a human carriage model. Older adults had elevated Th1 and lower Th17 frequencies and failed to generate Th17 memory. During pneumonia, pro-inflammatory subsets increased with age, but Treg frequency and function were maintained. In conclusion, failure of pneumococcal carriage to generate immune memory, together with altered neutrophil responses to \(S.\) \(pneumoniae\) and high frequencies of inflammatory Th subsets in older adults who succumb to infection, could contribute to their increased susceptibility to pneumococcal infection.

Pseudomonas aeruginosa is a common industrial contaminant associated with costly recalls of home and personal care(HPC) products. Preservation systems are used to prevent bacterial contamination and protect consumers, but little is known about the mechanisms of preservative resistance in P. aeruginosa. The aim of this research was to map genetic and metabolic pathways associated with preservative resistance and bacterial growth in HPC products. The genome of the industrial strain P. aeruginosa RW109 was sequenced, functionally annotated, and compared to other strains of the species. This revealed the first complete genome of a P. aeruginosa isolate from the HPC industry. Comparative analysis with 102 P. aeruginosa strains from various sources, showed industrial strains’ genomes to be significantly larger than clinical and environmental strains and RW109’s genome was the largest of the species (7.8 Mbp) and included two plasmids. Identification of differentially expressed genes by RNA-Seq (more informative than mini-Tn5-luxCDABE mutagenesis), revealed complex genetic networks utilised by RW109 when exposed to benzisothiazolone(BIT), phenoxyethanol (POE) and a laundry detergent formulation. Differential expression of five sets of genes was consistently observed in response to these industry relevant conditions - MexPQ-OpmE efflux pump, sialic acid transporter and isoprenoid biosynthesis (gnyRDBHAL) genes were frequently upregulated; whereas phnBA and pqsEDCBA genes encoding PQS production and quorum-sensing, respectively, were consistently down-regulated. Genome-scale metabolic network reconstruction of RW109, the first with a P. aeruginosa industrial strain, along with integration of transcriptomic data, predicted essential pathways for RW109’s preservative resistance (e.g. cell membrane phospholipid biosynthesis as a key pathway for POE resistance). This study highlights the utility of integrating genomic, transcriptomic and metabolic modelling approaches to uncover the basis of industrial bacterial resistance to preservative and product formulations. The ability to predict the metabolic basis of P. aeruginosa preservative resistance will inform the development of targeted industrial preservation systems, enhancing product safety and minimising future resistance development.

The subject matter of this thesis is the role of nuclear non histone proteins in the control of gene expression. To understand how these proteins may be able to regulate gene expression we first look at their molecular environment, viz. the structure of chromatin; some of the evidence linking non histone (chromatin) proteins with gene expression is then examined, together with the background of the experimental work reported here. In the first experimental chapter, the binding of a labelled carcinogen ([14C]-acetylaminofluorene) to rat liver nuclear protein fractions was examined. Most of the recovered carcinogen (80%) was bound to the nonhistone protein fraction. After two weeks the label was more evenly distributed; the highest specific activity being in the protein fraction most tightly bound to DNA. Pre-feeding rats with sodium sulphate and phenobarbitone reduced binding to all nuclear fractions. Labelled proteins were further analysed by isoelectric focusing which revealed a complex binding pattern. In the second experimental chapter the nucleoplasmic and non-histone chromatin proteins from four related and two unrelated mouse plasmacytoma cell lines were analysed by biosynthetic labelling with [35S]-methionine followed by one and two dimensional electrophoresis. In this work in clonal cell lines it was hoped that by looking at cells that were "frozen" at one stage of differentiation, viz. the plasma cell, that it would be possible to both limit the differences between the cell lines and correlate particular proteins with phenotypic differences. The majority of proteins are common to all of the cell lines studied as would be expected if the majority of nuclear proteins are concerned with functions common to all plasma cells. There are however both qualitative and quantitative differences in the nuclear protein patterns of mutant and parent cell lines which appear to correlate with differences in gene expression. The turnover of nuclear proteins in two of the cell lines, MOPC 315.40 (IgA) producer and MOPC 315.32 (λ2 chain producer), which exhibited these differences, was studied using pulse-chase techniques and the half lives of both nuclear protein fractions and nuclear proteins which differed between the cell lines were calculated. Finally, the results of these experiments are discussed in the context of findings by other authors working in similar areas and in the light of recent developments concerning the role of these proteins in gene expression.

Shiga toxin (Stx) producing E. coli (STEC) infections represent an important public health problem given the severity of the disease and sequelae associated to it. Since the use of antibiotics enhances the virulence of STEC, new therapeutic strategies are urgently required. Thus, the main aim of this project is the study of small molecules that are able to block expression of Shiga toxin in Escherichia coli O157:H7. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimized compound interferes with the SOS response by directly affecting the activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants. As a result of the successful identification of a small molecule capable of inhibiting Stx2 expression in E. coli O157:H7, an additional high-throughput screening (HTS) of small molecules was performed. Two new compounds with activity against Stx2 production were successfully identified and characterised in biological assays. Finally, we describe the use of a small molecule with previously reported anti-quorum sensing activity. Our findings suggest that the compound furanone C-30 blocks stx expression in vitro.

The research described in this thesis was aimed at the characterization of the Candida adhesin, and elucidation of the nature of the human epithelial cell receptor with which it combines. Previous data have shown that the fibrillar mannoprotein layer, produced when Candida albicans is grown in high concentrations of galactose, contains the proteinaceous adhesin. Different Candida species from leukoplakia patients were assayed for their ability to adhere to buccal cells. The results supported previous conclusions that there is a relationship between the ability of different Candida albicans strains to adhere to epithelial cells and their capacity for cell surface modification. Extracellular polymeric material (EPM) was isolated from culture supernates of C. albicans after growth in medium containing 500 mM galactose. When used to pretreat buccal epithelial cells, EPM inhibited adherence, which suggested that it contains an adhesin that binds to, and blocks epithelial cell receptors. Fractionation of EPM by affinity chromatography was performed. An index, the adhesion inhibition index (AII) was used to compare the various "lectin-like" components isolated from crude EPM. These studies indicated that use of different buccal cell donors gave different results, with the same C. albicans strain. Attempts to resolve EPM by SDS-polyacrylamide gel electrophonesis had previously proved unsuccessful. However here, success was achieved using the silver-stain technique. Chemical and enzymatic digestion of the EPM indicated that the protein portion , of the glycoprotein was more important than the carbohydrate at inhibiting adherence. N-Glycanase, papain, mild alkali treatment of EPM, followed by Synsorb-H-2 affinity adsorption chromatography, resulted in a purification of the yeast adhesin of more than 220 fold, relative to the crude EPM (on a protein weight basis). The nature of the epithelial cell receptor for C. albicans was investigated with potential receptor analogues such as sugars, lectins, monoclonal antibodies and saliva. The adhesion of C. albicans to the buccal cells of blood groups A and O, was found to be significant with respect to secretor status but not blood group. Caution should be shown in the interpretation of sugar inhibition tests. Nevertheless, N-acetyl-D- galactosamine was the most effective single sugar as an inhibitor of adhesion for buccal cell donors of blood group A; N-acetyl-D-galactosamine is the immunodominant blood group sugar for group A cells and the possibility exists that the blood group oligosaccharide on the buccal cell surface functions as the receptor for yeast adhesion. Further work would be needed to establish whether the purified adhesin had any therapeutic value in treating Candida infections.

The research in this thesis was aimed at determining the chemical nature of the surface structures on Candida albicans and on host epithelial cells that are involved in yeast-epithelial adhesion. From previous studies it was known that C. albicans from active infections (I strains) were more adherent to buccal and vaginal epithelial cells after growth in medium containing 500 mM galactose than after growth on 50 mM glucose. Extracellular polymeric material (EPM) was isolated from culture supernates of C. albicans after growth in medium containing 500 mM galactose. EPM was composed of carbohydrate (70%), protein (10%) and phosphorus (0.05%) and was therefore considered to be glycoprotein. When used to pretreat buccal epithelial cells, EPM inhibited adherence, which suggested that it contains an adhesin that binds to, and blocks, epithelial-cell receptors. Fractionation of EPM by gel filtration and ion-exchange chromatography gave five components, all of which were capable of inhibiting adherence but to different extentso An index, the adherence inhibition index (All) was devised to compare activities of the various components. Fractionation of EPM by affinity chromatography on Con A-Sepharose and application of Con A-bound material to a DEAE-cellulose column yielded material which was 30 times more active as an inhibitor than crude EPM. Attempts to resolve EPM by SDS-polyacrylamide gel electrophoresis proved unsuccessful even though a variety of buffer and staining procedures was investigated. Chemical and enzymic treatment of EPM indicated that the protein portion of the (postulated) glycoprotein was more important than the carbohydrate at inhibiting adherence. Proteolytic enzymes, mild acid and heat treatment of EPM abolished its ability to inhibit adherence. Periodate and a-mannosidase had no effect, whereas papain and mild alkaline treatment of EPM enhanced its ability to inhibit adherence. The nature of the epithelial cell receptor for C. albicans was investigated with potential receptor analogues such as sugars and lectins and inhibitors. The adhesion of C. albicans GDH 2346 to buccal, cells was inhibited by L-fucose and pretreatment of the buccal cells with winged-pea lectin (which is specific for L-fucose), However, the adhesion of another C. albicans strain (GDH 2023) to buccal cells was inhibited by N-acetyl-D-glucosamine and by pretreatment of the buccal cells with wheat-germ agglutinin (which is specific for N-acetyl-D-glucosamine). These experiments indicated that glycosides function as epithelial cell receptors for C. albicans, but with the sugar residue varying between strains. Samples of EPM from various strains of C. albicans bound to affinity columns of Sepharose containing coupled sugars. The bound fractions were capable of inhibiting adherence, suggesting that they contained an adhesin component. These results indicate that 'lectin-like' interactions may be involved in yeast adherence to host epithelial cells.

No description available.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis. It affects approximately 0.05% of the general population presenting as peripheral arthritis, axial disease or a combination of both. About 10% of patients with psoriasis suffer from PsA. Currently available therapies have limited efficacy with associated toxicity. PsA is therefore a common arthropathy for which novel therapies are urgently required. Our understanding of the role of the immune system in the pathogenesis of rheumatic disease has led to the development of specific targeted therapies. The identification of tumour necrosis factor alpha (TNFα) as a key player in the inflammatory response has been a major advance in the field of rheumatology. The effects of blocking this pro inflammatory molecule have resulted in dramatic clinical effects across the spectrum of rheumatic diseases. Unfortunately however there remain a proportion of non-responders and concerns have been raised surrounding the adverse effects of these therapies (Furst, Keystone et al. 2001). It is therefore important to continue to elucidate other key players in the inflammatory cascade that can be modified to achieve clinical response. A critical advance in understanding the immune system's role in driving inflammation has been the recognition of different functional subsets of T helper (Th) cells according to their cytokine profile. Techniques have evolved that enable us to achieve clarification of a true in vivo Th1 response by identifying cells in SF that spontaneously secrete IFNγ. In this thesis, spontaneously secreting cells were identified in SF of patients with active PsA. That such IFNγ expressing cells may have a pathogenic role in PsA is supported by the following observations: IFNγ is the prototype macrophage activator of TNFα that drives inflammation in psoriasis and PsA. IFNγ when tested as a potential treatment for psoriasis induced synovitis in a subset of patients. (O'Connell, Gerber et al. 1992). The injection of IFNγ into the skin induces hyperkeratosis (Fierlbeck, Rassner et al. 1990). Autologous Natural Killer T (NKT) cells, potent producers of IL-4 and IFNγ, are able to induce psoriasis in 'normal skin' grafted onto a SCID mouse (Nickoloff, Wrone- Smithetal. 1999). Those factors regulating the expression of IFNγ may therefore be of considerable interest as they may offer potential targets to modify Th1 responses in PsA. Cytokines such as interleukin 18 (IL-18) and interleukin 12 (IL-12) have been shown to regulate Th1 responses in animal models of arthritis (Leung, McInnes et al. 2000). Whether such cytokines are present and indeed induce inflammation in PsA is unknown. The first part of this thesis focuses on the characterisation of the predominant Th response in PsA and the identification of key regulatory cytokines such as IL-12 and IL-18 that may drive TNFα production and as such lead to inflammation. The presence of other regulatory cells capable of driving the inflammatory response was explored briefly. NKT cells are a subset of T cell, which secrete IFNγ and IL-4 depending on the predominant cytokine milieu (Hafner, Falk et al. 1999). A pathogenic role for NKT cells in psoriasis has been suggested. (Bonish, Jullien et al. 2000). The ability of synovial fluid mononuclear cells (SF MC) to proliferate and secrete TNFα in response to glycolipid presented to NK T cells was investigated. Although TNFα contributes directly to synovial inflammation, the blockade of TNFα does not protect completely against cartilage damage (Joosten, Helsen et al. 1999). IFNγ and IL- 17 have opposing effects on bone resorption. IFNγ inhibits bone resorption whereas IL-17 enhances resorption through the degradation or enhancement of TNF receptor associated factor 6 (TRAF6) respectively (Chabaud, Lubberts et al. 2001). The expression of IL-17 and the functional effects of this cytokine on MMP3 expression by fibroblasts were therefore studied as a final section in this thesis.

Innate lymphoid cells (ILCs) mainly reside at mucosal sites, where they produce effector cytokines upon activation. ILCs are also found, at lower frequencies, in secondary lymphoid tissues. ILCs in the lymph nodes (LN) are located in the interfollicular area, which is rich in migratory dendritic cells (DCs) and recently activated T cells and B cells. We recently discovered that some intestinal ILCs traffic from the intestinal lamina propria to mesenteric lymph nodes (MLN). However, the functions of ILCs in the LNs are hardly understood. Here we provide for the first time a detailed description of the migratory ILCs in intestinal lymph of mice in homeostasis and immunity. To isolate migratory ILCs, we obtained lymph by thoracic duct cannulations of mice with or without a previous mesenteric lymphadenectomy. The ILC populations were then analysed by flow cytometry, and their transcriptomic profile was assessed by performing RNA sequencing. Our results show that a small number of migratory ILCs continuously traffic from the intestine to the MLNs. Whole genome analyses in the steady state revealed a shared global ILC signature between migratory ILCs in lymph and intestinal resident ILCs. We then demonstrated that all subsets of ILCs migrate in intestinal lymph, with ILC1 and ILC2 being most frequent. We compared the transcriptomic profile of migratory DCs, T cells and ILCs in afferent lymph and clearly identified a core migratory signature shared by all cell types. In order to investigate the impact of inflammation on this migratory ILC population, we used several infection and inflammation models, and assessed changes in the migratory ILC populations. We detected phenotypic changes of migratory ILCs following Salmonella infections of mice. Furthermore, although Salmonella infection did not increase total numbers of migratory ILCs in lymph, we observed an increase in T-bet+Roryt+ co-expressing ILCs in lymph of infected mice. After infection, changes in ILCs transcriptomic profile indicate an activated phenotype. Corresponding to the lymph ILC data, we also observed an accumulation of T-bet+Roryt+ co-expressing ILCs in the colonic draining LN (cMLN). Our data clearly demonstrate, for the first time, that inflammation can alter migratory ILC populations. This might be important for the initiation and regulation of adaptive immune responses in the draining LN. This first characterisation of migratory ILCs is important as it helps to understand how they contribute to shaping adaptive immune responses in the interfollicular area of the LNs.

The emergence of vancomycin-resistant enterococci (VRE) since the 1980s has turned this Gram-positive bacterium into a serious and growing clinical challenge. Enterococci have acquired resistance to a number of different antibiotics and are intrinsically resistant to cephalosporin b-lactam drugs. As a result, they are now placed in the World Health Organisation list of priority pathogens for which new antibiotics are urgently needed. An important risk factor for the emergence of VRE is treatment with cephalosporins, suggesting a connection between the origins of resistance between these two different types of antibiotics, the mechanistic basis for which was unknown. In this study I demonstrate that cephalosporin resistance is significantly enhanced in the well characterized E. faecalis OG1RF strain containing the Tn1549 transposon conferring the VanB type vancomycin resistance. Cephalosporin resistance is shown to be enhanced to the presence of the Tn1549 transposon and a single, chromosomally encoded Serine/Threonine (ST) kinase gene called ireK. Complementation experiments and fluorescence microscopy with a range of biophysical techniques to characterise the protein. Deletion of the gene results in cephalosporin sensitivity. Moreover, this phenotype can be chemically simulated by treatment of E. faecalis with inhibitors of cytoplasmic ST kinases such as staurosporine, demonstrating a requirement for ST kinase activity for cephalosporin resistance. This also demonstrates a therapeutic potential for targeting ST kinases in Gram-positive pathogens with small molecule inhibitors to restore cephalosporin sensitivity which may have clear translational significance. IreK was identified as a key protein involved in enhanced cephalosporin resistance and led to examination of its extracellular and intracellular domains to understand its signal transduction mechanisms and its linkage to resistance. The extracellular domain of IreK was essential for cellular location. Further biophysical experiments identified ligands for the extracellular domains include sugar motifs such as the glycan backbone of peptidoglycans and sugar containing antibiotics. This has led to a new functional model for ST kinases in enterococci.