Staphylococcus aureus is the most common cause of hospital-acquired infection and contributes significantly to patient morbidity and mortality. The ability of S. aureus to switch to an alternative phenotype in the presence of antimicrobial agents is clearly favourable. One of these alternatives are small colony variants (SCVs). The novel phenotypes include changes to colony morphology, antibiotic susceptibility, haemolytic activity and many other physiological activities. It is now recognised that SCVs have a deficiency in electron transport, owing to mutations affecting its efficacy. This study investigated SCVs in various ways. In the evolution component changes (mutations) occurring sequentially in successive cycling (15 cycles), were identified. In this experiment selection was made for sequentially SCV mutants and wild type revertants. Two sequenced clinical MRSA strains COL and N315 were chosen so changes in sequence in SCVs and wild type revertants could be compared. Selection for SCVs was made independently for triclosan and gentamicin for both strains. The final SCV and WT strains isolated were compared physiologically and genetically and showed differences in frequency, biochemical profiles, pigment production, haemolysis, catalase, coagulase, levels of intracellular ATP and phage yield. The genomic sequence of the final 4 cycle isolates (SCV15) showed numerous and diverse mutations occurred COL and N315 SCVs. Over 70 mutations were found and 33 were determined as historic mutations and the rest were termed novel mutations. The novel mutations occurred during the cycling process. The historic mutations occurred prior to the experiment and these mutations were acquired during growth in laboratory culture. Only one mutation was found to be common between COL and N315 and this was in the fabI gene. These data indicate mutations occurring in ~1.3% of the genome (~ 40 Kb) can generate mutants with the SCV phenotype. Susceptibility to phage 80α and transduction of S. aureus wild type and their SCVs 1-3 was studied. Wild type strain of S. aureus and SCV3 both yielded a high number of lysogens (~68%) the remaining being resistant mutants. SCV 1 and SCV 2 provided a much lower proportion of lysogens (4-10%). There was no obvious relationship between cellular ATP levels and lysogen formation. Consequently the frequency of lysogen formation (or that of resistance mutants) cannot be related to energy status. Transduction of ciprofloxacin resistance (grlA) was observed into COL wild type at a 5-10-fold higher frequency than into SCV1. Transduction of rifampicin resistance (rpoB) into SCVs was reduced almost 10-fold. As transduction was significantly decreased into SCVs it is hypothesised this process was influenced by ATP levels. The data thus suggests that SCV strains will be less efficient in gene exchange by transduction in vivo. Three SCVs previously isolated from S. aureus COL on the basis of different growth rate were further studied. Results clearly support the hypothesis that there is a physiological diversity in SCV populations. Sensitivity of S. aureus wild type and SCVs strains to various antimicrobial was determined. The SCV strains were more sensitive to some antibiotics and heavy metals than the wild type strain.

Campylobacter is a major cause of acute bacterial gastroenteritis worldwide, with the highest number of infections being attributed to Campylobacter jejuni. C. jejuni is a Gram negative, spiral, motile bacterium that belongs to the campylobacterales order and is related to both Helicobacter spp. and Wolinella sp. It has long been established that proton pump inhibitors (PPIs) and other benzimidazole derivatives display anti-Helicobacter activity in vitro. PPIs have in the past been shown to affect Helicobacter pylori growth, survival, motility, morphology, adhesion/invasion potential and susceptibility to conventional antibiotics. PPIs are highly effective drugs that are well tolerated, safe for prolonged daily use and are therefore in high demand. Both the PPIs omeprazole and lansoprazole featured in the top ten drugs prescribed in England in 2014. In 2014 Campylobacter was also the most commonly diagnosed gastrointestinal infection in Scotland, in England and Wales and also in Europe. It has previously been generally accepted that patients who are being treated with PPIs are more susceptible to enteric infections such as Campylobacter than people not taking PPIs. The effect of PPI exposure on H. pylori has been investigated rigorously in the past. A single previous study has hinted that PPIs may also be capable of affecting the related organism C. jejuni,but investigations have been extremely limited in comparison to those investigating the effect of PPIs on H. pylori. This study has investigated the in vitro effects of direct contact with PPIs on the biology ofC. jejuni. Exposure to the PPI pantoprazole was found to affect C. jejuni growth/survival, motility, morphology, biofilm formation, invasion potential and susceptibility to some conventional antibiotics. Microarray studies showed that the cmeA and Cj0561c genes were significantly up-regulated in response to pantoprazole exposure and a CmeABC deficient mutant was found to be significantly more susceptible to killing by pantoprazole than was the parent strain. Proteomic analysis indicated that the oxidative stress response of C. jejuni was induced following exposure to sub-lethal concentrations of pantoprazole. C. jejuni gene expression was assessed using qRT-PCR and the genes encoding for thiol peroxidase and GroEL co-chaperonin (both involved in the C. jejuni oxidative stress response) were found to be around four times higher in response to exposure to sub-lethal concentrations of pantoprazole. Experiments using the oxidative stress inhibitors thiourea (a hydroxyl radical quencher) and bipyridyl (a ferrous iron chelator) showed that killing by pantoprazole was not mediated by hydroxyl radical production.

Monoclonal antibodies are a class of therapeutic that is an expanding area of the lucrative biopharmaceutical industry. These complex proteins are predominantly produced from large cultures of mammalian cells; the industry standard cell line being Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed that a further bottleneck in biosynthesis is in protein folding and assembly within the secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was generated by researchers at the pharmaceutical company UCB that stably overexpressed two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was confirmed to have a high yield of secreted antibody. The work presented in this thesis further characterises CHO-S XE, with the aim of using the information gained to lead the generation of novel host cell lines with more optimal characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE had improved production of two other secreted proteins: one with a simple tertiary structure and one complex multi-domain protein; and higher levels of a number of endogenous protein chaperones. As a more controlled system of gene expression to unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE, CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the Unfolded Protein Response, GADD34, were generated. From these cell lines, it was shown that more antibody was secreted by cells with induced overexpression of XBP1; however, Ero1α and GADD34 overexpression did not improve antibody yield. Further investigation revealed that endogenous XBP1 splicing was downregulated in the presence of an abundance of the active form of XBP1. This result indicated a novel aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease responsible for XBP1 splicing. Overall, the work described in this thesis confirms that the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO cells; information which could contribute to the development of host cells with a greater capacity for antibody production.

Streptococcus pneumoniae is a major human pathogen and causes a significant burden of disease in both developed and developing countries. Currently, two pneumococcal vaccines are available, a polysaccharide conjugate vaccine for children <2 years of age and an adult polysaccharide vaccine for ‘at risk’ groups such as the elderly and immunocompromised. Unfortunately, due to the vast variation and highly recombinant nature of the pneumococcus vaccine escape through serotype replacement is significantly decreasing the efficacy of pneumococcal vaccines globally. New cost-effective and protective pneumococcal vaccines are urgently required. Pneumolysin (PLY) is a 53Kd cholesterol-dependent cytolysin that is largely conserved in all strains of Streptococcus pneumoniae, making it an ideal candidate for inclusion in a broad spectrum vaccine. It has been shown that PLY is not only a protective immunogen but also has potent adjuvant properties and stimulates both IgG and IgA antibody responses to antigens genetically coupled to the toxin (Douce et al., 2010). Both systemic and mucosal responses are induced when PLY is used as an adjuvant which may prevent colonization and therefore provide non-serotype specific herd immunity to Streptococcus pneumoniae. The cytolytic activity of PLY prevents its inclusion in a human vaccine; a non-lytic deletion mutant 76PLY was created for this purpose which retains adjuvanticity, albeit slightly reduced. The aim of this study was to elucidate the mechanism(s) of PLY/Δ6PLY adjuvanticity, it will be essential to have a basic model of adjuvant activity before PLY-based vaccines can be advanced to human clinical trials. This project used a combination of high-throughput methods such as protein pulldowns and gene expression profiling to examine the abilities of PLY, 76PLY and the truncation mutants D123PLY and D4PLY to bind to and be internalized by host cells and to differentially regulate gene expression. These studies highlighted specific and direct interactions between PLY variants and the host cytoskeleton that could mediate antigen/PLY uptake; they also revealed a pattern of gene expression that is similar to those of other adjuvants and could provide the basis for a model of adjuvanticity. Finally, through the use of reporter cell lines and transgenic TLR4-/- BMDM, the relationship between PLY and TLR4 has been further defined. A novel method for preparing vehicle controls provided evidence that the ligation of TLR4 in this system is PLY-dependent and is not an artefact caused by contaminating TLR ligands such as LPS. Once this was established it was possible to further investigate the role of TLR4 in the adjuvant activity of PLY, in particular the PLYdependent production of IL-1@. Through these studies a surprising role for TLR4 in in vitro PLY-dependent cytokine production was discovered. Additionally, it was found that complement has an essential role in the PLY-dependent production of IL-1@. The role(s) of complement and IL-1@ in the adjuvant activity were further investigated using an in vivo immunization model and the biological basis for the difference in adjuvant activity of PLY and 76PLY was defined.

No description available.

Background: There are a large number of viruses spread by mosquitoes, many of which cause debilitating, often fatal, neurological disease such as acute encephalitis. In this study we have used two different neurotropic viruses: Semliki Forest virus (SFV), and West Nile virus (WNV), both of which can cause severe panencephalitis in the mouse. The influx of leukocytes into the infected tissues is mediated by chemokines and is believed to be important for virus clearance. To date, we have only limited insights into the precise nature of chemokine involvement, and an improved understanding of these important axes provides a new target for the development of novel therapies. Hypothesis: Based on previous studies investigating the role of chemokines during neuroinflammation it was hypothesised that chemokines and other cytokines are highly upregulated during viral encephalitis, and the blockade of selected chemokine receptors would lead to altered disease outcome. It was also hypothesised that chemokine receptors would present plausible targets for the treatment of viral encephalitis. Results: To test these hypotheses, the chemokine expression pattern and the kinetics of chemokine mediated leukocyte recruitment during viral encephalitis were analysed in unprecedented detail by TaqMan low density array, and flow cytometry, respectively, and key chemokine receptor were identified as therapeutic targets. Both SFV and WNV exhibited a similar pattern of chemokine upregulation, although WNV induced significantly higher fold expression. The key chemokines upregulated were CCL2, 3, 5, 7, CXCL9 and CXCL10. The upregulation of chemokines coincided with leukocyte influx into the CNS. After identifying the key chemokines upregulated during viral encephalitis, next a selected panel of chemokine receptor antagonists was utilized to evaluate the hierarchy and relative importance of distinct chemokine receptors for CNS leukocyte influx, viral clearance, neuropathogenesis and host survival. We identified the CXCR3 axis as being the key instigator of CNS inflammation in response to alphavirus infection, placing it at the top of a hierarchal cascade that is followed by CCR2 and CCR5. Critically, inhibition of both CXCR3 and CCR2 simultaneously, significantly improved host survival to otherwise lethal encephalitis. Conclusion: These data suggest that chemokine receptors represent plausible therapeutic targets for viral encephalitis.

Coinfection with bacteria and viruses is an understudied area of microbiology, despite its potential to modulate pathogen abundance and host survival. We investigated the effect of bacteria on virus infection and developed an in vitro system to study the first step: viral internalization. Our studies show that multiple bacterial species promote the entry of a diverse panel of viruses into lung and gut epithelial cells. Bacteria expressing the toll-like receptor (TLR)5 agonist, flagellin, are most efficient at inducing viral uptake and studies using recombinant flagellin or aflagellate bacterial strains confirm that flagellin has pro-viral activity. Flagellin promotes epithelial cells to support virus entry via TLR5-dependent activation of NF-KB. To extend these observations and study the role of flagellin in the complete viral replicative lifecycle, we studied human immunodeficiency virus (HIV)-1 replication in T cells. Flagellin augments HIV-1 entry and promoter activity and increases the production of extracellular virus. The data presented in this thesis highlight a new role for bacterial flagellin to promote diverse virus infection of epithelial barriers and enhance the spread of HIV-1. This has significant implications for understanding how exposure to multiple pathogens can alter susceptibility to infection and its associated pathogenesis.

Outer membrane vesicles (OMVs) are nanosized proteoliposomes ubiquitously released from the outer membrane of Gram negative bacteria, and are known to contribute to immune priming and disease pathogenesis. However, the current understanding of their interactions with host cells is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery. This work has developed a highly sensitive method to study vesicle entry into host cells in real-time using a genetically encoded, vesicle targeted probe. Using this approach, it was found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of O polysaccharide in lipopolysaccharide creates a bias towards non-receptor mediated endocytosis, which enhances both the rate and efficiency of entry into host cells. This work indicates that the composition of the bacterial cell wall influences the behaviour of OMVs, and is therefore implicated in secretion-system independent delivery of bacterial virulence factors during Gram negative infection.

Hepatocytes can capture dead cells. This phenomenon is called efferocytosis. Furthermore, our lab previously observed live CD4+ T cells captured by hepatocytes. This was reminiscent of entosis. This project aimed to further the understanding of the mechanisms and consequences of these processes. In vitro experimentation showed that efferocytosis could be modulated through cytokine treatment and using macropinocytosis inhibitors. Captured cells were also shown to associate with the uncharacterised receptor, SCARF2. Furthermore, efferocytosis was shown to cause multinucleation in hepatocytes. This was demonstrated in vitro, in vivo using mouse models of acute injury, and ex vivo with cauterised donor human tissue. Increased multinucleation was also associated with hepatocellular carcinoma and vascular invasion. Live CD4+ T cell capture occurred at a lower frequency than efferocytosis and required alternative membrane rearrangements. This process also did not share defining characteristics of entosis, such as E-cadherin association or susceptibility to Rho-kinase inhibitors. Furthermore, anti-inflammatory T-regulatory cells were more likely to enter acidic compartments within their captors in vitro. This project has unearthed novel aspects regarding the regulation and molecular processes of hepatocyte cell-in-cell structure formation. Further understanding into the mechanisms of these processes may provide future targets for therapeutic intervention of inflammatory disease and cancer.

Invariant natural killer T (iNKT) cells are endowed with features of both innate and adaptive immunity. They are activated by the recognition of glycolipid agonists presented by CD1d which makes them excellent candidates for cellular therapies. In order to investigate the ability of iNKT cells to suppress experimental acute Graft versus host disease (GVHD), iNKT cells were first expanded in vitro, which is likely to be required prior to their use as a cellular therapeutic. Interestingly, the expanded cells showed increased frequencies of IL-10, IL-13 and IL-17 producing cells and were found to robustly suppress alloreactive T cell proliferation in vitro compared to freshly isolated cells. However, in a model of acute GVHD induced by alloantigen-reactive TCR-transgenic T cells neither freshly isolated nor expanded iNKT cells suppressed GVHD, although some survival benefit was seen following the activation of host iNKT cells. These data indicate that iNKT cells can be expanded ex vivo, that they can acquire different functional properties and that such cells robustly suppress alloreactive T cell proliferation in vitro. Therefore, further investigation into the suppressive behaviour of these cells is warranted despite a failure to suppress acute GVHD in the current study.