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Neisseria meningitidis is the most common cause of bacterial meningitis, a disease that kills thousands of people yearly. Asymptomatic colonisation of the oropharynx (i. e. carriage) occurs in 10% – 30% of humans. Significant features of meningococcal genomes are simple sequence repeats (SSR), which have been shown to control gene expression in a reversible process known as phase variation (PV). This study investigated the consequence of “switching-OFF” of two haemoglobin-acquisition systems (hpuAB and hmbR) during disease and also explored potential associations between PV state of the immunodominant meningococcal antigen, PorA, and the adaptive immune response during carriage. Using an ex vivo human whole blood model, hmbR-OFF mutants of strain MC58 (hpuAB-negative) exhibited a pattern of growth similar to wild-type. Conversely, an inability to utilise transferrin (ΔtbpBA) significantly affected growth but not survival in blood. Five recombinant versions of these Hb receptors were prepared in this study and subsequently used as antigens for the generation of polyclonal and monoclonal antibodies in mice. The polyclonal antirHpuA, anti-rHpuB and anti-rHmbR antisera were reactive with homologous receptors in lysates of diverse meningococcal strains. Surface expression of HpuA and HmbR was detected by flow cytometry but all antisera were incapable of mediating killing of iron-restricted meningococci. An immunodetection assay employed in this study revealed the induction of variant-specific anti-PorA IgG antibodies following acquisition of carriage. These antibodies may have contributed to subsequent loss of carriage but a role for PV in immune escape in vivo was not established. This study posits that HmbR is less important than TbpBA and HpuAB during disease and that phase variable expression of surface receptors is irrelevant for immune evasion during carriage. Further studies are recommended to confirm the proposed importance of HpuAB over HmbR during invasive disease and to investigate the opsonophagocytic property of the anti-HpuA and anti-HmbR antisera.

Bioluminescence, the emission of light from living organisms, occurs throughout nature. In bacteria, bioluminescence is directly coupled to electron transport making it an excellent reporter of bacterial metabolic activity. The full lux operon from the terrestrial bioluminescent species, Photorhabdus IUl11inescens was transferred via a plasmid to the human pathogenic species, Staphylococcus alfrelfS and Neisseria meningitidis. The resulting transformants produced light constitutively and without the need for addition of exogenous substrate. Bioluminescent S. Ql~lreUS was challenged with the antimicrobial agent linezolid, and the inhibitory effect of the antibiotic on the organism at 6, 13 and 20 mg/L (Cmin, Cint and Cnax) along with its recovery following the withdrawal of linezolid was observable in real-time, by monitoring bacterial light output. Bacterial metabolism recovered more rapidly than bacterial replication (l.5 hours vs 3.5 hours at Cint) and the bacteriostatic nature of linezolid was confirmed since low light levels were detected throughout exposure to the drug. Bioluminescent Streptococcus pneul110niae (transformed prior to this study) was applied to a model blood-brain barrier (BBB) comprising a co-culture of cells grown on opposing sides of a polycarbonate membrane. By monitoring bioluminescence, it was possible to observe the anti-pneumococcal activity of gemifloxacin across the BBB in real-time, and non-invasively. Above the gemifloxacin MIC (0.16 mg/L), inhibition of metabolism was observed and a reduction in bacterial metabolism by sub-MIC gemifloxacin concentrations (0.03 and 0.13 mg/L) across the BBB was also recorded. Bioluminescent S. aureus and S. pneumoniae both demonstrated the potential of lux-expressing bacteria of clinical importance to be used as a novel, rapid and real time method of assessing antimicrobial efficacy when challenged directly, or in the presence of eukaryotic cell lines such as the model BBB. It was also possible to explore, using bioluminescent S. pneumoniae, factors which may damage BBB integrity and which are exploitable by bacteria. Bacterial penetration of the BBB is the key step in the pathogenesis of bacterial meningitis, but remains one of the most poorly understood aspects of the disease. Bioluminescent N. meningitidis was applied to the model BBB and, using low light camera equipment, it was possible to observe meningococcal penetration of the BBB in real time and in situ. By optimising growth conditions, BBB penetration time by N. meningitidis was reduced from 50 hours to 9 hours. This application has shown bioluminescence to be a valuable reporter of bacterial location and activity and has the potential to yield new information about the pathogenesis of meningococcal disease and meningitis in general

Neisseria meningitidis is the major cause of bacterial meningitis worldwide. The genome of this pathogen contains >40 phase variable loci whose expression is regulated by tandem DNA repeat tracts. Majority of these loci encode OMPs, which are potential targets for host innate and adaptive immune responses. An analysis for the distribution, frequency and role of PV of these genes is relevant in determining their virulence association and suitability as a future vaccine candidate. The project investigated the combined distribution, frequency and PV status of two important genes, hpuAB and hmbR, in disease (n=221) and carriage (n=305) isolates. Strains with both genes or only hmbR were present at similar frequencies among disease isolates as compared with carriage isolates. However, >90 % of isolates from CC5, CC8 and CC11 (CCs with the highest disease to carriage ratios) contained both genes. Strains with only hpuAB gene were under-represented among disease isolates, possibly due to the receptor having a high level of immunogenicity or being inefficient in iron acquisition during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. Further, one or both genes were found in an ON state in 96 % of disease and 66 % of carriage isolates. This suggests that expression of at least one Hb receptor is of major importance for disease, and that the presence of both receptors contributes to virulence in some strains. The experimental findings also revealed the ability of strain 8047 to escape from MAb P1.2 mediated killing due to PV in porA promoter sequences. We conclude that incubation in the presence of antibody selected for phase variants (10C or 9C) with >3 fold lower PorA surface expression than wild type. This escape in the presence of a specific immune response is an elegant demonstration of the importance of PV for adaptation, and have implications in the development of PorA based vaccines. Finally, the bactericidal activity of HpuA polyclonal antisera generated during this project was evaluated and possible reasons for a lack of its activity are discussed.

Central nervous system (CNS) infection remains a major cause of mortality and morbidity worlwide. This thesis focuses on the causes, prognostic markers, and pathogenesis of meningitis in adults in Viet Nam and explores the pharmacokinetic properties of fluoroquinolones in the treatment of tuberculous meningitis (TBM). Chapter One provides an introduction to infections of the CNS in Viet Nam. Chapter Two describes the clinical studies of CNS infections at the Hospital for Tropical Diseases in Viet Nam which underpin this thesis. Mycobacterium tuberculosis is the most common cause of CNS infections with the three commonest causes of acute pyogenic meningitis Streptococcus suis, Streptococcus pneumoniae and Neisseria meningitidis. In Chapter Three, I report on a study of the pathogenesis of meningitis and encephalitis in particular the role for Metalloproteinase/Tissue Inhibitor of Metalloproteinase. One of the most challenging situations is to distinguish partially treated pyogenic meningitis from TBM and viral meningitis and encephalitis. I identified two major clinical factors that can help distinguish TBM from pyogenic meningitis- gingivo-herpes and deafness are very much more common in pyogenic meningitis than in TBM (Odds Ratio 32). In Chapter Four, I develop a clinical aligorithm and in Chapter Five, a prognostic system to determine which variables can be used to predict the clinical outcome.TBM remains very difficult to treat. In Chapter Six, I report the results of a pharmacokinetic study aimed to identify the optimal fluoroquinolone to be used for the treatment of TBM. My results demonstrate levofloxacin has a better pharmacokinetic profile than ciprofloxacin or gatifloxacin for the treatment of TBM.

Meningococcal meningitis is a major public health problem in an area of sub-Saharan Africa known as the meningitis belt. In this region, thousands of people fall ill each year during highly seasonal meningitis outbreaks. At frequent but unpredictable times, large epidemics of meningitis sweep the belt. Despite the gravity of the problem, much is still unknown about what drives this pattern of epidemics. The aim of this thesis is to use mathematical models to gain insights into the epidemiology of meningococcal meningitis in the meningitis belt. Firstly, several simple deterministic models are analysed. They highlight the importance of immunity following both asymptomatic carriage and invasive meningitis. For a broad range of plausible parameter values, seasonal variation of the rate of meningococcal transmission results at long, irregular intervals. Realistic sized repeated epidemics are not possible if only the ration of cases to carriers changes seasonally.

In sub-Saharan Africa bacterial meningitis is common and carries a high mortality, Steroid adjuvant therapy reduces mortality in aduhs in the developed world but has not been adequately tested in developing countries or in the context of advanced HIV. The mechanism of action of adjunctive steroids in bacterial meningitis is incompletely understood but may involve down regulation of the host's proinflammatory cytokine response. Bacterial meningitis in this setting presents late and is strongly associated with HIV co-infection. Dexamethasone adjuvant therapy for bacterial meningitis in aduhs did not reduce mortality or morbidity. Intramuscular ceftriaxone was not inferior to intravenous ceftriaxone for the treatment of bacterial meningitis. Cytokine expression was unaffected by the use of steroid adjuvant therapy.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, it gains access to the bloodstream and causes septicaemia and subsequently meningitis. T-cell stimulating protein A (TspA) is an immunogenic, conserved, T -cell and B-cell stimulating protein of N meningitidis that is required for optimal adhesion to human cells. The role of TspA in the adhesion process is thought to be indirect since it is predicted that the N-terminus is localised to the periplasmic space with an inner membrane- spanning domain linking it to a cytoplasmic C-terminal domain; the aim of this study was to identify proteins within the meningococcal envelope that interact with TspA. Four overlapping recombinant fragments of TspA were expressed and purified as fusion proteins with the pGEX-2T-encoded glutathione-S- transferase protein and pQE70. A receptor activity-directed affinity tagging protocol was employed to identify TspA-interacting meningococcal proteins. Interactions between TspA and candidate proteins were investigated further using ELISA and surface plasmon resonance. Four putative TspA-interacting proteins were identified: PilQ and PilT (components of the type IV pilus machinery); the major outer membrane protein, PorA, and the protein chaperone, ClpB. Furthermore, the portion of TspA responsible for interaction with PorA was confirmed to be the N- terminus. Together, findings show that periplasmic domains of TspA interact with several outer membrane proteins which have a key role in meningococcal pathogenesis and adhesion to host cells.

Neisseria meningitidis, a nasopharyngeal commensal and the causative agent of meningococcal disease in humans, is exposed to nitrosative stress in its biological niche. The genome of the meningococcus contains the genes aniA and norB, coding for a nitrite reductase and a nitric oxide reductase, respectively. Together these constitute a partial denitrification pathway, which the meningococcus uses to supplement its growth under microaerobic conditions.

Cryptococcus neofomans causes up to 80% of cryptococcal meningitis cases in HIV positive individuals, resulting in significant morbidity and mortality within this patient group. Heat-shock proteins (Hsps) are a group of proteins play vital functions in the folding and unfolding or translocation of proteins, as well as in the assembly and disassembly of protein complexes. Many heat-shock proteins have been identified in fungi, such as Hsp70 in C. neoformans and Hsp90 in C. albicans. Mycograb® is a recombinant antibody derived against the LKVIRKNIV epitope of candidal Hsp90. In the present thesis, anti-Hsp90 polyclonal antibody was used in the immunoelectron microscopy and immunoblot assays to demonstrate the cellular localization and induction of Hsp90 in C. neoformns under various stress factors.