Pagliuca, Chiara (2010) Role of glutamate in the meningococcal pathogenesis: mouse model of meningococcal meningitis and innovative therapeutic strategy. [Tesi di dottorato] (Unpublished)

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Neisseria meningitidis (meningococcus) is a human-specific Gram-negative β-proteobacterium, recognized as the leading cause of bacterial meningitis globally. The meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject; in particular circumstances it acquires invasive ability: penetrates the mucosal barrier, invades the bloodstream causing septicaemia and, in about half of bacteremic persons, N. meningitidis crosses the blood-brain barrier and invades the cerebrospinal fluid effecting fulminant meningitis. All the pathological consequences of meningococcal infection depend on the bacterium’s ability to acquire nutrients for replication within the systemic circulation while at the same time avoiding exclusion by host innate immune mechanism. In vitro experimental data have been demonstrated that the L-glutamate ABC transporter GltT is essential for the survival of the meningococcus, microrganism naturally auxotrophs for L-glutamate. However, animal infection models are of great importance for a complete understanding of meningococcal disease. Several experimental model systems have been evaluated over the last decades, but no models was able to reproduce the characteristic pathological events of meningococcal disease. In the present work, it was developed a mouse model of meningococcal meningitis based on intracisternal inoculation of bacteria. Survival and clinical parameters of infected mice and microbiological and histological analysis of the brain have demonstrated the establishment of meningitis with features comparable to those of the disease in humans. When using low bacterial inocula, meningococcal replication in the brain was very efficient, with a 1,000-fold increase of viable counts in 18 h. Mouse passage was a key step to develop the model. Compared to laboratory strain, mouse-passaged meningococci were also found in the blood, spleens, and livers of infected mice. As glutamate uptake from the host has been implicated in meningococcal virulence, mice were infected intracisternally with an isogenic strain deficient in the ABC-type L-glutamate transporter GltT. Noticeably, the mutant was attenuated in virulence in mixed infections, indicating that wild-type bacteria outcompeted the GltT-deficient meningococci. The data show that the GltT transporter plays a role in meningitis and concomitant systemic infection, suggesting that meningococci may use L-glutamate as a nutrient source in the murine host. To assess whether the transporter GltT may represent a potential candidate for the development of new vaccines, protein components have been expressed and purified to obtain specific immune sera anti-GltT. Moreover, it has been shown that animals and patients suffering from bacterial meningitis present high levels of L-glutamate in cerebrospinal fluid. In the present work it was analyzed the possible therapeutic role of L-glutamate analogues and/or NMDA receptor antagonists on the replication and survival of the meningococcus and on reduction of neuronal damage.

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