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Investigation of the function of meningococcal genes : NMB0711, NMB0768, NMB1048, NMB1525, NMB1898, NMB1948 and NMB1966Chow, Noel Yuet Sung January 2007 (has links)
This thesis describes the construction and evaluation of six knockout mutants in Neisseria meningitidis serogroup B strain MC58. The genes that were inactivated were NMB0711, NMB1048, NMB1525, NMB1898, NMB1948 and NMB1966. Attempts to inactivate a seventh gene, NMB0768, were not successful. These genes were chosen as they had been observed previously to be up-regulated following incubation in whole blood using Differential Fluorescence Induction. Mutant strains were constructed by allelic exchange with a plasmid construction in which a kanamycin resistance cassette had been incorporated within the coding sequence of each cloned target gene. Confirmation of successful allelic exchange was achieved by Southern blotting. The phenotype of all mutant strains were evaluated by assessment of in vitro growth and in the infant mouse model of infection. Of the six mutants, all except that involving NMB1966, showed no differences compared with wild-type. The mutant knockout of NMB1966 showed (1) impaired growth beyond the mid-logarithmic phase in shaking broth culture but normal growth on solid medium, (2) reduced virulence in a mouse model of infection, (3) impairment in its capacity to invade (although not adhere to) cultured human bronchial epithelial cells, and (4) more rapid killing in ex vivo human blood. NMB1966 is predicted to encode the ATP-binding subunit of an ABC transporter and, after experiments for this thesis had been completed, it was demonstrated, by others, that this ABC transporter is responsible for uptake of L-Glutamate at low sodium concentrations. It is likely that defective uptake of L-Glutamate explains the observed defect in shaking broth culture and intracellular survival, both of which are associated with low ambient concentrations of sodium. However, it is not certain if this mechanism explains the observed defect in survival in human blood and in the infant mouse model which test predominantly extracellular survival and represent environments with high sodium concentrations.
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