Genome-wide transcriptional profiling of Neisseria Meningitidis in ex vivo models of Septicaemia

Neisseria meningitidis is a Gram negative bacterium that commonly colonizes the nasopharyngeal mucosa of 10% of the population at any given time. Infrequently it invades to cause life threatening septicaemia and/or meningitis. I have used microarray technology to study the repertoire of meningococcal gene expression in the course of prolonged incubation in human plasma and blood, with the aim of identifying genes that are progressively up-regulated during sepsis, especially those encoding surface antigens. I hypothesize that any surface exposed gene products that are expressed at a higher level in the bloodstream than on the mucosal surface, where immune selection pressure drives antigenic diversity, should be less variable, and so, attractive as cross-protective vaccine candidates. In my thesis research I explored experimental methods for modelling meningococcal bacteraemia ex vivo. I characterised the transcriptional response of a serogroup B strain of N. meningitidis during prolonged incubation (up to 4 hours) in fresh frozen non-bactericidal plasma from two individuals. Subsequently the meningococcal transcriptome was examined during incubation in fresh, anticoagulated non-bactericidal whole human blood for di erent periods up to 4 hours, using microarrays and realtime PCR technology. The cellular localisation of proteins encoded by genes con rmed to be signi cantly di erentially expressed in plasma and/or blood, both by microarray technology and by real-time PCR, was explored using bioinformatic resources, and those predicted (or established) to be surface-exposed were brought forward for further evaluation. Amongst these, I selected genes up-regulated in bacteria incubated in plasma and/or blood, but down-regulated (according to data previously obtained) for organisms isolated from an experimental system modelling the nasopharyngeal carriage state, for examination of sequence diversity. Using serogroup B meningococcal strains of established (multi locus sequence type) diversity; I have used direct sequencing to address the starting hypothesis that these are comparatively invariant genes