Genome-wide transcriptional profiling of Neisseria Meningitidis in ex vivo models of Septicaemia
Author(s)
Hedman, Asa Katarina
Type
Thesis
Abstract
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.
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.
Date Issued
2009-02
Date Awarded
2009
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Creator
Hedman, Asa Katarina
Publisher Department
Department of Paediatrics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)