Genome wide transcriptional profiling of Neisseria meningitidis in an epithelial cell line model of human nasopharyngeal colonization
Author(s)
Hey, Ariann
Type
Thesis
Abstract
Neisseria meningitidis (the meningococcus) is a Gram negative diplococcus which asymptomatically colonizes
the human nasopharynx in about 10%-30% of the population. In rare cases it can invade the bloodstream and
cross the blood brain barrier to cause sepsis and meningitis. Serogroup B strains are the major cause of
disease in the UK.
To gain insight into the process of colonization, I have studied the meningococcal transcriptome in a model of
human nasopharyngeal colonization. As meningococcal colonization can last for weeks to months,
investigating the meningococcal transcriptome over an extended period of time offers potentially important
new insights into meningococcal biology unexplored in short time-course experiments as undertaken by
others.
Confluent layers of a human bronchial epithelial cell line (16HBE14) were infected with a serogroup B
meningococcal strain (MC58) for different time periods (4h, 24h, 96h and 21d) prior to bacterial RNA
extraction. Using microarray technology, the transcriptome of host cell-associated meningococci was
investigated and expression of selected genes (some previously shown to be involved in host cell
interaction/colonization, and others surface expressed vaccine candidates) was validated with quantitative
real time PCR.
The transcription profile at late time points (24 h, 96 h and 21 d) was compared to that at 4 h to identify genes
potentially involved in prolonged bacteria host-cell association. A set of seven genes (NMB034230348) was
identified which was up-regulated at later time points. By use of RT-PCR the operon structure of the locus was
defined: one set of four and one pair of these genes are co-transcribed. MC58 kanR insertion mutants in five
of these seven genes were prepared and studied in quantitative adherence and invasion assays. An impaired
adherence and reduced invasion profile compared to the wild type suggests a potential role in sustained host
cell association.
the human nasopharynx in about 10%-30% of the population. In rare cases it can invade the bloodstream and
cross the blood brain barrier to cause sepsis and meningitis. Serogroup B strains are the major cause of
disease in the UK.
To gain insight into the process of colonization, I have studied the meningococcal transcriptome in a model of
human nasopharyngeal colonization. As meningococcal colonization can last for weeks to months,
investigating the meningococcal transcriptome over an extended period of time offers potentially important
new insights into meningococcal biology unexplored in short time-course experiments as undertaken by
others.
Confluent layers of a human bronchial epithelial cell line (16HBE14) were infected with a serogroup B
meningococcal strain (MC58) for different time periods (4h, 24h, 96h and 21d) prior to bacterial RNA
extraction. Using microarray technology, the transcriptome of host cell-associated meningococci was
investigated and expression of selected genes (some previously shown to be involved in host cell
interaction/colonization, and others surface expressed vaccine candidates) was validated with quantitative
real time PCR.
The transcription profile at late time points (24 h, 96 h and 21 d) was compared to that at 4 h to identify genes
potentially involved in prolonged bacteria host-cell association. A set of seven genes (NMB034230348) was
identified which was up-regulated at later time points. By use of RT-PCR the operon structure of the locus was
defined: one set of four and one pair of these genes are co-transcribed. MC58 kanR insertion mutants in five
of these seven genes were prepared and studied in quantitative adherence and invasion assays. An impaired
adherence and reduced invasion profile compared to the wild type suggests a potential role in sustained host
cell association.
Date Issued
2012-02
Date Awarded
2012-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Kroll, John
Sponsor
HPA
Creator
Hey, Ariann
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)