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Title: Genome wide transcriptional profiling of Neisseria meningitidis in an epithelial cell line model of human nasopharyngeal colonization
Author: Hey, Ariann
ISNI:       0000 0004 2714 7889
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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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.
Supervisor: Kroll, John Sponsor: HPA
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral