Award details

Whole genome screen to identify virulence factors of the porcine pathogen Actinobacillus pleuropneumoniae

Reference	BB/C508193/1
Principal Investigator / Supervisor	Professor Paul Langford
Co-Investigators / Co-Supervisors	Professor John Kroll, Professor Andrew Rycroft
Institution	Imperial College London
Department	Dept of Medicine
Funding type	Research
Value (£)	262,943
Status	Completed
Type	Research Grant
Start date	14/03/2005
End date	13/05/2008
Duration	38 months

Abstract

Actinobacillus pleuropneumoniae (APL) is a bacterium that causes a world-wide economically important severe life-threatening lung disease of pigs ¿ it is only known natural host. There is a real need for an effective vaccine. Unfortunately very little is known about the mechanisms by which the bacterium causes disease, the understanding of which is an important step in the search for a vaccine. In this work we propose to use a new technique (transposon screen by microarray; TSM) to identify those genes of APL that are essential for survival in vivo on a genome-wide scale. TSM is now possible because of the recent availability of genome sequences of APL allowing the construction of whole genome arrays on which the method depends. We will initially determine the most random method of transposon mutagenesis (Tn5 or Tn10) for APL and optimise TSM in vitro with a model system (thiol stress). An advantage of the method is that large numbers of random mutants (500+) can be assessed in a single experiment. The method involves PCR amplification, including a fluorescent labelling step, from chromosomal DNA from input (inoculum) and output (end of experiment) pools and probing of a whole genome microarray. Comparison of the fluorescent signals obtained for individual genes from the input and output pools allows the identification of genes that are essential for growth in the environment under investigation. Once optimised in vitro, TSM will be used to identify genes where interruption by the transposon leads to attenuation in vivo. Giving priority to conserved hypothetical genes and novel genes that are prevalent in the 15 known serotypes of APL, defined mutants will be constructed in these genes of interest and their attenuation confirmed in comparative virulence studies with the wild-type parent strain. Subsequently, the most promising mutants will be evaluated as live vaccine strains for their ability to protect against APL infection.

Summary

unavailable

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Animal Health, Immunology, Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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