Award details

Molecular and functional characterisation of a type III secretion system (TTSS) of the obligate intracellular bacterium Lawsonia intracellularis

Reference	BB/C510532/1
Principal Investigator / Supervisor	Professor David Smith
Co-Investigators / Co-Supervisors	Dr Maria Alberdi
Institution	University of Glasgow
Department	Veterinary Pathology
Funding type	Research
Value (£)	306,334
Status	Completed
Type	Research Grant
Start date	01/06/2005
End date	31/05/2009
Duration	48 months

Abstract

Although the pathological features of proliferative enteropathy, the disease caused by Lawsonia intracellularis, are well-described, little is understood of the bacterial determinants involved in pathogenicity. Recently we have identified in L. intracellularis genes conserved among bacterial Type III Secretion Systems (TTSS). These are contact-dependent protein secretion systems that deliver bacterial proteins into host cells leading to changes in normal cellular function, for instance, cytoskeletal reorganisation, cytotoxicity, modulation of host defences. The presence of a TTSS in L. intracellularis is a significant finding and we propose to characterise this locus to improve our understanding of the pathogenicity of this bacterium. So far we are the only group to have successfully taken molecular approaches to characterise L. intracellularis and this project will build on the experience we have gained. Genomic, proteomic and functional studies will be combined as follows: i) genomes from two L. intracellularis strains are being sequenced and this data will be accessed. A series of analysis tools will be used to predict ORFs and non-coding regions within the TTSS pathogenicity island of this bacterium. Comparison will be made to other genomes to predict the function of determinants encoded by this locus. Complementation in heterologous host bacteria and ablation of expression in L. intracellularis will be attempted to confirm function in secretion. ii) TTSS gene expression will be monitored during infection in vivo and in vitro using qRT-PCR. Where feasible, promoter regions from initial genes in operons will be cloned into a dual GFP-CAT reporter systems that we have developed ¿ transcriptional activities will be confirmed by fluoresent and/or enzymatic detection. iii) expression and secretion of TTSS proteins will be established by immunoblotting and immunofluorescent microscopy. Proteins secreted by L. intracellularis will be assessed by radiolabelling and selection of corresponding proteins on parallel 2d gels for characterisation by MALDI-ToF MS. iv) function of selected secreted factors will be assessed in vitro and in vivo. Characterisation of intracellular localisation (by immunofluorescence microscopy), protein-protein interactions (immunoprecipitation followed by MALDI-MS) and phenotypic assessment (e.g. interference in apoptosis and cell proliferation) will be carried out ¿ precise procedures will depend on specific characteristics of secreted proteins as identified in preceding investigations. This project represents a detailed examination of L. intracellularis pathogenicity determinants taking combined genomic, proteomic and functional approaches. Our study will not only significantly advance understanding of this bacterium, but additionally will provide comparison with other TTSS thus improving comprehension of these important bacterial determinants.

Summary

unavailable

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Animal Health, Immunology, Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	Proteomics and Cell Function (PCF) [2003-2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search