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A Proteomic Approach to Identify Virulence Determinants of EHEC 0157. - OZ0705(1)

Escherichia coli O157:H7 is a major food-borne human pathogen. Bovines are thought to be the most important reservoir and source of infection for man. The mechanisms by which cattle become and remain infected are not understood. Oral infection of neonate calves results in so-called attaching and efacing gut lesions but this is strain dependant. Moreover, older animals do not display such lesions implying that other genetic determinants are likely to be responsible for persistence. Given that the genome of O157 is some 20% larger than that of the laboratory strain K-12, there is considerable genetic capacity for novel pathogenic determinants and some of these loci are likely to contribute to persistence.

The key aim of this proposal is to use a sensitive and rapid proteomic approach to identify novel proteins and their encoding genes that are induced in the bovine gut. Proteins from Escherichia coli O157:H7 cells grown in appropriate environments will be analysed by high resolution two-dimensional gel electrophoresis and image analysis. Proteins of interest will then be identified using a rapid and highly-sensitive proteomic approach involving the electrospray mass spectrometry and peptide mass fingerprinting. Standard genetic methodology will be used to disrupt the genes for such proteins and resulting mutant Escherichia coli O157:H7 strains will be tested for colonisation.

The proposed studies will shed light on the behaviour of Escherichia coli O157:H7 in an infected host animal of relevance to the food industry. In particular, it will provide detailed information on the physiological status of the pathogen in the bovine gut and will identify novel components that are likely to be involved in colonisation and persistence. Mutant strains lacking these components may be attenuated in virulance. Such strains may be of use in the construction of live vaccines, e.g. for use in farm host animals.
01 11/1999 To construct refence maps of EHEC 0157 proteins induced and repressed in response to specific environmental conditions. For base line studies, organsims will be grown in vitro under conditions likely to mimic the stressess associated with the bovine gut environment and protein profiles will determined by 2-D gel electrophoresis and image analysis. These profiles will be compared to those of organisms recovered from the intralumenal bovine ligated gut to detect proteins uniquely induced in the latter environment.

02 04/2000 To create global regulatory mutants of EHEC 0157 for proteomic studies and correlation with proteins identified in objective 01. To further define the proteins detected in objective 01 and the environmental stimuli controlling their expression, we will create global regulatory mutants for proteomic analysis. Growth of these mutants will be as described above.

03 07/2000 To examine the presence and levels of expression of proteins identified in objetives 01 and 02 in field and clinical isolates of EHEC. The studies thus far will utilise a single well defined clinical isolate of human origin. The need is to compare and contrast the protein profiles of this strain with those associated with EHEC 0157 isolated from a range of environments throughout the food chain. Also to assess the levels of expression of proteins of interest and where possible relate this to virulence.

04 12/2000 To identify putative virulence factors by peptide mass fingerprinting and de novo sequencing. It is anticipated that we will unambigously identify up to 200 gut induced proteins by state-of-the-art mass spectrometric techniques.

05 06/2001 To generate mutants of genes encoding putative virulence factors described in objective 04. The data generated in objective 01, 02 and 03 will indicate proteins likely to be involved gut colonisation; the sequence data from objective 04 will enable cloning of the relevant genes and their insertional inactivation to produce null mutants.

06 03/2002 To assess gut coloisation and persdistence of mutants in a suitable bovine model. Normal weaned calves of 6-8 wks of age will be dosed orally with pooled mutants (no more than three, each individually marked) and their persistence will be assessed in competition experiments.
Time-Scale and Cost
From: 1999

To: 2002

Cost: £237,995
Contractor / Funded Organisations
University - Southampton
Animal Health              
E.coli O157              
GM Non-Food              
Plants and Animals              
Fields of Study
Animal Health