Research, Technology and Development I

RTD Leader: Francois Lefevre, INRA, France

 

WP1 Bioimaging platform, Optical Tweezers and Convergent Evolution

WP Leader: Henrik Siegumfeldt, LIFE, Denmark

This WP will establish techniques for studying matrix interactions for pathogens and protective cultures, to be used in WP8, WP9 and WP11. The techniques are bioimaging, Fluorescent Ratio Imaging Microscopy (FRIM), Optical Tweezers and convergent evolution.

For in situ time-lapse studies of interactions between individual microbial cells and solid matrices, the FRIM technique for intracellular pH (pHi) analysis will be applied.

The Optical Tweezers consists of a strongly focused laser beam, which has the ability to catch and hold particles in a size range from nm to µm. It will be used to determine the minute forces between a cell and a solid surface can be determined.

A standardised chamber system for the investigation of solid surfaces will be constructed and used for both FRIM and the Optical Tweezers. The chamber system will be created in such a way that a plane surface inside the chamber can be coated to mimic feeds and foods and representative contact surfaces. Furthermore the chamber will be constructed to allow for controlled exposure to liquid stresses such as acids, bases, ethanol and bacteriocins; the composition of the atmosphere (CO2) as well as heating and cooling.

Peptide convergent evolution using phage display will be used for identification of proteins that bind to targets of interest. The targets of interest are epithelial cells and food contact surfaces, whilst the native proteins we seek to identify are microbial adhesion proteins. The technique relies on bioinformatics and the availability of comprehensive microbial genome sequences. These are available for both bio-protective (Lactobacillus plantarum and Bifidobacterium longum) and pathogenic species (C. jejuni, E. coli (STEC) and L. monocytogenes) as well as S. cerevisiae.

The selection of target molecules and how they are presented during phage display screening requires careful consideration. Where food contact surfaces are targets, surfaces of stainless steel and selected polymers will be used. For the identification of proteins involved in adhesion to epithelial cells, phage displayed peptides will be screened against the functional cells models. The adhesion molecules identified will be used for design of prebiotics which can prevent pathogen adhesion.

 

The WP will include the following tasks:

Task 1.1        Adaptation of Fluorescence Ratio Imaging Microscopy (FRIM) for studies of microbial interaction with food matrices and food contact surfaces.

Task 1.2        Develpoment of a chamber system for controlled exposure to different environments

Task 1.3        Development of Optical Tweezers studies of attachment and detachment of single microbial cells to food matrices and food contact surfaces.

Task 1.4        Development of convergent evolution for identification of surface molecules responsible for microbial attachment to food matrices and food contact surfaces.

 

Partner 1, Partner 2, Partner 3 and Partner 10 will be involved in this WP.

 

 

WP2 Functional Genomic Platform

WP Leader: Lene Jespersen, LIFE, Denmark

This WP will establish methods of transcriptomics and proteomics for pathogens and their interactions with solid matrices (WP8) and intestinal epithelial cells of pigs, chickens and ruminants (WP9). It will adapt transcriptomics for the functional cell model developed in WP3. The results obtained will also be used for production of DNA-arrays (WP5) and for development of culture independent techniques (WP4).

 For C. jejuni, E. coli (STEC), L. monocytogenes and S. cerevisiae gene expressions studies related to adhesion, stress and virulence traits will be carried out. Genes involved in formation of adhesion proteins for these bacteria will be searched for by use of bio-informatics, databases and convergent evolution (WP1). Information on adhesion, stress and virulence genes will be made available for development of culture independent technique (WP4) and microarrays (WP5).

For S. cerevisiae studies will be carried out to elucidate how matrix interaction, as mentioned above, and environmental factors relevant to the food chain can influence the transcription of genes playing a role during adhesion and invasive growth and indicate how yeast normally regarded as safe may turn into emerging pathogens. S. cerevisiae is fully sequenced and by use of already available microarray techniques genome-wide transcriptional studies will be performed and genes related to adhesion, invasive growth and stress will be identified for clinical isolates of S. cerevisiae. Information on stress genes in S. cerevisiae and information on genes involved in adhesion, invasion and stress will be made available for development and production of microarrays (WP5) and for the application of culture independent methods (WP4).

For protective and probiotic bacteria, genes expressing desired technological characters such as multifunctional protective cultures against pathogenic bacteria (e.g. Listeria and Campylobacter) will be identified in selected fully genome sequenced protective lactic acid bacteria and bifidobacteria. Together with highly conserved stress genes, the genes will be made available for construction of DNA-arrays (WP5). Genes responsible for adhesion will be identified by use of convergent evolution (WP1)

For the OTA producing fungi, information on genes involved in synthesis of OTA will be made available for production of DNA-arrays. Genes that are differentially expressed during ochratoxin biosynthesis will be characterized and sequenced. For that purpose P. nordicum will be grown in media, which are restrictive or permissive for ochratoxin production. Differentially expressed genes under permissive conditions will be identified and compared to public gene databases. Sequences with homology to putative ochratoxin biosynthetic enzymes will be used for generation of micro arrays specific for ochratoxigenic genes (WP5).

For studies of host-pathogen interactions the macro and microarrays corresponding to the animal species studied i.e. pig, chicken and ruminant will be available from INRA as part of the French AGENAE project (Analyze du Génome des Animaux d´Elevage, Analysis of the Genome of Farm Animals). They will be adapted to the functional cell model developed in this project (WP9).

The WP will include the following tasks:

Task 2.1        Transcriptomics for C. jejuni, E. coli (STEC) and L. monocytogenes.

Task 2.2        Transcriptomics and proteomics for S. cerevisiae.

Task 2.3        Transcriptomics for protective and probiotic cultures.

Task 2.4        Transcriptomics for ochratoxin A producing filamentous fungi

Task 2.5        Adaptation of transcriptomics for the functional cell model developed in WP3.

 

Partner 1, Partner 4 and Partner 5 will be involved in this WP.

 

 

WP3 Functional Cell Model

WP Leader: Avrelija Cencic, UM, Slovenia

This WP will establish functional cell models based upon intestinal enterocyte cell lines of porcine, ruminant and poultry origin, respectively. As the primary source of TBEV infection by oral route seems to be ruminant milk, a ruminant mammary epithelial cell line will be established. The models will be used for adaptation of transcriptomics to the three cell lines (WP2) and for studying host-pathogen interactions and the influence of protective and probiotic bacteria on their interactions (WP9).

The functional mammalian and avian cell models to be established will mimic the intestinal tract of ruminant, chicken and pigs. Cell lines will be used for the functional cell model. The most common cells in the intestinal epithelium are enterocytes. They are polarised cells with a distinct apical and basolateral cytoplasmatic membrane. Enterocytes are separated from one another by junctions, which form a tight epithelial barrier. Good in vitro cell models must satisfy two basic requirements: 1) retention of tissue characteristics to support interpretation of results for the in vivo situation and 2) availability and easy handling for high-throughput testing. Such models will be established

The WP includes the following tasks:

Task 3.1        Development of a porcine cell model

Task 3.2        Development of a ruminant cell model

Task 3.3        Development of a chicken cell model

 

Partner 4 and Partner 7 will be involved in this WP.