IIBR has developed comprehensive tools and know-how for the global inspection of genomes, transcriptomes and proteomes of pathogenic microorganisms. These tools are being applied in diverse R&D areas, including vaccine development, drug discovery, pathogenesis/virulence studies, identification of specific and multiple-pathogen genetic markers and characterization of immune response.
Global inspection of pathogen genomes:
The availability of the genomic sequences of human pathogens, together with the development of computational tools for data mining, analysis and processing, allow for characterization of the complete repertoire of genes expressed by a given organism. The microbial genome research at the IIBR bioinformatics unit focuses on selection of various genes of interest (e.g., genes encoding for potential virulent factors, for secreted or membrane-associated proteins, for regulatory elements, and for species specific genes) from various pathogens.
The application of advanced methodologies together with high-power computer capabilities enable the researchers to perform a state-of-the-art, large-scale genomic analysis, which includes:
- DNA and protein sequence analysis
- Sequence similarity searches
- Motif, domain, and structure prediction
- Database mining and functional assignments
- Database construction, integration and management
- Cross-genome analysis and comparative genomics studies
- Immunoinformatics (T-cell and B-cell epitope mapping)
Identification of relevant genes through systematic computational whole genome screening is then followed by analysis of the encoded proteins through classical or high throughput functional in vitro and in vivo screening, employing immuno-PCR expression elements and DNA and polypeptide vaccine approaches.
Global inspection of gene expression by microarray analysis.
State-of-the-art functional genomics by microarray analysis is carried out aimed at depicting the response of the pathogen and host at various stages of infection. Functional profiling of the genes of interest is investigated in silico. Regulons (groups of co-regulated genes) identified by microarray analysis are individually interrogated by Quantitative Real-time RT-PCR.
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Global inspection of pathogen proteome:
The actual expression of the potential coding repertoire of the pathogen is tested by proteomics analysis, performed on microorganisms exposed to various environments (in vitro and in vivo).
The proteomic capability at IIBR allows for establishing characteristic protein maps and direct identification of proteins. Hundreds of cellular proteins from cultured pathogens are separated by high-resolution 2-dimensional electrophoresis, establishing a protein signature characteristic of the particular biological sample analyzed. MALDI-TOF mass spectrometry analysis of individual protein "spots", combined with direct peptide sequencing by tandem mass spectroscopy, serves for identification of proteins in genomic databank.
The arsenal of proteomic methods at IIBR includes fluorescent multiplex differential quantitative imaging and analysis of protein samples by DIGE (Differential Gel Electrophoresis). This approach is exploited for the selection of proteins possibly involved in the interaction of pathogenic bacteria with their host, as well as for identification of vaccine candidate antigens and strain specific biomarkers for early detection and also for development of diagnostic and forensic tools.
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