Comparative proteomics of bacterial pathogens

1. Comparative proteomics of bacterial pathogens (Proteomics 2001, 1, 461-472) M.G.L 2001-20487 Kim Sang Hoon

2. Contents • Introduction • Proteomics approaches • Reference mapping • Technological limitations and alternative technologies • Sub-cellular fractionation • Membrane protein, Extracellular protein, Basic protein • Metabolic pathway analysis • Application • Conclusion

3. Introduction • Monitoring gene expression at the mRNA level • Advantages – easily scalable to cover near-to-total prokaryotic and simple eukaryotic genomes • Disadvantages – not completely represent the post-transcriptional, co-translational, and degradative modification of proteins

4. Introduction • Transcriptomics • Used to identify specific response of infection or drug treatment. • The global analysis of protein expression from a genome under a given set of condition is best summerizd by the term ‘proteomics’. • Mycobacterium tuberculosis. Helicobacter pylori, Chlamydia.. etc.

6. Proteomics approaches • Reference mapping-1 • Organism grown under average conditions can be a useful starting point for comparative proteomics. • Understanding the entire genetic complement of a bacterium has been shown to be essential for beginning to predict their metabolism and physiology. • Provides a central point from which all exp -eriment strategies can then be compared

7. Proteomics approaches • Reference mapping-2 • 2-DE combined with MS will account for 30-65% of the genome set. • In most cases the number of identified genes remains lower than 10% of the total genome • Alternative technology => isotope-coded affinity tags(ICATs), monoclonal antibody • Used narrow-range pH gradient

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9. Helicobacter pylori 26695: Pseudo-2D Gel http://www.tigr.org/tigr-scripts/CMR2/Pseudo2DGel.spl?db_data_id=61

10. Proteomics approaches • Technological limitations and alternative technologies • An inability to solubilise and separate highly hydrophobic proteins prior to IEF • The lack of suitable buffers to reproducibly separate very basic proteins • The acrylamide pore size of both the first dimension IPG strip and second dimension slab gel excluding high mass proteins and protein complexes

11. Sub-cellular fractionation • Membrane protein • Provides a benchmark for pathogenicity, antibiotic resistance, therapeutic targets. • Hydrophobic region and transmembrane spanning region lead to difficulties in solubilising. • In silico approaches – predict membrane protein

13. Sub-cellular fractionation • Extracellular protein • Mapping of whole cell protein lysates and extracellular fractions from these species allows for the characterisation of specific pathogenicity factors that can be correlated with phenotype and thus provide subsets of proteins suitable for further analysis • Difficult due to contamination protein

14. Sub-cellular fractionation • Basic protein • Annotation of such molecules remains problematic. • Denature (5M urea, 0.1% CHAPS)- separate complex mixtures into two fractions (pI > 9.5, pI < 9.5) • Account for less than 10% of the total protein

16. Functional pathway analysis • Nearest functional neighbour analysis • proteins are predicted to be nearest functional neighbours where the product of an initial protein reaction is the substrate for a following reaction and one of these proteins has been identified from proteomic analyses. • This process allows for the prediction of further proteins based on the identification of up-or down-stream neighbours

17. Application • Strain comparisons • be compared at both the genomic and proteomic levels for genes and gene-products that correlate with a known phenotype. • This approach has been successful in determining potential markers in Neisseria meningitidis, Mycobacterial species, H,pylori

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19. Application • Environmental influences • Subtractive analysis or differential display of protein expression can be utilised to decipher pathways associated with any given set of conditions. • Such responses are particularly important for pathogenic bacteria within the host, as enviornmental sensing is necessary for the production of cell surface pathogencity, factors, a variety of toxins.

20. Application • Genetic modification • Proteins with altered cellular levels may therefore be those that interact with the given target gene-product. • The analysis of global protein expression in response to genetic manipulation can provide additional information with respect to protein function in association with altered cellular phenotypes.

21. Conclusion • Technical improvements allowing high-throughput characterisation of microbial proteomes is leading to rapid developments of novel therapeutics and diagnostics against bacterial pathogens. • As such proteomics is now in a position to define regulatory and stimulatory networks associated with in vivo biological processes in pathogenic and nonpathogenic microbial species.