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Bacterial Physiology A Proteomic Approach to Oral Diseases

Bacterial Physiology A Proteomic Approach to Oral Diseases. Peter Zilm Microbiology Laboratory Dental School The University of Adelaide. Genomics versus Proteomics. Post Genomic era - Reading of the human genome sequence.

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Bacterial Physiology A Proteomic Approach to Oral Diseases

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  1. Bacterial Physiology A Proteomic Approach to Oral Diseases Peter Zilm Microbiology Laboratory Dental School The University of Adelaide

  2. Genomics versus Proteomics • Post Genomic era- Reading of the human genome sequence • Relatively few medical breakthroughs derived from genetic research • - Can cellular processes be understood by screening genomes? • - The organisation and timing of cellular events is not a projection • of the genome and its transcription. • Proteomics - relies on genomics to facilitate protein identification • - which genes are important and under which circumstances • combination of proteomic and genomic information will • likely lead to the understanding of fundamental processes • such as cell development and growth, cell differentiation, • Cell signaling and cell death.

  3. Post-translational processing

  4. Agrobacterium tumefaciens Bacillus subtilis Bifidobacterium longum Borrelia burgdorferi Brucella suis Buchnera aphidicola Campylobacter jejuni Caulobacter crescentus Chlamydophila pneumoniae Chlamydia trachomatis Chlorobium tepidum TLS Clostridium perfringens Clostridium tetani E88 Corynebacterium efficiens YS-314 Escherichia coli K12 Fusobacterium nucleatum Haemophilus influenzae Helicobacter pylori 26695 Lactococcus lactis subsp. lactis Mycobacterium tuberculosis H37Rv Mycobacterium leprae Mycoplasma pneumoniae Neisseria meningitidis MC58 Pasteurella multocida Porphyromonas gingivalis Pseudomonas aeruginosa Rickettsia conorii Salmonella typhimurium LT2 Salmonella typhi Shigella flexneri Staphylococcus aureus N315 Staphylococcus epidermidis Streptococcus mutans UA159 Streptococcus pneumoniae Streptococcus pyogenes Streptomyces coelicolor Treponema pallidum Tropheryma whipplei Thermotoga maritima Ureaplasma urealyticum Vibrio cholerae Vibrio vulnificus Xanthomonas campestris Wigglesworthia brevipalpis Xanthomonas citri Xylella fastidiosa Temecula1 Yersinia pestis CO92 112 Complete Microbial Genomes - Revised March 10, 2003 Bacteria – 96 species Archaea - 16 species Aeropyrum pernix K1 Archaeoglobus fulgidus Halobacterium sp. Methanobacterium thermoautotrophicum Methanococcus jannaschii Methanopyrus kandleri AV19 Methanosarcina acetivorans str.C2A Methanosarcina mazei Goe1 Pyrobaculum aerophilum Pyrococcus abyssi Pyrococcus furiosus Pyrococus horikoshii Sulfolobus solfataricus Sulfolobus tokodaii Thermoplasma acidophilum Thermoplasma volcanium

  5. Global Characterisation of proteome • Characterisation of specific sub-sets of • the proteome. • Regulons or stimulons • Macromolecular complex or sub-cellular • compartment • Immunogenic proteins • Problems with specific post-translational • modification State1 (Healthy) State 2 (disease) Profile 1 Profile 2 Proteomic Applications APPROACHES Profiling Functional Structural Perturbation (signal)

  6. Sample preparation Publish results 2D-P.A.G.E Data analysis Protein identification Image analysis Spot cutting & Mass spec. analysis Spot identification Step by step Proteomics

  7. 2-Dimensional Gel Electrophoresis Iso- Electric Focusing • In the 1st dimension, proteins are separated according to their charge. • Electrophoretic migration is dependent upon • pH charge dependence and “iso-electricity. • Since the 1990’s the position of proteins within • gels and their position within the pH gradient • could be correlated with the amino acid • composition of polypeptides. P.A.G.E. • In the 2nd dimension proteins are separated according to their relative • mass. • Thousands of proteins can be displayed in a single experiment.

  8. Iso- Electric Focusing Mol Mass

  9. Protein Staining Techniques • Sensitive protein identification methods exist which are compatible with • the resolving power of 2D-PAGE.

  10. ExPASy Molecular Biology Server SWISS-2DPAGE Map Selection Escherichia coli(4.5-5.5)

  11. : P26427 1 protein has been found in the clicked spot (2D-0015D5): View entry in original SWISS-2DPAGE format Entry name AHPC_ECOLI Primary accession number P26427 Entered in SWISS-2DPAGE in Release 02, August 1995 Last modified in Release 16, May 2003 Description Alkyl hydroperoxide reductase C22 protein (EC 1.6.4.-) (SCRP-23) (Sulfate starvation-induced protein 8) (SSI8). Gene name(s) AHPC OR B0605 OR C0694 OR Z0749 OR ECS0644 OR SF0524 From Escherichia coli. [TaxID: 562] Taxonomy Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia. [1]   MAPPING ON GEL. MEDLINE=96314059; PubMed=8740179;[NCBI, ExPASy, EBI, Israel, Japan]Pasquali C., Frutiger S., Wilkins M.R., Hughes G.J., Appel R.D., Bairoch A., Schaller D., Sanchez J.-C., Hochstrasser D.F.; "Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2DPAGE database."; Electrophoresis 17:547-555(1996).

  12. The Mechanism of Plaque Formation

  13. Plaque as a Biofilm

  14. Growth Changes & Cellular Fractionation • Growth of F. nucleatumby continuous culture- maintain growth parameters while changing a • single factor of interest. • Growth conditions examined – growth rate • growth temperature • redox potential • growth pH • presence of chlorhexidine (antimicrobial) • nutrient availability • biofilm growth • Sample preparation- • a) consideration of mol. Wt. and pI. • b) reduce the complexity of the protein mixture, (cytoplasmic and • cell envelope). • c) degradation of proteins by proteases • d) removal of nucleic acids • e) staining- determined by amount of protein • f) protein contamination

  15. Increasing solubility

  16. Protein recovery-sequential protein extraction of the cell envelope of F. nucleatum ATCC 10953 Extract 1 - 8M Urea, 50mM DTT, 4% CHAPS Extract 2 – 7M Urea, 50mM DTT, 2M Thiourea, 4% CHAPS

  17. Iso-electric focusing- considerations for the novice • salt, protein solubility and ampholyte concentration • What size format? – 7cm, 11cm, 17cm • pH range – 10 possible • Protein concentration during rehydration. • Active or passive rehydration

  18. pH range and IPG size pH 3 pH 10 11cm IPG pH 3 pH10 7cm IPG

  19. pH 4 pH7 11cm IPG 200 kDa 14.4 kDa

  20. Cytosolic fraction of F. nucleatum pH 4-7 Master 39oC BHI 11cm pH 8.0 Control CHX

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