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

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.

Post-translational processing

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 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 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 CO Complete Microbial Genomes 112 Complete Microbial Genomes - Revised March 10, 2003 Archaea - 16 species Bacteria – 96 species

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

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

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

Iso- Electric Focusing Mol Mass

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

ExPASy Molecular Biology Server SWISS-2DPAGE Map Selection Escherichia coli( )

: P26427P 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 ) (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]562 Taxonomy Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia. [1] MAPPING ON GEL. MEDLINE= ; PubMed= ;[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: (1996).NCBIExPASyEBIIsraelJapan

The Mechanism of Plaque Formation

Plaque as a Biofilm

Growth Changes & Cellular Fractionation Growth of F. nucleatum by 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 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

Increasing solubility

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

Iso-electric focusing 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

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

pH 4 pH7 200 kDa 14.4 kDa 11cm IPG

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