Bacterial Physiology Bacterial Physiology A Proteomic Approach to A Proteomic Approach to

Oral DiseasesOral Diseases

Peter ZilmMicrobiology LaboratoryDental SchoolThe University of Adelaide

Genomics versus ProteomicsGenomics 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 willlikely lead to the understanding of fundamental processes such as cell development and growth, cell differentiation,Cell signaling and cell death.

Post-translationalprocessing

Aeropyrum pernix K1Archaeoglobus fulgidus Halobacterium sp. Methanobacterium thermoautotrophicum Methanococcus jannaschii Methanopyrus kandleri AV19 Methanosarcina acetivorans str.C2A Methanosarcina mazei Goe1 Pyrobaculum aerophilumPyrococcus abyssiPyrococcus furiosusPyrococus horikoshii Sulfolobus solfataricusSulfolobus tokodaiiThermoplasma acidophilumThermoplasma 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 26695 Lactococcus lactis subsp. lactis Mycobacterium tuberculosis H37Rv Mycobacterium leprae Mycoplasma pneumoniae Neisseria meningitidis MC58 Pasteurella multocida

Porphyromonas gingivalisPseudomonas 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 Genomes112 Complete Microbial Genomes - Revised March 10, 2003

Archaea - 16 speciesArchaea - 16 species Bacteria – 96 speciesBacteria – 96 species

Proteomic ApplicationsProteomic Applications

APPROACHESAPPROACHESProfilingProfiling

FunctionalFunctionalStructuralStructural

Characterisation of specific sub-sets ofCharacterisation of specific sub-sets of the proteomethe proteome.

• Regulons or stimulons• Macromolecular complex or sub-cellular compartment• Immunogenic proteins• Problems with specific post-translational modification

Global CharacterisationGlobal Characterisationof proteomeof proteome

State1(Healthy)

State 2 (disease)

Profile 1 Profile 2

Perturbation (signal)

Step by step ProteomicsStep by step Proteomics

Sample preparation

2D-P.A.G.E

Image analysis

Spotidentification

Data analysisProtein

identification

Spot cutting &Mass spec.

analysis

PublishPublishresultsresults

Iso- Electric FocusingIso- Electric Focusing

• In the 1st dimension, proteins are separated according to their charge.

2-Dimensional Gel Electrophoresis2-Dimensional Gel Electrophoresis

P.A.G.E.P.A.G.E.

• In the 2In the 2ndnd dimension proteins are separated according to their relative 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 withinSince the 1990’s the position of proteins within gels and their position within the pH gradientgels 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 FocusingIso- Electric Focusing

Mol MassMol Mass

Protein required for detection

0

1000

2000

3000

4000

5000

ponceauS

amidoblack

CBB India ink colloidalsilver

stain

ng

pro

t

Protein Staining TechniquesProtein 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(4.5-5.5)

: P26427

1 protein has been found in the clicked spot (2D-0015D5): View entry in original SWISS-2DPAGE formatEntry nameAHPC_ECOLI

Primary accession numberP26427

Entered in SWISS-2DPAGE inRelease 02, August 1995

Last modified inRelease 16, May 2003DescriptionAlkyl 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 SF0524FromEscherichia coli. [TaxID: 562]TaxonomyBacteria; 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).

The Mechanism of Plaque FormationThe Mechanism of Plaque Formation

Plaque as a Biofilm

Growth Changes & Cellular FractionationGrowth 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 preparationSample 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

Increasingsolubility

Growth Condition Extract 1 Extract 2 SDS

BHI 8.7 0.53 1.27Oxygen 4.8 0.41 0.57Control 4.8 0.26 0.11

CHX 8.7 0.71 0.1239oC 7.7 0.53 0.98

pH 8.0 8.3 0.64 0.26

Protein mg/ml

Protein recovery-sequential protein extraction of the cell envelope of F. nucleatum ATCC 10953

Extract 1 - 8M Urea, 50mM DTT, 4% CHAPSExtract 2 – 7M Urea, 50mM DTT, 2M Thiourea, 4% CHAPS

Iso-electric focusingIso-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 sizepH range and IPG size

pH 3 pH 10

pH 3 pH10

11cm IPG

7cm IPG

pH 4 pH7200 kDa

14.4 kDa

11cm IPG

39oC BHI

CHXpH 8.0 Control

Master11cm

Cytosolic fraction of Cytosolic fraction of F. nucleatumF. nucleatum pH 4-7 pH 4-7