Proteomics in Analysis of Bacterial Pathogens

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Proteomics in Analysis of Bacterial Pathogens
Tina Guina University of Washington, Seattle
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Outline
Postgenomic studies of Pseudomonas in context of
lung infection in patients with cystic
fibrosis Study of bacterial posttranslational
regulation by monitoring changes in protein
subcellular localization
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Pseudomonas aeruginosa and Cystic Fibrosis
Gram-negative environmental bacterium (soil,
water) Invades plants, animals causes disease
in immunocompromised humans and chronic lung
disease in cystic fibrosis patients Cystic
fibrosis (CF) most common genetic disease in
Caucasians caused by a mutation in chloride
channel CFTR Chronic Pseudomonas lung infection
is a major cause of morbidity in CF
patients Bacteria persist and multiply in lung
(up to 109 cfu/g of sputum)
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Environmental P. aeruginosa
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Environmental P. aeruginosa
Unknown Innate immune defect
CFTR-
PA colonization - ASYMPTOMATIC
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Environmental P. aeruginosa
Bacterial Adaptation
Unknown Innate immune defect
CFTR-
Innate Immune Selective Pressure
PA colonization - ASYMPTOMATIC
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Environmental Pseudomonas
Increased airway inflammation Resistance to
antimicrobials
Bacterial Adaptation Unique surface
modifications
Chronic Lung Disease
Unknown Innate immune defect
CFTR-
Innate Immune Selective Pressure
Increased bacterial burden - SYMPTOMATIC
PA colonization - ASYMPTOMATIC
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Chronic Lung Disease
Bacterial Adaptation
?
Increased bacterial burden - SYMPTOMATIC
PA colonization - ASYMPTOMATIC
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Intervention
Chronic Lung Disease
Bacterial Adaptation
?
Increased bacterial burden - SYMPTOMATIC
PA colonization - ASYMPTOMATIC
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Questions
Can we characterize stages of bacterial
adaptation to the lung ? Can we use
characteristics of these stages to develop assays
to predict CF patients clinical outcome ? Can
drugs be developed that would arrest adaptation
? Can Pseudomonas staging be used for therapy ?
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Approaches for Studying Pseudomonas Adaptation
in CF Lung

• Analysis of laboratory-adapted Pseudomonas
  strains grown under
• conditions that promote phenotypes typical to
  the clinical isolates
• Analysis of Pseudomonas clinical isolates from CF
  airway
• - serial isolates from young children with CF
• - isolates from patients with mild vs. severe
  disease symptoms
• Analysis of bacterial phenotypes morphology,
  surface properties,
• production of secreted factors
• Postgenomic analysis whole genome sequencing,
  genome typing,
• transcriptional profiling, protein expression
  profiling

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Analysis of Pseudomonas Clinical Isolates From
Young Children With CF

• Natural history study to determine infection and
  inflammation in young children, three centers in
  US
• Early isolates from 29 children, 4 to 36 months
  of age,
• 2 to 30 isolates for each patient
• Later isolates from 11/29 children enrolled into
  the original study, currently up to 9 years of
  age
• Isolates from upper airway (OP) and lower airway
  (BAL)

(Rosenfeld et al. 2001)
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Postgenomic Analysis of Pseudomonas in CF
Environmental isolates Clinical CF Isolates
Microarray Analysis
Proteomic Analysis
Genomic Analysis
Phenotypic Analysis
Bioinformatics
Identification of CF-unique Characteristics
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Pseudomonas Adapt to the Cystic Fibrosis Lung
Environment
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CF Isolate-Specific Characteristics Outer
Membrane LPS Modifications
1) Increased Antimicrobial Peptide Resistance
2) Increased Proinflammatory Signaling
Through Tlr4
LPS modifications are induced in - all early
isolates from infants with CF (as early as 4
months of age) - laboratory-adapted strain PAO1
during magnesium limitation and anaerobic growth
(Ernst et al. 1999, Hajjar et al. 2002)
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I. Adaptation to the CF Lung Is Genomic
Organization of Pseudomonas CF Infant and
Environmental Isolates Similar?
Whole genome analysis using DNA microarrays -
13 CF, 4 environmental, and 3 clinical non-CF
isolates - 38 common chromosomal islands
divergent or absent (N 1) when compared to
PAO-1 Results Suggest no selection of a
Pseudomonas subpopulation from the environment in
colonization of the CF airways.
(Ernst et al. 2003)
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II. Adaptation to the CF Lung Is Genomic
Organization of Longitudinal Pseudomonas CF
Isolates Similar?
Sequencing of parentally-related Pseudomonas
isolates from a CF patient
Isolates from 6 months to 8 years of age CF416 (6
months) 4.0 X coverage CF5296 (8 years) 4.0 X
coverage Results 40 point mutations/deletions
between early and late isolate
(Smith, Olson et al.)
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Analysis of 40 Chromosomal RegionsComparison of
Longitudinal CF Isolates
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III. Adaptation to the CF Lung Is There a Gene
Expression Pattern Unique to the Infant CF
Isolates?
Transcriptional (mRNA) profiling using DNA
microarrays
of patients (N5)

• CF-activated genes
• PA1290 probable transcriptional regulator 5
• PA5095 ABC transporter permease 5
• CF-repressed genes
• PA1008 bacterioferritin comigratory protein 5
• PA1244 hypothetical gene 5
• PA1708 popB - translocator protein 5
• PA1752 hypothetical gene 5
• PA2461 hypothetical gene 5

Results Mode of regulation for 7 genes is unique
to a subset of clinical isolates
(Ernst et al.)
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Cellular Protein Levels Do Not Always Correlate
With Levels of the Corresponding Gene Transcripts
Anaerobic regulation in PAO1 Postgenomic Analysis
Regulated Genes 209
Regulated Proteins 122
Quantified Proteins 553
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IV. Adaptation to the CF Lung Is There a
Protein Expression Pattern Unique to the Infant
CF Isolates?
Quantitative protein profiling of differentially
labeled whole cell protein
Strain/Condition A
Whole cell protein ICAT
mLC-MS/MS in silico analysis
Combine and proteolyze
Protein X in A Protein X in B
Strain/Condition B
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Pseudomonas aeruginosa Proteome Analysis
Regulation by Low Magnesium Stress Induces CF
isolate- Specific Surface Modifications
Laboratory-adapted Pseudomonas strain PAO-1
8 mM Mg2 CF-like phenotype
1 mM Mg2
Differential protein labeling
MS/in silico protein identification and
quantitative analysis
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Postgenomic Analysis of Pseudomonas During Mg
Limitation
Transcriptional Profiling 2250 (40) genes
expressed 650 genes regulated
Qualitative proteomic analysis 1331 proteins
identified Quantitative analysis (ICAT)
546 proteins quantified 76
proteins induced 69 proteins
repressed 50 correlation with transcriptional
profiling data
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Selected Proteins Induced During Growth of
Pseudomonas in Low Mg
Fold increase Conserved low
Mg stress-response proteins
two-component response regulator PhoP
10.3 magnesium transport ATPase
MgtA 5.8 MgtC homologue 4.0 CF-specific
surface modifications, resistance to
antimicrobial peptides PmrH homologue 2.8 PmrF
homologue 2.3 PmrI homologue 6.1
Enzymes for synthesis of quorum sensing signal
PQS PA0996, PA0997, PA0998, PA0999
1.5 - 2.0
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Quorum Sensing Bacterial Intercellular
Communication Via Small Signaling
Molecules
C4-HSL C12-HSL PQS
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Quorum Sensing Secretion of Toxins, Virulence
Factors
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Quorum Sensing Biofilm, Antibiotic Resistance
AB
AB
AB
AB
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PQS
b-keto-decanoic acid
Butyryl-ACP
C4-HSL
RhlI
S-adenosylmethionine (SAM)
Acyl-homoserine lactones
LasI
C12-HSL
Dodecanoyl-ACP
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PQS Production by Laboratory Strain of
Pseudomonas Is Increased During Growth in Low Mg
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High Levels of PQS Are Produced by CF Pseudomonas
Isolates Grown in High Mg
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PQS Production by Pseudomonas Isolates
From Infants with Cystic Fibrosis
190 isolates from 25 children up to 3 years of
age analyzed for PQS production Bacteria were
grown in medium with high Mg2
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PQS Production by Isolates from Infants with CF
Similar to CF-specific surface modifications,
most Pseudomonas clinical isolates from young
children with CF produce high PQS levels
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Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Environmental Pseudomonas
Lung Disease
Bacterial Adaptation

• Alginate/mucoidy
• Auxotrophy

• surface modifications
• Increased PQS
• (biofilm, virulence,
• antibiotic resistance)

Innate Immune Selective Pressure
Increased bacteria - SYMPTOMATIC
PA colonization-ASYMPTOMATIC
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Natural History Study Infant patients
isolates, 8-yr vs. early isolates
Mild vs. Severe Study
Genome sequencing DNA Microarray, Proteomic
Analyses To Identify Additional Markers
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Natural History Study Infant patients
isolates, 8-yr vs. early isolates
Mild vs. Severe Study
Genome sequencing DNA Microarray, Proteomic
Analyses To Identify Additional Markers
Develop tests for broad screening of large CF
populations to validate markers specific for
Pseudomonas adaptation
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Natural History Study Infant patients
isolates, 8-yr vs. early isolates
Mild vs. Severe Study
Genome sequencing DNA Microarray, Proteomic
Analyses To Identify Additional Markers
Develop tests for broad screening of large CF
populations to validate markers specific for
Pseudomonas adaptation
Correlate with the disease outcome
Disease outcome prediction Vaccine/drug
development
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Bacterial Posttranslational Regulation
Study Pseudomonas Envelope Remodeling During
Growth In Low Mg
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Gram-negative Bacterial Membrane
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Magnesium Stabilizes Gram-negative Outer Membrane
O
O
O
O
O
O
O
P
HO
O
P
-O
O
O
O
O
O
O
NH
NH
OH
HO
O
Lipid A
OH
O
HO
O
O
O-
O
O
P
OH
O
O
P
O
NH
Mg
O
NH
O
O
O
O
O
OH
O
O
OH
OH
O
OH
OH
OH
Growth in low magnesium
Membrane stress Membrane remodeling
Growth in low magnesium
Membrane stress Membrane remodeling
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Gram-Negative Envelope Remodeling During
Magnesium Limitation
Alteration in outer membrane proteins
Lipid A acylation
Proteases
PagC PagN
PagP
PgtE
OprH
OM
IM
PmrF
MgtA
MgtC
PhoQ
PmrB
Small molecule transport Nutrient acquisition
Environmental sensing
LPS modifications
Modulation and resistance to the host innate
immune defense
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ICAT Analysis of Pseudomonas Membrane and Whole
Cell Protein During Mg Limitation
Pseudomonas strain PAO-1
8 mM Mg2 membrane
8 mM Mg2 whole cell
1 mM Mg2 membrane
1 mM Mg2 whole cell
ICAT analysis
ICAT analysis
163 proteins
486 proteins
106 proteins were quantified in both
experiments Compare relative protein levels in
membrane vs. in whole cell
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Pseudomonas Metabolic Enzymes and Protein
Translation Machinery Concentrate at the Membrane
During Growth in Low Magnesium
FI membrane/FI whole
cell Energy metabolism succinate dehydrogenase
(A, B subunits) 1.6 - 2.4
2-oxoglutarate dehydrogenase (E1 subunit)
SucA 3.0 phosphoenolpyruvate synthase 3.1 ATP
synthase subunits 1.5
1.8 cytochrome c5 1.6 GroEL
chaperone 3.0 Translation machinery 30S
ribosomal proteins (S2, S4, S13, S5) 1.5
1.8 elongation and ribosome recycling factor
G 2.0
FI fold induction
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Bacterial ribosomal fractions
Cytoplasmic
Membrane-associated
Soluble protein synthesis
Membrane and secreted protein synthesis
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Bacterial ribosomal fractions
Low Mg2 membrane stress
Cytoplasmic
Membrane-associated
Low Mg2 membrane stress
Soluble protein synthesis
Increased membrane and secreted protein synthesis
Formation of stress-induced multienzyme complexes
Membrane lipid and protein remodeling Decreased
membrane permeability Resistance to various
antimicrobials
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Proteomic Analysis in Studying Bacterial
Pathogens Summary

• Advantages
• Useful tool for analysis of bacteria for which
  there are little
• or no genetic tools available
• Analysis of posttranscriptional regulation
• Analysis of protein compartmentalization,
  posttranslational regulation
• Disadvantages
• Still expensive, time/labor intensive
• Need for dishwasher-like technology, for
  improved data analysis software

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Acknowledgements
Manhong Wu Robert Ernst Hai Nguyen Sam
Miller Jane Burns Eric Smith Maynard Olson
David Goodlett Sam Purvine Ruedi Aebersold Jimmy
Eng
CFF NIH